WO2013192543A2 - Enzymes and methods for styrene synthesis - Google Patents

Enzymes and methods for styrene synthesis Download PDF

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Publication number
WO2013192543A2
WO2013192543A2 PCT/US2013/047098 US2013047098W WO2013192543A2 WO 2013192543 A2 WO2013192543 A2 WO 2013192543A2 US 2013047098 W US2013047098 W US 2013047098W WO 2013192543 A2 WO2013192543 A2 WO 2013192543A2
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Prior art keywords
seq
cinnamic acid
styrene
acid decarboxylase
host cell
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PCT/US2013/047098
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French (fr)
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WO2013192543A3 (en
Inventor
Mohammad Wadud BHUIYA
Hui Chen
Xianpeng CAI
Jixiang Han
Xiaodan Yu
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Phytogene, Inc.
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Application filed by Phytogene, Inc. filed Critical Phytogene, Inc.
Priority to EP13806939.8A priority Critical patent/EP2864491B1/en
Priority to BR112014031942A priority patent/BR112014031942A2/en
Priority to CN201380033044.8A priority patent/CN104487581B/en
Priority to DK13806939.8T priority patent/DK2864491T3/en
Priority to US14/409,600 priority patent/US20150337336A1/en
Priority to ES13806939T priority patent/ES2709217T3/en
Priority to LTEP13806939.8T priority patent/LT2864491T/en
Publication of WO2013192543A2 publication Critical patent/WO2013192543A2/en
Publication of WO2013192543A3 publication Critical patent/WO2013192543A3/en
Priority to US15/705,361 priority patent/US10752921B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/002—Preparation of hydrocarbons or halogenated hydrocarbons cyclic
    • C12P5/005—Preparation of hydrocarbons or halogenated hydrocarbons cyclic aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01024—Phenylalanine ammonia-lyase (4.3.1.24)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01005—Phenylalanine ammonia-lyase (4.3.1.5)

Definitions

  • the subject technology generally relates to enzymes and methods for biosynthesis of styrene.
  • Styrene (vinyl benzene) is an organic compound with a chemical formula of CsH 8 .
  • This cyclic hydrocarbon is a colorless, oily liquid that evaporates easily and has a sweet rubber-like smell. At higher concentrations, styrene confers a less pleasant odor.
  • Styrene is named after the styrax trees (Styrax platanifolius) from which sap (a type of benzoin resin) can be extracted. Low levels of styrene occur naturally in several plant species. A variety of foods such as fruits, vegetables, nuts, beverages, and meats also contain styrene.
  • styrene is the precursor to polystyrene and several copolymers.
  • the presence of the vinyl group allows styrene to polymerize. Approximately 15 billion pounds are produced annually.
  • the production of styrene in the United States increased dramatically during the 1940s, when it was popularized as a feedstock for synthetic rubber.
  • ABS acrylonitrile butadiene styrene
  • SBR styrene-butadiene
  • SIS styrene-isoprene-styrene
  • S-EB-S styrene-ethylene/butylene-styrene
  • S-DVB styrene-divinylbenzene
  • SAN styrene-acrylonitrile resin
  • Styrene is produced in industrial quantities mostly from ethylbenzene, which is in turn prepared on a large scale by alkylation of benzene with ethylene. It is one of the most important petrochemical products. There are several methods to produce styrene. Dehydrogenation of ethylbenzene is the most common way of production. Ethylbenzene is mixed in the gas phase with 10-15 times its volume in high-temperature steam, and passed over a solid catalyst bed. Most ethylbenzene
  • dehydrogenation catalysts are based on iron(III) oxide, promoted by several percent potassium oxide or potassium carbonate. Steam serves several roles in this reaction. It is the source of heat for powering the endothermic reaction, and it removes coke that tends to form on the iron oxide catalyst through the water gas shift reaction. The potassium promoter enhances this decoking reaction. The steam also dilutes the reactant and products, shifting the position of chemical equilibrium towards products.
  • a typical styrene plant consists of two or three reactors in series, which operate under vacuum to enhance the conversion and selectivity. Typical per-pass conversions are ca. 65% for two reactors and 70-75% for three reactors. Selectivity to styrene is 93-97%>.
  • the main byproducts are benzene and toluene. Because styrene and ethylbenzene have similar boiling points (145 and 136 °C, respectively), their separation requires tall distillation towers and high return/reflux ratios. At its distillation temperatures, styrene tends to polymerize. To minimize this problem, early styrene plants added elemental sulfur to inhibit the polymerization. During the 1970s, new free radical inhibitors consisting of nitrated phenol-based retarders were developed. More recently, a number of additives have been developed that exhibit superior inhibition against polymerization.
  • the subject technology generally relates to the biosynthesis of styrene.
  • Some embodiments of the subject technology are based, in part, on the recognition that phenylalanine can be converted to styrene by a two-step pathway of deamination and decarboxylation, with trans-cinnamic acid (tCA) as the intermediate.
  • Two types of enzymes are directly involved in this process, phenylalanine ammonia lyase (PAL), which converts phenylalanine to tCA, and cinnamic acid decarboxylase, which coverts tCA to styrene.
  • PAL phenylalanine ammonia lyase
  • cinnamic acid decarboxylase which coverts tCA to styrene.
  • Host cells expressing these two types of enzymes can be cultured in a bioreactor to produce styrene from renewable substrates such as glucose.
  • the subject technology relates to a fusion protein comprising: (a) a first domain that comprises a phenylalanine ammonia lyase, and (b) a second domain that comprises a cinnamic acid decarboxylase.
  • the phenylalanine ammonia lyase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces .
  • the phenylalanine ammonia lyase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof.
  • the cinnamic acid decarboxylase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
  • the cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; and a functional fragment or variant thereof.
  • the cinnamic acid decarboxylase may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38.
  • a functional variant of a cinnamic acid decarboxylase may comprise an amino acid sequence that is about 85%, about 90%, about 95%o, about 96%o, about 97%o, about 98%o, about 99%o, or even 100% identical to any one of SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38.
  • a functional variant of a cinnamic acid decarboxylase can comprise: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
  • the cinnamic acid decarboxylase can comprise a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, or a combination thereof.
  • the mutant cinnamic acid decarboxylase can comprise a mutation at an amino acid residue position corresponding to one of the following positions: 175, 190, 193 of SEQ ID NO:8, and a combination thereof.
  • the cinnamic acid decarboxylase can comprise a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8 selected from: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441, and a combination thereof.
  • an amino acid residue at one the of the following positions: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441 of SEQ ID NO:8 can be substituted with another amino acid residue.
  • the mutant cinnamic acid decarboxylase comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 175, 190, 193, and a combination thereof.
  • the fusion protein of the subject technology further comprises a linker covalently linking the first domain and the second domain.
  • the linker of the fusion protein described herein may be a peptide linker, e.g., a peptide linker comprising 2 to 15 amino acids.
  • Peptide linkers can include those shown in Table 1, for example.
  • nucleic acids encoding the fusion proteins described herein
  • a vector comprising the nucleic acid encoding the fusion protein
  • host cells comprising the vector described herein.
  • the subject technology relates to a method for producing styrene comprising (a) contacting a host cell with a fermentable substrate (preferably carbon substrate or nitrogen substrate), the host cell comprises a fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase, and (ii) a second domain that comprises a cinnamic acid decarboxylase; and (b) culturing the cell in a culture medium for a time sufficient to produce styrene.
  • the method can further comprise harvesting styrene from the cell culture. Any one of the fusion proteins described herein can be used to produce styrene.
  • the subject technology relates to a cinnamic acid decarboxylase comprising (a) any one of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38; (b) an amino acid sequence that is about 85%, about 90%>, about 95%o, about 97%, about 98%, about 99%, or even 100% identical to any one of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38, with the proviso that said
  • the cinnamic acid decarboxylase comprises: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
  • the cinnamic acid decarboxylase comprises any one of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38.
  • the subject technology also relates to a mutant cinnamic acid decarboxylase comprising a mutation at an amino acid residue position corresponding to one of the following positions: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, and a combination thereof.
  • the mutant cinnamic acid decarboxylase can comprise a mutation at an amino acid residue position corresponding to one of the following positions: 175, 190, 193 of SEQ ID NO:8, and a combination thereof.
  • the mutant cinnamic acid decarboxylase comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441, and a combination thereof.
  • the mutation is a substitution.
  • the mutant cinnamic acid decarboxylase comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 175, 190, 193, and a combination thereof.
  • nucleic acids encoding the cinnamic acid decarboxylases described herein, host cells comprising the cinnamic acid decarboxylases described herein, and host cells comprising the nucleic acids described herein.
  • the subject technology also relates to a method for the production of styrene comprising: (a) contacting a host cell with a fermentable substrate (preferably carbon substrate or nitrogen substrate), the host cell comprising (i) a phenylalanine ammonia lyase as described herein; and (ii) a cinnamic acid decarboxylase as described herein; and (b) culturing the cell in a culture medium for a time sufficient to produce styrene.
  • a fermentable substrate preferably carbon substrate or nitrogen substrate
  • the subject technology also relates to a host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase as described herein; (b) a recombinantly expressed cinnamic acid decarboxylase as described herein; and (c) a recombinantly expressed membrane-bound transporter as described herein.
  • the phenylalanine ammonia lyase and the cinnamic acid decarboxylase can be expressed as two separate proteins, or can be covalently linked by a linker as described herein.
  • the membrane-bound transporter is an ATP-binding cassette transporter (ABC transporter).
  • ABC transporter is a bacterial ABC transporter, such as one derived from Pseudomonas putida.
  • the ABC transporter is a solvent resistance efflux pump, such as the SrpABC pump derived from Pseudomonas putida SI 2.
  • the subject technology also provides a method for screening candidate proteins for mutated cinnamic acid decarboxylase activity, the method comprising:(a) providing a protein sample comprising a candidate protein, and a substrate selected from the group consisting of phenylalanine, trans- cinnamic acid, tyrosine, coumaric acid, and combinations thereof; (b) combining the protein sample and the substrate sample to form a mixture, and incubating the mixture under a condition that allows a mutated cinnamic acid decarboxylase to convert the substrate to a product selected from the group consisting of styrene, 4-hydroxystyrene, and combination thereof; and (c) exposing the mixture to a detection material that comprises a polymeric resin that absorbs the product vapor.
  • the mutated cinnamic acid decarboxylase is capable of converting trans-cinnamic acid to styrene, at a rate that is comparable to or higher than the wild type enzyme (e.g. FDC1). In another embodiment, the mutated cinnamic acid decarboxylase is capable of converting coumaric acid to 4-hydroxystyrene, at a rate that is comparable to higher than the wild type enzyme.
  • the candidate protein comprises a fusion protein comprising a mutated cinnamic acid decarboxylase.
  • the subject technology also relates to a method for screening candidate proteins for cinnamic acid decarboxylase activity, comprising: (a) providing a protein sample comprising the candidate protein, and a substrate sample comprising trans-cinnamic acid; (b) combining the protein sample and substrate sample to form a mixture, and incubating the mixture under a condition that allows a cinnamic acid decarboxylase to convert trans-cinnamic acid to styrene; and (c) exposing the mixture to a detection material that comprises a polymeric resin that absorbs styrene vapor.
  • the detection material further comprises a detectable marker that causes a color change in the presence of styrene.
  • the detectable marker can be 4- nitrobenzyl-pyridine.
  • the detection material can be attached to a solid support.
  • the polymeric resin comprises an aromatic functional group.
  • the change of color can be detected by spectrophotometry.
  • the change of color can be detected by measuring the absorbance of the sample at about 600 nm wavelength.
  • the screening method described herein can be used to simultaneously screen a plurality of candidate proteins.
  • the subject technology also relates to a method of isolating a recombinantly produced cinnamic acid decarboxylase, comprising (a) providing a bacterial host comprising a nucleic acid that encodes a cinnamic acid decarboxylase operably linked to a promoter sequence; (b) culturing the bacterial host in a culture medium to express the cinnamic acid decarboxylase in the host cell, therein the host cell is cultured at a temperature that is from about 10 °C to about 25 °C; and ( c) isolating the cinnamic acid decarboxylase from the host cell, wherein the isolation is conducted in an anaerobic environment.
  • one or more buffer solutions used for isolating the cinnamic acid decarboxylase comprises a reducing agent, such as Tris(2-carboxyethyl) phosphine (TCEP), ⁇ - mercaptoethanol, and a combination thereof.
  • a reducing agent such as Tris(2-carboxyethyl) phosphine (TCEP), ⁇ - mercaptoethanol, and a combination thereof.
  • the subject technology also relates to a method of crystallizing a cinnamic acid decarboxylase, the method comprising: (a) providing a cinnamic acid decarboxylase solution at a concentration of from about 1 mg/ml to about 50 mg/ml; (b) mixing the cinnamic acid decarboxylase solution with a reservoir solution at a volume ratio of from about 1 : 10 to about 10:1 ; and (c) maintaining the mixture of the cinnamic acid decarboxylase solution and the reservoir solution at a temperature suitable for the formation of the cinnamic acid decarboxylase crystals.
  • the subject technology also relates to a crystal of cinnamic acid decarboxylase, wherein the cinnamic acid decarboxylase is in a complex with 3-hydroxyl cinnamic acid.
  • the subject technology also relates to a method for producing styrene, the method comprising: (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) a cinnamic acid decarboxylase; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene, wherein the vapor of the styrene product is absorbed by an absorbing material.
  • a method for simultaneously screening phenylalanine ammonia lyase and cinnamic acid decarboxylase activities comprising: (a) providing a fusion protein comprising: (i) a first domain comprising a phenylalanine ammonia lyase, and (ii) a second domain comprising a cinnamic acid decarboxylase; (b) mixing the fusion protein with a substrate under a condition that allows the fusion protein to convert the substrate to a product; and (c) detecting the amount of the remaining substrate, or the amount of the product, or a combination thereof.
  • the screening comprises detecting the loss of the substrate, or the amount of a product, such as trans-cinnami acid, styrene, and the downstream derivatives of styrene.
  • the screening can comprise detecting the amount of trans -cinnamic acid, or styrene, or a combination thereof.
  • a fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase, and (ii) a second domain that comprises a cinnamic acid decarboxylase; and (iii) a linker that covalently links the first domain and the second domain.
  • said phenylalanine ammonia lyase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
  • cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441 of SEQ ID NO: 8.
  • cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
  • cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a substitution of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
  • a host cell comprising any one of the fusion protein of clauses 1-16.
  • a host cell comprising the nucleic acid of clause 17.
  • a method for the producing styrene comprising: (a) contacting a host cell with a fermentable carbon substrate, said host cell comprises a fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase; (ii) a second domain that comprises a cinnamic acid decarboxylase; and (iii) a linker that covalently links the first domain and the second domain; (b) culturing said cell in a culture medium for a time sufficient to produce styrene.
  • cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
  • a cinnamic acid decarboxylase comprising (i) any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38; (ii) an amino acid sequence that is about 85% identical to any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38, with the proviso that said amino acid sequence is not SEQ ID NO: 8; or (iii) a functional fragment of (i) or (ii).
  • a mutant cinnamic acid decarboxylase comprising a mutation at an amino acid residue position corresponding to one of the following positions: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441 of SEQ ID NO:8.
  • 41 The mutant cinnamic acid decarboxylase of clause 40, comprising a mutation at an amino acid residue position corresponding to one of the following positions: 175 or 190 of SEQ ID NO: 8.
  • a mutant cinnamic acid decarboxylase comprising a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
  • mutant cinnamic acid decarboxylase of clause 42 comprising a substitution of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
  • mutant cinnamic acid decarboxylase of clause 42 comprising a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 175 or 190.
  • a method for the production of styrene comprising: (a) contacting a host cell with a fermentable carbon substrate, said host comprises (i) a phenylalanine ammonia lyase; and (ii) a cinnamic acid decarboxylase of any one of clauses 37-44; (b) culturing said cell in a culture medium for a time sufficient to produce styrene.
  • a host cell comprising: (i) a recombinantly expressed phenylalanine ammonia lyase; (ii) a recombinantly expressed cinnamic acid decarboxylase; and (iii) a recombinantly expressed AB C -transporter .
  • phenylalanine ammonia lyase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
  • a method for screening a candidate proteins for cinnamic acid decarboxylase activity comprising: (a) providing a protein sample comprising said candidate protein, and a substrate sample comprising trans-cinnamic acid; (b) combining said protein sample and substrate sample to form a mixture, and incubating said mixture under a condition that allows a cinnamic acid decarboxylase to convert trans-cinnamic acid to styrene; and (c) exposing said mixture to a detection material that comprises (i) polymeric resin that absorbs styrene vapor; and (ii) a detectable marker that causes a color change in the presence of styrene; wherein a change of the color of said detection material indicates that said candidate protein has cinnamic acid decarboxylase activity.
  • a method of isolating a recombinantly produced cinnamic acid decarboxylase comprising: (a) providing a bacterial host comprising a nucleic acid that encodes a cinnamic acid decarboxylase operably linked to a promoter sequence; (b) culturing said bacterial host in a culture medium to express said cinnamic acid decarboxylase in said host cell, therein said host cell is cultured at a temperature that is from about 10 °C to about 25 °C; and (c) isolating said cinnamic acid decarboxylase from said host cell, wherein said isolation is conducted in an anaerobic environment.
  • Figure 1 A is a schematic illustration of the predicted structure of yeast FDC 1.
  • Figure IB shows four possible tCA binding sites in FDCl (sites A-D).
  • Figure 1C shows the predicted substrate (tCA) binding site in yeast FDCl.
  • Figure 2 A shows the expression of FDCl in E. coli at 37°C, using three different buffers. The recombinantly produced FDCl only accumulated in the insoluble pellet fractions (inclusion bodies).
  • Figure 2B shows the expression of FDCl in E. coli at 16°C. Soluble FDCl was detected in the supernatant.
  • Figure 3 shows the SDS-PAGE analysis of purified FDCl .
  • Figure 4 shows the effect of pH on FDCl activity.
  • the enzyme showed maximum activity at pH 6.5, and is considered as having 100% relative activity.
  • the x-axis represents pH values of different reaction buffers.
  • Figure 5 shows the pH stability of FDCl .
  • the enzyme exhibited good pH stability. It did not lose any activity when incubated for 30 minutes at pH 6.0 prior to the reaction, and is considered as having 100% relative activity.
  • the x-axis represents pH values at which the protein was incubated for 30 minutes prior to enzymatic reaction.
  • Figure 6 shows the effect of temperature on FDCl activity.
  • the enzyme showed maximum activity when the reaction was carried out at 50 °C, and is considered as having 100% relative activity.
  • the x-axis represents temperatures at which the enzymatic reactions were carried out.
  • Figure 7 shows the temperature stability of FDCl.
  • the enzyme showed maximum activity at 50 °C, and did not lose any activity when incubated at 50 °C for 30 minutes prior to the reaction (which is considered as having 100% relative activity).
  • the x-axis represents temperatures at which the enzyme was incubated prior to enzymatic reaction.
  • Figure 8 shows the temperature stability of FDCl at 50°C for various time periods.
  • the enzyme showed maximum activity after being incubated at 50°C for 30 minutes prior to the reaction, and is considered as having 100% relative activity.
  • the x-axis represents the duration (in minutes) in which the enzyme was incubated at 50°C prior to enzymatic reaction.
  • Figure 9 shows the effect of cofactors on FDCl activity. Specific activity is shown as nmol of styrene produced per mg enzyme per minute.
  • Figure 10A shows the effect of metal ions on FDCl activity.
  • Figure 10B shows the effect of Zn 2+ and Fe + on FDCl activity. Specific activity is shown as nmol of styrene produced per mg enzyme per minute.
  • Figure 11 shows the substrate specificity of FDC 1.
  • the enzyme showed maximum activity when i-cinnamic acid is used as a substrate, and is considered as having 100% relative activity.
  • Figures 12A and 12B depict the kinetic analysis of FDCl.
  • the x-axis represents the enzymatic reaction with various concentrations of substrate. Specific activity is shown as nmol of styrene produced per mg enzyme per minute.
  • Figure 13 depicts the process of random mutagenesis to create FDC mutants, and the high throughput colorimetric screening of FDCl mutants.
  • Figure 14 shows styrene production by FDCl mutants.
  • the Y-axis represents amount of styrene produced by FDCl mutants.
  • the X-axis represents changed amino acids of the FDCl mutants.
  • Figure 15 shows the activities of FDC-PAL fusion proteins.
  • the X-axis shows the lengths of the linkers.
  • Figure 16 is a schematic illustration of the predicted structure of FDC-PAL fusion protein.
  • Figure 17 shows that the production of styrene was significantly increased with the co- expression of an ABC-transporter.
  • Figure 18A shows the production of styrene and tCA from glucose.
  • Figure 18B shows production of styrene from trans-cinnamic acid or phenylalanine.
  • Figure 19 shows the use of STRATA-X® resin on top of a culture tube (left) or on top of a 96-well culture plate (right).
  • Figure 20 shows the SDS-PAGE analysis of purified FDCl.
  • Figure 21A shows a typical electron density map with initial model of the crystalline structure of FDC(K190E).
  • Figure 21B shows an asymmetric unit of the crystalline structure of FDC(K190E).
  • the subject technology generally relates to biosynthesis of styrene. Certain embodiments of the subject technology is based, in part, on the recognition that phenylalanine can be converted to styrene by a two-step pathway of deamination and decarboxylation, with trans-cinnamic acid (tCA) as the intermediate.
  • tCA trans-cinnamic acid
  • Two types of enzymes are directly involved in this process, phenylalanine ammonia lyase (PAL), which converts phenylalanine to tCA, and cinnamic acid decarboxylase, which coverts tCA to styrene.
  • PAL phenylalanine ammonia lyase
  • Host cells expressing these two types of enzymes can be cultured in bioreactor to produce styrene from renewable substrates such as glucose.
  • fusion proteins have been designed in which phenylalanine ammonia lyase and cinnamic acid decarboxylase are covalently linked by a linker.
  • the fusion protein takes advantage of the "substrate channeling" phenomenon.
  • Substrate channeling refers to a phenomenon in which substrates are efficiently delivered from enzyme to enzyme without equilibration with other pools of the same substrates. In effect, this creates local pools of metabolites at high concentrations relative to those found in other areas of the cell.
  • phenylalanine ammonia lyase trans-cinnamic acid
  • cinnamic acid decarboxylase proximity between these two enzymes (by covalently linking the two enzymes in the form of a fusion protein) would provide for a more efficient use of the substrate.
  • fusion proteins linking these two enzymes benefit from the substrate channeling phenomenon, and can reduce production costs and increase the number of enzymatic reactions that occur during a given time period.
  • a protein complex in which the phenylalanine ammonia lyase and cinnamic acid decarboxylase form a protein complex via non-covalent interaction can also be used.
  • the subject technology provides a fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase, and (ii) a second domain that comprises a cinnamic acid decarboxylase.
  • Host cells comprising fusion proteins described herein, as well as method of using the fusion protein for styrene production are also provided.
  • mutant cinnamic acid decarboxylases have been designed, and screened for their respective activities. Mutant cinnamic acid decarboxylases showing higher catalytic activities, as compared to that of wild type, were identified. Some of the mutant cinnamic acid decarboxylases described herein increased the styrene production by two- to three-fold. These mutant cinnamic acid decarboxylases can be introduced to host cells to promote the bioproduction of styrene. [0150] In another aspect, a high throughput, colorimetric screening method can allow for large- scale screening of mutant cinnamic acid decarboxylases or fusion proteins.
  • the colorimetric screening described herein is highly reproducible and can screen about 1 ,000 mutant cinnamic acid decarboxylases and fusion proteins in a single day. This can provide fast and recursive screening of enzymes involved in biosynthesis of styrene and related compounds, providing an important method for improvement of styrene biosynthesis.
  • the subj ect technology provides mutant cinnamic acid decarboxylases, host cells comprising a mutant cinnamic acid decarboxylase, as well as method of using the mutant cinnamic acid decarboxylases for styrene production. Also provided herein are libraries of mutant cinnamic acid decarboxylases, and method of screening a candidate protein for cinnamic acid decarboxylase activity.
  • Another issue that limits the bioproduction of styrene is the toxicity of styrene to host cells.
  • the accumulation of hydrophobic aromatics within the cytoplasmic membrane is known to disrupt its integrity.
  • an ABC-transporter was introduced into the host cell - an efflux pump that removes organic solvent from the cell.
  • styrene production by E. coli cells expressing the ABC-transporter was enhanced nearly four times.
  • the subject technology provides a host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase; (b) a recombinantly expressed cinnamic acid decarboxylase; and (c) a recombinantly expressed ABC-transporter.
  • a host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase; (b) a recombinantly expressed cinnamic acid decarboxylase; and (c) a recombinantly expressed ABC-transporter.
  • Method of using the ABC- transporter for styrene production is also provided.
  • CA cinnamic acid
  • tCA Trans-cinnamic acid
  • cinnamic acid decarboxylase refers to an enzyme that catalyzes the conversion of trans-cinnamic acid to styrene.
  • the term encompasses wild type or naturally occurring cinnamic acid decarboxylase, as well as functional fragments or variants of a wild type cinnamic acid decarboxylase.
  • the Saccharomyces cerevisiae cinnamic acid decarboxylase described herein is also termed ferulic acid decarboxylase (FDC or FDC1). Ferulic acid is also a phenylacrylic acid.
  • control refers to a sample that provides a basis for comparison.
  • a "control” can be a parallel sample comprising a cinnamic acid decarboxylase whose activity has been characterized (e.g., a wild type cinnamic acid decarboxylase, a specific mutant cinnamic acid decarboxylase, etc.).
  • a control may be a pre -determined threshold value, or a value that is present in a database (e.g., a table, electronic database, spreadsheet, etc.).
  • amino acid position "corresponding to" a reference position is a position that aligns with a reference sequence, as identified by aligning the amino acid sequences. Such alignments can be done by hand or by using well-known sequence alignment programs such as ClustalW2, Blast 2, etc.
  • a protein is "derived” from an organism when the protein is isolated from that organism, or modified or generated (e.g., chemically synthesized or recombinantly produced) using information of the protein from that organism
  • detectable marker refers to a chemical compound that is added to (or coated onto) a styrene-absorption material, in an amount effective to detect styrene vapor.
  • Preferred detectable markers are chemical compounds that undergo a chemical reaction in the presence of styrene, and produce a colorimetric species. The chemical response of the detectable marker is preferable concentration dependent.
  • fermentable carbon substrate refers to a carbon source capable of being metabolized by host cells as described herein, and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, one-carbon substrate, and a combination thereof.
  • the term "functional fragment" of protein refers to refers to a peptide fragment that is a portion of the full length protein, and has substantially the same biological activity, or carries out substantially the same function as the full length protein (e.g., carrying out the same enzymatic reaction).
  • a functional fragment of a PAL can catalyze the phenylalanine to cinnamic acid conversion
  • a functional fragment of a FDC can catalyze the cinnamic acid to styrene conversion.
  • the term "functional variant" of protein refers to a protein in which one or more amino acid residues have been changed without altering the overall conformation and function of the reference protein.
  • Functional variants includes, e.g., replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable.
  • isoleucine, a hydrophobic amino acid may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide.
  • homologous in all its grammatical forms and spelling variations refers to the relationship between polynucleotides or proteins that possess a "common evolutionary origin,” including polynucleotides or proteins from superfamilies and homologous polynucleotides or proteins from different species (Reeck et al., Cell 50:667, 1987). Such polynucleotides or proteins have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or the presence of specific amino acids or motifs at conserved positions. For example, two homologous proteins can have amino acid sequences that are about 80%, about 85%, about 90%>, about 95%o, about 96%o, about 97%o, about 98%, about 99%, or even 100% identical.
  • similarity means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed “percent similarity” may then be determined between the compared polypeptide sequences.
  • Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded therein, and comparing this to a second amino acid sequence.
  • identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • Two or more polynucleotide sequences can be compared by determining their "percent identity", as can two or more amino acid sequences.
  • the programs available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.), for example, the GAP program are capable of calculating both the identity between two polynucleotides and the identity and similarity between two polypeptide sequences, respectively. Other programs for calculating identity or similarity between sequences are known by those skilled in the art.
  • mutant refers to a deletion, an insertion, or a substitution of a nucleotide or an amino acid residue of a wild type sequence.
  • a wild type sequences refers to the most frequent sequence found in nature, against which mutants are defined.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • phenylalanine ammonia lyase refers to an enzyme that catalyzes the conversion of phenylalanine to trans-cinnamic acid.
  • the term encompasses wild type or naturally occurring phenylalanine ammonia lyase, as well as functional fragments or variants of a wild type phenylalanine ammonia lyase.
  • mutated cinnamic acid decarboxylase refers to an enzyme that catalyzes the conversion of trans-cinnamic acid to styrene, or the conversion of coumaric acid to 4-hydroxystyrene.
  • the term “recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature.
  • the term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids.
  • a reference to an element in the singular is not intended to mean “one and only one” unless specifically stated, but rather “one or more.”
  • Pronouns in the masculine include the feminine and neuter gender (e.g., her and its) and vice versa.
  • the term “some” refers to one or more.
  • Underlined, italicized and/or boldface headings and subheadings are used for convenience only, do not limit the subject technology, and are not referred to in connection with the interpretation of the description of the subject technology.
  • percent identity of two polypeptide or polynucleotide sequences refers to as the percentage of identical amino acid residues or nucleotides across the entire length of the shorter of the two sequences.
  • Phenylalanine ammonia lyase (EC 4.3.1.24; formally EC 4.3.1.5) is an enzyme that catalyzes the chemical reaction:
  • PAL is a non-mammalian enzyme widely distributed in plants and yeast.
  • a representative list of PALs include (identified by Genbank accession number and species): Q9ATN7 Agastache rugosa; 093967 Amanita muscaria (Fly agaric); P35510, P45724, P45725, Q9SS45, Q8RWP4 Arabidopsis thaliana (Mouse-ear cress); Q6ST23 Bambusa oldhamii (Giant timber bamboo); Q42609 Bromheadia finlaysoniana (Orchid); P45726 Camellia sinensis (Tea); Q9MAX1 Catharanthus roseus (Rosy periwinkle) (Madagascar periwinkle); Q9SMK9 Cicer arietinum (Chickpea); Q9XFX5, Q9XFX6 Citrus Clementina x Citrus reticulate; Q42667 Citrus Ziman (Lemon); Q8H6V9, Q8H6W0 Coffea
  • Rhodosporidium toruloides also known as Rhodotorula glutinis
  • PAL is a homotetramer, each subunit having a "seahorse" shape that interlocks head-to-tail with two other subunits, thereby maximizing adjacent subunit interactions and yielding a close-fitting tetramer.
  • the tetramer assembly leads to a cluster of four vicinal cysteines (residues 140, 455, 467, and 530), with the sulfur atoms of Cys467 and Cys530 separated by 3.62 angstroms.
  • the structure of the main body of PAL has a central core of nearly parallel a-helices of varying lengths. There is only one section of ⁇ -sheet longer than three residues (strands of residues 231-237 and 240-246); it resides in the funnel region leading to the active site. MacDonald, et al., A modern view of phenylalanine ammonia lyase, Biochem Cell Biol. 2007; 85(3):273-82.
  • At3g 10340 can be purchased from Arabidopsis Biological Resource Center (ABRC) at the Ohio State University under the stock numbers G10120 (in pENTR223.1 vector), G12256 (in pENTR223.1 vector), and U24927 (in pENTR/D-TOPO vector), respectively.
  • the phenylalanine ammonia lyase of the subject technology is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
  • Other preferred source organisms include, e.g., yeasts such as Rhodotorula, Rhodosporidium, and Sporobolomyces; bacteria such as Streptomyces; and plants such as pea, potato, rice, eucalyptus, pine, corn, petunia, arabidopsis, tobacco, and parsley.
  • the subject technology also encompasses homologs (including orthologs), functional fragments, or functional variants of the exemplary PALs described herein.
  • Methods of obtaining homologs and variants of PALs are well known in the art, including for example, sequence-dependent protocols.
  • Exemplary sequence-dependent protocols include, e.g., nucleic acid hybridization, DNA and RNA amplification (e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR)), etc.
