Classifications

• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • 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
  • C12N9/10—Transferases (2.)
  • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
  • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
  • C12Y205/01054—3-Deoxy-7-phosphoheptulonate synthase (2.5.1.54)
• • 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
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

• the invention relates to a novel DAHP synthase. Further, the invention relates to a method for in vivo screening of engineered enzyme variants.
• Microbes have been extensively engineered to produce value-added compounds from renewable feedstock (Becker et al, 2015; Lee and Kim, 2015; Liao et ak, 2016). For this purpose, the engineering of biosynthetic pathways plays a crucial role.
• Several strategies based on the adjustment of gene expression and enzyme concentrations have been developed (Alper et al., 2005; Blazeck and Alper, 2013; Hwang et al., 2018; Zhou and Zeng, 2015). However, they are not able to overcome some inherent limitations associated with enzymes themselves.
• protein engineering consists of three distinct steps: (i) construction of a gene variant library; (ii) screening of the library and (iii) further characterization of candidate enzyme variants (Boville et al., 2018; Buller et al., 2018) (Fig. lb). It has been widely and successfully used for improving the performance of many enzymes (Chen et al., 201 la; Rees et al., 2017;
• mutant strains harboring enzyme variants obtained from conventional protein engineering approaches may be phenotypically hardly distinguishable, making screening and characterization of desired enzyme variants not only rather challenging and time consuming (Ren et al., 2018), but also make it hard to identify the best performer among apparently improved enzyme variants (Ren et al., 2015).
• the subsequent in vitro and in vivo characterizations of the best performer identified from screening may not be relevant to a real bioproduction process using the host microorganism. For these reasons, it is of great interest to have a method for identifying enzyme variants having improved properties and being suitable for a real bioproduction process.
• CRISPR/Cas9 is a suitable genome editing tool for quickly and effectively integrating gene variants of a target enzyme into the chromosome of a production strain (Guo et al., 2018).
• L-Tryptophan is an essential amino acid that is widely used in medicine, food industry, animal feed industry (Becker et al., 2015), and also as a precursor for other products (Huccetogullari et al., 2019; Noda and Kondo, 2017; Vargas-Tah et al., 2015; Wendisch, 2014). It is therefore of great interest to be able to produce Tryptophan in a simple and cost-efficient manner.
• the invention provides a mutated DAHP synthase (AroG) variant, having the sequence of SEQ ID NO: 1, with the proviso that the amino acids at positions 6 and 7 are not both aspartic acid.
• the DAHP synthase of the invention is not or significantly less feedback-inhibited by phenylalanine than the wildtype enzyme and exhibits a higher specific enzyme activity than that of a reference variant AroG sl80F known from the prior art in the presence of 40 mM Phe.
• engineered enzyme variants means enzymes the amino acid sequences of which have been biotechnologically altered in comparison to the wild type enzyme.
• one or more amino acids at given positions in the wild type enzyme may, for example, purposefully or randomly be replaced by other amino acids.
• mutated enzymes may also be used for such enzyme variants.
• auxotroph for an organic compound relates to the inability of a cell or organism to synthesize a particular organic compound required for its growth.
• recombineering system also“recombination system” relates to the component or the components of a homologous recombination systems, i.e. a system for recombination- mediated genetic engineering, which are necessary for homologous recombination in vivo.
• a recombineering system is the lamda-red (l-red) recombineering system.
• the lamda-red recombineering system consists of three components necessary for homologous recombination of dsDNA, namely the proteins Exo, Beta and Gam.
• Another example for a recombineering system is the Rec E/T system, composed of the proteins Rec E and Rec T.
• expressible in relation to a coding DNA sequence means that the DNA sequence can be transcribed under the control of an inducible or constitutive promoter into an RNA transcript and translated into the corresponding protein product.
• the term is to be understood as meaning the formation of an RNA transcript from the DNA sequence under control of an inducible or constitutive promoter.
• the term“expressible CRISPR guide RNA” relates to a DNA sequence encoding a CRISPR guide RNA, i.e. a DNA sequence the transcription of which leads to a RNA transcript being a CRISPR guide RNA.
• reporter molecule is synthesized in the presence of the organic compound.
• A“reporter molecule” is a molecule that can easily be detected, e.g. visualized.
• a reporter molecule can, for example, be a fluorescent molecule.
• the synthesis of the reporter molecule may directly or indirectly depend on the presence of the organic compound.
• An example of a reporter molecule is green fluorescent protein (GFP) or variants thereof, e.g. eGFP.
• GFP green fluorescent protein
• Cas protein refers to a CRISPR associated protein.
• CRISPR is an abbreviation for the term“Clustered Regularly Interspaced Short Palindromic Repeats” used to denote sections of prokaryotic DNA containing palindromic repeat sequences, interspaced by segments of variable spacer DNA.
• the term encompasses a family of proteins being part of the CRISPR- Cas system, which is divided in two classes (class 1 and 2) and several types (I to VI) and subtypes.
• Class 1 encompasses types I, III, and IV, class 2 types II, V, and VI.
• the Cas types are often characterized by a signature protein.
• Cas9 for example, is a signature protein for Cas type II. See, e.g., Makarova et al. 2015; Koonin et al.
• Cas proteins have nuclease activity and use CRISPR RNAs (crRNAs) to guide a Cas nuclease component to the target nucleic acid molecule to be cleaved.
• crRNAs CRISPR RNAs
• Cas9 protein or intendCas9“ refers to the CRISPR associated protein 9.
• Cas9 is an RNA- guided DNA endonuclease enzyme, e.g. from Streptococcus pyogenes.
• the term sketchCas9“ as used herein also encompasses all Cas9 orthologs and also recombinant, i.e. engineered variants thereof.
• the Cas9 nuclease is active when it forms a complex with a guide RNA, which can be composed of two separate RNA molecules, the tracrRNA and the crRNA, or a single RNA molecule, consisting of tracrRNA and crRNA fused together. Part of the crRNA sequence defines the specificity of the nuclease by complementary base pairing with the target DNA sequence. By specifying the targeting sequence of the crRNA it is possible to direct the
• Cas9-mediated DNA targeting is the presence of a short (e.g. 2-6 base pairs) and conserved protospacer adjacent motif (PAM) in the immediate vicinity of the target site, e.g. immediately downstream of the target site.
• PAM sequence differs for different Cas proteins and can also be adapted by engineering the Cas protein.
• crRNA refers to CRISPR RNA, and refers to an RNA molecule being able to complement with a tracrRNA.
• the crRNA confers target specificity to a Cas protein, e.g. Cas9.
• the RNA:RNA duplex composed of crRNA and tracrRNA, which may be fused together to a single RNA molecule, is also called guide RNA (gRNA) and binds to the Cas protein.
• gRNA guide RNA
• the crRNA is found in the CRISPR repeat/spacer section and consists of the spacer, which is complementary to the target, and the repeat that complements with the tracrRNA.
• tracrRNA refers to trans-activating RNA, a small trans-encoded RNA that is partially complementary to and base pairs with crRNA, thus forming an crRNA:tracrRNA duplex.
• CRISPR guide RNA also“CRISPR gRNA” or“gRNA”, refers to a
• crRNA:tracrRNA duplex crRNA and tracrRNA may or may not be fused to a single RNA molecule.
• the term“CRISPR guide RNA” thus encompasses the term“CRISPR single guide RNA” (sgRNA).
• base-pair or“hybridize” relate to the formation of a duplex by base pairing between complementary nucleic acid molecules, e.g. between two RNA or DNA molecules or an RNA and a DNA molecule.
• genomic DNA is used herein synonymously to chromosomal DNA and does not refer to extrachromosomal DNA like plasmid DNA.