  • genes encoding homologs of a PAL can be isolated directly by using all or a portion of the known sequences as DNA hybridization probes to screen libraries from any desired plant, fungi, yeast, or bacteria, using techniques well known to those skilled in the art.
  • Specific oligonucleotide probes based upon the literature nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, infra).
  • the entire sequences can be used directly to synthesize DNA probes by methods known to a skilled artisan, such as random primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems.
  • primers can be designed and used to amplify a part of or full-length of a target sequences.
  • the resulting amplification products can be labeled directly during amplification reactions, or labeled after amplification reactions, and used as probes to isolate full-length cDNA or genomic fragments under conditions of appropriate stringency.
  • two short segments of the literature sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA.
  • the polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the literature sequences, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding bacterial genes.
  • the second primer sequence may be based upon sequences derived from the cloning vector.
  • the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the literature sequences. Using commercially available 3' RACE or 5' RACE systems, specific 3' or 5' cDNA fragments can be isolated (Ohara et al., Proc. Natl. Acad. Sci. USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
  • nucleic acid and protein sequences of a homolog or a variant can further be identified by using a "query sequence" to perform a search against public databases to identify other family members or related sequences.
  • the phenylalanine ammonia lyase of the subject technology comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof.
  • SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 are the amino acid sequences of Arabidopsis PALI, PAL2, and PAL4, respectively.
  • Variants of PAL may include those polypeptide sequences comprising an amino acid sequence that is about 60%, about 70%o, about 75%o, about 80%o, about 85%o, about 90%o, about 95%o, about 96%, about 97%, or about 98%, about 99%, or even 100% identical to any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or a fragment thereof.
  • the fragment may comprise about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 amino acid residues of the full length PAL.
  • the phenylalanine ammonia lyase of the subject technology comprises an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, and a fragment or variant thereof.
  • SEQ ID NOs:l, SEQ ID NO:3, and SEQ ID NO:5 are the nucleotide sequences encoding Arabidopsis PALI, PAL2, and PAL4, respectively.
  • Variants of PAL-coding sequence may include those polynucleotide sequences that is about 60%o, about 70%o, about 75%o, about 80%o, about 85%o, about 90%o, about 95%o, about 96%, about 97%, about 98%, about 99% , or even 100% identical to any one of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, or a fragment thereof.
  • the fragment may comprise about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 2100 nucleotides of the full length PAL-coding sequence.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence and non-homologous sequences can be disregarded for comparison purposes).
  • an alignment is a global alignment, (i.e., the percentage of identical amino acid residues or nucleotides across the entire length of the shorter of the two sequences). If a local alignment is desired, preferably, the length of a sequence aligned for comparison is about 30%o, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the length of the shorter of the two sequences.
  • Cinnamic acid decarboxylase is an enzyme that catalyzes the conversion of trans- cinnamic acid to styrene.
  • the cinnamic acid decarboxylase of the subject technology is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
  • the cinnamic acid decarboxylase of the subject technology is derived from Saccharomyces cerevisiae.
  • Saccharomyces cerevisiae cinnamic acid decarboxylase described herein is also termed ferulic acid decarboxylase (FDC or FDC1).
  • FDC ferulic acid decarboxylase
  • the sequence of FDC/FDC1 is disclosed in U.S. Patents Nos. 5,955,137 and 6,468,566.
  • full length cinnamic acid decarboxylase from Saccharomyces cerevisiae comprises 503 amino acids (SEQ ID NO:8).
  • a computer model of full length FDC1 is shown in Figure 1A; and four possible tCA binding sites are shown in Figure IB, with site C being the most likely site. Further analysis of site C shows that 1173, A174, R175, V188, 1189, K190 (not shown for figure clarification), 1194, E280, M286, F291 and F440 are involved with substrate binding ( Figure 1C).
  • the nucleotide sequence encoding FDC1 is shown as SEQ ID NO:7.
  • cinnamic acid decarboxylase is 3-octaprenyl-4- hydroxybenzoate carboxy-lyase from Aspergillus niger (OHBA1; SEQ ID NO: 10).
  • OHBA1 3-octaprenyl-4- hydroxybenzoate carboxy-lyase from Aspergillus niger
  • SEQ ID NO: 10 The nucleotide sequence encoding OHBA1 is shown as SEQ ID NO:9.
  • the subject technology also encompasses homologs (including orthologs), functional fragments, or functional variants of the exemplary cinnamic acid decarboxylases described herein.
  • Homologs and variants of cinnamic acid decarboxylases can be obtained using methods described above.
  • a functional variant may be a sequence that (i) is about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100% identical to SEQ ID NO:8, (ii) comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the following residues: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO:
  • Amino acid residues that "correspond to" a particular position of SEQ ID NO: 8 can be identified by aligning the target sequence with SEQ ID NO: 8. As an example, alignment of FDC1 and OHBA1 is shown below (FDC1 is the query sequence; OHBA1 is the subject sequence).
  • the cinnamic acid decarboxylase of the subject technology comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:10, and a functional fragment or variant thereof.
  • Variants of cinnamic acid decarboxylase may include those polypeptide sequences comprising an amino acid sequence that is about 50%, about 60%, about 70%o, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100% identical to any one of SEQ ID NO:8, SEQ ID NO: 10, or a fragment thereof.
  • the fragment may comprise about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 amino acid residues of the full length cinnamic acid decarboxylase.
  • the cinnamic acid decarboxylase of the subject technology comprises an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:9, and a fragment or variant thereof.
  • Variants of cinnamic acid decarboxylase-coding sequence may include those polynucleotide sequences that is about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100% identical to any one of SEQ ID NO:7, SEQ ID NO:9, or a fragment thereof.
  • the fragment may comprise about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 2100 nucleotides of the full length coding sequence.
  • the subject technology provides a mutant cinnamic acid decarboxylase, wherein a mutation is introduced at an amino acid residue position corresponding to one of the following positions: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, and a combination thereof.
  • the mutation may be a deletion, an insertion, or a substitution mutation.
  • the mutant cinnamic acid decarboxylase comprises a mutation at an amino acid residue position corresponding to residue 190 of SEQ ID NO: 8.
  • the residue corresponding to 190 is replaced with one of the following: E, C, D, V, N, L, H.
  • the mutant cinnamic acid decarboxylase comprises a mutation at an amino acid residue position corresponding to residue 175 of SEQ ID NO: 8.
  • the residue corresponding to 175 is replaced with I.
  • the mutant cinnamic acid decarboxylase is a mutant FDCl, wherein a mutation is introduced to one of the following positions in SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441, and a combination thereof.
  • the mutant cinnamic acid decarboxylase is a mutant FDCl, wherein a mutation is introduced to that one of the following positions in SEQ ID NO:8: 175, 190, 193, and a combination thereof.
  • the mutant cinnamic acid decarboxylase is a mutant FDC1, comprising one of the mutations in SEQ ID NO:8: K190E, K190C, K190H, K190P, K190L, K190R, K190D, K190V, K190S, K190N, R175I, H193P, and a combination thereof.
  • the mutant cinnamic acid decarboxylase comprises any one of the sequences selected from the group consisting of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38.
  • the cinnamic acid decarboxylase of the subject technology may also comprise a functional fragment or variant of the mutant cinnamic acid decarboxylase described herein.
  • Variants of cinnamic acid decarboxylase may include those polypeptide sequences comprising an amino acid sequence that is about 50%, about 60%o, about 70%o, about 75%o, about 80%o, about 85%o, about 90%o, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100% identical to any one of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; and a fragment thereof.
  • the fragment may comprise about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550
  • the variants may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 the following residues: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
  • the library comprises a plurality of mutant cinnamic acid decarboxylases.
  • the library may comprise cinnamic acid decarboxylase mutants that represent about 15, about 16, about 17, about 18, or about 19 different mutations at particular target position.
  • the library may comprise about 19 different mutants in which K190 of FDC1 (SEQ ID NO:8) is replaced with each one of the other 19 amino acids.
  • the library may comprise cinnamic acid decarboxylase mutants that represents about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19 mutations at different positions.
  • an Alanine scan may be used to create mutant libraries in which amino acid residues at different positions are replaced with Alanine.
  • Changes to the amino acid sequence may be generated by changing the nucleic acid sequence encoding the amino acid sequence.
  • a nucleic acid sequence encoding a mutant cinnamic acid decarboxylase may be prepared by methods known in the art using the guidance of the present specification for particular sequences. These methods include, but are not limited to, preparation by site- directed (or oligonucleotide-mediated) mutagenesis (Coombs et al., Proteins (1998), 259-311, 1 plate. Editor(s): Angeletti, Ruth Hogue.
  • in vivo mutagenesis may be employed using commercially available materials such as E. coli XL 1 -Red strain, and the Epicurian coli XL 1 -Red mutator strain from E. coli XL 1 -Red strain.
  • STRATAgene (STRATAgene, La Jolla, Calif, Greener and Callahan, Strategies 7:32-34) (1994)). This strain is deficient in three of the primary DNA repair pathways (mutS, mutD and mutT), resulting in a mutation rate 5000-fold higher than that of wild type.
  • the subject technology provides a method for screening a candidate proteins for cinnamic acid decarboxylase activity, comprising (a) providing a protein sample comprising the candidate protein, and a substrate sample comprising trans-cinnamic acid; (b) combining the protein sample and substrate sample to form a mixture, and incubating the mixture under a condition that allows a cinnamic acid decarboxylase to convert trans-cinnamic acid to styrene; and (c) exposing the mixture to a detection material that comprises (i) polymeric resin that absorbs styrene vapor; and (ii) a detectable marker that causes a color change in the presence of styrene. A change of the color of the detection material indicates that the candidate protein has cinnamic acid decarboxylase activity.
  • the subject technology provides a spectroscopic- based colorimetric assay method.
  • a candidate protein and tCA are incubated under a condition that allows a cinnamic acid decarboxylase to convert trans-cinnamic acid to styrene.
  • the incubation condition should allow a cinnamic acid decarboxylase to exhibit optimal enzymatic activity (for example, at a temperature from about 10 °C to about 65 °C, from about 25 °C to about 55 °C, from about 35 °C to about 60 °C, or from about 35 °C to about 55 °C; at a pH between 6-10, between 6-8, or between 5.5-7.5; in the presence of metal ions from about 1 mM to about 20 mM; etc.).
  • optimal enzymatic activity for example, at a temperature from about 10 °C to about 65 °C, from about 25 °C to about 55 °C, from about 35 °C to about 60 °C, or from about 35 °C to about 55 °C; at a pH between 6-10, between 6-8, or between 5.5-7.5; in the presence of metal ions from about 1 mM to about 20 mM; etc.
  • the candidate protein and tCA can be incubated for about 30 minutes, about 1 hour; about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours, to allow sufficient amount of styrene to be produced.
  • the reaction mixture is exposed to a detection material that comprises (i) polymeric resin that absorbs styrene vapor; and (ii) a detectable marker that causes a color change in the presence of styrene. Because styrene vaporizes, the detection material can be placed on top of the reaction mixture to absorb styrene vapor (without actually dipping into the reaction mixture). Accordingly, the detection material can be attached to a solid support, and can be inverted during the detection process to absorb styrene vapor. See, e.g., Figure 19.
  • Preferred polymeric resins for absorbing styrene vapor include, e.g., reversed phase hydrophobic resins, which has a hydrocarbon or aromatic functional group (e.g., an aromatic benzene ring) that can bind with polar and non -polar compounds. More preferably, the hydrophobic resins have a hydrocarbon or aromatic functional group and do not have any polar group. Examples of the reversed phase hydrophobic resins include CI 8, C8, phenyl, SDB-L sorbents resins, and combinations thereof.
  • Exemplary polymeric resins that can be used to absorb volatile organic compounds include, for example, STRATA-X® polymeric resins (Phenomenex), Amberlite polymeric resins ( Sigma- Aldrich), DOWEXTM polymeric resins or AMBERJETTM polymeric resins (The Dow Chemical Company), CelletTM polymeric resins, PuroSorbTM polymeric resins, or Chromalite® resins (PuroLite), etc.
  • the surface area and pore size distribution of the resin should suitable for absorbing volatile organic compounds. Because a polymeric resin is made with few functional groups (often it is the single dominant functional group which gives the surface its adsorption characteristics), one can ascertain the affinity (e.g., Hanson solubility parameter) and predict the adsorption capacity based on
  • thermodynamics Various studies have shown that adsorption capacities of a variety of solutes, and polymeric resins can be correlated with solubility parameters.
  • Other materials suitable for absorbing styrene vapor include activated carbon and cellulosic materials.
  • activated carbon For example, the cotton burrs disclosed in US 2002/0151622 as cellulosic materials can be used in the present invention to absorb volatile organic compounds.
  • the detection material also comprises a detectable marker that causes a color change in the presence of styrene.
  • a detectable marker is 4-nitrobenzyl-pyridine (NBP).
  • NBP 4-nitrobenzyl-pyridine
  • the color change of the detectable material can be determined by a quantitative assay, such as by spectrophotometry. The color change may also be determined qualitatively. Sometimes, the color change would be apparent to an observer.
  • the activity of the candidate protein can be compared with a control.
  • a control can be a parallel sample comprising a cinnamic acid decarboxylase whose activity has been characterized (e.g., a wild type cinnamic acid decarboxylase, or a specific mutant cinnamic acid decarboxylase that serves as the base sequence for further mutations).
  • a control may be a pre-determined threshold value, or a value that is present in a database (e.g., a table, electronic database, spreadsheet, etc.).
  • the screening assay can be conducted for more than one round, and can be combined with computer modeling to rationally design and improve the activities of cinnamic acid decarboxylases.
  • mutated cinnamic acid decarboxylase capable of converting converting trans-cinnamic acid to styrene, or converting coumaric acid to 4-hydroxystyrene, can be screened by the method described herein.
  • the candidate protein being screened can be a fusion protein comprising a mutated cinnamic acid decarboxylase.
  • the subject technology also provides a method of recombinant production and purification of cinnamic acid decarboxylase, such as FDC.
  • the subject technology provides a method of isolating a recombinantly produced cinnamic acid decarboxylase, comprising: (i) providing a bacterial host comprising a nucleic acid that encodes a cinnamic acid decarboxylase operably linked to a promoter sequence; (ii) culturing the bacterial host in a culture medium to express the cinnamic acid decarboxylase in the host cell, therein the host cell is cultured at a temperature that is from about 10 °C to about 25 °C; and (iii) isolating the cinnamic acid decarboxylase from the host cell, wherein the isolation is conducted in an anaerobic environment.
  • culturing the bacterial host cell e.g., E. coli
  • the cell culture is grown at a temperature of about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 °C.
  • Reducing agents such as Tris(2-carboxyethyl) phosphine (TCEP) or ⁇ -mercaptoethanol, can also be used in the method of recombinantly producing cinnamic acid decarboxylase, such as FDC1.
  • one or more buffer solutions used for isolating the cinnamic acid decarboxylase may comprise a reducing agent, such as TCEP at a concentration of about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or 100 mM; or ⁇ -mercaptoethanol at a concentration of about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or 100 mM; and a combination thereof.
  • Samples and cell extracts containing the recombinant cinnamic acid decarboxylase (such as FDC1) maintained in an anaerobic environment also contain cinnamic acid decarboxylase activity.
  • the recombinantly produced cinnamic acid decarboxylase (such as FDC1) maintains cinnamic acid decarboxylase activity in the presence of metal ions.
  • metal ions include, e.g., calcium, zinc, magnesium, iron, manganese ion, or a combination thereof.
  • the metal ion may be present at a concentration of from about 0.1 mM to about 100 mM, such as about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM; from about 0.1 mM to about 50 mM, from about 0.1 mM to about 25 mM, from about 0.1 mM to about 20 mM, from about 0.1 mM to about 10 mM, from about 1 mM to about 50 mM, from about 1 mM to about 25 mM, from about 1 mM to about 20 mM, from about 1 mM to about 15 mM, or from about 1 m
  • the purified cinnamic acid decarboxylase described herein is suitable for crystallization.
  • the subject technology provides a method of crystallizing a cinnamic acid
  • the method comprising (a) providing a cinnamic acid decarboxylase solution at a concentration of from about 1 mg/ml to about 50 mg/ml; (b) mixing the cinnamic acid decarboxylase solution with a reservoir solution at a volume ratio of from about 1 : 10 to about 10:1 ; and (c) maintaining the mixture of step (b) at a temperature suitable for the formation of the cinnamic acid decarboxylase crystals.
  • the protein concentration in step (a) is from about 2 mg/ml to about 20 mg/ml, more preferably from about 5 mg/ml to about 10 mg/ml.
  • the volume ratio of the cinnamic acid decarboxylase solution to the reservoir solution is preferably from about 1 :5 to about 5: 1, more preferably from about 1 :2 to about 2:1.
  • 1 to 2 ⁇ of the cinnamic acid decarboxylase solution protein concentration at about 5.0 to 10.0 mg/ml
  • 1 to 2 ⁇ of the cinnamic acid decarboxylase solution is mixed with 1 to 2 ⁇ of a reservoir solution in a drop on glass.
  • the mixture is put upside down on a well, containing the reservoir solution (typically about 500 ⁇ ) and the system is incubated at a temperature of from about 1°C to about 20°C, preferably from about 4°C to about 12°C.
  • the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water evaporates from the drop and transfers to the reservoir, the precipitant concentration increases to a level for crystallization. Since the system is in equilibrium, these conditions are maintained until the crystallization is complete.
  • the crystallization of the cinnamic acid decarboxylase can be improved when the protein forms a complex with a small molecule.
  • the small molecule may be added to the protein solution at step (a) or to the protein/reservoir mixture at step (b) to allow the formation of a protein-small molecule complex.
  • the final concentration of the small molecule in the mixture of step (b) is from about 0.01% (w/v) to about 1% (w/v), preferably from about 0.05%> (w/v) to about 0.5%> (w/v), more preferably from about 0.08%> (w/v) to about 0.2%> (w/v).
  • Suitable small molecules include substrates of cinnamic acid decarboxylase (e.g. trans- cinnamic acid) and its analogs, such as 3-hydroxyl cinnamic acid, ferulic acid, 2-methylcinnamic acid, 4- hydroxy-cinnamic acid, 3,4-dimethoxycinnamic acid, 2,5-dimethoxy-cinnamic acid, and combinations thereof.
  • cinnamic acid decarboxylase e.g. trans- cinnamic acid
  • its analogs such as 3-hydroxyl cinnamic acid, ferulic acid, 2-methylcinnamic acid, 4- hydroxy-cinnamic acid, 3,4-dimethoxycinnamic acid, 2,5-dimethoxy-cinnamic acid, and combinations thereof.
  • the crystallization method can be further improved by including one or more additives in the solution, such as Mn 3 ⁇ 4 at a concentration of from about 0.001M to about 0.1M, preferably from about 0.005M to about 0.02M; polyvinylpyrrolidone K15 at a concentration of from about 0.1% (w/v) to about 2.5%> (w/v), preferably from about 0.25%> (w/v) to about 1% (w/v); Non-detergent Sulfobetaine 201 (NDSB-201, CsHnNC S) at a concentration of from about 0.02M to about 1M , preferably from about 0.1M to about 0.3M; benzamidine hydrochloride at a concentration of from about 0.2% (w/v) to about 10% (w/v), preferably from about 1% (w/v) to about 3%> (w/v); all concentrations being measured in the protein-reservoir mixture of step (b) .
  • additives in the solution such as Mn 3
  • the subject technology provides a fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase, and (ii) a second domain that comprises a cinnamic acid decarboxylase.
  • a phenylalanine ammonia lyase and a cinnamic acid decarboxylase can form a protein complex, via non-covalent interaction(s).
  • the fusion protein or protein complex described herein takes advantage of the "substrate channeling" phenomenon.
  • Substrate channeling refers to a phenomenon in which substrates are efficiently delivered from enzyme to enzyme without equilibration with other pools of the same substrates.
  • the fusion protein or protein complex can channel intermediates between sequential enzymes, and control the flux of substrates into competing branches of the pathway. In effect, this creates local pools of metabolites at high concentrations relative to those found in other areas of the cell.
  • the fusion protein further comprises a linker covalently linking the first domain (PAL) and the second domain (cinnamic acid decarboxylase).
  • the linker preferably comprises one or more amino acid residues (e.g., an amino acid linker or a peptide linker).
  • the amino acid residues of the linker may comprise L-amino acid(s), D-amino acid(s), amino acid analogues, or a combination thereof.
  • linkers include, e.g., a covalent bond, C1-C6 alkyl, a cycloalkyl such as a cyclopentyl or cyclohexyl, a cycloalkenyl, aryl, or heteroaryl moiety.
  • a linker may also comprise a combination of one or more amino acid(s) with another linking moiety (such as C1-C6 alkyl-, cycloalkyl-(C5, C6), aryl, or heteroaryl moieties).
  • the linker is a peptide linker.
  • the peptide linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, or 40 amino acid long.
  • the length of the linker is from 2 to 15 amino acids.
  • the linker is a glycine/serine linker, i.e., a peptide linker consisting essentially of glycine and serine.
  • the linker comprises GS or GSG.
  • the linker comprises the Gly-Ser-Gly (GSG) motif, such as GGSG (SEQ ID NO:39), (GS)x3 (SEQ ID NO:40), (GGSG)x2 (SEQ ID NO:41), SGGSGGSGG (SEQ ID NO:42), GGSGGGSGGGSG (SEQ ID NO:43), (GGGGS)x3 (SEQ ID NO:44), as described in Table 1 below.
  • Fusion proteins described herein can be produced using techniques well known in the art.
  • the linker is a peptide linker
  • the fusion protein can be produced using standard recombinant DNA technology.
  • Other types of linker can be attached by, e.g., standard conjugation techniques. See, e.g., Hermanson et al., Bioconjugate Techniques, 2nd Ed., 2008; Academic Press.
  • the fusion protein may comprise multiple units of phenylalanine ammonia lyase and cinnamic acid decarboxylase.
  • the fusion protein can be PAL-FDC, PAL-FDC-PAL-FDC, or PAL-FDC-FDC-PAL, etc.
  • the FDC can be at the 5' terminal of the DNA encoding the fusion protein
  • the PAL can be at the 3 ' terminal of the DNA encoding the fusion protein.
  • the PAL can be at the 5 ' terminal of the DNA encoding the fusion protein
  • the FDC can be at the 3' terminal of the DNA encoding the fusion protein.
  • the cinnamic acid decarboxylase unit of the fusion protein can be wild type (WT) or mutant proteins, such as any of the mutant FDC proteins described above.
  • the fusion protein can be PAL-FDC(WT) or PAL-FDC(K190E).
  • the phenylalanine ammonia lyase and the cinnamic acid decarboxylase can form a protein complex, via non-covalent interaction(s).
  • a "protein complex” refers to an association of more than one protein.
  • the proteins of the complex may be associated by e.g., functional, stereochemical, conformational, biochemical, or electrostatic association. It is intended that the term encompass associations of any number of proteins.
  • the phenylalanine ammonia lyase and the cinnamic acid decarboxylase can be modified to include an "interacting moiety."
  • one enzyme can comprise an antibody, and the other can comprise a cognate antigen; or one enzyme can comprise a ligand, and the other can comprise a cognate receptor; or one enzyme can comprise biotin, and the other can comprise avidin, etc.
  • the interacting moieties can interact with each other, thereby brining the two enzymes in proximity to each other. Any pairs of interacting moieties can be used.
  • a host cell can be derived from a bacterium, fungus (e.g., yeast), protist (e.g., algae), plant, insect, amphibian, fish, reptile, bird, mammal (including human), or can be a hybridoma cell.
  • Host cells can be unmodified cells or cell lines, or cell lines which have been genetically modified (e.g., to facilitate production of styrene).
  • the host cell is a cell line that has been modified to allow for growth under desired conditions, such as at a lower temperature.
  • suitable host cells that express phenylalanine ammonia lyase or cinnamic acid decarboxylase can be cultured, sometimes in large scale (i.e., about 1 liter, about 10 liters, at les 100 liters, etc.), to produce commercially useful amounts of styrene or other compounds downstream from styrene (compounds which will result from further processing of styrene in these microorganisms via enzymatic or biological pathways).
  • the methods described herein can be applied to any size of cell culture flask and/or bioreactor.
  • the methods can be applied in bioreactors or cell cultures of 1 L, 10 L, 30 L, 50 L, 100 L, 150 L, 200 L, 300 L, 500 L, 1000 L, 2000 L, 3000 L, 4000 L, 5000 L, 10,000 L or larger.
  • the pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not.
  • the cultures can be grown batchwise, semibatchwise or continuously.
  • Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously.
  • Media components include, e.g., buffer, amino acid content, vitamin content, salt content, mineral content, serum content, carbon source content, lipid content, nucleic acid content, hormone content, trace element content, ammonia content, co-factor content, indicator content, small molecule content, hydrolysate content and enzyme modulator content.
  • the culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981 ; the entirety of which is hereby incorporated herein by reference). These media which can be employed in accordance with the subject technology usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
  • Carbon sources for use in the culture media of the host cells comprise sugars, such as mono-, di- or polysaccharides.
  • sugars such as mono-, di- or polysaccharides.
  • Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffmose, starch or cellulose.
  • Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous.
  • oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds.
  • nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used individually or as a mixture.
  • the inorganic salt compounds that are present in the culture media comprise about one of the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper or iron.
  • Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing derivatives of styrene.
  • Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.
  • Other components that may be added to the culture medium in order to keep the metal ions in solution comprise dihydroxy phenols such as catechol or protocatechuate and organic acids such as citric acid.
  • the culture medium may comprise one or more metal ions, such as calcium, zinc, magnesium, iron, manganese ion, and a combination thereof.
  • the metal ion may be present at a concentration of from about 0.1 mM to about 100 mM, such as about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM; from about 0.1 mM to about 50 mM, from about 0.1 mM to about 25 mM, from about 0.1 mM to about 20 mM, from about 0.1 mM to about 10 mM, from about 1 mM to about 50 mM, from about 1 mM to about
  • the fermentation media used according to the subject technology for culturing the host cells of the subject technology usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.
  • growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium.
  • the exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the culture of media can be found in the textbook "Applied Microbial. Physiology, A Practical Approach" (Editors P. M.
  • Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
  • All media components are sterilized, either by heat (20 min at 1.5 bar and 121° C.) or by filter sterilization.
  • the components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.
  • the temperature of the cell culture will be selected based primarily on the range of temperatures at which the cell culture remains viable, at which a high level of polypeptide is produced, at which misfolding and/or aggregation of the polypeptide are reduced, at which the polypeptide exhibits a more extensive or otherwise more desirable post-translational modification (e.g., glycosylation, phosphorylation, etc.), or any combination of these or other factors deemed important by the practitioner.
  • most host cells grow well and can produce high levels or protein or polypeptide within a range of about 15 °C to 45 °C.
  • the cell culture is grown at a temperature of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 °C at one or more times during the cell culture process.
  • a temperature of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 °C at one or more times during the cell culture process.
  • the culture may be subjected to one or more temperature shifts during the course of the culture.
  • the temperature shift may be relatively gradual. For example, it may take several hours or days to complete the temperature change.
  • the temperature shift may be relatively abrupt.
  • the temperature may be steadily increased or decreased during the culture process.
  • the temperature may be increased or decreased by discrete amounts at various times during the culture process.
  • the subsequent temperature(s) or temperature range(s) may be lower than or higher than the initial or previous temperature(s) or temperature range(s).
  • the temperature may be shifted once (either to a higher or lower temperature or temperature range), the cells maintained at this temperature or temperature range for a certain period of time, after which the temperature may be shifted again to a new temperature or temperature range, which may be either higher or lower than the temperature or temperature range of the previous temperature or temperature range.
  • the temperature of the culture after each discrete shift may be constant or may be maintained within a certain range of temperatures.
  • the temperature or temperature range of the cell culture after the temperature shift(s) is generally selected based primarily on the temperature(s) at which the cell culture remains viable, the range in which a high level of protein is produced.
  • a bacterial cell culture may be grown at 37 °C after seeding to encourage cell proliferation; once the cells reach a desired density, the expression of a recombinant protein is induced, and the temperature is shifted to 25 °C to reduce misfolding or aggregation of the recombinantly produced protein.
  • Anaerobic condition is also contemplated.
  • exemplary anaerobic bacterial hosts include, e.g., Bacteroids, Fusobacterium, Clostridium, Propionibacterium, Lactobacillus, Peptococcus,
  • the styrene produced in the host cells can further undergo enzymatic reaction and be converted to a downstream derivative of styrene such as toluene, xylene, polystyrene, ABS, styrene-butadiene (SBR) rubber, styrene-butadiene latex, SIS (styrene-isoprene-styrene ), S-EB-S (styrene-ethylene/butylene-styrene ), styrene-divinylbenzene (S-DVB), styrene-acrylonitrile resin (SAN) and unsaturated polyesters and the like.
  • styrene such as toluene, xylene, polystyrene, ABS, styrene-butadiene (SBR) rubber, styrene-butadiene latex, SIS (styrene-isopren
  • the host cells of the subject technology can convert certain percentage of the styrene produced to a downstream derivative of styrene.
  • the host cell can convert about 5% of the styrene to a downstream derivative, or from about 5% to about 15% of the styrene to a downstream derivative, or from about 10% to about 25% of the styrene to a downstream derivative, or from about 20% to about 35% of the styrene to a downstream derivative, or from about 30% to about 45% of the styrene to a downstream derivative, or from about 40% to about 55% of the styrene to a downstream derivative, or from about 50% to about 65% of the styrene to a downstream derivative, or from about 60% to about 75% of the styrene to a downstream derivative, or from about 70% to about 85% of the styrene to a downstream derivative, or from about 80% to about 95% of the styren
  • Microorganisms useful for the production of styrene may include bacteria, such as enteric bacteria (Escherichia, and Salmonella for example), Bacillus, Acinetobacter, Actinomycetes (such as Streptomyces), Corynebacterium, Methanotrophs (such as Methylosinus), Methylomonas,
  • Rhodococcus Pseudomona, Cyanobacteria (such as Rhodobacterand, Synechocystis), Klebsiella, Pantoea, Corynebacterium, Clostridium, etc.; yeasts, such as Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia and Torulopsis; filamentous fungi such as Aspergillus, Arthrobotrys; algae, etc.
  • mutant strains of bacteria that over-produce phenylalanine.
  • "Phenylalanine overproducing" strain is a mutant microbial strain that produces higher level of phenylalanine as compared to that of the wild-type strain that does not have the mutation. Phenylalanine is naturally present in micro-organisms.
  • a host cell preferably overproduces phenylalanine such that the substrate level does not limit styrene production by the host cell.
  • Methods to increase aromatic amino acid synthesis in a micro-organism are known in the art.
  • E. coli phenylalanine over-producer is the E. coli strain NST74 (U.S. Pat. No. 4,681,852).
  • Others suitable Phenylalanine overproducing strains include, e.g., Corynebacterium glutamicum (Ikeda, M. and Katsumata, R. Metabolic engineering to produce tyrosine or phenylalanine in a tryptophan-producing Corynebacterium glutamicum strain, Appl. Environ. Microbial. (1992), 58(3), pp.
  • phenylalanine overproducing strains can be found, e.g., in Maiti et al, Supra and Metabolic Engineering For Microbial Production Of Aromatic Amino Acids And Derived Compounds, J. Bongaertes et al, Metabolic Engineering vol 3, 289-300), 2001.
  • the host cell may also be selected for increased resistance against a toxic analogue of an aromatic amino acid (e.g., phenylalanine).
  • a toxic analogue of an aromatic amino acid e.g., phenylalanine
  • mutant micro-organisms can be selected for resistance to toxic (m-fluoro-)analogues of phenylalanine. These insensitive mutants often produce high levels of phenylalanine and tyrosine (GB 1071935; U.S. Pat. No. 3,709,785).