• genomic genetic material i.e. genetic material on the chromosome and not to genetic material on plasmids, for example.
• growing the cell, in the absence of the organic compound, in a growth medium suitable for growing the cell means growing the cell in a solid, liquid or semi-solid medium lacking the organic compound, but containing nutrients supporting growth of the cell in the absence of the organic compound.
• the cell will thus be able to grow on the medium if it is able to synthesize the organic compound and will otherwise not be able to grow.
• the growth medium may be a minimal medium containing the minimum nutrients possible for growth of the cell.
• the term“Determining the growth of the cell” relates to the qualitative and/or quantitative determination of growth parameter, e.g. growth rate, growth efficiency (yield coefficient), or maximum biomass, preferably to the growth rate. Growth efficiency relates to the relation between the amount of carbon source consumed and the biomass produced.
• the term“Determining the synthesis of the reporter molecule” relates to the qualitative and/or quantitative determination of the synthesis of the reporter molecule. This may, for example, include the measurement of the fluorescence of a fluorescent reporter molecule. In particular, the term relates to the determination of the strength of the signal generated by the reporter molecule, e.g. the fluorescence intensity.
• DAHP synthase relates to a phospho-2-dehydro-3-deoxyheptonate aldolase (EC 2.5.1.54; also 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) catalyzing the synthesis of DAHP (3-deoxy-D-arabino-hept-2-ulosonate-7-phosphate) from phosphoenolpyruvate and D-erythrose 4-phosphate.
• the term“AroG” relates to a DAHP synthase isoform from E. coli which is feedback-inhibited by the amino acid phenylalanine (Phe), and may also be used here synonymously for the term“DAHP synthase”.
• aromatic amino acid relates to an amino acid having an aromatic ring, in particular to the amino acids phenylalanine, tryptophan, and tyrosine.
• the mutated DAHP synthase has one of the sequences of SEQ ID NO: 2, 3 or 4.
• AroG D6G D7A (SEQ ID NO: 2)
• AroG D6L D7P (SEQ ID NO: 3)
• AroG D6P D71 (SEQ ID NO: 4)
• the enzymes of the invention are not or significantly less feedback-inhibited by phenylalanine than the wildtype enzyme and exhibits higher specific enzyme activities than that of a reference variant AroG sl80F known from the prior art in the presence of 40 mM Phe.
• the replacement of AroG sl80F with the best-performing newly identified AroG D6G D7A in a Trp-producing strain significantly improved the Trp production by 38.5% (24.03 ⁇ 1.02 g/L at 36h) in a simple fed-batch fermentation.
• the invention further relates to a bacterial cell being genetically engineered to express a DAHP synthase of the invention.
• the bacterial cell is an E. coli cell.
• the invention relates to the use of a bacterial cell of the invention for the production of an aromatic amino acid, in particular tryptophan.
• the bacterial cell which preferably is an E. coli cell, is preferably grown in a suitable medium in a bioreactor.
• the invention further relates to a method for the biosynthesis of an aromatic amino acid, comprising the step of growing a bacterial cell according to the invention in a medium and under conditions suitable for growing the bacterial cell.
• the method of the invention further comprises the step of isolating the aromatic amino acid produced by the bacterial cell from the medium.
• the bacterial cell grown is an E. coli cell.
• the aromatic acid produced is preferably tryptophan.
• the invention also relates to a method for in vivo screening of engineered enzyme variants, comprising
• (v) being genetically engineered to include, at the target site on the chromosome, a DNA sequence comprising the target sequence for the CRISPR guide RNA, a PAM sequence and an expressible target gene,
• a donor DNA comprising a donor gene coding for a variant of the enzyme necessary for the synthesis of the organic compound
• the method of the invention combines CRISPR/Cas-facilitated engineering of target gene(s) directly on the chromosome with growth-coupled and biosensor-guided in vivo screening and characterization of protein variants.