  • It is also possible to obtain a recombinant host cell with increased phenylalanine production by overexpression of one or more key genes in the biosynthesis of phenylalanine Ikeda 2003. Amino acid production processes. P. 1-35. in T. Scheper (Ed.), Advances in Biochemical
  • Standard recombinant DNA methodologies may be used to obtain a nucleic acid that encodes a protein described herein (e.g., phenylalanine ammonia lyase, cinnamic acid decarboxylase, or a fusion protein), incorporate the nucleic acid into an expression vector, and introduce the vector into a host cell, such as those described in Sambrook, et al. (eds), Molecular Cloning; A Laboratory Manual, Third Edition, Cold Spring Harbor, (2001); Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons (1995).
  • a protein described herein e.g., phenylalanine ammonia lyase, cinnamic acid decarboxylase, or a fusion protein
  • a nucleic acid encoding a protein may be inserted into an expression vector or vectors such that the nucleic acids are operably linked to transcriptional and translational control sequences (such as a promoter sequence, a transcription termination sequence, etc.).
  • transcriptional and translational control sequences such as a promoter sequence, a transcription termination sequence, etc.
  • the expression vector and expression control sequences are generally chosen to be compatible with the expression host cell used.
  • the expression of proteins in a microbial host described herein can be further improved by codon-optimization. For example, modifying a less-common codon with a more common codon may affect the half-life of the mRNA or alter its structure by introducing a secondary structure that interferes with translation of the message. All or a portion of a coding region can be optimized. In some cases the desired modulation of expression is achieved by optimizing essentially the entire gene. In other cases, the desired modulation will be achieved by optimizing part of but not entire sequence of the gene.
  • the codon usage of any coding sequence can be adjusted to achieve a desired property, for example high levels of expression in a specific cell type.
  • the starting point for such an optimization may be a coding sequence with 100% common codons, or a coding sequence which contains a mixture of common and non-common codons.
  • Two or more candidate sequences that differ in their codon usage can be generated and tested to determine if they possess the desired property.
  • Candidate sequences can be evaluated by using a computer to search for the presence of regulatory elements, such as silencers or enhancers, and to search for the presence of regions of coding sequence which could be converted into such regulatory elements by an alteration in codon usage. Additional criteria may include enrichment for particular nucleotides, e.g., A, C, G or U, codon bias for a particular amino acid, or the presence or absence of particular mRNA secondary or tertiary structure. Adjustment to the candidate sequence can be made based on a number of such criteria.
  • the codon optimized nucleic acid sequence can express its protein, at a level which is about 110%, about 150%, about 200%o, about 250%o, about 300%>, about 350%o, about 400%o, about 450%o, or about 500%o, of that expressed by nucleic acid sequence that has not been codon optimized.
  • the expression vector may additionally carry regulatory sequences that control the expression of the protein in a host cell, such as promoters, enhancers or other expression control elements that control the transcription or translation of the nucleic acid(s).
  • regulatory sequences are known in the art (see, e.g., Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the recombinant expression vectors of the subject technology may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the expression vector(s) encoding the protein may be transformed or transfected into a host cell by standard techniques, such as electroporation, calcium-phosphate precipitation, or DEAE- dextran trans fection.
  • a classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired microorganism or microorganisms and fermentation is permitted to occur adding nothing to the system. Typically, however, the concentration of the carbon source in a "batch" fermentation is limited and attempts are often made at controlling factors such as pH and oxygen concentration.
  • the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped.
  • cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in the log phase generally are responsible for the bulk of production of end product or intermediate.
  • a variation on the standard batch system is the Fed-Batch system.
  • Fed-Batch fermentation processes are also suitable in the subject technology and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses.
  • Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as C0 2 .
  • Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, T. D.; Biotechnology: A Textbook of Industrial Microbiology, 2nd ed.; Sinauer Associates:
  • Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in their log phase of growth.
  • Continuous fermentation allows for modulation of any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by the medium turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium removal must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
  • Plant cells may also be used as hosts for producing styrene.
  • Preferred plant hosts will be any variety that support a high expression level of phenylalanine ammonia lyase and/or cinnamic acid decarboxylase.
  • Suitable green plants will include, e.g., soybean, rapeseed (Brassica napus, B.
  • Suitable plants also include biofuel, biomass, and bioenergy crop plants.
  • exemplary plants include Arabidopsis thaliana, lice (Oryza sativa), switchgrass (Panicum vigratum), Brachypodium spp, Brassica spp., and Crambe abyssinica.
  • the plant cell is an Arabidopsis plant cell, a tobacco plant cell, a petunia plant cell, or a cell from an oilseed crop (including, e.g., a soybean plant cell, a canola plant cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, etc.).
  • an oilseed crop including, e.g., a soybean plant cell, a canola plant cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, etc.
  • Suitable host cells can be genetically engineered to express phenylalanine ammonia lyase and cinnamic acid decarboxylase.
  • nucleic acid encoding phenylalanine ammonia lyase or cinnamic acid decarboxylase can be operably linked to promoters capable of directing expression of a protein in the desired tissues at the desired stage of development. Any suitable promoter and/or terminator capable of inducing expression of a coding region may be used.
  • Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV) genes.
  • High level plant promoters that may be used in the subject technology include the promoter of the small subunit (ss) of the ribulose-l,5-bisphosphate carboxylase for example from soybean (Berry-Lowe et al., J. Molecular and App. Gen., 1 :483-498) (1982)), and the promoter of the chlorophyll a/b binding protein. These two promoters are known to be light-induced in plant cells (see, for example, Genetic Engineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum, N.Y. (1983), pages 29-38); Coruzzi, G. et al., The Journal of Biological Chemistry, 258: 1399 (1983), and Dunsmuir, P. et al., Journal of Molecular and Applied Genetics, 2:285 (1983)).
  • Standard recombinant DNA methodologies may be used to obtain a nucleic acid that encodes a protein described herein, incorporate the nucleic acid into an expression vector and introduce the vector into a host cell.
  • the choice of vector depends upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the vector. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., EMBO J. 4:2411-2418) (1985); De Almeida et al., Mol. Gen.
  • the expression of proteins in a plant host described herein can be further improved by codon-optimization, as described above.
  • the codon optimized nucleic acid sequence can express its protein, at a level which is about 110%, about 150%, about 200%o, about 250%o, about 300%, about 350%, about 400%o, about 450%o, or about 500%o, of that expressed by nucleic acid sequence that has not been codon optimized.
  • the subject technology also provides transgenic host cells or host cells that have been transformed with one or more of nucleic acids disclosed herein.
  • the nucleic acid molecule can be stably integrated into the genome of the cell, or the nucleic acid molecule can also be present as an
  • extrachromosomal molecule Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or subjects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • nucleic acid of the subject technology into a plant cell can be performed by a variety of methods known to those of ordinary skill in the art including, but not limited to, insertion of a nucleic acid sequence of interest into an Agro bacterium rhizogenes Ri or Agrobacterium tumefaciens Ti plasmid, microinjection, electroporation, or direct precipitation.
  • transient expression of a nucleic acid sequence or gene of interest can be performed by agro-infiltration methods.
  • a suspension of Agrobacterium tumefaciens containing a nucleic acid sequence or gene of interest can be grown in culture and then injected into a plant by placing the tip of a syringe against the underside of a leaf while gentle counter-pressure is applied to the other side of the leaf.
  • the Agro bacterium solution is then injected into the airspaces inside the leaf through stomata. Once inside the leaf, the Agro bacterium transforms the gene of interest to a portion of the plant cells where the gene is then transiently expressed.
  • transformation of a vector or nucleic acid of interest into a plant cell can be performed by particle gun bombardment techniques.
  • a suspension of plant embryos can be grown in liquid culture and then bombarded with plasmids or nucleic acids that are attached to gold particles, wherein the gold particles bound to the plasmid or nucleic acid of interest can be propelled through the membranes of the plant tissues, such as embryonic tissue.
  • the transformed embryos can then be selected using an appropriate antibiotic to generate new, clonally propagated, transformed embryogenic suspension cultures.
  • the subject technology provides a host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase; (b) a recombinantly expressed cinnamic acid decarboxylase; and (c) a recombinantly expressed membrane-bound transporter.
  • a membrane-bound transporter e.g. an efflux pump
  • an efflux pump can be introduced into the host cell to remove organic solvent from the cell.
  • the host cell displays a tolerant phenotype towards hydrophobic solvents.
  • the membrane-bound transporter can be an ABC-transporters (ATP- binding cassette transporters), which are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out certain biological processes including translocation of various substrates across membranes and nontransport-related processes such as translation of RNA and DNA repair. They transport a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids and sterols, and drugs. Proteins are classified as ABC transporters based on the sequence and organization of their ATP-binding cassette (ABC) domain(s).
  • ABC-transporters ATP- binding cassette transporters
  • the ABC-transporter is a solvent-resistant pump.
  • Solvent-resistant pumps conferring resistance or tolerance towards organic solvents have been shown to possess very broad specificity, taking organic compounds that by virtue of their chemical- physical characteristics (e.g., accumulating in the bacterial membrane), such as aromatics, alcohols, alkanes etc., as substrates (Kieboom et al. 1998. J. Biol. Chem. 273:85-91). Aromatic compounds also partition effectively to the cell membrane where they act as substrates for solvent-resistant pumps.
  • a host cell comprises a member of the proton-dependent resistance/nodulation/cell division (RND) family of efflux pumps.
  • RND-type efflux pumps belong to the multidrug resistance (MDR) pumps. They have an extremely broad substrate specificity and protect bacterial cells from the actions of antibiotics on both sides of the cytoplasmic membrane. Members of this family have been shown to be involved in export of antibiotics, metals, and oligosaccharides involved in nodulation signalling.
  • RND-type efflux pumps usually function as three- component assemblies spanning the outer and cytoplasmic membranes and the periplasmic space of Gram-negative bacteria.
  • the host cell comprises the solvent resistance pump srpABC of P. putida S12 (Isken et al. 1996 J. Bacterial. 178:6056; Kieboom et al. 1998. J. Biol. Chem. 273:85-91).
  • the deduced amino acid sequences of the proteins encoded by the srpABC genes have extensive homology with those of the RND family of efflux pumps. It is composed of three protein components that together span the inner and outer membranes of Gram-negative bacteria: an inner membrane transporter (SrpB analogues), an outer membrane channel (SrpC analogues), and a periplasmic linker protein (SrpA analogues). Dendrograms showing the phylogenetic relationship of SrpA, SrpB, and SrpC to other proteins involved in multidrug resistance are shown in Kieboom et al. (1998 J. Biol. Chem. 273:85-91).
  • the srpABC-encoded proteins show the most homology with those for the mexAB/oprM-encoded multidrug resistance pump found in Pseudomonas aeruginosa.
  • SrpA, SrpB, and SrpC are 57.8, 64.4, and 58.5% identical to MexA, MexB, and OprM, respectively.
  • a host cell comprises an efflux pump consisting of an inner membrane transporter, an outer membrane channel, and a periplasmic linker protein belonging to the RND-family of efflux pumps wherein the proteins show a homology of about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95%, about 98%, about 99%o, or even 100%o sequence identity to the SrpA, SrpB or SrpC proteins of P. putida S12. Any functional equivalents of known solvent efflux pumps that can use an aromatic compound as a substrate can be used.
  • the host cell can convert the fermentable carbon substrate into an aromatic amino acid (e.g., phenylalanine), which is subsequently converted into styrene.
  • an efflux pump preferably by a member of the proton- dependent resistance/nodulation/cell division (RND) family of efflux pumps, such as srpABC.
  • the bacterium P. putida S12 has also been engineered as a solvent tolerance platform for the biosynthesis of both p-hydroxybenzoate and p-hydroxystyrene (Verhoef et al., 2007,
  • the engineered strain can be used for the bioproduction of styrene.
  • Other hosts may be engineered in a similar fashion to increase tolerance to organic compounds.
  • Styrene can be harvested from the cell culture using conventional methods. For example, host cells can be removed by filtration or centrifugation. Oil phase and water phase may be separated by centrifugation or chromatography. Additional adsorption, distillation, and microfiltration techniques may be used to further purify styrene. A general scheme of purification involves removing polymerization inhibitors with alkaline water solution (usually 5-10% NaOH), washing in stilled water, drying and fractional distillation under reduced pressure, microfiltration of the styrene monomer in the gas state, and a combination thereof.
  • alkaline water solution usually 5-10% NaOH
  • the subject technology provides an inexpensive biological route to the production of styrene which is useful in a variety of commercial materials including polystyrene, ABS, styrene- butadiene (SBR) rubber, styrene-butadiene latex, SIS (styrene-isoprene-styrene), S-EB-S (styrene- ethylene/butylene-styrene), styrene-divinylbenzene (S-DVB), styrene-acrylonitrile resin (SAN) and unsaturated polyesters.
  • SBR styrene-butadiene
  • SIS styrene-isoprene-styrene
  • S-EB-S styrene- ethylene/butylene-styrene
  • S-DVB styrene-divinylbenzene
  • SAN styrene-acrylonit
  • styrene can be produced by incubating a substrate described herein (such as glucose, phenylalanine, or trans -cinnamic acid) with the host cell comprising any cinnamic acid decarboxylase described herein or any fusion proteins described herein.
  • a substrate described herein such as glucose, phenylalanine, or trans -cinnamic acid
  • the concentration of glucose in the incubation typically is from about 0.02% to about 3%, preferable from about 0.05% to about 1.5%, more preferably from about 0.1% to about 1%.
  • the concentration of trans-cinnamic acid in the incubation typically is from about 0.02% to about 0.5%, preferable from about 0.05% to about 0.2%.
  • the concentration of phenylalanine in the incubation typically is from about 0.02% to about 0.5%, preferable from about 0.05% to about 0.2%.
  • the host cells is cultured continuously for about 10 to about 72 hours, preferably from about 15 to about 36 hours, at a temperature of from about 16°C to about 37°C, preferably from 22°C to about 30°C.
  • the subject technology provides a method of producing styrene, the method comprising (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising a fusion protein as described above; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene.
  • the subject technology also provides a method of producing styrene, the method comprising (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) a mutant cinnamic acid decarboxylase as described above; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene.
  • the subject technology also provides a host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase; (b) a recombinantly expressed cinnamic acid decarboxylase; and (c) a recombinantly expressed membrane-bound transporter. Accordingly, the subject technology also provides a method for the production of styrene, the method comprising: (a) contacting the above host cell with a fermentable carbon substrate; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene.
  • styrene biosynthesis is accomplished in closed containers to prevent styrene from evaporating from the container.
  • This closed system cannot maintain the facultative anaerobic conditions (optimal conditions) for a biological system to produce styrene.
  • styrene product accumulates in the closed container and the styrene biosynthesis eventually stops due to the toxicity effect imposed by styrene on the biosynthetic system (e.g. a host cell).
  • the inclusion of an absorbing material to remove the styrene vapor from the biosynthesis process not only enables a reliable detection of the styrene product (e.g. by the method of screening FDC mutant activities as described above), but also improves the yield of styrene biosynthesis.
  • the subject technology also provides a method for producing styrene, the method comprising: (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) a cinnamic acid decarboxylase; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene, wherein the vapor of the styrene product is absorbed by an absorbing material.
  • the phenylalanine ammonia lyase and the cinnamic acid decarboxylase may include both wild type proteins and mutant proteins.
  • the cinnamic acid decarboxylase may be a mutant FDC protein as described above.
  • the phenylalanine ammonia lyase and the cinnamic acid decarboxylase may be present as separate proteins or as a fusion protein, such as the PAL- FDC(WT) or PAL-FDC(K190E) fusion proteins described above.
  • the styrene vapor is absorbed by a polymeric resin that is capable of absorbing organic molecules, while air is allowed to flow freely into the biosynthesis system (e.g. a host cell).
  • a polymeric resin that is capable of absorbing organic molecules
  • Any absorbing material used in the high-throughput screening process for cinnamic acid decarboxylase activity described above can also be employed in the styrene biosynthesis process with similar devices ( Figure 19).
  • STRATA-X column containing reverse phase polymeric resin
  • STRATA-X column can be used to absorb the styrene vapor produced from a host cell (thus reducing toxicity to the host cell), while allowing oxygen to pass through the resin to maintain facultative anaerobic conditions for cell growth.
  • this method allows styrene to be produced at level of greater than 1 g/L.
  • All the method of producing styrene described above can also be used to produce 4-hydroxystyrene.
  • tyrosine or coumaric acid or both can be used as substrate in the methods described above to produce 4-hydroxystyrene.
  • the subject technology provides a method for simultaneously screening phenylalanine ammonia lyase and cinnamic acid decarboxylase activities, the method comprising: (a) providing a fusion protein comprising: (i) a first domain comprising a phenylalanine ammonia lyase, and (ii) a second domain comprising a cinnamic acid decarboxylase; (b) mixing the fusion protein with a substrate under a condition that allows the fusion protein to convert the substrate to a product; and (c) detecting the amount of the remaining substrate, or the amount of the product, or both.
  • the subject technology also provides a method for simultaneously screening phenylalanine ammonia lyase and cinnamic acid decarboxylase activities, the method comprising: (a) providing a fusion protein comprising: (i) a first domain comprising a phenylalanine ammonia lyase, and (ii) a second domain comprising a cinnamic acid decarboxylase; (b) providing a substrate selected from the group consisting of phenylalanine, trans-cinnamic acid, tyrosine, coumaric acid, and combinations thereof; (c) incubating the fusion protein and the substrate under a condition that allows the fusion protein to convert the substrate to a product selected from the group consisting of styrene, 4-hydroxystyrene, and combination thereof; and (d) detecting the amount of the remaining substrate, or the amount of the product, or both.
  • the fusion protein may be prepared using any of the above-described PALs and cinnamic acid decarboxylases, such as PAL-FDC or PAL-FDC(K190E).
  • a substrate such as phenylalanine or trans-cinnamic acid
  • the activities of the PAL and cinnamic acid decarboxylase in the fusion protein can be simultaneously detected by measuring the amount of the remaining substrate, or the amount of the product, or both.
  • the amount of trans-cinnami acid and styrene as products can be measured.
  • the concentration of cinnamic acid reflects both its production from phenylalanine (by PAL) and its conversion to styrene (by cinnamic acid decarboxylases), and the concentration of styrene reflects the overall styrene production by the fusion protein from any substrate ( Figure 18).
  • trans-cinnamic acid is mixed with the fusion protein for conversion to styrene, both the remaining amount of trans-cinnamic acid substrate and the amount of styrene product can be measured.
  • the simultaneously screening for phenylalanine ammonia lyase and cinnamic acid decarboxylase activities can be performed under similar conditions used for the high- throughput screening assays for cinnamic acid decarboxylase activity described herein.
  • the detection of styrene comprises exposing the mixture of the fusion protein and the substrate to a polymeric resin that absorbs styrene vapor. Suitable resins include hydrophobic resins, such as CI 8, C8, phenyl, SDB-L sorbents resins, and combinations thereof.
  • FDCl was expressed in E. coli under various conditions. Plasmid pDESTl 7- FDCl was transformed into E. Coli strain BL21 (DE3) competent cells. Terrific-broth media supplemented with ampicillin (100 g/mL) was inoculated with a 1000-time dilution of an overnight culture. The bacteria were cultured at 37°C until OD 6 oo reached 0.8 to 1.0, at which point isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and continued to be cultured for 3 hours at the same temperature.
  • IPTG isopropyl ⁇ -D-l-thiogalactopyranoside
  • Buffer A contained 25 mM potassium phosphate buffer pH 7.5, 500 mM sodium chloride, 10 mM imidazole, 20mM ⁇ , 0.5 mM PMSF, 10 mM MgCl 2 , 2 g/mL DNAse, 2 g/mL RNAse, and 4 g/mL lysozyme.
  • Buffer B contained 50 mM potassium phosphate pH buffer 7.0, 1 mM DTT, 50 mM sodium thiosulfate, 20% glycerol, 500 mM NaCl, 10 mM MgCl 2 , 20 mM imidazole, 2 g/mL DNAse, 2 g/mL RNAse, and 4 g/mL lysozyme.
  • Buffer C was composed of Buffer A containing 50 mM sodium thiosulfate and 20% glycerol.
  • the cells were disrupted by ultra-sonication and the supernatant was collected by centrifugation at 15,000 g for 20 min at 4°C. The resulting supernatant was filtered through a 0.45 ⁇ PES filter and then applied to Ni + -agarose affinity column (GE Healthcare), equilibrated with the buffer A. The column was washed well with buffer A until all the non-specific binding proteins eluted from the column. The protein was eluted with buffer A containing 250 mM imidazole. The protein content was determined by the spectrophotometer at 280 nm. The protein was fairly pure (Figure 3).
  • Recombinant FDC1 that can convert tCA to styrene was purified. However, the protein lost its activity very fast during purification. The protein activity can be maintained when the purification was conducted under an anaerobic condition, such as by adding higher amounts of reducing agents (50 mM Na 2 S203, 25 mM TCEP, and 20 mM ⁇ -mercaptoethanol) in the buffers. 3. Activity Assays for Recombinantly Produced FDC1
  • the standard reaction mixture for decarboxylation consisted of 25 mM potassium phosphate buffer (pH 6.5) containing 5 mM dithiothreitol, 1.4 mM trans-cinnamic acid and enzyme (0.50 mg) to a final volume of 1.0 mL.
  • the reaction was started by the addition of the enzyme and was incubated at 30°C for 5 min.
  • the reaction was stopped by adding 24 ⁇ of glacial acetic acid (17.4N) after which 2-propanol was added in equal volume to the reaction mixture in order to solubilize the product.
  • the amount of styrene produced was measured by HPLC using a Dionex Ultimate3000 UHPLC equipped with an auto sampler, diode array (UVVVis) detector, and reverse phase Acclaim 120 CI 8 column (2.1x150 mM Dionex USA). Samples (10 ⁇ ) were injected for analyses at a total constant flow rate of 0.6 ml/minute.
  • the samples were resolved in 0.15% acetic acid (A) with an increasing concentration gradient of acetonitrile containing 0.15% acetic acid (B) for 0 to 4 min, 5%; 4 to 5 minutes, 5 to 40 %; 5 to 7 min, 40 to 45%; 7 to 8 min, 45 to 85%; 8 to 12 min, 85 to 95%; 12 to 14 minutes, 95 to 5% at a flow rate of 1 ml/min.
  • the specific activity was expressed in U (nmol styrene) , mg "1, min "1 .
  • Buffer pH is one of the main factors that can influence an enzymatic reaction. Reactions in buffers at pH 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively, were carried out to evaluate optimal pH for FDC1 activity. Potassium phosphate buffer was used for pH values 6.0 to 7.5 and Tris-HCl buffer was used for 8.0 and 8.5. The reaction mixture was composed of 200 ⁇ cinnamic acid, 5 mM DTT, 100 mM various pH buffers, and 150 L FDC1 crude extract to a final volume of 1 mL. The reaction mixtures were incubated for 30 minutes at 30°C. The styrene was extracted with 100 L of butanol.
  • the experimental negative control used the same conditions except the reaction mixture contained no substrate.
  • the optimal pH for the decarboxylation of FDC1 crude extract was found to be about 6.5.
  • Our results showed that buffer had a significant impact on FDC1 activity, and FDC1 showed highest activity at pH 6.5 ( Figure 4). This information is useful for industrial production of styrene.
  • Buffer also affects the stability of FDC1 and the reaction rate. To study the stability of FDC1, the protein was incubated in buffers of different pH values, from 5.0 to 11.0.
  • the protein (crude extract, 586 ⁇ ) was added to 112 ⁇ of 500 mM buffers at pH values of 5, 6, 7, 8, 9, 10, and 11, and incubated at 30°C for 30 minutes. After treated in various pH buffers, an aliquot of protein (42 mg) was added to a final concentration of 100 mM potassium phosphate buffer pH 6.5, 5 mM DTT, 1.4 mM cinnamic acid and brought up to a final volume of 2 mL. The reaction mixture was then incubated for 8 minutes at 30°C. The styrene was extracted by the addition of 250 L butanol. The experimental negative control used the same conditions except the reaction mixture contained no substrate.
  • the FDC1 crude extract was found to be stable at a pH ranging from 6 to 10 ( Figure 5). This protein is stable and active in a wide range of pH and can be used for industrial production of styrene.
  • the enzyme showed its maximum activity at a higher temperature as compared with other yeast enzymes. This information is useful for industrial production of styrene because a significant amount of cost associated with fermentation is cooling the fermentation system. Since FDC enzyme is active at higher reaction temperature, temperature range for fermentation can be controlled for increased yield and reduced cost on cooling.
  • reaction mixtures containing 25 mM potassium phosphate buffer pH 6.5, 5 mM DTT, 0.5mM tCA, 0.5mM cofactor, and 0.5 mg purified FDC1 to a volume of 1 mL were incubated for 30 minutes at 30°C. Styrene was extracted with 250 L butanol.
  • the experimental control used the same conditions except the reaction mixture contained no cofactors.
  • the experimental negative control used the same conditions except the reaction mixture contained no substrate.
  • the experimental control used the same conditions except the reaction mixture contained no metal ions.
  • the experimental negative control used the same conditions except the reaction mixture contained no substrate.
  • Ca 2+ , Mg 2+ , Zn 2+ , and Fe 3+ ions increased the activity of FDC1 ( Figure 10A).
  • the experimental negative control used the same conditions except the reaction mixture contained no substrate.
  • the reactions containing the metal ion and EDTA had a similar activity as compared to the control ( Figure 1 OB), indicating that Zn 2+ and Fe + increased the activity of FDC1.
  • enzyme activity of FDC1 can be increased by metal ions and it was not due to experimental artifact.
  • this piece of information suggests that including certain metal ions in the culture media can be beneficial to styrene biosynthesis.
  • tCA and its substrate analogues such as ferulic acid, 2-methylcinnamic acid, 2- hydroxycinnamic acid, 3-hydroxycinnamic acid, 4-hydroxycinnamic acid, 3,4-dimethoxycinnamic acid, and 2,5-dimethoxycinnamic acid were tested.
  • the reaction mixtures of 25 mM potassium phosphate buffer pH 6.5 containing 5 mM DTT, 0.2 mM substrate, and 0.5 mg purified FDC1 were adjusted to a final volume of 1 mL. The reaction mixtures were then incubated for 5 minutes at 30°C.
  • the experimental negative control used the same conditions except the reaction mixture contained no substrate.
  • the substrates ferulic acid, 2- methylcinnamic acid, and 4-hydroxycinnamic acid showed activities of 57%, 35%, and 68%>, respectively, compared with that of tCA ( Figure 11).
  • the enzyme did not show any activity for substrates 2-hydroxycinnamic acid, 3-hydroxycinnamic acid, 3,4-dimethoxycinnamic acid, and 2,5-dimethoxycinnamic acid ( Figure 11).
  • the enzyme did not show strict specificity for tCA; instead, it showed moderate activity for ferulic acid, 2-methylcinnamic acid and 4-hydroxycinnamic acid.
  • the K m for wild type FDCl was found to be 688 ⁇ and the V max was 6.17 nmol'mg " '•min 1 .
  • the catalytic efficiency of this protein was found to be 8.4 M _1 S _1 which is lower compared with that of other natural enzymes. Lower catalytic efficiency of FDCl is the major obstacle for production of styrene. Structure guided protein engineering will be a good approach for molecular evolution of FDCl for increasing activity.
  • Saturation mutagenesis allows the change of one amino acid to 19 other alternative amino acid residues.
  • Saturation mutagenesis was performed at sites 155-156, 159, 162-164, 172-175, 187-196, 226-227, 285-287, 291, 326, 331, 360-361, 395-396, 398, and 440-441 of FDCl by following the QuickChang site-directed mutagenesis strategy (STRATAgene, CA) using NNK degenerate primers (N represents the mixture of A, T, G, C, and K for G/T).
  • STRATAgene, CA QuickChang site-directed mutagenesis strategy
  • N represents the mixture of A, T, G, C, and K for G/T.
  • the codon NNK has 32-fold degeneracy and encodes all 20 amino acids without rare codons.
  • the QuikChange PCR products were examined by agarose gel electrophoresis and then 15 ⁇ of PCR products were digested with 1 ⁇ Dpnl (New England Biolabs) at 37 °C for 4 hours to remove the template plasmid. Aliquots of (2 ⁇ ) digestive products were transformed into BL21-Gold (DE3) competent cells (STRATAgene, CA) and inoculated on Luria-Bertani (LB) containing kanamycin. The quality of the library by DNA sequencing was confirmed. The library covers 90% of mutagenesis (17 mutants out of 19). To cover 100% (19 out of 19 mutant), we screened 150 mutants for each site.
  • the transformants (Example II.2) were inoculated in 96-well plates (NUNC, Roskilde, Denmark) containing 100 ⁇ LB broth per well and incubated at 37 °C, overnight. The cultures were then mixed with equal amount of 50% glycerol and stored at -80°C as the master plate. An aliquot of 10 ⁇ culture was inoculated in 2 ml deep-well plates (USA scientific) with 1 ml TB broth per well and incubated at 37 °C until the OD 600 reached 1.0. The cultures were then induced by 0.2 mM IPTG and incubated at 18 °C for 20 hours with shaking at 250 rpm.
  • NBP 4- nitrobenzyl-pyridine
  • Styrene was mixed with cytochrome P450 BM-3 in 50 mM phosphate buffer pH 8.0.
  • the styrene oxidation reaction was started by adding 0.2 mM NADPH, and the reaction mixture was incubated at room temperature for 5 minutes with shaking.
  • the NBP solution was added to the mixture and incubated at room temperature for further 5 minutes with shaking which gives white precipitate.
  • the tube or plate containing reaction mixtures were heated at 70 °C for 30 minutes and chilled on ice for 5 minutes.
  • reaction mixture After adding Dimethylformamide (150 ⁇ ) and 25 ⁇ of 1M K 2 CO 3 , the reaction mixture gave blue color that can be measured spectrophotometrically at 600 nm immediately.
  • the unpaired electron of NBP reacts with the oxirane ring of styrene oxide to yield a blue chromophore a blue color that can be monitored spectrophotometrically at 600 nm.
  • mutants showing higher activity were confirmed by HPLC using an ACQUITY UPLC BEH CI 8 column (1.7- ⁇ , 2.1x50-mm, Waters, USA).
  • the samples were resolved in 30% acetonitrile (A) with an increasing concentration gradient of acetonitrile (B) for 1 min, 95%; then to 1 min, 30%; at a flow rate of 1 ml/min.
  • UV absorption was monitored at 254-, 280-, and 310-nm.
  • the changed amino acid residues in the mutants showing the higher activity were confirmed by DNA sequence.
  • Mutagenesis at different sites showed an additive or synergistic effect on protein evolution of methyltransferase (Bhuiya et al., 2010, Journal of Biological Chemistry, 285: 277-285.). Mutagenesis at K190 and R175 sites of FDC1 may further increase the production of styrene.
  • Wild type FDC1 produced half the amount of styrene in an anaerobic condition, as compared to that of an aerobic condition.
  • STRATA-X® column we can trap the volatile styrene and can maintain aerobic conditions.
  • FDC-PAL2 fusion protein in E. coli. Artificial channels were included in the styrene production. To construct the fusion protein of PAL2 and FDC using Gateway technology, four primers were designed for PCR amplification.
  • the FDC-5' primer contained CACC sequence and the FDC-3' primer had a 9-amino acid linker, and two restriction sites Ncol and Pstl at the 3'-end.
  • the PAL2-5' primer correspondingly had an Ncol site and the PAL2-3' primer with a Pstl site.
  • the FDC gene ( ⁇ 1.5 kb) and the PAL2 gene (2.2 kb) were amplified using the above primers and cloned into the Gateway vector pDEST17.
  • the constructs of the fusion protein of PAL2 and FDC were transformed to BL21(DE3). Fusion proteins of FDC mutant and PAL also can be obtained by the above process.
  • the use of a linker for fusion proteins was evaluated based on the information from the resveratrol biosynthetic pathway. Previous results showed that fusion protein produced 15 fold more product compared with the expression two individual proteins (Yechun Wang et al (2011), JACS, 133: 20684-20687).
  • linker plays an important role in fusion protein for improvement of biosynthetic pathway.
  • Different length of linker 2, 3, 4, 6, 8, 9, 12 and 15 amino acid lengths were designed using GSG motif and examined the effect of linkers for resveratrol biosynthetic pathway.