• cell growth e.g. the growth rate
• a biosensor e.g. the signal strength of the reporter molecule
• the DAHP synthetase of the invention described above has been identified.
• a better or best performer among improved enzyme variants can be identified by determining the growth of the cell and the synthesis of the reporter molecule.
• the results as regards cell growth and synthesis of the reporter molecule for different enzyme variants are taken to identify the best performing enzyme variants.
• a first cell expressing a first enzyme variant and growing better e.g.
• the amount of the reporter molecule produced can, for example, be detected by measuring fluorescence in case the reporter molecule is a fluorescent molecule. It is particular preferred to take the growth rate, growth yield or growth efficiency and the production rate, production yield, or yield efficiency of the reporter molecule.
• Growth yield may, for example, be measured by measuring the O ⁇ boo, i.e. the optical density measured in a spectrophotometer at 600 nm, at the end of the logarithmic growth phase.
• the method of the invention makes use of a cell, which is genetically engineered in a specific manner in order to allow the efficient in vivo screening and characterization of enzyme variants.
• the cell is auxotrophic for an organic compound necessary for growth of the cell, e.g. an essential amino acid.
• the cell can be naturally auxotrophic for the organic compound, but it is preferred that the cell is genetically engineered to render it auxotrophic for the organic compound.
• the cell is genetically engineered to include genes for a Cas protein.
• a Cas protein e.g. a Cas9 protein
• the gene encoding the Cas protein is contained in a plasmid, although it is also possible to incorporate the Cas protein in the genome of the cell.
• the Cas protein may be put under the control of a constitutive or an inducible promoter. It is preferred that the Cas protein is under the control of a constitutive promoter.
• the CRISPR guide RNA is preferably introduced on a plasmid separate from the plasmid carrying the Cas protein (see Jiang et al., 2015), i.e. the Cas protein is introduced on a first plasmid and the CRISPR guide RNA on a second plasmid. It is further preferred that the CRISPR guide RNA is introduced into the cell together with the donor DNA.
• the gRNA and the donor DNA may be contained in the same plasmid.
• the gRNA can be under the control of a constitutive or inducible promoter, and is preferably under the control of a constitutive promoter.
• the gRNA is able to base-pair to a target sequence at the target site on the chromosome into which the donor DNA, i.e. the DNA comprising the gene encoding an enzyme variant, shall be introduced, and to guide the Cas protein to the target site.
• the guide RNA can be a duplex of two separate RNA molecules, i.e. crRNA and tracrRNA, it is preferred to use a single RNA molecule composed of the crRNA and the tracrRNA, i.e. a single guide RNA (sgRNA), in the method of the invention.
• the gRNA composed of crRNA and tracrRNA, fused to a single molecule or not, can be a synthetic RNA, which is, for example, shorter than naturally occurring gRNA.
• the synthetic gRNA may be chemically modified, e.g. by replacing nucleotides naturally occurring in gRNA with nucleotides not naturally occurring in gRNA, e.g. in order to stabilize the gRNA (see, for example, WO 2016/100951 A2).
• the cell further includes a target gene at a target site on the chromosome as a placeholder for the enzyme variant DNA to be inserted into the genome.
• the target gene may, for example, be an antibiotic resistance gene.
• the target gene can, for example, be introduced at the site of the gene for the wildtype enzyme, such that the gene for the wildtype enzyme is disrupted by or replaced with the target gene.
• the target site also contains a DNA sequence comprising a target sequence for the CRISPR guide RNA, i.e. a sequence to which the CRISPR guide RNA can hybridize (base-pair), and a PAM sequence in order to allow the Cas protein to cut the target sequence.
• the cell is genetically engineered to synthesize a reporter molecule depending on the presence of a functional gene encoding an enzyme necessary for the synthesis of the organic compound.
• the cell may be engineered to include, preferably genomically, a reporter gene the expression of which depends on the presence of the organic compound.
• a reporter gene is tnaC , encoding the leader sequence of the tnaCAB operon, with eGFP fused upstream of tnaC.
• eGFP will be expressed in the presence of tryptophan.
• the production of eGFP can be determined qualitatively or quantitatively, preferably quantitatively, by measuring fluorescence, preferably fluorescence intensity.
• the production of the reporter molecule preferably correlates, for example linearly, with the production of the organic compound.
• a reporter system suitable for detecting the presence of another organic compound in a cell can readily be designed by the skilled person (Xu et al. 2014, Fang et al. 2016).
• a donor DNA is introduced into the cell by biotechnological means, e.g. via electroporation.
• the donor DNA comprises a donor gene coding for a variant of the enzyme to be inserted into the cell.
• the donor gene is introduced into the cell such that the donor gene can be expressed constitutively or inducibly.
• a recombinant cell is thus generated expressing an enzyme variant necessary for the synthesis of the organic compound.
• the donor DNA preferably is a double-stranded linear or circular DNA (dsDNA).
• the donor DNA is inserted in a plasmid.
• the plasmid also carries the gRNA for guiding the Cas protein to the target site.
• the donor DNA comprising the donor gene can be introduced into the cell, e.g. via electroporation, and the donor gene can be inserted into the genome of the cell at the target site via CRISPR/Cas-facilitated recombination.
• the donor gene may be flanked by“homology arms”, i.e. sequences homologous to the target site on the chromosome for homologous recombination.
• the Cas protein is a class II Cas protein, preferably a Cas9 protein.
• the Cas protein may be a recombinant Cas protein, preferably a recombinant type II Cas protein, e.g. a recombinant Cas9 protein.
• the gene for the Cas protein is preferably contained in a plasmid, and is preferably under the control of a constitutive promoter.
• the cell can further be genetically engineered to also include genes for a recombineering system.
• the genes for the Cas protein and the recombineering system may both be contained in the same or separate plasmids. It is however, also possible that the recombineering system, in particular in case of a lamda-red recombineering system, is inserted in the genome. It is preferred to have the gene encoding the Cas protein and the genes coding for the
• the recombineering system in the same plasmid. It is also possible to incorporate the Cas protein in the genome of the cell.
• the recombineering system is preferably under the control of an inducible promoter, e.g. a temperature-sensitive promoter.
• an inducible promoter e.g. a temperature-sensitive promoter.
• the donor gene is preferably flanked by“homology arms”, i.e.
• biotechnologically introduced into the cell thus comprises a donor gene coding for a variant of the enzyme necessary for the synthesis of the organic compound, and flanking sequences homologous to the target gene on the chromosome.
• the recombineering system if included in the cell used in the method, is a lambda-red (l-red) recombineering system, comprising the components Exo, Beta and Gam.
• the lambda-red recombineering system is used for mediating homologous recombination in order to insert the donor gene into the genome of the cell.
• the l-red recombineering system is preferably incorparted in the genome. It is however, also possible to arrange the recombineering system in a plasmid, e.g. the plasmid carrying the gene for the Cas protein. It is preferred that the l-red recombineering system is expressed under the control of an inducible promoter, e.g. a temperature-sensitive promoter.
• the cell used in the method of the invention is preferably a microbial cell, further preferred a bacterial cell, further preferred an enterobacterial cell, and especially preferred an Escherichia coli cell.
• the method of the invention can be used for the in vivo screening and characterization of a library of enzyme variants.
• a plurality of cells as defined above is
• the cells differing in the enzyme variant they are complemented with are grown in the absence of the organic compound, and the growth of the cells and the synthesis of the reporter molecule is determined in order to identify an enzyme variant with superior properties compared to the wild-type enzyme.
• the best performing enzyme variant(s) can be identified by taking, for example, the growth rate and the signal strength of the reporter molecule, e.g. the fluorescence intensity, as parameters.