  • 9 amino acid linker showed the highest yield, as compared to that of other linker ( Figure 15). Based on our findings, we designed 9 amino acid linkers for our PAL-FDC fusion proteins.
  • Some bacteria have developed multiple mechanisms to adapt unfavorable conditions.
  • Pseudomonas strains have about three strategies for addressing organic solvent toxicity, i.e. modified membrane structure, active efflux pumps, and enzymatic detoxification.
  • a recombinant E. coli host expressing an efflux pump (called solvent-resistant-pump) was produced.
  • the pump a member of the ABC-transporter family, is composed of three subunits, srpA, periplasmic linker; srpB, inner membrane transporter; and srpC, out-membrane channels.
  • the full-length of the srpABC pump sequence ( ⁇ 6 kb) was cloned from the genomic DNA of P. putida. The clone was first inserted to pENTR vector with Zeocin resistance and then to E. coli expression vector pCONA-2-DEST with Kanamycin resistance.
  • the pump was transformed to the E. coli strain BL21 (DE3) containing the fusion of PAL2 and FDC which is resistant to Ampicillin.
  • E. coli strain BL21 DE3 containing the fusion of PAL2 and FDC which is resistant to Ampicillin.
  • one clone was chosen for further experiment with fermenter. Fifty milliliters of overnight culture were inoculated in 1.5 L LB medium containing appropriate antibiotics. When cells reach an OD600 ⁇ 0.8, 5 mM of IPTG was added to the culture for induction. After 2 hrs the substrate Phe was added to a final concentration 5 g/L of cell culture. The styrene vapor was tracked in a bottle containing 250 ml of butanol.
  • a vector comprising fusion protein FDC1(K190E)- PAL was constructed, transformed it into phenylalanine producing strains (ATCC 31884 and HG), which was grown in LB containing ampicillin and/or kanamycin.
  • the cultures were grown at 30°C for 12 hours, after that the cultures were harvested and an aliquot of cultures were inoculated in M9 medium.
  • the cultures were grown at 30 °C until the OD 6 oo reached to 0.8 and then they were induced with 0.2 mM IPTG and continued to culture at 30°C or 37°C for 48hrs.
  • the volatile styrene was collected by
  • STRATA-X® column The amount of phenylalanine, tCA, and styrene was measured by HPLC.
  • the ATCC 31884 strains produced 177 mg/L of styrene from glucose at 30°C; no accumulation of tCA or phenylalanine was observed ( Figure 18A).
  • the HG strain produced 13.1 mg/L of styrene and 5.0 mg/1 of tCA from glucose at 30 °C, no accumulation of phenylalanine was observed ( Figure 18 A).
  • the BL-21(DE3) strains produced 182 mg/L of styrene from glucose by co-expression of FDC -PAL fusion protein and phenylalanine producing vector (HGTM) at 37°C.
  • HGTM phenylalanine producing vector
  • STRATA-X® column produced 25 fold higher styrene (125 mg/L), as compared with that of in anaerobic condition and in absence of STRATA-X® column (5mg/L), when FDC -PAL produced styrene from phenylalanine.
  • STRATA-X® column maintain aerobic condition, trap volatile styrene and remove styrene that ultimately increase styrene production and remove toxicity effect on culture.
  • the cells transformed with FDC wild type and FDC were fed with 0.1% trans-cinnamic acid.
  • the cells transformed with FDC (WT)-PAL and FDC (K190E)-PAL fusion proteins were fed with either 0.1% trans-cinnamic acid or 0.1 %> L-phenylalanine.
  • the fed cells were continuously cultured for 36 hours.
  • STRATA X column was used to collect styrene.
  • the product was eluted from the column by butanol and the amount was quantified by HPLC.
  • the cells transformed with FDC (K190E) mutant produced higher amount of styrene (758 mg/L) than that of FDC wild type (175 mg/ml). In comparison,
  • FDC(K190E) fused with PAL produced slightly lower amount of styrene from either trans-cinnamic acid or L-phenylalanine than that produced by FDC wild type fused with PAL.
  • FDCl that has 6-His and SUMO at the N-terminal end was constructed.
  • the DNA encoding FDCl were transformed into E. coli (Rosetta 2) competent cells and grown in LB containing ampicillin. The cultures were grown in LB at 37°C for overnight. The cultures were inoculated in terrific-broth media and grown at 37°C until the OD6 00 reached 0.8 to 1.0 at which point IPTG was added to a final concentration of 0.2 mM, and continued to culture at 16°C for 16 hours. Cells were harvested by centrifugation for 15 minutes at 4°C at 4500 rpm.
  • the cell pellet was suspended in buffer A (50 mM potassium phosphate buffer, 50 mM sodium thiosulfate, 50 mM TCEP-HC1, 500 mM NaCl, 0.5 mM PMSF, 10 mM MgCl 2 , 10 mM imidazole, 20 mM ⁇ , and 20% glycerol adjusted to pH 7.5) containing 0.1% triton X-100, and 2 g/mL DNAse, RNAse, and 4 g/mL lysozyme.
  • the cells were disrupted by ultra-sonication 10 times at amplitude 15, process time 5 seconds, 1 second pulse on/off.
  • the supernatant was collected by centrifugation for 15 minutes at 15,000 rpm at 4°C.
  • the supernatant was filtered through a 0.45 ⁇ PES filter and applied to a Ni + -agarose affinity column (GE Healthcare).
  • the column was washed with buffer A until non-specific binding proteins eluted from the column.
  • FDCl was eluted with 50 mM potassium phosphate, 50 mM sodium thiosulfate, 50 mM TCEP-HC1, 500 mM sodium chloride, 0.5 mM PMSF, 10 mM magnesium chloride, 250 mM imidazole, 20 mM ⁇ , and 20% glycerol adjusted to pH 8.0.
  • a total of 45.0 mg protein was found after Ni + -agarose affinity column.
  • Hydrolase (0.5 mg) was added to cleave SUMO from the FDCl . Digestion was carried out at 4°C by dialysis of the protein overnight in 1 L of 25 mM potassium phosphate, 50 mM sodium thiosulfate, 500 mM NaCl, 5 mM DTT, and 20% glycerol adjusted to pH 7.5. Dialysis was continued in a fresh liter of dialysis buffer for 2-4 hours the next morning.
  • the protein was dialyzed overnight in 1 L Q-column buffer A (25 mM potassium phosphate, 5 mM DTT, and 25 mm sodium thiosulfate adjusted to pH 7.5).
  • Q-column buffer A 25 mM potassium phosphate, 5 mM DTT, and 25 mm sodium thiosulfate adjusted to pH 7.5.
  • Anion- exchange chromatography The anion-exchange Q-column (GE Healthcare) was equilibrated with Q-column buffer A. The protein was loaded into the column, and washed with buffer A until all the non-specific binding proteins were eluted from the column. The protein was eluted with Q- column buffer A containing 1 M sodium chloride. Elution started at 27% Q-column buffer B. The fractions containing FDC1 were used for SDS-PAGE to check the purity of the protein.
  • a total of 5.0 mg protein was found after anion-exchange chromatography.
  • the protein was dialyzed overnight in size exclusion buffer (50 mM sodium phosphate pH 7.5, 150 mM NaCl, 5 mM DTT, and 25 mM sodium thiosulfate).
  • FDC mutants for example, FDC (K190E) having an amino acid sequence as set forth in SEQ ID NO: 16, can be purified by the same process as FDC1 as described above.
  • Molecular replacement (Phaser) in CCP4i suite was used to solve the structure of FDC(K190E) mutant using 3-octaprenyl-4-hydroxybenzoate decarboxylase (2IDB) structure as a model template.
  • the initial model of the structures was built by manual building using the COOT and the model was refined using Refmac5.
  • a typical electron density with current initial model of FDC is provided in Figure 21 A.
  • An asymmetric unit of the crystal structure containing 3 chains is shown in Figure 21B.
  • FDC(K190E) molecules A and B form a dimer that is biologically active
  • FDC(K190E) molecule C forms another dimer with its partner of another asymmetric unit (not shown).

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Abstract

The subject technology generally relates to biosynthesis of styrene. Certain embodiments of the subject technology is based, in part, on the recognition that phenylalanine can be converted to styrene by a two-step pathway of deamination and decarboxylation, with trans-cinnamic acid (tCA) as the intermediate. Two types of enzymes are directly involved in this process, phenylalanine ammonia lyase (PAL), which converts phenylalanine to tCA, and cinnamic acid decarboxylase, which coverts tCA to styrene. Host cells expressing these two types of enzymes can be cultured in bioreactor to produce styrene from renewable substrates such as glucose.

Description

ENZYMES AND METHODS FOR STYRENE SYNTHESIS
INCORPORATION OF SEQUENCE LISTING
[0001] A paper copy of the Sequence Listing and a computer readable form of the Sequence Listing containing the file named "32990-5_ST25.txt", which is 151 ,674 bytes in size (as measured in MS-DOS), are provided herein and are herein incorporated by reference. This Sequence Listing consists of SEQ ID NOs: 1-44.
BACKGROUND
[0002] The subject technology generally relates to enzymes and methods for biosynthesis of styrene.
[0003] Styrene (vinyl benzene) is an organic compound with a chemical formula of CsH8. This cyclic hydrocarbon is a colorless, oily liquid that evaporates easily and has a sweet rubber-like smell. At higher concentrations, styrene confers a less pleasant odor. Styrene is named after the styrax trees (Styrax platanifolius) from which sap (a type of benzoin resin) can be extracted. Low levels of styrene occur naturally in several plant species. A variety of foods such as fruits, vegetables, nuts, beverages, and meats also contain styrene.
[0004] Industrially, styrene is the precursor to polystyrene and several copolymers. The presence of the vinyl group allows styrene to polymerize. Approximately 15 billion pounds are produced annually. The production of styrene in the United States increased dramatically during the 1940s, when it was popularized as a feedstock for synthetic rubber. Today, commercially significant products include polystyrene, acrylonitrile butadiene styrene (ABS), styrene-butadiene (SBR) rubber, styrene-butadiene latex, SIS (styrene-isoprene-styrene), S-EB-S (styrene-ethylene/butylene-styrene), styrene-divinylbenzene (S-DVB), styrene-acrylonitrile resin (SAN) and unsaturated polyesters. These materials are used in rubber, plastic, insulation, fiberglass, pipes, automobile and boat parts, food containers, and carpet backing.
[0005] Styrene is produced in industrial quantities mostly from ethylbenzene, which is in turn prepared on a large scale by alkylation of benzene with ethylene. It is one of the most important petrochemical products. There are several methods to produce styrene. Dehydrogenation of ethylbenzene is the most common way of production. Ethylbenzene is mixed in the gas phase with 10-15 times its volume in high-temperature steam, and passed over a solid catalyst bed. Most ethylbenzene
dehydrogenation catalysts are based on iron(III) oxide, promoted by several percent potassium oxide or potassium carbonate. Steam serves several roles in this reaction. It is the source of heat for powering the endothermic reaction, and it removes coke that tends to form on the iron oxide catalyst through the water gas shift reaction. The potassium promoter enhances this decoking reaction. The steam also dilutes the reactant and products, shifting the position of chemical equilibrium towards products. A typical styrene plant consists of two or three reactors in series, which operate under vacuum to enhance the conversion and selectivity. Typical per-pass conversions are ca. 65% for two reactors and 70-75% for three reactors. Selectivity to styrene is 93-97%>. The main byproducts are benzene and toluene. Because styrene and ethylbenzene have similar boiling points (145 and 136 °C, respectively), their separation requires tall distillation towers and high return/reflux ratios. At its distillation temperatures, styrene tends to polymerize. To minimize this problem, early styrene plants added elemental sulfur to inhibit the polymerization. During the 1970s, new free radical inhibitors consisting of nitrated phenol-based retarders were developed. More recently, a number of additives have been developed that exhibit superior inhibition against polymerization.
[0006] Since styrene is an essential petrochemical used in many chemical products, alternative production methods, especially ones that do not require fossil fuels as feed stock, are urgently needed. Hence, despite the availability of methods for producing styrene, there is a continuing need for new methods for producing styrene monomers that are efficient and less expensive.
SUMMARY
[0007] The subject technology generally relates to the biosynthesis of styrene. Some embodiments of the subject technology are based, in part, on the recognition that phenylalanine can be converted to styrene by a two-step pathway of deamination and decarboxylation, with trans-cinnamic acid (tCA) as the intermediate. Two types of enzymes are directly involved in this process, phenylalanine ammonia lyase (PAL), which converts phenylalanine to tCA, and cinnamic acid decarboxylase, which coverts tCA to styrene. Host cells expressing these two types of enzymes can be cultured in a bioreactor to produce styrene from renewable substrates such as glucose.
[0008] In one aspect, the subject technology relates to a fusion protein comprising: (a) a first domain that comprises a phenylalanine ammonia lyase, and (b) a second domain that comprises a cinnamic acid decarboxylase.
[0009] In certain embodiments, the phenylalanine ammonia lyase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces . In an exemplary embodiment, the phenylalanine ammonia lyase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof.
[0010] In certain embodiments, the cinnamic acid decarboxylase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces. In an exemplary embodiment, the cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; and a functional fragment or variant thereof. For example, the cinnamic acid decarboxylase may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38.
[0011] A functional variant of a cinnamic acid decarboxylase may comprise an amino acid sequence that is about 85%, about 90%, about 95%o, about 96%o, about 97%o, about 98%o, about 99%o, or even 100% identical to any one of SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38. Alternatively, or in addition, a functional variant of a cinnamic acid decarboxylase can comprise: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
[0012] In certain embodiments, the cinnamic acid decarboxylase can comprise a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, or a combination thereof. For example, the mutant cinnamic acid decarboxylase can comprise a mutation at an amino acid residue position corresponding to one of the following positions: 175, 190, 193 of SEQ ID NO:8, and a combination thereof.
[0013] In certain embodiments, the cinnamic acid decarboxylase can comprise a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8 selected from: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441, and a combination thereof. For example, an amino acid residue at one the of the following positions: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441 of SEQ ID NO:8 can be substituted with another amino acid residue. In another example, the mutant cinnamic acid decarboxylase comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 175, 190, 193, and a combination thereof. [0014] In one embodiment, the fusion protein of the subject technology further comprises a linker covalently linking the first domain and the second domain. The linker of the fusion protein described herein may be a peptide linker, e.g., a peptide linker comprising 2 to 15 amino acids. Peptide linkers can include those shown in Table 1, for example.
[0015] Also provided are nucleic acids encoding the fusion proteins described herein, a vector comprising the nucleic acid encoding the fusion protein, and host cells comprising the vector described herein.
[0016] In one aspect, the subject technology relates to a method for producing styrene comprising (a) contacting a host cell with a fermentable substrate (preferably carbon substrate or nitrogen substrate), the host cell comprises a fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase, and (ii) a second domain that comprises a cinnamic acid decarboxylase; and (b) culturing the cell in a culture medium for a time sufficient to produce styrene. The method can further comprise harvesting styrene from the cell culture. Any one of the fusion proteins described herein can be used to produce styrene.
[0017] In one aspect, the subject technology relates to a cinnamic acid decarboxylase comprising (a) any one of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38; (b) an amino acid sequence that is about 85%, about 90%>, about 95%o, about 97%, about 98%, about 99%, or even 100% identical to any one of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38, with the proviso that said amino acid sequence is not SEQ ID NO: 8; or (c) a functional fragment of (a) or (b).
[0018] In certain embodiments, the cinnamic acid decarboxylase comprises: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
[0019] In certain embodiments, the cinnamic acid decarboxylase comprises any one of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38. [0020] The subject technology also relates to a mutant cinnamic acid decarboxylase comprising a mutation at an amino acid residue position corresponding to one of the following positions: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, and a combination thereof. For example, the mutant cinnamic acid decarboxylase can comprise a mutation at an amino acid residue position corresponding to one of the following positions: 175, 190, 193 of SEQ ID NO:8, and a combination thereof.
[0021] In certain embodiments, the mutant cinnamic acid decarboxylase comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441, and a combination thereof. In certain embodiments, the mutation is a substitution.
[0022] In certain embodiments, the mutant cinnamic acid decarboxylase comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 175, 190, 193, and a combination thereof.
[0023] Also provided are nucleic acids encoding the cinnamic acid decarboxylases described herein, host cells comprising the cinnamic acid decarboxylases described herein, and host cells comprising the nucleic acids described herein.
[0024] The subject technology also relates to a method for the production of styrene comprising: (a) contacting a host cell with a fermentable substrate (preferably carbon substrate or nitrogen substrate), the host cell comprising (i) a phenylalanine ammonia lyase as described herein; and (ii) a cinnamic acid decarboxylase as described herein; and (b) culturing the cell in a culture medium for a time sufficient to produce styrene.
[0025] The subject technology also relates to a host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase as described herein; (b) a recombinantly expressed cinnamic acid decarboxylase as described herein; and (c) a recombinantly expressed membrane-bound transporter as described herein. The phenylalanine ammonia lyase and the cinnamic acid decarboxylase can be expressed as two separate proteins, or can be covalently linked by a linker as described herein.
[0026] In one embodiment, the membrane-bound transporter is an ATP-binding cassette transporter (ABC transporter). For example, the ABC transporter is a bacterial ABC transporter, such as one derived from Pseudomonas putida. Preferably, the ABC transporter is a solvent resistance efflux pump, such as the SrpABC pump derived from Pseudomonas putida SI 2. [0027] The subject technology also provides a method for screening candidate proteins for mutated cinnamic acid decarboxylase activity, the method comprising:(a) providing a protein sample comprising a candidate protein, and a substrate selected from the group consisting of phenylalanine, trans- cinnamic acid, tyrosine, coumaric acid, and combinations thereof; (b) combining the protein sample and the substrate sample to form a mixture, and incubating the mixture under a condition that allows a mutated cinnamic acid decarboxylase to convert the substrate to a product selected from the group consisting of styrene, 4-hydroxystyrene, and combination thereof; and (c) exposing the mixture to a detection material that comprises a polymeric resin that absorbs the product vapor. In one embodiment, the mutated cinnamic acid decarboxylase is capable of converting trans-cinnamic acid to styrene, at a rate that is comparable to or higher than the wild type enzyme (e.g. FDC1). In another embodiment, the mutated cinnamic acid decarboxylase is capable of converting coumaric acid to 4-hydroxystyrene, at a rate that is comparable to higher than the wild type enzyme. In certain embodiments, the candidate protein comprises a fusion protein comprising a mutated cinnamic acid decarboxylase.
[0028] In one embodiment, the subject technology also relates to a method for screening candidate proteins for cinnamic acid decarboxylase activity, comprising: (a) providing a protein sample comprising the candidate protein, and a substrate sample comprising trans-cinnamic acid; (b) combining the protein sample and substrate sample to form a mixture, and incubating the mixture under a condition that allows a cinnamic acid decarboxylase to convert trans-cinnamic acid to styrene; and (c) exposing the mixture to a detection material that comprises a polymeric resin that absorbs styrene vapor.
[0029] In certain embodiments, the detection material further comprises a detectable marker that causes a color change in the presence of styrene. For example, the detectable marker can be 4- nitrobenzyl-pyridine. The detection material can be attached to a solid support. In certain embodiments, the polymeric resin comprises an aromatic functional group.
[0030] In certain embodiments, the change of color can be detected by spectrophotometry. For example, the change of color can be detected by measuring the absorbance of the sample at about 600 nm wavelength.
[0031] The screening method described herein can be used to simultaneously screen a plurality of candidate proteins.
[0032] The subject technology also relates to a method of isolating a recombinantly produced cinnamic acid decarboxylase, comprising (a) providing a bacterial host comprising a nucleic acid that encodes a cinnamic acid decarboxylase operably linked to a promoter sequence; (b) culturing the bacterial host in a culture medium to express the cinnamic acid decarboxylase in the host cell, therein the host cell is cultured at a temperature that is from about 10 °C to about 25 °C; and ( c) isolating the cinnamic acid decarboxylase from the host cell, wherein the isolation is conducted in an anaerobic environment. [0033] In certain embodiments, one or more buffer solutions used for isolating the cinnamic acid decarboxylase comprises a reducing agent, such as Tris(2-carboxyethyl) phosphine (TCEP), β- mercaptoethanol, and a combination thereof.
[0034] The subject technology also relates to a method of crystallizing a cinnamic acid decarboxylase, the method comprising: (a) providing a cinnamic acid decarboxylase solution at a concentration of from about 1 mg/ml to about 50 mg/ml; (b) mixing the cinnamic acid decarboxylase solution with a reservoir solution at a volume ratio of from about 1 : 10 to about 10:1 ; and (c) maintaining the mixture of the cinnamic acid decarboxylase solution and the reservoir solution at a temperature suitable for the formation of the cinnamic acid decarboxylase crystals.
[0035] The subject technology also relates to a crystal of cinnamic acid decarboxylase, wherein the cinnamic acid decarboxylase is in a complex with 3-hydroxyl cinnamic acid.
[0036] The subject technology also relates to a method for producing styrene, the method comprising: (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) a cinnamic acid decarboxylase; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene, wherein the vapor of the styrene product is absorbed by an absorbing material.
[0037] A method for simultaneously screening phenylalanine ammonia lyase and cinnamic acid decarboxylase activities, the method comprising: (a) providing a fusion protein comprising: (i) a first domain comprising a phenylalanine ammonia lyase, and (ii) a second domain comprising a cinnamic acid decarboxylase; (b) mixing the fusion protein with a substrate under a condition that allows the fusion protein to convert the substrate to a product; and (c) detecting the amount of the remaining substrate, or the amount of the product, or a combination thereof. In certain embodiments, the screening comprises detecting the loss of the substrate, or the amount of a product, such as trans-cinnami acid, styrene, and the downstream derivatives of styrene. For example, the screening can comprise detecting the amount of trans -cinnamic acid, or styrene, or a combination thereof.
[0038] The subject technology is illustrated, for example, according to various aspects described below. Various examples of aspects of the subject technology are described as numbered clauses (1, 2, 3, etc.) for convenience. These are provided as examples and do not limit the subject technology. It is noted that any of the dependent clauses may be combined in any combination, and placed into a respective independent clause, e.g., clause 1 or clause 17. The other clauses can be presented in a similar manner.
[0039] 1. A fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase, and (ii) a second domain that comprises a cinnamic acid decarboxylase; and (iii) a linker that covalently links the first domain and the second domain. [0040] 2. The fusion protein of clause 1, wherein said phenylalanine ammonia lyase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
[0041] 3. The fusion protein of clause 1 or 2, wherein said phenylalanine ammonia lyase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof.
[0042] 4. The fusion protein of any one of clauses 1-3, wherein said cinnamic acid decarboxylase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
[0043] 5. The fusion protein of any one of clauses 1-4, wherein said cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and a functional fragment or variant thereof.
[0044] 6. The fusion protein of clause 5, wherein said cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.
[0045] 7. The fusion protein of clause 5, wherein said functional variant comprises an amino acid sequence that is about 85% identical to any one of SEQ ID NOs: 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.
[0046] 8. The fusion protein of clause 5 or 7, wherein said functional variant comprises: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
[0047] 9. The fusion protein of any one of clauses 1-4, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441 of SEQ ID NO: 8.
[0048] 10. The fusion protein of any one of clauses 1-4, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following positions: 175 or 190 of SEQ ID NO:8. [0049] 11. The fusion protein of any one of clauses 1-4, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
[0050] 12. The fusion protein of any one of clauses 1-4, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a substitution of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
[0051] 13. The fusion protein of any one of clauses 1-4, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8:175 or 190.
[0052] 14. The fusion protein of any one of clauses 1-13, wherein said linker is a peptide linker comprising 2 to 15 amino acids.
[0053] 15. The fusion protein of any one of clauses 1-14, wherein said linker is a peptide linker consisting essentially of glycine and serine.
[0054] 16. The fusion protein of any one of clauses 1-15, wherein said linker comprises an amino acid sequence as set forth in any one of SEQ ID NOs. 39-44.
[0055] 17. A nucleic acid encoding any one of the fusion protein of clauses 1-16.
[0056] 18. A host cell comprising any one of the fusion protein of clauses 1-16.
[0057] 19. A host cell comprising the nucleic acid of clause 17.
[0058] 20. A method for the producing styrene comprising: (a) contacting a host cell with a fermentable carbon substrate, said host cell comprises a fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase; (ii) a second domain that comprises a cinnamic acid decarboxylase; and (iii) a linker that covalently links the first domain and the second domain; (b) culturing said cell in a culture medium for a time sufficient to produce styrene.
[0059] 21. The method of clause 20, further comprising harvesting styrene from said cell culture.
[0060] 22. The method of clause 20 or 21, wherein said phenylalanine ammonia lyase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces. [0061] 23. The method of any one of clauses 20-22, wherein said phenylalanine ammonia lyase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof.
[0062] 24. The method of any one of clauses 20-23, wherein said cinnamic acid decarboxylase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
[0063] 25. The method of any one of clauses 20-24, wherein said cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and a functional fragment or variant thereof.
[0064] 26. The method of clause 25, wherein said cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.
[0065] 27. The method of clause 25, wherein said functional variant comprises an amino acid sequence that is about 85% identical to any one of SEQ ID NOs: 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.
[0066] 28. The method of clause 25 or 27, wherein said functional variant comprises: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
[0067] 29. The method of any one of clauses 20-24, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441 of SEQ ID NO: 8.
[0068] 30. The method of any one of clauses 20-24, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following positions: 175 or 190 of SEQ ID NO:8.
[0069] 31. The method of any one of clauses 20-24, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
[0070] 32. The method of any one of clauses 20-24, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a substitution of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
[0071] 33. The method of any one of clauses 20-24, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 175 or 190.
[0072] 34. The method of any one of clauses 20-33, wherein said linker is a peptide linker comprising 2 to 15 amino acids.
[0073] 35. The method of any one of clauses 20-34, wherein said linker is a peptide linker consisting essentially of glycine and serine.
[0074] 36. The method of any one of clauses 20-35, wherein said linker comprises an amino acid sequence as set forth in any one of SEQ ID NOs. 39-44.
[0075] 37. A cinnamic acid decarboxylase comprising (i) any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38; (ii) an amino acid sequence that is about 85% identical to any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38, with the proviso that said amino acid sequence is not SEQ ID NO: 8; or (iii) a functional fragment of (i) or (ii).
[0076] 38. The cinnamic acid decarboxylase of clause 37, wherein said amino acid sequence comprises: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
[0077] 39. The cinnamic acid decarboxylase of clause 37 or 38, comprising any one of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.
[0078] 40. A mutant cinnamic acid decarboxylase comprising a mutation at an amino acid residue position corresponding to one of the following positions: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441 of SEQ ID NO:8. [0079] 41. The mutant cinnamic acid decarboxylase of clause 40, comprising a mutation at an amino acid residue position corresponding to one of the following positions: 175 or 190 of SEQ ID NO: 8.
[0080] 42. A mutant cinnamic acid decarboxylase comprising a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
[0081] 43. The mutant cinnamic acid decarboxylase of clause 42, comprising a substitution of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
[0082] 44. The mutant cinnamic acid decarboxylase of clause 42, comprising a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 175 or 190.
[0083] 45. A nucleic acid encoding the cinnamic acid decarboxylase of clause 37-44.
[0084] 46. A host cell comprising the cinnamic acid decarboxylase of clause 37-44.
[0085] 47. A host cell comprising the nucleic acid of clause 45.
[0086] 48. A method for the production of styrene comprising: (a) contacting a host cell with a fermentable carbon substrate, said host comprises (i) a phenylalanine ammonia lyase; and (ii) a cinnamic acid decarboxylase of any one of clauses 37-44; (b) culturing said cell in a culture medium for a time sufficient to produce styrene.
[0087] 49. The method of clause 48, further comprising harvesting styrene from said cell culture.
[0088] 50. The method of clause 48 or 49, wherein said phenylalanine ammonia lyase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
[0089] 51. The method of any one of clauses 48-50, wherein said phenylalanine ammonia lyase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof.
[0090] 52. A host cell comprising: (i) a recombinantly expressed phenylalanine ammonia lyase; (ii) a recombinantly expressed cinnamic acid decarboxylase; and (iii) a recombinantly expressed AB C -transporter . [0091] 53. The host cell of clause 52, wherein said phenylalanine ammonia lyase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
[0092] 54. The host cell of clause 52 or 53, wherein said phenylalanine ammonia lyase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof.
[0093] 55. The host cell of any one of clauses 52-54, wherein said cinnamic acid decarboxylase is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
[0094] 56. The host cell of any one of clauses 52-55, wherein said cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and a functional fragment or variant thereof.
[0095] 57. The host cell of clause 56, wherein said cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.
[0096] 58. The host cell of clause 56, wherein said functional variant comprises an amino acid sequence that is about 85% identical to any one of SEQ ID NOs: 8, 10, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38.
[0097] 59. The host cell of clause 56 or 58, wherein said functional variant comprises: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
[0098] 60. The host cell of any one of clauses 52-55, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following positions: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441 of SEQ ID NO:8.
[0099] 61. The host cell of any one of clauses 52-55, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441 of SEQ ID NO: 8.
[0100] 62. The host cell of any one of clauses 52-55, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a mutation at an amino acid residue position corresponding to one of the following positions: 175 or 190 of SEQ ID NO:8.
[0101] 63. The host cell of any one of clauses 52-55, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
[0102] 64. The host cell of any one of clauses 52-55, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a substitution of an amino acid residue at one of the positions of SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187,
188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, or 441.
[0103] 65. The host cell of any one of clauses 52-55, wherein said cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase that comprises a deletion, a substitution, or an addition of an amino acid residue at one of the positions of SEQ ID NO:8: 175 or 190.
[0104] 66. The host cell of any one of clauses 52-65, wherein said phenylalanine ammonia lyase and said cinnamic acid decarboxylase are covalently linked by a linker.
[0105] 67. The host cell of clause 66, wherein said linker is a peptide linker comprising 2 to 15 amino acids.
[0106] 68. The host cell of clause 66 or 67, wherein said linker is a peptide linker consisting essentially of glycine and serine.
[0107] 69. The host cell of any one of clauses 66-68, wherein said linker comprises an amino acid sequence as set forth in SEQ ID NOs. 39-44.
[0108] 70. The host cell of any one of clauses 52-69, wherein said ABC transporter is a bacterial ABC transporter.
[0109] 71. The host cell of clause any one of clauses 52-70, wherein said ABC transporter is derived from Pseudomonas putida.
[0110] 72. The host cell of clause any one of clauses 52-71, wherein said ABC transporter is a solvent resistance efflux pump. [0111] 73. The host cell of clause any one of clauses 52-72, wherein said ABC transporter is SrpABC pump derived from Pseudomonas putida SI 2.
[0112] 74. A method for screening a candidate proteins for cinnamic acid decarboxylase activity, comprising: (a) providing a protein sample comprising said candidate protein, and a substrate sample comprising trans-cinnamic acid; (b) combining said protein sample and substrate sample to form a mixture, and incubating said mixture under a condition that allows a cinnamic acid decarboxylase to convert trans-cinnamic acid to styrene; and (c) exposing said mixture to a detection material that comprises (i) polymeric resin that absorbs styrene vapor; and (ii) a detectable marker that causes a color change in the presence of styrene; wherein a change of the color of said detection material indicates that said candidate protein has cinnamic acid decarboxylase activity.
[0113] 75. The method of clause 74, further comprising comparing the activity of said candidate protein with a control.
[0114] 76. The method of clause 74 or 75, wherein said detection material is attached to a solid support.
[0115] 77. The method of any one of clauses 74-76, said polymeric resin comprises an aromatic functional group.
[0116] 78. The method of any one of clauses 74-77, wherein said change of color is detected by spectrophotometry.
[0117] 79. The method of any one of clauses 74-78, wherein said detectable marker is 4- nitrobenzyl-pyridine.
[0118] 80. The method of clause 79, wherein said change of color is detected by measuring the absorbance of the sample at about 600 nm wavelength.
[0119] 81. The method of any one of clauses 74-80, wherein a plurality of candidate proteins are screening simultaneously.