• FIG. 1 Simplified schematic representation of an embodiment of the method of the invention.
• A Complementation of a cell being auxotrophic for an organic compound with a donor DNA comprising a functional gene necessary for the synthesis of the organic compound.
• B Complementation of a cell being auxotrophic for an organic compound with a donor DNA comprising a functional gene necessary for the synthesis of the organic compound.
• Complemented cell with donor gene introduced into the genome of the cell via CRISPR/Cas9- assisted recombineering
• CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening and characterization (CGSSC) approach (a, solid line) compared to the conventional screening and characterization approach (b, dotted line) in rational protein engineering.
• CGSSC CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening and characterization
• FIG. 3 Design and implementation of the method of the invention in screening and characterization of feedback-resistant AroG (AroG 0 ”) enzyme variants.
• DAHP synthase AroG, AroF, and AroH
• AAAs aromatic amino acids
• An AAAs-auxotrophic strain strain WS002 was constructed by disrupting the DAHP synthase and used as a platform for screening aroG gene variants, which were individually integrated into the chromosome of E. coli using the
• Figure 4 Comparison of the growth and fluorescence of the two strains WS003 and WS004 generated by introducing aroG WT and aroG sl80F into the chromosome of the strain WS002, respectively, under different growth conditions. Left, complex medium (LB-agar); middle, M9- agar (without any amino acids); right, M9-agar with 25 mM Phe.
• FIG. 8 Map of the plasmid pCm-aroG.
• the donor DNA contained in this plasmid is composed of a part of CmR gene, the whole wildtype aroG gene, and a part of serA gene.
• FIG. 9a Amino acid sequences (in one-letter code) of the DAHP synthase of the invention (Fig. 9a) and preferred embodiments (Fig. 9b-c) thereof.
• X any amino acid, but not both aspartic acid (D).
• Figure 1 shows in a simplified and schematic way an embodiment of the method of the invention.
• a cell preferably a bacterial cell like an E. coli cell, which is auxotrophic for an organic compound due to lack of a functional gene encoding an enzyme necessary for the synthesis of the organic compound, is complemented by
• a donor DNA comprising a donor gene coding for an enzyme necessary for the synthesis of the organic compound (Fig. 1 A).
• the donor gene is introduced into the genome of the cell via CRISPR/Cas-assisted recombineering.
• the cell is, in this embodiment, genetically engineered to include (a) an expressible recombineering system, (b) an expressible gene coding for a Cas protein, here Cas9, (c) an expressible CRISPR guide RNA (single guide RNA, sgRNA) being configured to base-pair to a target sequence (“protospacer”) at a target site on the chromosome of the cell and to guide the Cas protein to the target sequence, and, (d) at the target site on the chromosome, a DNA sequence comprising the target sequence (“protospacer”) for the CRISPR guide RNA, a PAM sequence and an expressible target gene, for example a gene conferring resistance against an antibiotic.
• the cell is further genetically engineered to synthesize a reporter molecule in the presence of the organic compound.
• the donor gene introduced into the cell is flanked by sequences homologous to the target gene on the chromosome.
• recombineering system here a lambda-red recombineering system, and the Cas protein
• recombineering system here a lambda-red recombineering system
• Cas protein the genes encoding the recombineering system and the Cas protein on individual plasmids or in the genome. The presence of the recombineering system is not necessary in the method of the invention.
• the donor gene introduced into the chromosome of the cell is expressed, such that a functional enzyme is produced, which is necessary for synthesizing the organic compound B, for which the cell was auxotrophic, from a precursor A.
• the organic compound is synthesized and a reporter molecule is formed in its presence (Fig. IB). Cell growth and the synthesis of the reporter molecule are determined.