[0120] 82. A method of isolating a recombinantly produced cinnamic acid decarboxylase, comprising: (a) providing a bacterial host comprising a nucleic acid that encodes a cinnamic acid decarboxylase operably linked to a promoter sequence; (b) culturing said bacterial host in a culture medium to express said cinnamic acid decarboxylase in said host cell, therein said host cell is cultured at a temperature that is from about 10 °C to about 25 °C; and (c) isolating said cinnamic acid decarboxylase from said host cell, wherein said isolation is conducted in an anaerobic environment.
[0121] 83. The method of clause 82, wherein one or more buffer solutions used for isolating said cinnamic acid decarboxylase comprises a reducing agent. [0122] 84. The method of clause 83, wherein said reducing agent is Tris(2-carboxyethyl) phosphine (TCEP) or β-mercaptoethanol.
BRIEF DESCRIPTION OF THE DRAWINGS
[0123] Figure 1 A is a schematic illustration of the predicted structure of yeast FDC 1. Figure IB shows four possible tCA binding sites in FDCl (sites A-D). Figure 1C shows the predicted substrate (tCA) binding site in yeast FDCl.
[0124] Figure 2 A shows the expression of FDCl in E. coli at 37°C, using three different buffers. The recombinantly produced FDCl only accumulated in the insoluble pellet fractions (inclusion bodies). Figure 2B shows the expression of FDCl in E. coli at 16°C. Soluble FDCl was detected in the supernatant.
[0125] Figure 3 shows the SDS-PAGE analysis of purified FDCl .
[0126] Figure 4 shows the effect of pH on FDCl activity. The enzyme showed maximum activity at pH 6.5, and is considered as having 100% relative activity. The x-axis represents pH values of different reaction buffers.
[0127] Figure 5 shows the pH stability of FDCl . The enzyme exhibited good pH stability. It did not lose any activity when incubated for 30 minutes at pH 6.0 prior to the reaction, and is considered as having 100% relative activity. The x-axis represents pH values at which the protein was incubated for 30 minutes prior to enzymatic reaction.
[0128] Figure 6 shows the effect of temperature on FDCl activity. The enzyme showed maximum activity when the reaction was carried out at 50 °C, and is considered as having 100% relative activity. The x-axis represents temperatures at which the enzymatic reactions were carried out.
[0129] Figure 7 shows the temperature stability of FDCl. The enzyme showed maximum activity at 50 °C, and did not lose any activity when incubated at 50 °C for 30 minutes prior to the reaction (which is considered as having 100% relative activity). The x-axis represents temperatures at which the enzyme was incubated prior to enzymatic reaction.
[0130] Figure 8 shows the temperature stability of FDCl at 50°C for various time periods. The enzyme showed maximum activity after being incubated at 50°C for 30 minutes prior to the reaction, and is considered as having 100% relative activity. The x-axis represents the duration (in minutes) in which the enzyme was incubated at 50°C prior to enzymatic reaction.
[0131] Figure 9 shows the effect of cofactors on FDCl activity. Specific activity is shown as nmol of styrene produced per mg enzyme per minute. [0132] Figure 10A shows the effect of metal ions on FDCl activity. Figure 10B shows the effect of Zn2+ and Fe + on FDCl activity. Specific activity is shown as nmol of styrene produced per mg enzyme per minute.
[0133] Figure 11 shows the substrate specificity of FDC 1. The enzyme showed maximum activity when i-cinnamic acid is used as a substrate, and is considered as having 100% relative activity.
[0134] Figures 12A and 12B depict the kinetic analysis of FDCl. The x-axis represents the enzymatic reaction with various concentrations of substrate. Specific activity is shown as nmol of styrene produced per mg enzyme per minute.
[0135] Figure 13 depicts the process of random mutagenesis to create FDC mutants, and the high throughput colorimetric screening of FDCl mutants.
[0136] Figure 14 shows styrene production by FDCl mutants. The Y-axis represents amount of styrene produced by FDCl mutants. The X-axis represents changed amino acids of the FDCl mutants.
[0137] Figure 15 shows the activities of FDC-PAL fusion proteins. The X-axis shows the lengths of the linkers.
[0138] Figure 16 is a schematic illustration of the predicted structure of FDC-PAL fusion protein.
[0139] Figure 17 shows that the production of styrene was significantly increased with the co- expression of an ABC-transporter.
[0140] Figure 18A shows the production of styrene and tCA from glucose.
[0141] Figure 18B shows production of styrene from trans-cinnamic acid or phenylalanine.
[0142] Figure 19 shows the use of STRATA-X® resin on top of a culture tube (left) or on top of a 96-well culture plate (right).
[0143] Figure 20 shows the SDS-PAGE analysis of purified FDCl.
[0144] Figure 21A shows a typical electron density map with initial model of the crystalline structure of FDC(K190E).
[0145] Figure 21B shows an asymmetric unit of the crystalline structure of FDC(K190E).
DETAILED DESCRIPTION
A. OVERVIEW
[0146] The subject technology generally relates to biosynthesis of styrene. Certain embodiments of the subject technology is based, in part, on the recognition that phenylalanine can be converted to styrene by a two-step pathway of deamination and decarboxylation, with trans-cinnamic acid (tCA) as the intermediate. Two types of enzymes are directly involved in this process, phenylalanine ammonia lyase (PAL), which converts phenylalanine to tCA, and cinnamic acid decarboxylase, which coverts tCA to styrene. Host cells expressing these two types of enzymes can be cultured in bioreactor to produce styrene from renewable substrates such as glucose.
Glucose
Figure imgf000020_0001
phenylalanine trans-cinnamic acid styrene
[0147] In particular, several approaches have been adopted to enhance the bioproduction of styrene. For example, as described and exemplified herein, fusion proteins have been designed in which phenylalanine ammonia lyase and cinnamic acid decarboxylase are covalently linked by a linker. The fusion protein takes advantage of the "substrate channeling" phenomenon. Substrate channeling refers to a phenomenon in which substrates are efficiently delivered from enzyme to enzyme without equilibration with other pools of the same substrates. In effect, this creates local pools of metabolites at high concentrations relative to those found in other areas of the cell. Because the product of phenylalanine ammonia lyase (trans-cinnamic acid) is consumed by cinnamic acid decarboxylase, proximity between these two enzymes (by covalently linking the two enzymes in the form of a fusion protein) would provide for a more efficient use of the substrate. As such, fusion proteins linking these two enzymes benefit from the substrate channeling phenomenon, and can reduce production costs and increase the number of enzymatic reactions that occur during a given time period. Alternatively, a protein complex in which the phenylalanine ammonia lyase and cinnamic acid decarboxylase form a protein complex via non-covalent interaction can also be used.
[0148] Accordingly, in one aspect, the subject technology provides a fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase, and (ii) a second domain that comprises a cinnamic acid decarboxylase. Host cells comprising fusion proteins described herein, as well as method of using the fusion protein for styrene production are also provided.
[0149] In another approach, a library of mutant cinnamic acid decarboxylases have been designed, and screened for their respective activities. Mutant cinnamic acid decarboxylases showing higher catalytic activities, as compared to that of wild type, were identified. Some of the mutant cinnamic acid decarboxylases described herein increased the styrene production by two- to three-fold. These mutant cinnamic acid decarboxylases can be introduced to host cells to promote the bioproduction of styrene. [0150] In another aspect, a high throughput, colorimetric screening method can allow for large- scale screening of mutant cinnamic acid decarboxylases or fusion proteins. The colorimetric screening described herein is highly reproducible and can screen about 1 ,000 mutant cinnamic acid decarboxylases and fusion proteins in a single day. This can provide fast and recursive screening of enzymes involved in biosynthesis of styrene and related compounds, providing an important method for improvement of styrene biosynthesis.
[0151] Accordingly, in another aspect, the subj ect technology provides mutant cinnamic acid decarboxylases, host cells comprising a mutant cinnamic acid decarboxylase, as well as method of using the mutant cinnamic acid decarboxylases for styrene production. Also provided herein are libraries of mutant cinnamic acid decarboxylases, and method of screening a candidate protein for cinnamic acid decarboxylase activity.
[0152] Another issue that limits the bioproduction of styrene is the toxicity of styrene to host cells. The accumulation of hydrophobic aromatics within the cytoplasmic membrane is known to disrupt its integrity. To reduce styrene toxicity and enhance production, an ABC-transporter was introduced into the host cell - an efflux pump that removes organic solvent from the cell. As described and exemplified herein, styrene production by E. coli cells expressing the ABC-transporter was enhanced nearly four times.
[0153] Accordingly, in another aspect, the subject technology provides a host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase; (b) a recombinantly expressed cinnamic acid decarboxylase; and (c) a recombinantly expressed ABC-transporter. Method of using the ABC- transporter for styrene production is also provided.
B. DEFINITIONS.
[0154] As used herein, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.
[0155] The terms "cinnamic acid" and "cinnamate" are used interchangeably in the specification, and are abbreviated as "CA." Trans-cinnamic acid is abbreviated as tCA.
[0156] The term "cinnamic acid decarboxylase" refers to an enzyme that catalyzes the conversion of trans-cinnamic acid to styrene. The term encompasses wild type or naturally occurring cinnamic acid decarboxylase, as well as functional fragments or variants of a wild type cinnamic acid decarboxylase. The Saccharomyces cerevisiae cinnamic acid decarboxylase described herein is also termed ferulic acid decarboxylase (FDC or FDC1). Ferulic acid is also a phenylacrylic acid.
[0157] The term "control" as used herein refers to a sample that provides a basis for comparison. For example, in a screening assay to determine cinnamic acid decarboxylase activity, a "control" can be a parallel sample comprising a cinnamic acid decarboxylase whose activity has been characterized (e.g., a wild type cinnamic acid decarboxylase, a specific mutant cinnamic acid decarboxylase, etc.). Alternatively, a control may be a pre -determined threshold value, or a value that is present in a database (e.g., a table, electronic database, spreadsheet, etc.).
[0158] An amino acid position "corresponding to" a reference position is a position that aligns with a reference sequence, as identified by aligning the amino acid sequences. Such alignments can be done by hand or by using well-known sequence alignment programs such as ClustalW2, Blast 2, etc.
[0159] A protein is "derived" from an organism when the protein is isolated from that organism, or modified or generated (e.g., chemically synthesized or recombinantly produced) using information of the protein from that organism
[0160] The term "detectable marker" refers to a chemical compound that is added to (or coated onto) a styrene-absorption material, in an amount effective to detect styrene vapor. Preferred detectable markers are chemical compounds that undergo a chemical reaction in the presence of styrene, and produce a colorimetric species. The chemical response of the detectable marker is preferable concentration dependent.
[0161] The term "fermentable carbon substrate" refers to a carbon source capable of being metabolized by host cells as described herein, and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, one-carbon substrate, and a combination thereof.
[0162] The term "functional fragment" of protein refers to refers to a peptide fragment that is a portion of the full length protein, and has substantially the same biological activity, or carries out substantially the same function as the full length protein (e.g., carrying out the same enzymatic reaction). For example, a functional fragment of a PAL can catalyze the phenylalanine to cinnamic acid conversion, and a functional fragment of a FDC can catalyze the cinnamic acid to styrene conversion.
[0163] The term "functional variant" of protein refers to a protein in which one or more amino acid residues have been changed without altering the overall conformation and function of the reference protein. Functional variants includes, e.g., replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide.
[0164] The term "homologous" in all its grammatical forms and spelling variations refers to the relationship between polynucleotides or proteins that possess a "common evolutionary origin," including polynucleotides or proteins from superfamilies and homologous polynucleotides or proteins from different species (Reeck et al., Cell 50:667, 1987). Such polynucleotides or proteins have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or the presence of specific amino acids or motifs at conserved positions. For example, two homologous proteins can have amino acid sequences that are about 80%, about 85%, about 90%>, about 95%o, about 96%o, about 97%o, about 98%, about 99%, or even 100% identical.
[0165] In this sense, techniques for determining amino acid sequence "similarity" are well known in the art. In general, "similarity" means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed "percent similarity" may then be determined between the compared polypeptide sequences. Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded therein, and comparing this to a second amino acid sequence. In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more polynucleotide sequences can be compared by determining their "percent identity", as can two or more amino acid sequences. The programs available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.), for example, the GAP program, are capable of calculating both the identity between two polynucleotides and the identity and similarity between two polypeptide sequences, respectively. Other programs for calculating identity or similarity between sequences are known by those skilled in the art.
[0166] The terms "mutation," or "mutant" as used herein, refer to a deletion, an insertion, or a substitution of a nucleotide or an amino acid residue of a wild type sequence. A wild type sequences refers to the most frequent sequence found in nature, against which mutants are defined.
[0167] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
[0168] The term "phenylalanine ammonia lyase," abbreviated PAL, refers to an enzyme that catalyzes the conversion of phenylalanine to trans-cinnamic acid. The term encompasses wild type or naturally occurring phenylalanine ammonia lyase, as well as functional fragments or variants of a wild type phenylalanine ammonia lyase. [0169] The term "mutated cinnamic acid decarboxylase" refers to an enzyme that catalyzes the conversion of trans-cinnamic acid to styrene, or the conversion of coumaric acid to 4-hydroxystyrene.
[0170] The term "recombinant" refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids.
[0171] A reference to an element in the singular is not intended to mean "one and only one" unless specifically stated, but rather "one or more." Pronouns in the masculine (e.g., his) include the feminine and neuter gender (e.g., her and its) and vice versa. The term "some" refers to one or more. Underlined, italicized and/or boldface headings and subheadings are used for convenience only, do not limit the subject technology, and are not referred to in connection with the interpretation of the description of the subject technology. All structural and functional equivalents to the elements of the various configurations described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and intended to be encompassed by the subject technology. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the above description.
[0172] Unless specified otherwise, the percent identity of two polypeptide or polynucleotide sequences refers to as the percentage of identical amino acid residues or nucleotides across the entire length of the shorter of the two sequences.
C. PHENYLALANINE AMMONIA LYASE
[0173] Phenylalanine ammonia lyase (EC 4.3.1.24; formally EC 4.3.1.5) is an enzyme that catalyzes the chemical reaction:
L-phenylalanine trans-cinnamate + NH3
Other names that are commonly used for this enzyme include tyrase, phenylalanine deaminase, tyrosine ammonia-lyase, L-tyrosine ammonia-lyase, phenylalanine ammonium-lyase, PAL, and L-phenylalanine ammonia-lyase. PAL is a non-mammalian enzyme widely distributed in plants and yeast.
[0174] A representative list of PALs include (identified by Genbank accession number and species): Q9ATN7 Agastache rugosa; 093967 Amanita muscaria (Fly agaric); P35510, P45724, P45725, Q9SS45, Q8RWP4 Arabidopsis thaliana (Mouse-ear cress); Q6ST23 Bambusa oldhamii (Giant timber bamboo); Q42609 Bromheadia finlaysoniana (Orchid); P45726 Camellia sinensis (Tea); Q9MAX1 Catharanthus roseus (Rosy periwinkle) (Madagascar periwinkle); Q9SMK9 Cicer arietinum (Chickpea); Q9XFX5, Q9XFX6 Citrus Clementina x Citrus reticulate; Q42667 Citrus Ziman (Lemon); Q8H6V9, Q8H6W0 Coffea canephora (Robusta coffee); Q852S1 Daucus carota (Carrot); 023924 Digitalis lanata (Foxglove); 023865 Daucus carota (Carrot); P27991 Glycine max (Soybean); 004058 Helianthus annuus (Common sunflower); P14166, (Q42858) Ipomoea batatas (Sweet potato); Q8GZR8, Q8W2E4 Lactuca sativa (Garden lettuce); 049835, 049836 Lithospermum erythrorhizon; P35511 , P26600 Lycopersicon esculentum (Tomato); P35512 Malus domestica (Apple) (Malus sylvestris); Q94C45, Q94F89 Manihot esculenta (Cassaya) (Manioc); P27990 Medicago sativa (Alfalfa); P25872, P35513, P45733 Nicotiana tabacum (Common tobacco); Q6T1C9 Quercus suber (Cork oak); P14717, P53443, Q7M1Q5, Q84VE0, Q84VE0 Oryza sativa (Rice); P45727 Persea americana (Avocado); Q9AXI5 Pharbitis nil (Violet) (Japanese morning glory); P52777 Pinus taeda (Loblolly pine); Q01861, Q04593 Pisum sativum (Garden pea); P24481, P45728, P45729 Petroselinum crispum (Parsley) (Petroselinum hortense); Q84L12 Phalaenopsis x Doritaenopsis hybrid cultivar; P07218, P19142, P19143 Phaseolus vulgaris (Kidney bean) (French bean); Q7XJC3, Q7XJC4 Pinus pinaster (Maritime pine); Q6UD65 Populus balsamifera subsp. trichocarpa x Populus deltoides; P45731, Q43052, 024266 Populus kitakamiensis (Aspen); Q8H6V5, Q8H6V6 Populus tremuloides (Quaking aspen); P45730 Populus trichocarpa (Western balsam poplar); 064963 Prunus avium (Cherry); Q94ENO Rehmannia glutinosa; PI 1544 Rhodosporidium toruloides (Yeast) (Rhodotorula gracilis); P10248 Rhodotorula rubra (Yeast) (Rhodotorula mucilaginosa); Q9M568, Q9M567 Rubus idaeus (Raspberry); P31425, P31426 Solanum tuberosum (Potato); Q6SPE8 Stellaria longipes (Longstalk starwort); P45732 Stylosanthes humilis (Townsville stylo); P45734 Trifolium subterraneum (Subterranean clover); Q43210, Q43664 Triticum aestivum (Wheat); Q96V77 Ustilago maydis (Smut fungus); P45735 Vitis vinifera (Grape); and Q8VXG7 Zea mays (Maize).
[0175] Crystal structures of several PALs are also known (identified by PDB accession code and species): 1T6J (Rhodosporidium toruloides); 1T6P (Rhodosporidium toruloides); 1W27
(Petroselinum crispum); 1Y2M (Rhodosporidium toruloides); 2NYF (Nostoc punctiforme); and 3nz4 (Taxus canadensis). PAL from Rhodosporidium toruloides (also known as Rhodotorula glutinis) is a homotetramer, each subunit having a "seahorse" shape that interlocks head-to-tail with two other subunits, thereby maximizing adjacent subunit interactions and yielding a close-fitting tetramer. The tetramer assembly leads to a cluster of four vicinal cysteines (residues 140, 455, 467, and 530), with the sulfur atoms of Cys467 and Cys530 separated by 3.62 angstroms. The structure of the main body of PAL has a central core of nearly parallel a-helices of varying lengths. There is only one section of β-sheet longer than three residues (strands of residues 231-237 and 240-246); it resides in the funnel region leading to the active site. MacDonald, et al., A modern view of phenylalanine ammonia lyase, Biochem Cell Biol. 2007; 85(3):273-82.
[0176] Genes or polynucleotides encoding PAL from both plant and microbial sources are known in the art. See, for example, EP 321488 (R. toruloides); WO 9811205 (Eucalyptus grandis and Pinus radiate); WO 9732023 (Petunia); JP 05153978 (Pisum sativum); WO 9307279 (potato, rice). The sequences of various PAL genes can be readily ascertained from literature, public databases (see for example GENBANK® Accession Nos. AJ010143 and X75967), or other commercial sources. For example, full-length cDNAs of Arabidopsis PALI (At2g37040), PAL2 (At3g53260) and PAL4
(At3g 10340) can be purchased from Arabidopsis Biological Resource Center (ABRC) at the Ohio State University under the stock numbers G10120 (in pENTR223.1 vector), G12256 (in pENTR223.1 vector), and U24927 (in pENTR/D-TOPO vector), respectively.
[0177] In certain embodiments, the phenylalanine ammonia lyase of the subject technology is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces. Other preferred source organisms include, e.g., yeasts such as Rhodotorula, Rhodosporidium, and Sporobolomyces; bacteria such as Streptomyces; and plants such as pea, potato, rice, eucalyptus, pine, corn, petunia, arabidopsis, tobacco, and parsley.
[0178] The subject technology also encompasses homologs (including orthologs), functional fragments, or functional variants of the exemplary PALs described herein. Methods of obtaining homologs and variants of PALs are well known in the art, including for example, sequence-dependent protocols. Exemplary sequence-dependent protocols include, e.g., nucleic acid hybridization, DNA and RNA amplification (e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR)), etc.
[0179] For example, genes encoding homologs of a PAL can be isolated directly by using all or a portion of the known sequences as DNA hybridization probes to screen libraries from any desired plant, fungi, yeast, or bacteria, using techniques well known to those skilled in the art. Specific oligonucleotide probes based upon the literature nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, infra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to a skilled artisan, such as random primers DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of or full-length of a target sequences. The resulting amplification products can be labeled directly during amplification reactions, or labeled after amplification reactions, and used as probes to isolate full-length cDNA or genomic fragments under conditions of appropriate stringency.
[0180] In addition, two short segments of the literature sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the literature sequences, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding bacterial genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the literature sequences. Using commercially available 3' RACE or 5' RACE systems, specific 3' or 5' cDNA fragments can be isolated (Ohara et al., Proc. Natl. Acad. Sci. USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
[0181] The nucleic acid and protein sequences of a homolog or a variant can further be identified by using a "query sequence" to perform a search against public databases to identify other family members or related sequences.
[0182] Accordingly, in an exemplary embodiment, the phenylalanine ammonia lyase of the subject technology comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof. SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 are the amino acid sequences of Arabidopsis PALI, PAL2, and PAL4, respectively. Variants of PAL may include those polypeptide sequences comprising an amino acid sequence that is about 60%, about 70%o, about 75%o, about 80%o, about 85%o, about 90%o, about 95%o, about 96%, about 97%, or about 98%, about 99%, or even 100% identical to any one of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or a fragment thereof. The fragment may comprise about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 amino acid residues of the full length PAL.
[0183] In another exemplary embodiment, the phenylalanine ammonia lyase of the subject technology comprises an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, and a fragment or variant thereof. SEQ ID NOs:l, SEQ ID NO:3, and SEQ ID NO:5 are the nucleotide sequences encoding Arabidopsis PALI, PAL2, and PAL4, respectively. Variants of PAL-coding sequence may include those polynucleotide sequences that is about 60%o, about 70%o, about 75%o, about 80%o, about 85%o, about 90%o, about 95%o, about 96%, about 97%, about 98%, about 99% , or even 100% identical to any one of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, or a fragment thereof. The fragment may comprise about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 2100 nucleotides of the full length PAL-coding sequence.
[0184] To determine the percent identity of two amino acid sequences or of two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence and non-homologous sequences can be disregarded for comparison purposes). Unless specified otherwise, an alignment is a global alignment, (i.e., the percentage of identical amino acid residues or nucleotides across the entire length of the shorter of the two sequences). If a local alignment is desired, preferably, the length of a sequence aligned for comparison is about 30%o, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the length of the shorter of the two sequences.
D. CINNAMIC ACID DECARBOXYLASE
[0185] Cinnamic acid decarboxylase is an enzyme that catalyzes the conversion of trans- cinnamic acid to styrene.
[0186] In certain embodiments, the cinnamic acid decarboxylase of the subject technology is derived from an organism selected from the group consisting of: an Arabidopsis, an Anabaena, a Nostoc, and a Saccharomyces.
1. FDC1 and OHBA1
[0187] In an exemplary embodiment, the cinnamic acid decarboxylase of the subject technology is derived from Saccharomyces cerevisiae. The Saccharomyces cerevisiae cinnamic acid decarboxylase described herein is also termed ferulic acid decarboxylase (FDC or FDC1). The sequence of FDC/FDC1 is disclosed in U.S. Patents Nos. 5,955,137 and 6,468,566.
[0188] As disclosed and exemplified herein, full length cinnamic acid decarboxylase from Saccharomyces cerevisiae comprises 503 amino acids (SEQ ID NO:8). A computer model of full length FDC1 is shown in Figure 1A; and four possible tCA binding sites are shown in Figure IB, with site C being the most likely site. Further analysis of site C shows that 1173, A174, R175, V188, 1189, K190 (not shown for figure clarification), 1194, E280, M286, F291 and F440 are involved with substrate binding (Figure 1C). The nucleotide sequence encoding FDC1 is shown as SEQ ID NO:7.
[0189] Kinetic studies show that the Km for wild type FDC is about 688 μΜ and the Vmax is about 6.17 nmolnng^nnin 1. In addition to tCA, FDC1 also binds to substrates including ferulic acid, 2- methylcinnamic acid, and 4-hydroxycinnamic acid.
[0190] Another exemplary cinnamic acid decarboxylase is 3-octaprenyl-4- hydroxybenzoate carboxy-lyase from Aspergillus niger (OHBA1; SEQ ID NO: 10). The nucleotide sequence encoding OHBA1 is shown as SEQ ID NO:9.
[0191] The subject technology also encompasses homologs (including orthologs), functional fragments, or functional variants of the exemplary cinnamic acid decarboxylases described herein.
Homologs and variants of cinnamic acid decarboxylases can be obtained using methods described above.
[0192] For example, a functional variant may be a sequence that (i) is about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100% identical to SEQ ID NO:8, (ii) comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the following residues: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8; and a combination of (i) and (ii).
[0193] Amino acid residues that "correspond to" a particular position of SEQ ID NO: 8 can be identified by aligning the target sequence with SEQ ID NO: 8. As an example, alignment of FDC1 and OHBA1 is shown below (FDC1 is the query sequence; OHBA1 is the subject sequence).
Query 8 LEFRDFIQVLKDEDDLIEITEEIDPNLEVGAIMRKftYESHLPAPLFKNLKGASKDLFSIL 67
I, FR F++ L ++DL+EI IDPNLE AI R+ E+÷ APLF NL G LF IL
Sbj ct 8 liCFRSFVEAL VDKDLVEINTPIDPNLEAAAITRRVCETND APIjFtllX'IilGM NGIjFRII, 67
Query 68 GCPAGl;RSKE GDHGRI HHLGl;DPKTTIKEI DYL.L;ECKE EPLPPITVPVSSAPC TK 127
G P LR +GR+A HI, L P +++EI+D +L + P+ V ÷ PCK +
Sbj ct 68 G PGSLR SSADRYGRLARHLALP'PTAS REILDK LSASDMPP'I - -- PPT VPTGPCKEN 125
Query 128 ILSEEKIH OS^PTPYLIHVSDGGKY^OTYGMW LO FDKKWTNWSIARG^A^DKHITG^ 187
L + ÷ L LP P +H SDGG Y+QTYGK I+Q+PD D H+TGL
Sbj ct 126 SLDDSEFDLTELPVPLIHKSDGGKYIQTYGMHIVQSPDGTWTNWSIARAMVHDKNHLTGL 185
■Que y 188 VIKPQHIRQIADS AAIGKANEIPFALCFGVPPAAILVSSMPIPEGVSESDYVGAILGE3 247
VI PQHI QI W G+t- ++P+AL FGVPPAAIt- SS PI P+GV+E+ YVGA+ G 3
Sbj ct 186 VI PPQHIWQIHQMWKKEGRS -DVPWALAFGVPPAAIMASSMPI PDGVTEAGYVGAMTGS3 244
Query 248 VP^'KCETNDLMVPA SEMVFEGTL3LTD HLEGPFGEMHGYVFK3QGHPCE^YTVKAM3 307
+ +VKC+TNDL VPATSE+V EGTL3+++T EGPFGEMHGY+F H Y V ++
Sbj ct 245 LELVK.CDTNDLYVPA SE VLEGTL3ISE GPEGPFGEMHGYIFPGDTHIGAKYK37NR. 304
Query 308 YRDNAILPVSNPGLCTDETHTLIGSLVATEAKELAIESGLPILDAFMPYEAQALWLILKV 367
+ AI+P+S+ G TDETHT+ GSL A E ++L ++ LPI DAF P+Et-Q W+ L+V
Sbj ct 305 Y"R]INAIMP SSCGRj-i E I-IT IGSj-iAAAEIR.K.IjCQQND.LjP'ITDAFA.PFESQVT VALRV 364
Query 368 DLKGLQALKTTPEEFCKKVGDIYFRTKVGFIVHEI ILVADDIDIFNFKEV WAYVTRHTP 427
D + L÷A+KTT E F K+VGD+ F K. G+ +H ++LV DDID++ K+Vt- At- TR P
Sbj ct 365 DTEKLRAMKTTSEGFRKRVGDVVFNHKAGYTIHRLVLVGDDIDVYEGKDVL AFSTRCRP 424
Que y 428 VADQMAFDDVTSFPLAPFVSQSSRSKTMKGGKCTTNCIFRQQYERSFDYITCNFEKGYPK 487
D-h F-DV FPL P-n- ■·■ +GGK V-n- + -Y ++ +F YP-t
Sbj ct 425 GMDETLFEDVRGFPLiPY7IGHGN-GPAHRGGKVVSDALMFTEYTTGRIiWEAADFNQSYPE 48.3
Query 488 GLVDKVNENWKRYGY 502
Figure imgf000029_0001
Sbj ct 484 DLKQKVLDNWTKMGF 498
[0194] In an exemplary embodiment, the cinnamic acid decarboxylase of the subject technology comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:10, and a functional fragment or variant thereof. Variants of cinnamic acid decarboxylase may include those polypeptide sequences comprising an amino acid sequence that is about 50%, about 60%, about 70%o, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100% identical to any one of SEQ ID NO:8, SEQ ID NO: 10, or a fragment thereof. The fragment may comprise about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 amino acid residues of the full length cinnamic acid decarboxylase.
[0195] In another exemplary embodiment, the cinnamic acid decarboxylase of the subject technology comprises an amino acid sequence encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:9, and a fragment or variant thereof. Variants of cinnamic acid decarboxylase-coding sequence may include those polynucleotide sequences that is about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100% identical to any one of SEQ ID NO:7, SEQ ID NO:9, or a fragment thereof. The fragment may comprise about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 1400, 1500, 1600, 1700, 1800, 1900, 2000, or 2100 nucleotides of the full length coding sequence.
2. Exemplary FDCl Mutants
[0196] Based on the structural analysis of yeast FDCl, residues 155-156, 159, 162-164, 172- 175, 187-196, 226-227, 280, 285-287, 291, 326, 331, 360-361, 395-396, 398, and 440-441 of FDCl (SEQ ID NO:8) are identified as potential sites for mutagenesis. Accordingly, in another aspect, the subject technology provides a mutant cinnamic acid decarboxylase, wherein a mutation is introduced at an amino acid residue position corresponding to one of the following positions: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, and a combination thereof. The mutation may be a deletion, an insertion, or a substitution mutation.
[0197] In an exemplary embodiment, the mutant cinnamic acid decarboxylase comprises a mutation at an amino acid residue position corresponding to residue 190 of SEQ ID NO: 8. Preferably, the residue corresponding to 190 is replaced with one of the following: E, C, D, V, N, L, H. In another exemplary embodiment, the mutant cinnamic acid decarboxylase comprises a mutation at an amino acid residue position corresponding to residue 175 of SEQ ID NO: 8. Preferably, the residue corresponding to 175 is replaced with I.
[0198] In certain embodiments, the mutant cinnamic acid decarboxylase is a mutant FDCl, wherein a mutation is introduced to one of the following positions in SEQ ID NO:8: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441, and a combination thereof.
[0199] In certain embodiments, the mutant cinnamic acid decarboxylase is a mutant FDCl, wherein a mutation is introduced to that one of the following positions in SEQ ID NO:8: 175, 190, 193, and a combination thereof. [0200] In certain embodiments, the mutant cinnamic acid decarboxylase is a mutant FDC1, comprising one of the mutations in SEQ ID NO:8: K190E, K190C, K190H, K190P, K190L, K190R, K190D, K190V, K190S, K190N, R175I, H193P, and a combination thereof.
[0201] In certain embodiments, the mutant cinnamic acid decarboxylase comprises any one of the sequences selected from the group consisting of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; and SEQ ID NO:38.
[0202] The cinnamic acid decarboxylase of the subject technology may also comprise a functional fragment or variant of the mutant cinnamic acid decarboxylase described herein. Variants of cinnamic acid decarboxylase may include those polypeptide sequences comprising an amino acid sequence that is about 50%, about 60%o, about 70%o, about 75%o, about 80%o, about 85%o, about 90%o, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100% identical to any one of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; and a fragment thereof. The fragment may comprise about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 amino acid residues of the full length cinnamic acid decarboxylase.
[0203] Alternatively, or in addition, the variants may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 the following residues: I at a position corresponding to residue 173 of SEQ ID NO: 8, A at a position corresponding to residue 174 of SEQ ID NO: 8, R at a position corresponding to residue 175 of SEQ ID NO: 8, V at a position corresponding to residue 188 of SEQ ID NO: 8, 1 at a position corresponding to residue 189 of SEQ ID NO: 8, K at a position corresponding to residue 190 of SEQ ID NO: 8, 1 at a position corresponding to residue 194 of SEQ ID NO: 8, E at a position corresponding to residue 280 of SEQ ID NO: 8, M at a position corresponding to residue 286 of SEQ ID NO: 8, F at a position corresponding to residue 291 of SEQ ID NO: 8, and F at a position corresponding to residue 440 of SEQ ID NO: 8.
[0204] Libraries of cinnamic acid decarboxylase mutants are also provided. The library comprises a plurality of mutant cinnamic acid decarboxylases. For example, the library may comprise cinnamic acid decarboxylase mutants that represent about 15, about 16, about 17, about 18, or about 19 different mutations at particular target position. For example, the library may comprise about 19 different mutants in which K190 of FDC1 (SEQ ID NO:8) is replaced with each one of the other 19 amino acids. Alternatively, the library may comprise cinnamic acid decarboxylase mutants that represents about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19 mutations at different positions. For example, an Alanine scan may be used to create mutant libraries in which amino acid residues at different positions are replaced with Alanine. [0205] Changes to the amino acid sequence may be generated by changing the nucleic acid sequence encoding the amino acid sequence. A nucleic acid sequence encoding a mutant cinnamic acid decarboxylase may be prepared by methods known in the art using the guidance of the present specification for particular sequences. These methods include, but are not limited to, preparation by site- directed (or oligonucleotide-mediated) mutagenesis (Coombs et al., Proteins (1998), 259-311, 1 plate. Editor(s): Angeletti, Ruth Hogue. Publisher: Academic, San Diego, Calif); PCR mutagenesis or error prone PCR (Melnikov et al., Nucleic Acids Research, (Feb. 15, 1999) Vol. 27, No. 4, pp. 1056-1062); cassette mutagenesis of an earlier prepared nucleic acid sequence; "gene shuffling" (U.S. Pat. No.
5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721 ; and U.S. Pat. No. 5,837,458); all of which are techniques well known in the art.
[0206] Alternatively, in vivo mutagenesis may be employed using commercially available materials such as E. coli XL 1 -Red strain, and the Epicurian coli XL 1 -Red mutator strain from
STRATAgene (STRATAgene, La Jolla, Calif, Greener and Callahan, Strategies 7:32-34) (1994)). This strain is deficient in three of the primary DNA repair pathways (mutS, mutD and mutT), resulting in a mutation rate 5000-fold higher than that of wild type.
3. Method of Screening
[0207] In another aspect, the subject technology provides a method for screening a candidate proteins for cinnamic acid decarboxylase activity, comprising (a) providing a protein sample comprising the candidate protein, and a substrate sample comprising trans-cinnamic acid; (b) combining the protein sample and substrate sample to form a mixture, and incubating the mixture under a condition that allows a cinnamic acid decarboxylase to convert trans-cinnamic acid to styrene; and (c) exposing the mixture to a detection material that comprises (i) polymeric resin that absorbs styrene vapor; and (ii) a detectable marker that causes a color change in the presence of styrene. A change of the color of the detection material indicates that the candidate protein has cinnamic acid decarboxylase activity.
[0208] In screening for FDC mutants, a high-throughput screening assay has been developed. Screening a large population of the protein library is the bottleneck for the molecular evolution of the protein. The functional characterization of decarboxylase routinely relies on analytic instruments, like HPLC or LC-MS. Although the HPLC is highly sensitive, it is time consuming, expensive, and generates waste like methanol or acetonitrile and not suitable for high-throughput applications.
[0209] To overcome these technical barriers, the subject technology provides a spectroscopic- based colorimetric assay method. First, a candidate protein and tCA are incubated under a condition that allows a cinnamic acid decarboxylase to convert trans-cinnamic acid to styrene. Typically, the incubation condition should allow a cinnamic acid decarboxylase to exhibit optimal enzymatic activity (for example, at a temperature from about 10 °C to about 65 °C, from about 25 °C to about 55 °C, from about 35 °C to about 60 °C, or from about 35 °C to about 55 °C; at a pH between 6-10, between 6-8, or between 5.5-7.5; in the presence of metal ions from about 1 mM to about 20 mM; etc.). The candidate protein and tCA can be incubated for about 30 minutes, about 1 hour; about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours, to allow sufficient amount of styrene to be produced.
[0210] Second, the reaction mixture is exposed to a detection material that comprises (i) polymeric resin that absorbs styrene vapor; and (ii) a detectable marker that causes a color change in the presence of styrene. Because styrene vaporizes, the detection material can be placed on top of the reaction mixture to absorb styrene vapor (without actually dipping into the reaction mixture). Accordingly, the detection material can be attached to a solid support, and can be inverted during the detection process to absorb styrene vapor. See, e.g., Figure 19.
[0211 ] Preferred polymeric resins for absorbing styrene vapor include, e.g., reversed phase hydrophobic resins, which has a hydrocarbon or aromatic functional group (e.g., an aromatic benzene ring) that can bind with polar and non -polar compounds. More preferably, the hydrophobic resins have a hydrocarbon or aromatic functional group and do not have any polar group. Examples of the reversed phase hydrophobic resins include CI 8, C8, phenyl, SDB-L sorbents resins, and combinations thereof.
[0212] Exemplary polymeric resins that can be used to absorb volatile organic compounds (such as styrene vapor) include, for example, STRATA-X® polymeric resins (Phenomenex), Amberlite polymeric resins ( Sigma- Aldrich), DOWEX™ polymeric resins or AMBERJET™ polymeric resins (The Dow Chemical Company), Macronet™ polymeric resins, PuroSorb™ polymeric resins, or Chromalite® resins (PuroLite), etc. The surface area and pore size distribution of the resin should suitable for absorbing volatile organic compounds. Because a polymeric resin is made with few functional groups (often it is the single dominant functional group which gives the surface its adsorption characteristics), one can ascertain the affinity (e.g., Hanson solubility parameter) and predict the adsorption capacity based on
thermodynamics. Various studies have shown that adsorption capacities of a variety of solutes, and polymeric resins can be correlated with solubility parameters.
[0213] Other materials suitable for absorbing styrene vapor include activated carbon and cellulosic materials. For example, the cotton burrs disclosed in US 2002/0151622 as cellulosic materials can be used in the present invention to absorb volatile organic compounds.
[0214] The detection material also comprises a detectable marker that causes a color change in the presence of styrene. One exemplary detectable marker is 4-nitrobenzyl-pyridine (NBP). The unpaired electron of NBP reacts with the oxirane ring of styrene oxide to yield a blue chromophore.
[0215] If desired, the color change of the detectable material can be determined by a quantitative assay, such as by spectrophotometry. The color change may also be determined qualitatively. Sometimes, the color change would be apparent to an observer. [0216] If desired, the activity of the candidate protein can be compared with a control. A control can be a parallel sample comprising a cinnamic acid decarboxylase whose activity has been characterized (e.g., a wild type cinnamic acid decarboxylase, or a specific mutant cinnamic acid decarboxylase that serves as the base sequence for further mutations). Alternatively, a control may be a pre-determined threshold value, or a value that is present in a database (e.g., a table, electronic database, spreadsheet, etc.).
[0217] As shown in Figure 13, the screening assay can be conducted for more than one round, and can be combined with computer modeling to rationally design and improve the activities of cinnamic acid decarboxylases.
[0218] In certain embodiments, mutated cinnamic acid decarboxylase capable of converting converting trans-cinnamic acid to styrene, or converting coumaric acid to 4-hydroxystyrene, can be screened by the method described herein. In certain embodiments, the candidate protein being screened can be a fusion protein comprising a mutated cinnamic acid decarboxylase.
4. Recombinant production and purification of cinnamic acid decarboxylase
[0219] In another aspect, the subject technology also provides a method of recombinant production and purification of cinnamic acid decarboxylase, such as FDC. In particular, the subject technology provides a method of isolating a recombinantly produced cinnamic acid decarboxylase, comprising: (i) providing a bacterial host comprising a nucleic acid that encodes a cinnamic acid decarboxylase operably linked to a promoter sequence; (ii) culturing the bacterial host in a culture medium to express the cinnamic acid decarboxylase in the host cell, therein the host cell is cultured at a temperature that is from about 10 °C to about 25 °C; and (iii) isolating the cinnamic acid decarboxylase from the host cell, wherein the isolation is conducted in an anaerobic environment.
[0220] In some embodiments, culturing the bacterial host cell (e.g., E. coli) at lower temperature can significantly promote the correct folding of the recombinantly produced cinnamic acid decarboxylase, such as FDC1. Lowering the culturing temperature also facilitates the conversion of aggregated or misfolded protein to a functionally soluble form. In certain embodiments, the cell culture is grown at a temperature of about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 °C.
[0221] Reducing agents, such as Tris(2-carboxyethyl) phosphine (TCEP) or β-mercaptoethanol, can also be used in the method of recombinantly producing cinnamic acid decarboxylase, such as FDC1. Accordingly, one or more buffer solutions used for isolating the cinnamic acid decarboxylase may comprise a reducing agent, such as TCEP at a concentration of about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or 100 mM; or β-mercaptoethanol at a concentration of about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, or 100 mM; and a combination thereof. [0222] Samples and cell extracts containing the recombinant cinnamic acid decarboxylase (such as FDC1) maintained in an anaerobic environment also contain cinnamic acid decarboxylase activity.
[0223] The recombinantly produced cinnamic acid decarboxylase (such as FDC1) maintains cinnamic acid decarboxylase activity in the presence of metal ions. Suitable metal ions include, e.g., calcium, zinc, magnesium, iron, manganese ion, or a combination thereof. The metal ion may be present at a concentration of from about 0.1 mM to about 100 mM, such as about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM; from about 0.1 mM to about 50 mM, from about 0.1 mM to about 25 mM, from about 0.1 mM to about 20 mM, from about 0.1 mM to about 10 mM, from about 1 mM to about 50 mM, from about 1 mM to about 25 mM, from about 1 mM to about 20 mM, from about 1 mM to about 15 mM, or from about 1 mM to about 10 mM. Preferably, the metal ion is an alkaline earth metal ion, or a transitional metal ion.
[0224] The purified cinnamic acid decarboxylase described herein is suitable for crystallization.
5. Preparation of Crystalline form of cinnamic acid decarboxylase.
[0225] To gain insight into the structure of cinnamic acid decarboxylase, a method to crystallize this protein was developed. Traditionally, hanging drop and sitting drop vapor diffusion methods are used for crystallization. Both methods require a closed system, that is, the system must be sealed off from the outside using an airtight container or high-vacuum grease between glass surfaces.
[0226] The subject technology provides a method of crystallizing a cinnamic acid
decarboxylase, the method comprising (a) providing a cinnamic acid decarboxylase solution at a concentration of from about 1 mg/ml to about 50 mg/ml; (b) mixing the cinnamic acid decarboxylase solution with a reservoir solution at a volume ratio of from about 1 : 10 to about 10:1 ; and (c) maintaining the mixture of step (b) at a temperature suitable for the formation of the cinnamic acid decarboxylase crystals.
[0227] Preferably, the protein concentration in step (a) is from about 2 mg/ml to about 20 mg/ml, more preferably from about 5 mg/ml to about 10 mg/ml. The volume ratio of the cinnamic acid decarboxylase solution to the reservoir solution is preferably from about 1 :5 to about 5: 1, more preferably from about 1 :2 to about 2:1. Typically, 1 to 2 μΐ of the cinnamic acid decarboxylase solution (protein concentration at about 5.0 to 10.0 mg/ml) is mixed with 1 to 2 μΐ of a reservoir solution in a drop on glass. The mixture is put upside down on a well, containing the reservoir solution (typically about 500 μΐ) and the system is incubated at a temperature of from about 1°C to about 20°C, preferably from about 4°C to about 12°C. Initially, the droplet of protein solution contains an insufficient concentration of precipitant for crystallization, but as water evaporates from the drop and transfers to the reservoir, the precipitant concentration increases to a level for crystallization. Since the system is in equilibrium, these conditions are maintained until the crystallization is complete.
[0228] The crystallization of the cinnamic acid decarboxylase can be improved when the protein forms a complex with a small molecule. For example, the small molecule may be added to the protein solution at step (a) or to the protein/reservoir mixture at step (b) to allow the formation of a protein-small molecule complex. Typically, the final concentration of the small molecule in the mixture of step (b) is from about 0.01% (w/v) to about 1% (w/v), preferably from about 0.05%> (w/v) to about 0.5%> (w/v), more preferably from about 0.08%> (w/v) to about 0.2%> (w/v).
[0229] Suitable small molecules include substrates of cinnamic acid decarboxylase (e.g. trans- cinnamic acid) and its analogs, such as 3-hydroxyl cinnamic acid, ferulic acid, 2-methylcinnamic acid, 4- hydroxy-cinnamic acid, 3,4-dimethoxycinnamic acid, 2,5-dimethoxy-cinnamic acid, and combinations thereof.
[0230] The crystallization method can be further improved by including one or more additives in the solution, such as Mn ¾ at a concentration of from about 0.001M to about 0.1M, preferably from about 0.005M to about 0.02M; polyvinylpyrrolidone K15 at a concentration of from about 0.1% (w/v) to about 2.5%> (w/v), preferably from about 0.25%> (w/v) to about 1% (w/v); Non-detergent Sulfobetaine 201 (NDSB-201, CsHnNC S) at a concentration of from about 0.02M to about 1M , preferably from about 0.1M to about 0.3M; benzamidine hydrochloride at a concentration of from about 0.2% (w/v) to about 10% (w/v), preferably from about 1% (w/v) to about 3%> (w/v); all concentrations being measured in the protein-reservoir mixture of step (b) .
E. FUSION PROTEINS AND PROTEIN COMPLEXES
[0231] In another aspect, the subject technology provides a fusion protein comprising: (i) a first domain that comprises a phenylalanine ammonia lyase, and (ii) a second domain that comprises a cinnamic acid decarboxylase. Alternatively, a phenylalanine ammonia lyase and a cinnamic acid decarboxylase can form a protein complex, via non-covalent interaction(s).
[0232] The fusion protein or protein complex described herein takes advantage of the "substrate channeling" phenomenon. Substrate channeling refers to a phenomenon in which substrates are efficiently delivered from enzyme to enzyme without equilibration with other pools of the same substrates. The fusion protein or protein complex can channel intermediates between sequential enzymes, and control the flux of substrates into competing branches of the pathway. In effect, this creates local pools of metabolites at high concentrations relative to those found in other areas of the cell.
[0233] Any one of the phenylalanine ammonia lyase or cinnamic acid decarboxylase described herein can be used to produce a fusion protein. In certain embodiments, the fusion protein further comprises a linker covalently linking the first domain (PAL) and the second domain (cinnamic acid decarboxylase). The linker preferably comprises one or more amino acid residues (e.g., an amino acid linker or a peptide linker). The amino acid residues of the linker may comprise L-amino acid(s), D-amino acid(s), amino acid analogues, or a combination thereof. Other possible linkers include, e.g., a covalent bond, C1-C6 alkyl, a cycloalkyl such as a cyclopentyl or cyclohexyl, a cycloalkenyl, aryl, or heteroaryl moiety. A linker may also comprise a combination of one or more amino acid(s) with another linking moiety (such as C1-C6 alkyl-, cycloalkyl-(C5, C6), aryl, or heteroaryl moieties).
[0234] In certain embodiment, the linker is a peptide linker. The peptide linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, or 40 amino acid long. Preferably, the length of the linker is from 2 to 15 amino acids.
[0235] In certain embodiments, the linker is a glycine/serine linker, i.e., a peptide linker consisting essentially of glycine and serine. In an exemplary embodiment, the linker comprises GS or GSG. In another exemplary embodiment, the linker comprises the Gly-Ser-Gly (GSG) motif, such as GGSG (SEQ ID NO:39), (GS)x3 (SEQ ID NO:40), (GGSG)x2 (SEQ ID NO:41), SGGSGGSGG (SEQ ID NO:42), GGSGGGSGGGSG (SEQ ID NO:43), (GGGGS)x3 (SEQ ID NO:44), as described in Table 1 below.
Table 1. Glycine/Serine Linkers
Figure imgf000037_0001
[0236] Fusion proteins described herein can be produced using techniques well known in the art. For example, when the linker is a peptide linker, the fusion protein can be produced using standard recombinant DNA technology. Other types of linker can be attached by, e.g., standard conjugation techniques. See, e.g., Hermanson et al., Bioconjugate Techniques, 2nd Ed., 2008; Academic Press.
[0237] The fusion protein may comprise multiple units of phenylalanine ammonia lyase and cinnamic acid decarboxylase. In certain embodiments, the fusion protein can be PAL-FDC, PAL-FDC-PAL-FDC, or PAL-FDC-FDC-PAL, etc. For example, the FDC can be at the 5' terminal of the DNA encoding the fusion protein, and the PAL can be at the 3 ' terminal of the DNA encoding the fusion protein. In another example, the PAL can be at the 5 ' terminal of the DNA encoding the fusion protein, and the FDC can be at the 3' terminal of the DNA encoding the fusion protein. In addition, the cinnamic acid decarboxylase unit of the fusion protein can be wild type (WT) or mutant proteins, such as any of the mutant FDC proteins described above. For example, the fusion protein can be PAL-FDC(WT) or PAL-FDC(K190E).
[0238] Alternatively or in addition, the phenylalanine ammonia lyase and the cinnamic acid decarboxylase can form a protein complex, via non-covalent interaction(s). A "protein complex" refers to an association of more than one protein. The proteins of the complex may be associated by e.g., functional, stereochemical, conformational, biochemical, or electrostatic association. It is intended that the term encompass associations of any number of proteins.
[0239] Alternatively, or in addition, the phenylalanine ammonia lyase and the cinnamic acid decarboxylase can be modified to include an "interacting moiety." For example, one enzyme can comprise an antibody, and the other can comprise a cognate antigen; or one enzyme can comprise a ligand, and the other can comprise a cognate receptor; or one enzyme can comprise biotin, and the other can comprise avidin, etc. The interacting moieties can interact with each other, thereby brining the two enzymes in proximity to each other. Any pairs of interacting moieties can be used.
F. HOST CELLS AND CELL CULTURES
[0240] Methods described herein use host cells to produce styrene. A host cell can be derived from a bacterium, fungus (e.g., yeast), protist (e.g., algae), plant, insect, amphibian, fish, reptile, bird, mammal (including human), or can be a hybridoma cell. Host cells can be unmodified cells or cell lines, or cell lines which have been genetically modified (e.g., to facilitate production of styrene). In some embodiments, the host cell is a cell line that has been modified to allow for growth under desired conditions, such as at a lower temperature.
[0241] As described herein, suitable host cells that express phenylalanine ammonia lyase or cinnamic acid decarboxylase can be cultured, sometimes in large scale (i.e., about 1 liter, about 10 liters, at les 100 liters, etc.), to produce commercially useful amounts of styrene or other compounds downstream from styrene (compounds which will result from further processing of styrene in these microorganisms via enzymatic or biological pathways).
[0242] The methods described herein can be applied to any size of cell culture flask and/or bioreactor. For example, the methods can be applied in bioreactors or cell cultures of 1 L, 10 L, 30 L, 50 L, 100 L, 150 L, 200 L, 300 L, 500 L, 1000 L, 2000 L, 3000 L, 4000 L, 5000 L, 10,000 L or larger.
[0243] The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batchwise, semibatchwise or continuously.
Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. [0244] Media components include, e.g., buffer, amino acid content, vitamin content, salt content, mineral content, serum content, carbon source content, lipid content, nucleic acid content, hormone content, trace element content, ammonia content, co-factor content, indicator content, small molecule content, hydrolysate content and enzyme modulator content.
[0245] The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981 ; the entirety of which is hereby incorporated herein by reference). These media which can be employed in accordance with the subject technology usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
[0246] Carbon sources for use in the culture media of the host cells comprise sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffmose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.
[0247] Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.
[0248] In an embodiment, the inorganic salt compounds that are present in the culture media comprise about one of the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper or iron.
[0249] Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing derivatives of styrene.
[0250] Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus. [0251] Other components that may be added to the culture medium in order to keep the metal ions in solution comprise dihydroxy phenols such as catechol or protocatechuate and organic acids such as citric acid.
[0252] The culture medium may comprise one or more metal ions, such as calcium, zinc, magnesium, iron, manganese ion, and a combination thereof. The metal ion may be present at a concentration of from about 0.1 mM to about 100 mM, such as about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM; from about 0.1 mM to about 50 mM, from about 0.1 mM to about 25 mM, from about 0.1 mM to about 20 mM, from about 0.1 mM to about 10 mM, from about 1 mM to about 50 mM, from about 1 mM to about 25 mM, from about 1 mM to about 20 mM, from about 1 mM to about 15 mM, or from about 1 mM to about 10 mM. Preferably, the metal ion is an alkaline earth metal ion, or a transitional metal ion.
[0253] The fermentation media used according to the subject technology for culturing the host cells of the subject technology usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the culture of media can be found in the textbook "Applied Microbial. Physiology, A Practical Approach" (Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73; the entirety of which is hereby incorporated herein by reference). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
[0254] All media components are sterilized, either by heat (20 min at 1.5 bar and 121° C.) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.
[0255] The temperature of the cell culture will be selected based primarily on the range of temperatures at which the cell culture remains viable, at which a high level of polypeptide is produced, at which misfolding and/or aggregation of the polypeptide are reduced, at which the polypeptide exhibits a more extensive or otherwise more desirable post-translational modification (e.g., glycosylation, phosphorylation, etc.), or any combination of these or other factors deemed important by the practitioner. In general, most host cells grow well and can produce high levels or protein or polypeptide within a range of about 15 °C to 45 °C. In certain embodiments, the cell culture is grown at a temperature of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 °C at one or more times during the cell culture process. Those of ordinary skill in the art will be able to select appropriate temperature or temperatures in which to grow cells, depending on the needs of the cells and the production requirements.
[0256] Furthermore, the culture may be subjected to one or more temperature shifts during the course of the culture. When shifting the temperature of the culture, the temperature shift may be relatively gradual. For example, it may take several hours or days to complete the temperature change.
Alternatively, the temperature shift may be relatively abrupt. The temperature may be steadily increased or decreased during the culture process. Additionally or alternatively, the temperature may be increased or decreased by discrete amounts at various times during the culture process. The subsequent temperature(s) or temperature range(s) may be lower than or higher than the initial or previous temperature(s) or temperature range(s). One of ordinary skill in the art will understand that multiple temperature shifts are encompassed by the subject technology. For example, the temperature may be shifted once (either to a higher or lower temperature or temperature range), the cells maintained at this temperature or temperature range for a certain period of time, after which the temperature may be shifted again to a new temperature or temperature range, which may be either higher or lower than the temperature or temperature range of the previous temperature or temperature range. The temperature of the culture after each discrete shift may be constant or may be maintained within a certain range of temperatures.
[0257] As with the initial temperature or temperature range, the temperature or temperature range of the cell culture after the temperature shift(s) is generally selected based primarily on the temperature(s) at which the cell culture remains viable, the range in which a high level of protein is produced. For example, a bacterial cell culture may be grown at 37 °C after seeding to encourage cell proliferation; once the cells reach a desired density, the expression of a recombinant protein is induced, and the temperature is shifted to 25 °C to reduce misfolding or aggregation of the recombinantly produced protein.
[0258] Anaerobic condition is also contemplated. Exemplary anaerobic bacterial hosts include, e.g., Bacteroids, Fusobacterium, Clostridium, Propionibacterium, Lactobacillus, Peptococcus,
Peptostreptococcus and Veillonella.
[0259] If desired, the styrene produced in the host cells can further undergo enzymatic reaction and be converted to a downstream derivative of styrene such as toluene, xylene, polystyrene, ABS, styrene-butadiene (SBR) rubber, styrene-butadiene latex, SIS (styrene-isoprene-styrene ), S-EB-S (styrene-ethylene/butylene-styrene ), styrene-divinylbenzene (S-DVB), styrene-acrylonitrile resin (SAN) and unsaturated polyesters and the like. Thus, in some embodiments the host cells of the subject technology can convert certain percentage of the styrene produced to a downstream derivative of styrene. For example, the host cell can convert about 5% of the styrene to a downstream derivative, or from about 5% to about 15% of the styrene to a downstream derivative, or from about 10% to about 25% of the styrene to a downstream derivative, or from about 20% to about 35% of the styrene to a downstream derivative, or from about 30% to about 45% of the styrene to a downstream derivative, or from about 40% to about 55% of the styrene to a downstream derivative, or from about 50% to about 65% of the styrene to a downstream derivative, or from about 60% to about 75% of the styrene to a downstream derivative, or from about 70% to about 85% of the styrene to a downstream derivative, or from about 80% to about 95% of the styrene to a downstream derivative.
1. Microbial Hosts
[0260] Microorganisms useful for the production of styrene may include bacteria, such as enteric bacteria (Escherichia, and Salmonella for example), Bacillus, Acinetobacter, Actinomycetes (such as Streptomyces), Corynebacterium, Methanotrophs (such as Methylosinus), Methylomonas,
Rhodococcus, Pseudomona, Cyanobacteria (such as Rhodobacterand, Synechocystis), Klebsiella, Pantoea, Corynebacterium, Clostridium, etc.; yeasts, such as Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia and Torulopsis; filamentous fungi such as Aspergillus, Arthrobotrys; algae, etc.
[0261] Although any of the above mentioned microorganisms would be useful in the production of styrene, preferred are mutant strains of bacteria that over-produce phenylalanine. "Phenylalanine overproducing" strain is a mutant microbial strain that produces higher level of phenylalanine as compared to that of the wild-type strain that does not have the mutation. Phenylalanine is naturally present in micro-organisms. However, for an optimal synthesis of styrene a host cell preferably overproduces phenylalanine such that the substrate level does not limit styrene production by the host cell. Methods to increase aromatic amino acid synthesis in a micro-organism are known in the art.
[0262] One specific example of an E. coli phenylalanine over-producer is the E. coli strain NST74 (U.S. Pat. No. 4,681,852). Others suitable Phenylalanine overproducing strains include, e.g., Corynebacterium glutamicum (Ikeda, M. and Katsumata, R. Metabolic engineering to produce tyrosine or phenylalanine in a tryptophan-producing Corynebacterium glutamicum strain, Appl. Environ. Microbial. (1992), 58(3), pp. 781-785); Microbacterium ammoniaphilum ATCC 10155; Corynebactrium lillium NRRL-B-2243, Brevibacterium divaricatum NRRL-B-2311 ; Arthrobacter citreus ATCC 11624.
Additional suitable phenylalanine overproducing strains can be found, e.g., in Maiti et al, Supra and Metabolic Engineering For Microbial Production Of Aromatic Amino Acids And Derived Compounds, J. Bongaertes et al, Metabolic Engineering vol 3, 289-300), 2001.
[0263] The host cell may also be selected for increased resistance against a toxic analogue of an aromatic amino acid (e.g., phenylalanine). For example, mutant micro-organisms can be selected for resistance to toxic (m-fluoro-)analogues of phenylalanine. These insensitive mutants often produce high levels of phenylalanine and tyrosine (GB 1071935; U.S. Pat. No. 3,709,785). [0264] It is also possible to obtain a recombinant host cell with increased phenylalanine production by overexpression of one or more key genes in the biosynthesis of phenylalanine (Ikeda 2003. Amino acid production processes. P. 1-35. in T. Scheper (Ed.), Advances in Biochemical
Engineering/Biotechnology, Vol. 79. Springer- Verlag, Berlin Heidelberg).
[0265] Standard recombinant DNA methodologies may be used to obtain a nucleic acid that encodes a protein described herein (e.g., phenylalanine ammonia lyase, cinnamic acid decarboxylase, or a fusion protein), incorporate the nucleic acid into an expression vector, and introduce the vector into a host cell, such as those described in Sambrook, et al. (eds), Molecular Cloning; A Laboratory Manual, Third Edition, Cold Spring Harbor, (2001); Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, John Wiley & Sons (1995). A nucleic acid encoding a protein may be inserted into an expression vector or vectors such that the nucleic acids are operably linked to transcriptional and translational control sequences (such as a promoter sequence, a transcription termination sequence, etc.). The expression vector and expression control sequences are generally chosen to be compatible with the expression host cell used.
[0266] The expression of proteins in a microbial host described herein can be further improved by codon-optimization. For example, modifying a less-common codon with a more common codon may affect the half-life of the mRNA or alter its structure by introducing a secondary structure that interferes with translation of the message. All or a portion of a coding region can be optimized. In some cases the desired modulation of expression is achieved by optimizing essentially the entire gene. In other cases, the desired modulation will be achieved by optimizing part of but not entire sequence of the gene.
[0267] The codon usage of any coding sequence can be adjusted to achieve a desired property, for example high levels of expression in a specific cell type. The starting point for such an optimization may be a coding sequence with 100% common codons, or a coding sequence which contains a mixture of common and non-common codons.
[0268] Two or more candidate sequences that differ in their codon usage can be generated and tested to determine if they possess the desired property. Candidate sequences can be evaluated by using a computer to search for the presence of regulatory elements, such as silencers or enhancers, and to search for the presence of regions of coding sequence which could be converted into such regulatory elements by an alteration in codon usage. Additional criteria may include enrichment for particular nucleotides, e.g., A, C, G or U, codon bias for a particular amino acid, or the presence or absence of particular mRNA secondary or tertiary structure. Adjustment to the candidate sequence can be made based on a number of such criteria. [0269] In certain embodiments, the codon optimized nucleic acid sequence can express its protein, at a level which is about 110%, about 150%, about 200%o, about 250%o, about 300%>, about 350%o, about 400%o, about 450%o, or about 500%o, of that expressed by nucleic acid sequence that has not been codon optimized.
[0270] In addition to the nucleic acid that encodes the protein, the expression vector may additionally carry regulatory sequences that control the expression of the protein in a host cell, such as promoters, enhancers or other expression control elements that control the transcription or translation of the nucleic acid(s). Such regulatory sequences are known in the art (see, e.g., Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
[0271 ] In addition to sequences encoding the protein and regulatory sequences, the recombinant expression vectors of the subject technology may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
[0272] The expression vector(s) encoding the protein may be transformed or transfected into a host cell by standard techniques, such as electroporation, calcium-phosphate precipitation, or DEAE- dextran trans fection.
[0273] Where commercial production of styrene is desired, a variety of fermentation methodologies may be applied. For example, large scale production may be effected by both batch or continuous fermentation.
[0274] A classical batch fermentation is a closed system where the composition of the media is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired microorganism or microorganisms and fermentation is permitted to occur adding nothing to the system. Typically, however, the concentration of the carbon source in a "batch" fermentation is limited and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in the log phase generally are responsible for the bulk of production of end product or intermediate.
[0275] A variation on the standard batch system is the Fed-Batch system. Fed-Batch fermentation processes are also suitable in the subject technology and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as C02. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, T. D.; Biotechnology: A Textbook of Industrial Microbiology, 2nd ed.; Sinauer Associates:
Sunderland, Mass., 1989; or Deshpande, M. V. Appl. Biochem. Biotechnol. 36:227, (1992).
[0276] Commercial production of styrene may also be accomplished with continuous fermentation. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in their log phase of growth.