• 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase is a key enzyme for engineering microbes for efficient biosynthesis of aromatic amino acids (AAAs): Trp, phenylalanine (Phe) and tyrosine (Tyr) (Chen and Zeng, 2017; Kim et al., 2015; Wu et al., 2018). It is under strong feedback inhibition by the end products (Ogino et al., 1982; Sprenger, 2006). For instance, in E.
• the strains and plasmids used in this study are listed in Table 1.
• the Trp producing strain S028Z the parent strain of S028 (Chen and Zeng, 2017) in which the temperature-sensitive l- red recombineering system had been removed from the chromosome, was used as starting strain.
• the DAHP synthase activity is offered only by the Phe-resistant AroG sl80F .
• the plasmid pCas (Jiang et al., 2015) was introduced into S028Z, resulting in the strain S028ZC (Table 1).
• trpR-sgRNA sgRNA with an N20 sequence for targeting the trpR locus.
• the encoding gene of aroG WT was isolated from the strain E. coli DY330 (Yu et ah, 2000) with primers aroG-His-Hindlll and Xbal-serA (Table 2). It was then inserted into the vector pET22b(+) at the sites Hindlll and Xbal to generate the plasmid pKT-aroG WT .
• the plasmids pET -aroG sl80F , pET -aroG D6G D7A , pET -aroG D6L D7P , and pET -aroG />6/’ ⁇ />7/ were generated by using mutagenic primers (Table 2) to amplify the whole plasmid pET -aroG WT .
• sgRNA single guide RNA
• a set of primers (Table 2) was used to amplify the pTagAmpR backbone (Fig. 8) through PCR.
• the 20bp spacer sequence specific for the target gene was selected with the aid of the web-based tool Cas-Designer (Bae et al., 2014; Park et al., 2015) and was synthesized within the primers (Table 2, shown in capital letter).
• the PCR products were then transformed into E.coli ToplO competent cells directly to obtain the desired sgRNA plasmids.
• sgRNA plasmids with donor DNAs, two 300- 500 bp homologous arms and the DNA fragment for substitution were separately amplified and then fused together by PCR. After gel-purification, the target PCR products were inserted into the desired sgRNA plasmids.
• the Trp-biosensor was integrated into the chromosome at the locus of the trpR gene, resulting the strain of WS001.
• the plasmid pN20 -trpR was constructed from the plasmid pTagAmpR (Fig.8), which is derived from the plasmid pTargetF (Jiang et al., 2015) with the change of spectinomycin resistance to ampicillin resistance, with the primers pTargR and TrpR-N20 (Table 2) for expressing gRNA targeting the trpR gene.
• the donor DNA fragment Trp-Sensor was amplified from the plasmid pSentrp (Fang et al., 2016) with the primers Tsen-trpR-IF and Tsen-trpR-IR.
• the plasmid pN20 -aroG was constructed at first from the plasmid pTagAmpR plasmid with the primers pTargR and AroG-N20 (Table 2) for expressing gRNA targeting the aroG gene.
• the donor DNA fragment P j23U9-r P sL-Cm R was amplified from the plasmid pJLC with the primers up-aroH-out and Cm-delG-R.
• pCm -aroG In order to construct pCm -aroG (Fig. 8), we constructed the plasmid pN20-CmR at first with the primers pTargR and Cm-N20 (Table 2) in the same way as constructing the plasmid pN20- aroG.
• the DNA fragment V-N20CmR was amplified from the plasmid pN20 -CnP using primers pN20VRCm and pN20VFSerA (Table 2). Meanwhile, the DNA fragments F -CnP, F- aroG , and F -serA were generated from the plasmid pJLC (Table 1), the genome DNA of E.
• the primer pairs were Cm-Cl/Cm-C2 , WaroG-FF/aroG-Fus-R, and serA-Fus-F/serA-Fus-R, respectively. Then four fragments ( ' V-N20CmR , F-Cm R , F-aroG , and F -serA) were fused together to construct the final plasmid pCrs ⁇ -aroG WT by using In-Fusion HD Cloning kits (Clontech® Laboratories, Inc.).