[0277] Continuous fermentation allows for modulation of any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by the medium turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium removal must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
2. Plant Hosts
[0278] Plant cells may also be used as hosts for producing styrene. Preferred plant hosts will be any variety that support a high expression level of phenylalanine ammonia lyase and/or cinnamic acid decarboxylase. Suitable green plants will include, e.g., soybean, rapeseed (Brassica napus, B. campestris), sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), wheat (Triticum sp ), barley (Hordeum vulgare), oats (Avena sativa, L), sorghum (Sorghum bicolor), rice (Oryza sativa), Arabidopsis, cruciferous vegetables (broccoli, cauliflower, cabbage, parsnips, etc.), melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses. Algal species include, e.g., commercially significant hosts such as Spirulina,
Haemotacoccus, and Dunalliela. Suitable plants also include biofuel, biomass, and bioenergy crop plants. Exemplary plants include Arabidopsis thaliana, lice (Oryza sativa), switchgrass (Panicum vigratum), Brachypodium spp, Brassica spp., and Crambe abyssinica. [0279] In some embodiments, the plant cell is an Arabidopsis plant cell, a tobacco plant cell, a petunia plant cell, or a cell from an oilseed crop (including, e.g., a soybean plant cell, a canola plant cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, etc.).
[0280] Suitable host cells can be genetically engineered to express phenylalanine ammonia lyase and cinnamic acid decarboxylase. For example, nucleic acid encoding phenylalanine ammonia lyase or cinnamic acid decarboxylase can be operably linked to promoters capable of directing expression of a protein in the desired tissues at the desired stage of development. Any suitable promoter and/or terminator capable of inducing expression of a coding region may be used. Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV) genes.
[0281] One type of efficient plant promoter that may be used is a high level plant promoter. High level plant promoters that may be used in the subject technology include the promoter of the small subunit (ss) of the ribulose-l,5-bisphosphate carboxylase for example from soybean (Berry-Lowe et al., J. Molecular and App. Gen., 1 :483-498) (1982)), and the promoter of the chlorophyll a/b binding protein. These two promoters are known to be light-induced in plant cells (see, for example, Genetic Engineering of Plants, an Agricultural Perspective, A. Cashmore, Plenum, N.Y. (1983), pages 29-38); Coruzzi, G. et al., The Journal of Biological Chemistry, 258: 1399 (1983), and Dunsmuir, P. et al., Journal of Molecular and Applied Genetics, 2:285 (1983)).
[0282] Standard recombinant DNA methodologies may be used to obtain a nucleic acid that encodes a protein described herein, incorporate the nucleic acid into an expression vector and introduce the vector into a host cell. The choice of vector depends upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the vector. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., EMBO J. 4:2411-2418) (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86) (1989)), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA blots (Southern, J. Mol. Biol. 98, 503, (1975)). Northern analysis of mRNA expression (Kroczek, J. Chromatogr. Biomed. Appl., 618 (12):133-145) (1993)), Western analysis of protein expression, or phenotypic analysis.
[0283] The expression of proteins in a plant host described herein can be further improved by codon-optimization, as described above. In certain embodiments, the codon optimized nucleic acid sequence can express its protein, at a level which is about 110%, about 150%, about 200%o, about 250%o, about 300%, about 350%, about 400%o, about 450%o, or about 500%o, of that expressed by nucleic acid sequence that has not been codon optimized.
[0284] The subject technology also provides transgenic host cells or host cells that have been transformed with one or more of nucleic acids disclosed herein. The nucleic acid molecule can be stably integrated into the genome of the cell, or the nucleic acid molecule can also be present as an
extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or subjects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
[0285] Introduction of a nucleic acid of the subject technology into a plant cell can be performed by a variety of methods known to those of ordinary skill in the art including, but not limited to, insertion of a nucleic acid sequence of interest into an Agro bacterium rhizogenes Ri or Agrobacterium tumefaciens Ti plasmid, microinjection, electroporation, or direct precipitation. By way of providing an example, in some embodiments, transient expression of a nucleic acid sequence or gene of interest can be performed by agro-infiltration methods. In this regard, a suspension of Agrobacterium tumefaciens containing a nucleic acid sequence or gene of interest can be grown in culture and then injected into a plant by placing the tip of a syringe against the underside of a leaf while gentle counter-pressure is applied to the other side of the leaf. The Agro bacterium solution is then injected into the airspaces inside the leaf through stomata. Once inside the leaf, the Agro bacterium transforms the gene of interest to a portion of the plant cells where the gene is then transiently expressed.
[0286] As another example, transformation of a vector or nucleic acid of interest into a plant cell can be performed by particle gun bombardment techniques. In this regard, a suspension of plant embryos can be grown in liquid culture and then bombarded with plasmids or nucleic acids that are attached to gold particles, wherein the gold particles bound to the plasmid or nucleic acid of interest can be propelled through the membranes of the plant tissues, such as embryonic tissue. Following bombardment, the transformed embryos can then be selected using an appropriate antibiotic to generate new, clonally propagated, transformed embryogenic suspension cultures.
[0287] For additional guidance regarding methods of transforming and producing transgenic plant cells, see U.S. Patent Nos. 4,459,355; 4,536,475; 5,464,763; 5,177,010; 5,187,073; 4,945,050; 5,036,006; 5,100,792; 5,371,014; 5,478,744; 5,179,022; 5,565,346; 5,484,956; 5,508,468; 5,538,877; 5,554,798; 5,489,520; 5,510,318; 5,204,253; 5,405,765; EP Nos. 267,159; 604,662; 672,752; 442,174; 486,233; 486,234; 539,563; 674,725; and, International Patent Application Publication Nos. WO
91/02071 and WO 95/06128. 3. Reducing Styrene Toxicity
[0288] In another aspect, the subject technology provides a host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase; (b) a recombinantly expressed cinnamic acid decarboxylase; and (c) a recombinantly expressed membrane-bound transporter.
[0289] One significant problem that limits the bioproduction of styrene is the toxicity of styrene to host cells. The accumulation of hydrophobic aromatics within the cytoplasmic membrane is known to disrupt its integrity. To reduce styrene toxicity and enhance production, a membrane-bound transporter (e.g. an efflux pump) can be introduced into the host cell to remove organic solvent from the cell.
Accordingly, the host cell displays a tolerant phenotype towards hydrophobic solvents.
[0290] In one embodiment, the membrane-bound transporter can be an ABC-transporters (ATP- binding cassette transporters), which are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out certain biological processes including translocation of various substrates across membranes and nontransport-related processes such as translation of RNA and DNA repair. They transport a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids and sterols, and drugs. Proteins are classified as ABC transporters based on the sequence and organization of their ATP-binding cassette (ABC) domain(s).
[0291 ] Provided herein are host cells that express an ABC-transporter, which allows the host cells to secrete styrene into the culture medium. In particular, the ABC-transporter is a solvent-resistant pump. Solvent-resistant pumps conferring resistance or tolerance towards organic solvents have been shown to possess very broad specificity, taking organic compounds that by virtue of their chemical- physical characteristics (e.g., accumulating in the bacterial membrane), such as aromatics, alcohols, alkanes etc., as substrates (Kieboom et al. 1998. J. Biol. Chem. 273:85-91). Aromatic compounds also partition effectively to the cell membrane where they act as substrates for solvent-resistant pumps.
[0292] In one embodiment of the subject technology, a host cell comprises a member of the proton-dependent resistance/nodulation/cell division (RND) family of efflux pumps. RND-type efflux pumps belong to the multidrug resistance (MDR) pumps. They have an extremely broad substrate specificity and protect bacterial cells from the actions of antibiotics on both sides of the cytoplasmic membrane. Members of this family have been shown to be involved in export of antibiotics, metals, and oligosaccharides involved in nodulation signalling. RND-type efflux pumps usually function as three- component assemblies spanning the outer and cytoplasmic membranes and the periplasmic space of Gram-negative bacteria. Examples of suitable RND-type efflux pumps for use in a method of the subject technology can be found in Tseng, T. T., Gratwick, K. S., Kollman, J., Park., D., Nies, D. H., Goffeau, A., & Saier Jr., M. H. (1999), J. Mol. Microbial. Biotechnol. 1 : 107-125. [0293] In one embodiment, the host cell comprises the solvent resistance pump srpABC of P. putida S12 (Isken et al. 1996 J. Bacterial. 178:6056; Kieboom et al. 1998. J. Biol. Chem. 273:85-91). The deduced amino acid sequences of the proteins encoded by the srpABC genes have extensive homology with those of the RND family of efflux pumps. It is composed of three protein components that together span the inner and outer membranes of Gram-negative bacteria: an inner membrane transporter (SrpB analogues), an outer membrane channel (SrpC analogues), and a periplasmic linker protein (SrpA analogues). Dendrograms showing the phylogenetic relationship of SrpA, SrpB, and SrpC to other proteins involved in multidrug resistance are shown in Kieboom et al. (1998 J. Biol. Chem. 273:85-91). The srpABC-encoded proteins show the most homology with those for the mexAB/oprM-encoded multidrug resistance pump found in Pseudomonas aeruginosa. SrpA, SrpB, and SrpC are 57.8, 64.4, and 58.5% identical to MexA, MexB, and OprM, respectively. In one embodiment, a host cell comprises an efflux pump consisting of an inner membrane transporter, an outer membrane channel, and a periplasmic linker protein belonging to the RND-family of efflux pumps wherein the proteins show a homology of about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95%, about 98%, about 99%o, or even 100%o sequence identity to the SrpA, SrpB or SrpC proteins of P. putida S12. Any functional equivalents of known solvent efflux pumps that can use an aromatic compound as a substrate can be used.
[0294] In one embodiment, the host cell can convert the fermentable carbon substrate into an aromatic amino acid (e.g., phenylalanine), which is subsequently converted into styrene. Once produced, styrene actively transported out of the host cell by an efflux pump, preferably by a member of the proton- dependent resistance/nodulation/cell division (RND) family of efflux pumps, such as srpABC.
[0295] The bacterium P. putida S12 has also been engineered as a solvent tolerance platform for the biosynthesis of both p-hydroxybenzoate and p-hydroxystyrene (Verhoef et al., 2007,
Bioproduction of p-hydroxybenzoate from renewable feed stockby solvent-tolerant Pseudomonas putida SI 2. Journal of Biotechnology 132, 49-56; Verhoef et al., 2009, Bioproduction of p-hydroxystyrene from glucose by the solvent-tolerant bacterium Pseudomonas putida S 12 in a two-phase water-decanol fermentation. Appliedand Environmental Microbiology 75, 931-936). The engineered strain can be used for the bioproduction of styrene. Other hosts may be engineered in a similar fashion to increase tolerance to organic compounds.
G. HARVESTING STYRENE FROM CELL CULTURE.
[0296] Styrene can be harvested from the cell culture using conventional methods. For example, host cells can be removed by filtration or centrifugation. Oil phase and water phase may be separated by centrifugation or chromatography. Additional adsorption, distillation, and microfiltration techniques may be used to further purify styrene. A general scheme of purification involves removing polymerization inhibitors with alkaline water solution (usually 5-10% NaOH), washing in stilled water, drying and fractional distillation under reduced pressure, microfiltration of the styrene monomer in the gas state, and a combination thereof.
[0297] Thus, the subject technology provides an inexpensive biological route to the production of styrene which is useful in a variety of commercial materials including polystyrene, ABS, styrene- butadiene (SBR) rubber, styrene-butadiene latex, SIS (styrene-isoprene-styrene), S-EB-S (styrene- ethylene/butylene-styrene), styrene-divinylbenzene (S-DVB), styrene-acrylonitrile resin (SAN) and unsaturated polyesters. These materials are used in rubber, plastic, insulation, fiberglass, pipes, automobile and boat parts, food containers, and carpet backing.
H. BIOSYNTHESIS OF STYRENE.
[0298] Typically, styrene can be produced by incubating a substrate described herein (such as glucose, phenylalanine, or trans -cinnamic acid) with the host cell comprising any cinnamic acid decarboxylase described herein or any fusion proteins described herein. The concentration of glucose in the incubation typically is from about 0.02% to about 3%, preferable from about 0.05% to about 1.5%, more preferably from about 0.1% to about 1%. The concentration of trans-cinnamic acid in the incubation typically is from about 0.02% to about 0.5%, preferable from about 0.05% to about 0.2%. The concentration of phenylalanine in the incubation typically is from about 0.02% to about 0.5%, preferable from about 0.05% to about 0.2%. In one embodiment, following the addition of the substrate, the host cells is cultured continuously for about 10 to about 72 hours, preferably from about 15 to about 36 hours, at a temperature of from about 16°C to about 37°C, preferably from 22°C to about 30°C.
[0299] Based on the above, the subject technology provides a method of producing styrene, the method comprising (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising a fusion protein as described above; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene.
[0300] The subject technology also provides a method of producing styrene, the method comprising (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) a mutant cinnamic acid decarboxylase as described above; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene.
[0301] The subject technology also provides a host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase; (b) a recombinantly expressed cinnamic acid decarboxylase; and (c) a recombinantly expressed membrane-bound transporter. Accordingly, the subject technology also provides a method for the production of styrene, the method comprising: (a) contacting the above host cell with a fermentable carbon substrate; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene. [0302] In traditional methods, styrene biosynthesis is accomplished in closed containers to prevent styrene from evaporating from the container. This closed system cannot maintain the facultative anaerobic conditions (optimal conditions) for a biological system to produce styrene. Thus, with the traditional method, styrene product accumulates in the closed container and the styrene biosynthesis eventually stops due to the toxicity effect imposed by styrene on the biosynthetic system (e.g. a host cell). Surprisingly, the inclusion of an absorbing material to remove the styrene vapor from the biosynthesis process not only enables a reliable detection of the styrene product (e.g. by the method of screening FDC mutant activities as described above), but also improves the yield of styrene biosynthesis.
[0303] Accordingly, the subject technology also provides a method for producing styrene, the method comprising: (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) a cinnamic acid decarboxylase; and (b) culturing the host cell in a culture medium for a time sufficient to produce styrene, wherein the vapor of the styrene product is absorbed by an absorbing material.
[0304] In this aspect, the phenylalanine ammonia lyase and the cinnamic acid decarboxylase may include both wild type proteins and mutant proteins. For example, the cinnamic acid decarboxylase may be a mutant FDC protein as described above. In addition, the phenylalanine ammonia lyase and the cinnamic acid decarboxylase may be present as separate proteins or as a fusion protein, such as the PAL- FDC(WT) or PAL-FDC(K190E) fusion proteins described above.
[0305] In one embodiment, the styrene vapor is absorbed by a polymeric resin that is capable of absorbing organic molecules, while air is allowed to flow freely into the biosynthesis system (e.g. a host cell). Any absorbing material used in the high-throughput screening process for cinnamic acid decarboxylase activity described above can also be employed in the styrene biosynthesis process with similar devices (Figure 19). For example, STRATA-X column (containing reverse phase polymeric resin) can be used to absorb the styrene vapor produced from a host cell (thus reducing toxicity to the host cell), while allowing oxygen to pass through the resin to maintain facultative anaerobic conditions for cell growth. Typically, this method allows styrene to be produced at level of greater than 1 g/L.
[0306] All the method of producing styrene described above, including the method involving a fusion protein, the method involving mutant cinnamic acid decarboxylase, the method involving membrane-bound transporter, and the method involving removal of styrene vapor by an absorbing material, can also be used to produce 4-hydroxystyrene. In one embodiment, tyrosine or coumaric acid or both can be used as substrate in the methods described above to produce 4-hydroxystyrene.
[0307] The capability to construct a fusion protein and to rapidly examine cinnamic acid decarboxylase activity in a large number of samples allows for simultaneously screening the activities of the PAL and cinnamic acid decarboxylase units in the fusion protein, such that an improved overall styrene yield can be achieved. This process is advantageous over the process of examining the PAL and cinnamic acid decarboxylase activities separately before building a fusion protein.
[0308] Accordingly, the subject technology provides a method for simultaneously screening phenylalanine ammonia lyase and cinnamic acid decarboxylase activities, the method comprising: (a) providing a fusion protein comprising: (i) a first domain comprising a phenylalanine ammonia lyase, and (ii) a second domain comprising a cinnamic acid decarboxylase; (b) mixing the fusion protein with a substrate under a condition that allows the fusion protein to convert the substrate to a product; and (c) detecting the amount of the remaining substrate, or the amount of the product, or both.
[0309] In one embodiment,, the subject technology also provides a method for simultaneously screening phenylalanine ammonia lyase and cinnamic acid decarboxylase activities, the method comprising: (a) providing a fusion protein comprising: (i) a first domain comprising a phenylalanine ammonia lyase, and (ii) a second domain comprising a cinnamic acid decarboxylase; (b) providing a substrate selected from the group consisting of phenylalanine, trans-cinnamic acid, tyrosine, coumaric acid, and combinations thereof; (c) incubating the fusion protein and the substrate under a condition that allows the fusion protein to convert the substrate to a product selected from the group consisting of styrene, 4-hydroxystyrene, and combination thereof; and (d) detecting the amount of the remaining substrate, or the amount of the product, or both.
[0310] The fusion protein may be prepared using any of the above-described PALs and cinnamic acid decarboxylases, such as PAL-FDC or PAL-FDC(K190E). Typically, a substrate (such as phenylalanine or trans-cinnamic acid) is mixed with the fusion protein, and the activities of the PAL and cinnamic acid decarboxylase in the fusion protein can be simultaneously detected by measuring the amount of the remaining substrate, or the amount of the product, or both. For example, when phenylalanine is mixed with the fusion protein as substrate, the amount of trans-cinnami acid and styrene as products can be measured. In this example, the concentration of cinnamic acid reflects both its production from phenylalanine (by PAL) and its conversion to styrene (by cinnamic acid decarboxylases), and the concentration of styrene reflects the overall styrene production by the fusion protein from any substrate (Figure 18). Similarly, when trans-cinnamic acid is mixed with the fusion protein for conversion to styrene, both the remaining amount of trans-cinnamic acid substrate and the amount of styrene product can be measured.
[0311] In one embodiment, the simultaneously screening for phenylalanine ammonia lyase and cinnamic acid decarboxylase activities can be performed under similar conditions used for the high- throughput screening assays for cinnamic acid decarboxylase activity described herein. In an exemplary embodiment, the detection of styrene comprises exposing the mixture of the fusion protein and the substrate to a polymeric resin that absorbs styrene vapor. Suitable resins include hydrophobic resins, such as CI 8, C8, phenyl, SDB-L sorbents resins, and combinations thereof. EXAMPLES
[0312] The subject technology is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the subject technology, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the subject technology, and without departing from the spirit and scope thereof, can make various changes and modifications of the subject technology to adapt it to various uses and conditions.
EXAMPLE I. RECOMBINATION PRODUCTION, PURIFICATION, AND
CHARACTERIZATION OF YEAST FDCl.
1. Expression of yeast FDCl in E. coli
[0313] Decarboxylation of trans-cinnamic acid (tCA) by ferulic acid decarboxylase (FDCl) is the last step in the styrene biosynthesis pathway to yield styrene. Though it was reported that expression of FDCl alone is insufficient to convert tCA to styrene (Jiang et al., 2005, Applied and Environmental Microbiology, 71 : 2962-2969; Clausen et al., 1994, Gene, 142: 107-112.), more recently, it was reported that expression of FDCl alone was sufficient to convert tCA to styrene in vivo (McKenna et al., 2011, Metabolic Engineering, 13 (5): 544-554). McKenna speculated that other proteins, like E. coli Ubix that is associated with FDCl in vivo, enable the conversion of tCA to styrene. There is no report that clearly shows that FDCl alone, without association of any other proteins, is sufficient to convert tCA to styrene.
[0314] To examine the activity of purified FDCl in vitro, an expression vector was constructed and FDCl was expressed in E. coli under various conditions. Plasmid pDESTl 7- FDCl was transformed into E. Coli strain BL21 (DE3) competent cells. Terrific-broth media supplemented with ampicillin (100 g/mL) was inoculated with a 1000-time dilution of an overnight culture. The bacteria were cultured at 37°C until OD6oo reached 0.8 to 1.0, at which point isopropyl β-D-l-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM, and continued to be cultured for 3 hours at the same temperature. Three different lysis buffers were used to check the expression of FDC 1. Buffer A contained 25 mM potassium phosphate buffer pH 7.5, 500 mM sodium chloride, 10 mM imidazole, 20mM βΜΕ, 0.5 mM PMSF, 10 mM MgCl2, 2 g/mL DNAse, 2 g/mL RNAse, and 4 g/mL lysozyme. Buffer B contained 50 mM potassium phosphate pH buffer 7.0, 1 mM DTT, 50 mM sodium thiosulfate, 20% glycerol, 500 mM NaCl, 10 mM MgCl2, 20 mM imidazole, 2 g/mL DNAse, 2 g/mL RNAse, and 4 g/mL lysozyme. Buffer C was composed of Buffer A containing 50 mM sodium thiosulfate and 20% glycerol. [0315] At 37°C, the recombinant FDC1 was not expressed as a soluble form in E. coli, and formed inclusion bodies, as shown in Figure 2A. The standard protocols for expressing recombinant proteins are not applicable for FDC1.
[0316] To produce functional FDC1 in E. coli, expression of FDC1 was induced at various temperatures (37°C, 30°C, 25°C, 18°C, and 16°C) in the presence of various concentrations of IPTG (0.2 mM, 0.5 mM). Concentrations of IPTG had no significant effect on expression; however, lower temperature improved expression and solubility of this protein. Optimal FDC1 expression was achieved after induction with 0.2 mM IPTG at 16°C for 16 hours (Figure 2B). The cells were harvested by centrifugation at 4500 rpm at 4°C and washed once with IX PBS buffer and then stored at -80°C.
[0317] Expression of FDC1 was examined by SDS-PAGE (10% acrylamide slab gel, 0.75 mm thick), using the Laemmli protocol. Coomassie brilliant blue R-250 was used to stain the protein band.
[0318] Our results indicated that expression of FDC 1 at lower temperature with lower concentration of IPTG played a role for the correct folding of the enzyme, and the conversion of aggregated or misfolded forms to a soluble, functional form.
2. Purification of Yeast FDC1
[0319] There is no report on the purification and characterization of yeast FDC1. To examine the activity and properties of FDC 1, functional FDC1 that was recombinantly produced from E. coli was purified.
[0320] Purification was carried out in an anaerobic environment at 4 °C. The cells were suspended in buffer A (composed of 50 mM Tris-HCl pH 8.0 containing 50 mM Na2S203, 25 mM TCEP, 500 mM NaCl, 0.5 mM PMSF, 20 mM β-mercaptoethanol, 20% glycerol, and 10 mM imidazole) containing 10 mM MgCl2, 0.2% Triton X-100, 2 g/mL DNAse, 2 g/mL RNAse, and 4 g/mL lysozyme. The cells were disrupted by ultra-sonication and the supernatant was collected by centrifugation at 15,000 g for 20 min at 4°C. The resulting supernatant was filtered through a 0.45 μιη PES filter and then applied to Ni+-agarose affinity column (GE Healthcare), equilibrated with the buffer A. The column was washed well with buffer A until all the non-specific binding proteins eluted from the column. The protein was eluted with buffer A containing 250 mM imidazole. The protein content was determined by the spectrophotometer at 280 nm. The protein was fairly pure (Figure 3).
[0321] Recombinant FDC1 that can convert tCA to styrene was purified. However, the protein lost its activity very fast during purification. The protein activity can be maintained when the purification was conducted under an anaerobic condition, such as by adding higher amounts of reducing agents (50 mM Na2S203, 25 mM TCEP, and 20 mM β-mercaptoethanol) in the buffers. 3. Activity Assays for Recombinantly Produced FDC1
[0322] To study the function of recombinant FDC1 in vitro, the effects of various conditions (e.g., buffers at a wide pH range, various substrate concentrations, and various organic solvents for product extraction) on the enzymatic activity of FDC1 were tested.
[0323] First, an enzymatic assay that is very accurate and reproducible was developed, which can measure FDC1 activity at a low concentration of substrate.
[0324] The standard reaction mixture for decarboxylation consisted of 25 mM potassium phosphate buffer (pH 6.5) containing 5 mM dithiothreitol, 1.4 mM trans-cinnamic acid and enzyme (0.50 mg) to a final volume of 1.0 mL. The reaction was started by the addition of the enzyme and was incubated at 30°C for 5 min. The reaction was stopped by adding 24 μΐ of glacial acetic acid (17.4N) after which 2-propanol was added in equal volume to the reaction mixture in order to solubilize the product. The amount of styrene produced was measured by HPLC using a Dionex Ultimate3000 UHPLC equipped with an auto sampler, diode array (UVVVis) detector, and reverse phase Acclaim 120 CI 8 column (2.1x150 mM Dionex USA). Samples (10 μΐ) were injected for analyses at a total constant flow rate of 0.6 ml/minute. The samples were resolved in 0.15% acetic acid (A) with an increasing concentration gradient of acetonitrile containing 0.15% acetic acid (B) for 0 to 4 min, 5%; 4 to 5 minutes, 5 to 40 %; 5 to 7 min, 40 to 45%; 7 to 8 min, 45 to 85%; 8 to 12 min, 85 to 95%; 12 to 14 minutes, 95 to 5% at a flow rate of 1 ml/min. The specific activity was expressed in U (nmol styrene),mg"1,min"1.
4. Effect of pH on FDC1 Activity
[0325] Buffer pH is one of the main factors that can influence an enzymatic reaction. Reactions in buffers at pH 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5, respectively, were carried out to evaluate optimal pH for FDC1 activity. Potassium phosphate buffer was used for pH values 6.0 to 7.5 and Tris-HCl buffer was used for 8.0 and 8.5. The reaction mixture was composed of 200 μΜ cinnamic acid, 5 mM DTT, 100 mM various pH buffers, and 150 L FDC1 crude extract to a final volume of 1 mL. The reaction mixtures were incubated for 30 minutes at 30°C. The styrene was extracted with 100 L of butanol. The experimental negative control used the same conditions except the reaction mixture contained no substrate. The optimal pH for the decarboxylation of FDC1 crude extract was found to be about 6.5. Our results showed that buffer had a significant impact on FDC1 activity, and FDC1 showed highest activity at pH 6.5 (Figure 4). This information is useful for industrial production of styrene.
5. pH Stability of FDC1
[0326] Buffer also affects the stability of FDC1 and the reaction rate. To study the stability of FDC1, the protein was incubated in buffers of different pH values, from 5.0 to 11.0.
[0327] The protein (crude extract, 586 μί) was added to 112 μί of 500 mM buffers at pH values of 5, 6, 7, 8, 9, 10, and 11, and incubated at 30°C for 30 minutes. After treated in various pH buffers, an aliquot of protein (42 mg) was added to a final concentration of 100 mM potassium phosphate buffer pH 6.5, 5 mM DTT, 1.4 mM cinnamic acid and brought up to a final volume of 2 mL. The reaction mixture was then incubated for 8 minutes at 30°C. The styrene was extracted by the addition of 250 L butanol. The experimental negative control used the same conditions except the reaction mixture contained no substrate. The FDC1 crude extract was found to be stable at a pH ranging from 6 to 10 (Figure 5). This protein is stable and active in a wide range of pH and can be used for industrial production of styrene.
6. Effect of Temperature on FDC1 Activity
[0328] Temperature can affect the stability of FDC1 and the reaction rate. The effect of temperature on reaction rate is described by the Arrhenius equation. As a rule of thumb, reaction rates for many reactions double or triple for every 10 degrees Celsius increase in temperature, though the effect of temperature may be much larger or smaller than this.
[0329] To evaluate the optimal temperature for FDC1 , reactions were carried out at temperatures of 25°C, 30°C, 32°C, 35°C, 40°C, 50°C, and 60°C, respectively, for 30 minutes. The protein (FDC 1 crude extract, 13 mg) was added to reaction mixtures containing 25 mM potassium phosphate pH 6.5, 5 mM DTT, and 1.4 mM cinnamic acid to a volume of 1 mL. After the reaction 1 mL of propanol was added to the reaction mixtures to dissolve styrene. The experimental negative control used the same conditions except the reaction mixture contained no substrate. The optimum temperature for FDC1 activity was about 50°C (Figure 6).
[0330] Unexpectedly, the enzyme showed its maximum activity at a higher temperature as compared with other yeast enzymes. This information is useful for industrial production of styrene because a significant amount of cost associated with fermentation is cooling the fermentation system. Since FDC enzyme is active at higher reaction temperature, temperature range for fermentation can be controlled for increased yield and reduced cost on cooling.
7. Temperature Stability of FDC1
[0331] To study the temperature stability of FDC 1, reactions were carried out at temperatures of 30°C, 50°C, 60°C, and 70°C, respectively, for 30 minutes. After incubation at various temperatures, an aliquot of protein (42 mg) was added to 25 mM potassium phosphate buffer pH 6.5, 5 mM DTT, and 1.4 mM tCA to a final volume of 1 mL. The reaction mixtures were incubated for 8 minutes at 30°C. The styrene was extracted with 250 μΐ butanol and quantified by HPLC using CI 8 column. The experimental negative control used the same conditions except the reaction mixture contained no substrate. The enzyme was stable at 50°C (Figure 7). [0332] The stability of FDC1 crude extract at 50°C was also tested by incubating the crude extract at 50°C for 10, 30, 60, and 120 minutes, respectively. After the protein was incubated for various times, an aliquot (42 mg) was added to 25 mM potassium phosphate buffer pH 6.5, 5 mM DTT, and 1.4 mM tCA to a final volume of 1 mL. The reaction mixtures were then incubated for 8 minutes at 30°C. Styrene was extracted by adding 250 L butanol to the reaction mixtures and quantified by HPLC. The experimental negative control used the same conditions except the reaction mixture contained no substrate. The protein was stable at 50°C for 2hr (Figure 8). This result demonstrates that the protein retained 100% of its activity even after incubation for about 2 hours. This piece of information is useful for industrial production of styrene, due to the thermo stability of FDC1.
8. Effect of Cofactors on FDC1 Activity
[0333] To examine the effect of cofactors on FDC1 enzymatic activity, reactions were carried out with various cofactors. Several cofactors, including thiamin pyrophosphate (TPP), biotin, and pyridoxal phosphate (PLP) were tested for their effects on FDC1 activity. Reaction mixtures containing 25 mM potassium phosphate buffer pH 6.5, 5 mM DTT, 0.5mM tCA, 0.5mM cofactor, and 0.5 mg purified FDC1 to a volume of 1 mL were incubated for 30 minutes at 30°C. Styrene was extracted with 250 L butanol. The experimental control used the same conditions except the reaction mixture contained no cofactors. The experimental negative control used the same conditions except the reaction mixture contained no substrate.
[0334] None of the cofactors increased the activity of FDC1. In fact, they were found to decrease FDC1 activity (Figure 9). This result showed that this enzyme does not require any commonly known cofactors for its activity.
9. Effect of Metal ions on FDC1 Activity
[0335] To study the effect of metal ions on FDC1 activity, reactions were carried out with various metal ions, including ZnS04 , FeCl3, MnCl2, MgS04 , CaCl2, MnS04 , and FeS04. Reaction mixtures of 25 mM potassium phosphate buffer pH 6.5 containing 1.4 mM tCA, 5 mM DTT, 10 mM metal ion, and 0.5 mg purified FDC1 adjusted to a final volume of 1 mL were incubated for 5 minutes at 30°C. After the reaction, 1 mL of propanol was added to the reaction mixtures to solubilize styrene. The experimental control used the same conditions except the reaction mixture contained no metal ions. The experimental negative control used the same conditions except the reaction mixture contained no substrate. Ca2+, Mg2+, Zn2+, and Fe3+ ions increased the activity of FDC1 (Figure 10A).
[0336] The effects of Zn2+ and Fe3+ on FDC1 activity were further investigated using EDTA to cancel out the effects of the metal ions. Reaction mixtures of 25 mM potassium phosphate buffer pH 6.5 containing 1.4 mM tCA, 5 mM DTT, 10 mM metal ion, 10 mM EDTA and 0.5 mg purified FDC1 were adjusted to a final volume of 1 mL, and were incubated for 5 minutes at 30°C. After the reaction, 1 mL of propanol was added to the reaction mixtures to solubilize styrene. The experimental control used the same conditions except the reaction mixture contained no metal ions or EDT A. The experimental negative control used the same conditions except the reaction mixture contained no substrate. The reactions containing the metal ion and EDTA had a similar activity as compared to the control (Figure 1 OB), indicating that Zn2+ and Fe + increased the activity of FDC1. Based on results presented in Figures 10A and 10B, enzyme activity of FDC1 can be increased by metal ions and it was not due to experimental artifact. For industrial production of styrene, this piece of information suggests that including certain metal ions in the culture media can be beneficial to styrene biosynthesis.
10. Substrate Specificity
[0337] To examine whether FDC1 is highly specific for tCA (or whether it shows substrate promiscuity), tCA and its substrate analogues, such as ferulic acid, 2-methylcinnamic acid, 2- hydroxycinnamic acid, 3-hydroxycinnamic acid, 4-hydroxycinnamic acid, 3,4-dimethoxycinnamic acid, and 2,5-dimethoxycinnamic acid were tested. The reaction mixtures of 25 mM potassium phosphate buffer pH 6.5 containing 5 mM DTT, 0.2 mM substrate, and 0.5 mg purified FDC1 were adjusted to a final volume of 1 mL. The reaction mixtures were then incubated for 5 minutes at 30°C. After the reaction 1 mL of propanol was added to the reaction mixtures to solubilize styrene. The experimental negative control used the same conditions except the reaction mixture contained no substrate. The substrates ferulic acid, 2- methylcinnamic acid, and 4-hydroxycinnamic acid showed activities of 57%, 35%, and 68%>, respectively, compared with that of tCA (Figure 11). The enzyme did not show any activity for substrates 2-hydroxycinnamic acid, 3-hydroxycinnamic acid, 3,4-dimethoxycinnamic acid, and 2,5-dimethoxycinnamic acid (Figure 11). The enzyme did not show strict specificity for tCA; instead, it showed moderate activity for ferulic acid, 2-methylcinnamic acid and 4-hydroxycinnamic acid.
[0338] Analyses of the substrate specificity will contribute to elucidate substrate binding site and the mechanism of enzyme activity. In addition, this substrate spectrum suggested that the same fermentation system can be used to produce different industrial monomers. In addition to converting cinnamic acid to styrene, 4-hydroxy, 3-methoxy-styrene (a.k.a. 4-vinylguajacol, 4VG) from ferulic acid; 2-methyl-styrene from 2-methylcinnamic acid; and 4-hydroxystyrene (a.k.a. 4-vinylphenol) from 4- hydroxycinnamic acid can now be produced.
11. Kinetics
[0339] To examine the catalytic efficiency of this protein, kinetic studies of FDC1 was conducted using tCA as the substrate. To measure the steady state kinetic constants of wild type FDC1, enzymatic activities were determined with different concentrations of tCA (100-2000 μΜ). The reactions were performed in a total of 1.0 mL standard reaction mixture with 0.5 mg purified protein, and were allowed to proceed for 5 min at 30°C. The styrene was extracted with 250 L butanol. The experimental negative control used the same conditions except the reaction mixture contained no substrate. Activities were quantified by standard assay method. Duplicate assays were performed and averaged. The max and Km were determined by nonlinear regression analysis of the velocity-concentration data fit to the Michaelis-Menten equation.
[0340] The Km for wild type FDCl was found to be 688 μΜ and the Vmax was 6.17 nmol'mg" '•min 1. The catalytic efficiency of this protein was found to be 8.4 M_1S_1 which is lower compared with that of other natural enzymes. Lower catalytic efficiency of FDCl is the major obstacle for production of styrene. Structure guided protein engineering will be a good approach for molecular evolution of FDCl for increasing activity.
EXAMPLE II. FDCl MUTANTS AND MUTANT LIBRARIES
1. FDCl Structure Models
[0341] There is no tertiary structure of FDCl that can be used for analyses of substrate binding sites. A homologous protein structure (3-Octaprenyl-4-Hydroxybenzoate decarboxylase, PDB code: 2IDB) showed only 20% identity with FDCl . To analyze substrate binding site, a model was built for truncated FDCl by SWISMODEL program and a model for full length FDCl by I-TASSER program. These two models are reliable as they superimpose very well. As the lower catalytic efficiency of FDCl is a bottleneck to produce higher amounts of styrene, we applied a combined method of molecular biology and structural biology for laboratory evolution of a protein based model of full length wild type FDCl (Figure 1A).
[0342] Docking substrate into FDCl. For laboratory evolution of protein, the substrate binding site of FDC 1 was examined. There is no report on the substrate binding site of FDC 1. Docking for tCA with FDCl was performed using the computer program SWISDOCK. After docking, it gave 4 possible binding sites (Figure IB).
[0343] After analysis of the structure, site C is studied for molecular evolution to improve the activity. Without limiting the scope of the subject technology, it is hypothesized that 1173, A174, R175, V188, 1189, K190 (not shown for figure clarification), 1194, E280, M286, F291 and F440 contribute to substrate binding (Figure 1C). Thus, the hydrophobic residues (A174, 1194 and VI 88) create a pocket for binding the phenyl ring of tCA; and positive charged Rl 75 makes hydrogen bonds with the carboxylic group of tCA and negative charged E280.
2. Saturation Mutagenesis of FDCl
[0344] As the conventional mutagenesis did not improve FDCl activity, site directed saturation mutagenesis was applied. Saturation mutagenesis allows the change of one amino acid to 19 other alternative amino acid residues. Saturation mutagenesis was performed at sites 155-156, 159, 162-164, 172-175, 187-196, 226-227, 285-287, 291, 326, 331, 360-361, 395-396, 398, and 440-441 of FDCl by following the QuickChang site-directed mutagenesis strategy (STRATAgene, CA) using NNK degenerate primers (N represents the mixture of A, T, G, C, and K for G/T). The codon NNK has 32-fold degeneracy and encodes all 20 amino acids without rare codons. The QuikChange PCR products were examined by agarose gel electrophoresis and then 15 μΐ of PCR products were digested with 1 μΐ Dpnl (New England Biolabs) at 37 °C for 4 hours to remove the template plasmid. Aliquots of (2 μΐ) digestive products were transformed into BL21-Gold (DE3) competent cells (STRATAgene, CA) and inoculated on Luria-Bertani (LB) agar plates containing kanamycin. The quality of the library by DNA sequencing was confirmed. The library covers 90% of mutagenesis (17 mutants out of 19). To cover 100% (19 out of 19 mutant), we screened 150 mutants for each site.
3. Colorimetric Method for High-Throughput Screening of FDC1 Mutant Library
[0345] Screening a large population of the protein library is the bottleneck for the molecular evolution of the protein. The functional characterization of decarboxylase routinely relies on analytic instruments, like HPLC or LC-MS. Although the HPLC is highly sensitive, it is time consuming, expensive, and generates waste like methanol or acetonitrile and not suitable for high-throughput applications. To overcome these technical barriers, a spectroscopic-based colorimetric assay method was developed, which was essentially based on detecting the enzymatically produced styrene from tCA. Detailed method has been given below.
[0346] The transformants (Example II.2) were inoculated in 96-well plates (NUNC, Roskilde, Denmark) containing 100 μΐ LB broth per well and incubated at 37 °C, overnight. The cultures were then mixed with equal amount of 50% glycerol and stored at -80°C as the master plate. An aliquot of 10 μΐ culture was inoculated in 2 ml deep-well plates (USA scientific) with 1 ml TB broth per well and incubated at 37 °C until the OD600 reached 1.0. The cultures were then induced by 0.2 mM IPTG and incubated at 18 °C for 20 hours with shaking at 250 rpm.
[0347] Cells were harvested and suspended with 0.25 ml PBS buffer at pH 7.0 and the substrate (tCA) was added at a final concentration of 1 g/L. The culture was incubated at 30° C for 4 hours and the volatile product styrene was collected by using 96 well STRATA-X® reversed phase plate containing 10 mg polymer based resin (Phenomenex) on top of the culture plate (Figure 19). To avoid styrene vapor diffusion, 96-square well silicone sealing mats with pre-slit were used between 96-well culture plate and 96 well STRATA-X® reversed phase plate. The product was collected by 200 μΐ propanol for each well and the amount of product was measured by colorimetric method.
[0348] Various chemicals were tested as an indicator for styrene detection, including NBP (4- nitrobenzyl-pyridine) as a preferred indicator. Styrene was mixed with cytochrome P450 BM-3 in 50 mM phosphate buffer pH 8.0. The styrene oxidation reaction was started by adding 0.2 mM NADPH, and the reaction mixture was incubated at room temperature for 5 minutes with shaking. The NBP solution was added to the mixture and incubated at room temperature for further 5 minutes with shaking which gives white precipitate. The tube or plate containing reaction mixtures were heated at 70 °C for 30 minutes and chilled on ice for 5 minutes. After adding Dimethylformamide (150 μΐ) and 25 μΐ of 1M K2CO3, the reaction mixture gave blue color that can be measured spectrophotometrically at 600 nm immediately. The unpaired electron of NBP reacts with the oxirane ring of styrene oxide to yield a blue chromophore a blue color that can be monitored spectrophotometrically at 600 nm.
[0349] The 96-well plate cultures that contain styrene were monitored directly using this high throughput method (Figure 13). This method can detect styrene as low as 1.0 mM. This colorimetric method is very fast, less expensive, reproducible and can measure 1000 colonies in a single day.
[0350] The mutants showing higher activity were confirmed by HPLC using an ACQUITY UPLC BEH CI 8 column (1.7-μιη, 2.1x50-mm, Waters, USA). The samples were resolved in 30% acetonitrile (A) with an increasing concentration gradient of acetonitrile (B) for 1 min, 95%; then to 1 min, 30%; at a flow rate of 1 ml/min. UV absorption was monitored at 254-, 280-, and 310-nm. The changed amino acid residues in the mutants showing the higher activity were confirmed by DNA sequence.
[0351] Several potential single mutants were identified (such as K190E, K190C, K190D, K190V, K190N, K190L, K190H, and R175I) that produced significantly higher amount of styrene, as compared to that of wild type (Figure 14). Mutants K190E, K190C, K190V produced 3 times more, and mutant R175I produced 1.5 times more styrene, as compared to that of wild type. As such, the FDC1 mutants produced a significantly higher amount of styrene than that of wild type. Mutagenesis at different sites showed an additive or synergistic effect on protein evolution of methyltransferase (Bhuiya et al., 2010, Journal of Biological Chemistry, 285: 277-285.). Mutagenesis at K190 and R175 sites of FDC1 may further increase the production of styrene.
[0352] Production of styrene by maintaining the culture in an aerobic environment was conducted. Wild type FDC1 produced half the amount of styrene in an anaerobic condition, as compared to that of an aerobic condition. Using the STRATA-X® column, we can trap the volatile styrene and can maintain aerobic conditions.
EXAMPLE III. FDC1-PAL FUSION PROTEINS
[0353] Expression of FDC-PAL2 fusion protein in E. coli. Artificial channels were included in the styrene production. To construct the fusion protein of PAL2 and FDC using Gateway technology, four primers were designed for PCR amplification. The FDC-5' primer contained CACC sequence and the FDC-3' primer had a 9-amino acid linker, and two restriction sites Ncol and Pstl at the 3'-end. To fuse PAL2 with FDC, the PAL2-5' primer correspondingly had an Ncol site and the PAL2-3' primer with a Pstl site. The FDC gene (~1.5 kb) and the PAL2 gene (2.2 kb) were amplified using the above primers and cloned into the Gateway vector pDEST17. The constructs of the fusion protein of PAL2 and FDC were transformed to BL21(DE3). Fusion proteins of FDC mutant and PAL also can be obtained by the above process. [0354] The use of a linker for fusion proteins was evaluated based on the information from the resveratrol biosynthetic pathway. Previous results showed that fusion protein produced 15 fold more product compared with the expression two individual proteins (Yechun Wang et al (2011), JACS, 133: 20684-20687). The results indicate that linker plays an important role in fusion protein for improvement of biosynthetic pathway. Different length of linker 2, 3, 4, 6, 8, 9, 12 and 15 amino acid lengths were designed using GSG motif and examined the effect of linkers for resveratrol biosynthetic pathway. We found that 9 amino acid linker showed the highest yield, as compared to that of other linker (Figure 15). Based on our findings, we designed 9 amino acid linkers for our PAL-FDC fusion proteins.
[0355] Based on above observation, a computer model was constructed to prove that the artificial linker indeed increased metabolic channeling. The 9 a.a. linker connected two proteins (Figure 16) in such a way that it prevent diffusion of intermediate (in this case cinnamic acid) so that intermediate can be uptake by second enzyme (FDC) efficiently and ultimately increase the final product styrene. The computer model predicted the distance between the two reaction centers (marked by the two arrows) are within 70 angstroms, forming a metabolic channel.
[0356] The expression analysis revealed that about 615 mg/L styrene was found in medium. This fusion protein produced styrene at an amount that was about 6-fold higher than that of FDC expressing in E. coli (95 mg/Lin medium). This amount of styrene was also much higher than that of non- fused proteins of PAL2 and FDC (expressed in yeast, with 69 mg/L styrene detected). These analyses suggest that the fusion protein of PAL2 and FDC can be used to improve the bioproduction of styrene.
EXAMPLE IV. EXPRESSION OF AN ABC-TRANSPORTER IN E. COLI
[0357] Some bacteria have developed multiple mechanisms to adapt unfavorable conditions. For example, Pseudomonas strains have about three strategies for addressing organic solvent toxicity, i.e. modified membrane structure, active efflux pumps, and enzymatic detoxification. Here, a recombinant E. coli host expressing an efflux pump (called solvent-resistant-pump) was produced. The pump, a member of the ABC-transporter family, is composed of three subunits, srpA, periplasmic linker; srpB, inner membrane transporter; and srpC, out-membrane channels. The full-length of the srpABC pump sequence (~ 6 kb) was cloned from the genomic DNA of P. putida. The clone was first inserted to pENTR vector with Zeocin resistance and then to E. coli expression vector pCONA-2-DEST with Kanamycin resistance.
[0358] Finally, the pump was transformed to the E. coli strain BL21 (DE3) containing the fusion of PAL2 and FDC which is resistant to Ampicillin. After the test expression of 5 clones in flasks, one clone was chosen for further experiment with fermenter. Fifty milliliters of overnight culture were inoculated in 1.5 L LB medium containing appropriate antibiotics. When cells reach an OD600 ~ 0.8, 5 mM of IPTG was added to the culture for induction. After 2 hrs the substrate Phe was added to a final concentration 5 g/L of cell culture. The styrene vapor was tracked in a bottle containing 250 ml of butanol. Samples from the overnight fermentation (~ 16 hrs) were taken and analyzed by HPLC in coupling with an Acclaim RSLC CI 8 column and detected at 280 nm. After 16 hrs fermentation, almost no styrene was found in medium, but high concentration of styrene can be found in butanol. Compared to the control that only contains the fusion of PAL2 and FDC, the clone containing triple genes (fusion of PAL2 and FDC plus srpABC) was able to produce nearly four times more product, i.e. 521 mg/L vis. 139 mg/L. (See Figure 17).
EXAMPLE V. BIOSYNTHESIS OF STYRENE.
[0359] Production of styrene from glucose. A vector comprising fusion protein FDC1(K190E)- PAL was constructed, transformed it into phenylalanine producing strains (ATCC 31884 and HG), which was grown in LB containing ampicillin and/or kanamycin. The cultures were grown at 30°C for 12 hours, after that the cultures were harvested and an aliquot of cultures were inoculated in M9 medium. The cultures were grown at 30 °C until the OD6oo reached to 0.8 and then they were induced with 0.2 mM IPTG and continued to culture at 30°C or 37°C for 48hrs. The volatile styrene was collected by
STRATA-X® column. The amount of phenylalanine, tCA, and styrene was measured by HPLC. The ATCC 31884 strains produced 177 mg/L of styrene from glucose at 30°C; no accumulation of tCA or phenylalanine was observed (Figure 18A). The HG strain produced 13.1 mg/L of styrene and 5.0 mg/1 of tCA from glucose at 30 °C, no accumulation of phenylalanine was observed (Figure 18 A).
[0360] Interestingly, the BL-21(DE3) strains produced 182 mg/L of styrene from glucose by co-expression of FDC -PAL fusion protein and phenylalanine producing vector (HGTM) at 37°C. The whole system for production of styrene from glucose is transferable to any host system.
[0361] STRATA-X® column produced 25 fold higher styrene (125 mg/L), as compared with that of in anaerobic condition and in absence of STRATA-X® column (5mg/L), when FDC -PAL produced styrene from phenylalanine. STRATA-X® column maintain aerobic condition, trap volatile styrene and remove styrene that ultimately increase styrene production and remove toxicity effect on culture.
[0362] Production of styrene from trans-cinnamic acid or L-phenylalanine. The nucleotides encoding FDC wild type, FDC mutant (K190E), FDC (WT)-PAL fusion protein, and FDC (K190E)-PAL fusion protein were transformed in E. coli BL-21 (DE3). The cells were grown in LB at 30°C for overnight. The cells were harvested and washed with M9 media. The cells were inoculated to 2.0 ml M9 media with the initial OD600 at 0.3 and were cultured at 30°C until the OD600 of the culture reached to 0.6 to 0.8. The cells were then induced with 0.2 mM IPTG for 8.0 hours at 30°C. The cells transformed with FDC wild type and FDC (K190E) were fed with 0.1% trans-cinnamic acid. The cells transformed with FDC (WT)-PAL and FDC (K190E)-PAL fusion proteins were fed with either 0.1% trans-cinnamic acid or 0.1 %> L-phenylalanine. The fed cells were continuously cultured for 36 hours. STRATA X column was used to collect styrene. The product was eluted from the column by butanol and the amount was quantified by HPLC. [0363] As shown in Figure 18B, the cells transformed with FDC (K190E) mutant produced higher amount of styrene (758 mg/L) than that of FDC wild type (175 mg/ml). In comparison,
FDC(K190E) fused with PAL produced slightly lower amount of styrene from either trans-cinnamic acid or L-phenylalanine than that produced by FDC wild type fused with PAL.
EXAMPLE VI. PURIFICATION OF CINNAMIC ACID DECARBOXYLASE FOR
CRYSTALLIZATION
[0364] An expression vector of FDCl that has 6-His and SUMO at the N-terminal end was constructed. The DNA encoding FDCl were transformed into E. coli (Rosetta 2) competent cells and grown in LB containing ampicillin. The cultures were grown in LB at 37°C for overnight. The cultures were inoculated in terrific-broth media and grown at 37°C until the OD600 reached 0.8 to 1.0 at which point IPTG was added to a final concentration of 0.2 mM, and continued to culture at 16°C for 16 hours. Cells were harvested by centrifugation for 15 minutes at 4°C at 4500 rpm. The cell pellet was suspended in buffer A (50 mM potassium phosphate buffer, 50 mM sodium thiosulfate, 50 mM TCEP-HC1, 500 mM NaCl, 0.5 mM PMSF, 10 mM MgCl2, 10 mM imidazole, 20 mM βΜΕ, and 20% glycerol adjusted to pH 7.5) containing 0.1% triton X-100, and 2 g/mL DNAse, RNAse, and 4 g/mL lysozyme. The cells were disrupted by ultra-sonication 10 times at amplitude 15, process time 5 seconds, 1 second pulse on/off. The supernatant was collected by centrifugation for 15 minutes at 15,000 rpm at 4°C. The supernatant was filtered through a 0.45 μΜ PES filter and applied to a Ni+-agarose affinity column (GE Healthcare). The column was washed with buffer A until non-specific binding proteins eluted from the column. FDCl was eluted with 50 mM potassium phosphate, 50 mM sodium thiosulfate, 50 mM TCEP-HC1, 500 mM sodium chloride, 0.5 mM PMSF, 10 mM magnesium chloride, 250 mM imidazole, 20 mM βΜΕ, and 20% glycerol adjusted to pH 8.0. A total of 45.0 mg protein was found after Ni+-agarose affinity column.
[0365] Hydrolase (0.5 mg) was added to cleave SUMO from the FDCl . Digestion was carried out at 4°C by dialysis of the protein overnight in 1 L of 25 mM potassium phosphate, 50 mM sodium thiosulfate, 500 mM NaCl, 5 mM DTT, and 20% glycerol adjusted to pH 7.5. Dialysis was continued in a fresh liter of dialysis buffer for 2-4 hours the next morning.
[0366] Subtr active Purification. The Ni+-agarose affinity column was washed with water, recharged with nickel sulfate and equilibrated with buffer A before loading the protein in the column. The protein was loaded in column, flow through was collected, and an addition 15 ml of buffer A was loaded which was collected as flow through. After all the FDCl had been collected, the column was washed with elution buffer to remove SUMO and hydrolase from the column. The flow through containing FDCl was combined and the amount of protein was measured. A total 27.5 mg was found after subtractive purification. The protein was dialyzed overnight in 1 L Q-column buffer A (25 mM potassium phosphate, 5 mM DTT, and 25 mm sodium thiosulfate adjusted to pH 7.5). [0367] Anion- exchange chromatography. The anion-exchange Q-column (GE Healthcare) was equilibrated with Q-column buffer A. The protein was loaded into the column, and washed with buffer A until all the non-specific binding proteins were eluted from the column. The protein was eluted with Q- column buffer A containing 1 M sodium chloride. Elution started at 27% Q-column buffer B. The fractions containing FDC1 were used for SDS-PAGE to check the purity of the protein. A total of 5.0 mg protein was found after anion-exchange chromatography. The protein was dialyzed overnight in size exclusion buffer (50 mM sodium phosphate pH 7.5, 150 mM NaCl, 5 mM DTT, and 25 mM sodium thiosulfate).
[0368] Size-exclusion chromatography. The protein was loaded onto the size exclusion column (GE Healthcare) and eluted with 50 mM sodium phosphate pH 7.5, 150 mM sodium chloride, 5 mM DTT, and 25 mM sodium thiosulfate. The fractions (tubes 33 through 38) containing FDC1 were combined and concentrated. A total of 3.5 mg purified protein was found after size-exclusion chromatography. The purity was checked by SDS-PAGE (Figure 20). The protein was more than 98% pure and ready for crystallization.
[0369] Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W. Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. et al., In Current Protocols in Molecular Biology, published by Greene Publishing and Wiley-Interscience, 1987.
[0370] FDC mutants, for example, FDC (K190E) having an amino acid sequence as set forth in SEQ ID NO: 16, can be purified by the same process as FDC1 as described above.
EXAMPLE VII. CRYSTALLIZATION OF CINNAMIC ACID DECARBOXYLASE
[0371] Crystals of mutant cinnamic acid decarboxylase FDC (K190E) in complex with 3- hydroxyl cinnamic acid were grown by the vapor diffusion method. Commercial screening Kit (Hampton research, Qiagen, Emerald Biosystems) was used for screening crystallization conditions. Different volume ratio of protein and reservoir were tested for crystallization. Hanging drops containing a 1 :1, 1 :2, or 2: 1 mixture of protein (5-10 mg/ml) and crystallization buffer (10% (w/v) polyethylene glycol (PEG) 6000, 5% (w/v) 2-methyl-2,4-pantanediol (MPD), 0.1 M HEPES, pH 7.5, and 2 mM DTT) were maintained at 4°C. In particular, well diffracted crystals were grown in 7-11% (w/v) polyethylene glycol (PEG) 6000, 3% (w/v) 2-methyl-2,4-pantanediol (MPD), pH 6.5-7.5, and 2 mM DTT with 0.1% 3- hydroxyl cinnamic acid. Crystal conditions were further improved by adding 0.01M MnC12, 0.5% (w/v) polyvinylpyrrolidone K15, 0.2M NDSB-201 or 2% (w/v) benzamidine hydrochloride as an additive.
[0372] The FDC(K190E) crystals grew in space group C2 with 3 chains per asymmetric unit. Unit cell dimensions for the crystal were a=249.5lA, b=120.67A, c=158.49A, β=94.9°; Diffraction data were collected from single crystals mounted in a cryoloop and flash frozen in a nitrogen stream at 100 K and reduced with the HKL suite. Crystals were diffracted at 2.15 A resolution. Molecular replacement (Phaser) in CCP4i suite was used to solve the structure of FDC(K190E) mutant using 3-octaprenyl-4-hydroxybenzoate decarboxylase (2IDB) structure as a model template. The initial model of the structures was built by manual building using the COOT and the model was refined using Refmac5. A typical electron density with current initial model of FDC is provided in Figure 21 A. An asymmetric unit of the crystal structure containing 3 chains is shown in Figure 21B. As illustrated in Figure 21B, FDC(K190E) molecules A and B form a dimer that is biologically active, and FDC(K190E) molecule C forms another dimer with its partner of another asymmetric unit (not shown).
[0373] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the subject technology and should not be construed to limit the scope of the subject technology. The skilled artisan readily recognizes that many other embodiments are encompassed by the subject technology. All publications, patents, sequences (including sequences that are identified by GenBank accession numbers) cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.
[0374] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the subject technology described herein. Such equivalents are intended to be encompassed by the embodiments.

Claims

WHAT IS CLAIMED IS:
1. A fusion protein comprising: (a) a first domain comprising a phenylalanine ammonia lyase, and (b) a second domain comprising a cinnamic acid decarboxylase.
2. The fusion protein of claim 1, wherein the phenylalanine ammonia lyase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof.
3. The fusion protein of claim 1, wherein the cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, and a functional fragment or variant thereof.
4. The fusion protein of claim 1, wherein the cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase comprising a mutation at an amino acid residue position corresponding to a position selected from the group consisting of: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, and combinations thereof.
5. The fusion protein of claim 4, wherein the mutation is at an amino acid residue position corresponding to a position selected from the group consisting of: 175 and 190 of SEQ ID NO:8, and combinations thereof.
6. The fusion protein of claim 1, further comprising a linker covalently linking the first domain and the second domain.
7. The fusion protein of claim 6, wherein the linker is a peptide linker comprising 2 to 15 amino acids.
8. The fusion protein of claim 7, wherein the linker is selected from the group consisting of: GS, GSG, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44.
9. A nucleic acid encoding the fusion protein of claim 1.
10. A vector comprising the nucleic acid of claim 9.
11. A host cell comprising the vector of claim 10.
12. A method for producing styrene, the method comprising:
(a) contacting a host cell with a fermentable carbon substrate, the host cell comprising a fusion protein of claim 1 ; and
(b) culturing the host cell in a culture medium for a time sufficient to produce styrene.
13. A cinnamic acid decarboxylase comprising a mutation at an amino acid residue position corresponding to a position selected from the group consisting of: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, and combinations thereof.
14. The cinnamic acid decarboxylase of claim 13, wherein the mutation is at an amino acid residue position corresponding to a position selected from the group consisting of: 175, 190, 193 of SEQ ID NO:8 and combinations thereof.
15. The cinnamic acid decarboxylase of claim 13, wherein the cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, and a functional fragment thereof.
16. An isolated nucleic acid encoding the cinnamic acid decarboxylase of claim 13.
17. A vector comprising the isolated nucleic acid of claim 16.
18. A host cell comprising the vector of claim 17.
19. A method for the production of styrene, the method comprising:
(a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) the cinnamic acid decarboxylase of claim 13; and
(b) culturing the host cell in a culture medium for a time sufficient to produce styrene.
20. A host cell comprising: (a) a recombinantly expressed phenylalanine ammonia lyase; (b) a recombinantly expressed cinnamic acid decarboxylase; and (c) a recombinantly expressed membrane- bound transporter.
21. The host cell of claim 20, wherein the cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase comprising a mutation at an amino acid residue position corresponding to a position selected from the group consisting of: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, and combinations thereof.
22. The host cell of claim 20, wherein the cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO:16; SEQ ID NO: 18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, and a functional fragment or variant thereof.
23. The host cell of claim 20, wherein the membrane transporter is a bacterial ABC transporter.
24. A method for the production of styrene, the method comprising:
(a) contacting the host cell of claim 20 with a fermentable carbon substrate; and
(b) culturing the host cell in a culture medium for a time sufficient to produce styrene.
25. A method for screening candidate proteins for mutated cinnamic acid decarboxylase activity, the method comprising:
(a) providing a protein sample comprising a candidate protein, and a substrate selected from the group consisting of phenylalanine, trans-cinnamic acid, tyrosine, coumaric acid, and combinations thereof;
(b) combining the protein sample and the substrate sample to form a mixture, and incubating the mixture under a condition that allows a mutated cinnamic acid decarboxylase to convert the substrate to a product selected from the group consisting of styrene, 4-hydroxystyrene, and combination thereof; and
(c) exposing the mixture to a detection material that comprises a polymeric resin that absorbs the product vapor.
26. The method of claim 25, wherein the detection material further comprises a detectable marker that causes a color change in the presence of the product.
27. The method of claim 26, wherein the detectable marker is 4-nitrobenzyl-pyridine.
28. The method of claim 25, wherein the polymeric resin is a hydrophobic resin.
29. The method of claim 25, wherein the candidate protein comprises a fusion protein comprising a mutated cinnamic acid decarboxylase.
30. A method of crystallizing a cinnamic acid decarboxylase, the method comprising
(a) providing a cinnamic acid decarboxylase solution at a concentration of from about 1 mg/ml to about 50 mg/ml;
(b) mixing the cinnamic acid decarboxylase solution with a reservoir solution at a volume ratio of from about 1 : 10 to about 10:1; and (c) maintaining the mixture of step (b) at a temperature suitable for the formation of the cinnamic acid decarboxylase crystal.
31. The method of claim 30, wherein the concentration of the cinnamic acid decarboxylase in step (a) is from about 5 mg/ml to about 10 mg/ml.
32. The method of claim 30, wherein the volume ratio in step (b) is from about 1 :2 to about 2:1.
33. The method of claim 30, wherein the cinnamic acid decarboxylase is in a complex with a small molecule selected from the group consisting of trans-cinnamic acid, 3-hydroxyl cinnamic acid, ferulic acid, 2-methylcinnamic acid, 4-hydroxy-cinnamic acid, 3, 4-dimethoxy cinnamic acid, 2,5- dimethoxy-cinnamic acid, and combinations thereof.
34. The method of claim 33, wherein the final concentration of the small molecule in the mixture of step (b) is from about 0.01% (w/v) to about 1% (w/v).
35. The method of claim 30, wherein the mixture of step (b) further comprises an additive selected from the group consisting of MnCl2, polyvinylpyrrolidone K15, Non-detergent Sulfobetaine 201, benzamidine hydrochloride, and combinations thereof.
36. The method of claim 30, wherein the temperature of step (c) is from about 4°C to about 12°C.
37. The method of claim 30, wherein the cinnamic acid decarboxylase comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:8.
38. A crystal of cinnamic acid decarboxylase, wherein the cinnamic acid decarboxylase is in a complex with 3-hydroxyl cinnamic acid.
39. The crystal of claim 38, wherein the cinnamic acid decarboxylase comprises an amino acid sequence as set forth in SEQ ID NO: 16.
40. The crystal of claim 39, wherein the crystal has unit cell parameters a=249.5lA, b=120.67A, c=158.49A, and =94.9°.
41. A method for producing styrene, the method comprising:
(a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) a cinnamic acid decarboxylase; and
(b) culturing the host cell in a culture medium for a time sufficient to produce styrene, wherein the vapor of the styrene product is absorbed by an absorbing material.
42. The method of claim 41, wherein the absorbing material is selected from the group consisting of polymeric resin, activated carbon, cellulosic material, and combination thereof.
43. The method of claim 42, wherein the polymeric resin is a hydrophobic resin.
44. The method of claim 42, wherein the polymeric resin is selected from the group consisting of CI 8, C8, phenyl, SDB-L sorbents resins, and combination thereof.
45. A method for simultaneously screening phenylalanine ammonia lyase and cinnamic acid decarboxylase activities, the method comprising:
(a) providing a fusion protein comprising: (i) a first domain comprising a phenylalanine ammonia lyase, and (ii) a second domain comprising a cinnamic acid decarboxylase;
(b) providing a substrate selected from the group consisting of phenylalanine, trans-cinnamic acid, tyrosine, coumaric acid, and combinations thereof;
(c) incubating the fusion protein and the substrate under a condition that allows the fusion protein to convert the substrate to a product selected from the group consisting of styrene, 4-hydroxystyrene, and combination thereof; and
(d) detecting the amount of the remaining substrate, or the amount of the product, or both.
46. The method of claim 45, wherein the detection of styrene comprises exposing the fusion protein and the substrate of step (c) to a polymeric resin that absorbs the product vapor.
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