• the plasmids of pCm -aroG sl80F and pCm -aroG fl>r were constructed using a corresponding pair of mutagenic primers (Table 2) to amplify the whole plasmid pCm -aroG WT .
• the plasmid pN202-C ? was firstly constructed as the same as other sgRNA plasmids but with primers pTargR and CmR-N202.
• the donor DNA fragment P j23U9-r P sL-tac- aroG D6G ⁇ D7A was obtained from two round PCR.
• the first round is for adding the tac promoter to the aroG D6G ⁇ DA 7 gene by amplifying the plasmid pET-AroG D6G DA7 with the primers NotF pTac-aroG and aroG-spel.
• the second round is for flanking the upstream homologous arm by using the primers u-rpsLp-tac and aroG-fus-R and using the PCR product from the first round as the template.
• the plasmid pN202-Cm / ⁇ and the fragment P j23ii9-r P sL-tac-aroG D6G D7A were co transformed into the strain WS005 with the plasmid pCas for generating the strain S028GM1.
• the cells were harvested by centrifugation at 4 °C and washed three times with precooled 10% glycerol or distilled water. Competent cells were re-suspended in 400 pL precooled 10% glycerol and divided into 200 pL for each reaction. The corresponding sgRNA plasmid (if required, plus donor dsDNA) eluted in water was mixed with the competent cells for transformation. The electroporation was done in 0.2 cm cuvette at 2.5 kV, and the cells were suspended in 1 ml SOB medium and recovered at 30 °C for 2 hours before plating. Plates were incubated more than 24 hours at 30 °C. Transformants were identified by colony PCR and DNA sequencing.
• AroG variant library p C m -ar ( l>6 '' ⁇ l> 7L (Fig.8, see above).
• the AroG variant library was transferred into the WS002 strain (containing pCas) and the cells were then washed with M9 medium (without any amino acids) three times after 2h incubation at 30 °C in SOB medium. The cells were spread onto M9 agar medium containing 25 mM Phe and 0.1 mM IPTG for screening. After incubation at 30 °C for more than 24 hours,
• FM-II medium II
• mutants containing chromosomal Trp sensor with reporter eGFP protein cultured the LB medium were harvested by centrifugation and individually washed three times with the M9 medium to remove the LB medium. Afterwards, each mutant was inoculated with the same amount of cells into 10 mL fresh M9 medium with 25 mM Phe in 50 mL conical tubes, and after a cultivation of 10 h cells were subjected to fluorescence analysis using flow cytometry.
• each culture was first washed three times using PBS buffer and diluted 100-fold and then eGFP fluorescence was monitored (MFI of > 10,000 events) using a flow cytometer (CytoFLEX, Beckman Coulter) at an excitation wavelength of 488 nm. All data were processed with the Beckman Flow software, and electronic gating was used to separate positive signals from instrument and water sample background. For fluorescent intensities, medium
• MFU fluorescence unit
• the complete reaction mixture contained 10 mM Bis-tris propane (BTP, pH 7.0), 50 mM MnSCE, 600 mM PEP, 500 pM E4P, and 25 pg purified enzyme in a total volume of 0.2 mL in cuvette at 25°C with or without inhibitor.
• the mixture (without PEP and E4P) and the substrates (PEP and E4P) were equilibrated to reaction temperature, separately, and the reaction was started by adding the substrates (PEP and E4P).
• the pre-culture and seed culture were performed under the same conditions as reported previously (Gu et al., 2012). Because the l recombination system integrated in the strain E. coli DY330 can cause temperature-sensitive cell growth at 42 °C, it was removed from each strain derived from DY330 before it was used for
• the Trp biosensor is composed of tnaC , which encodes the leader sequence of the tnaCAB operon (Bischoff et ak, 2014; Gong et ak, 2001); the eGFP protein was fused to the upstream of tnaC (Fang et ak, 2016).
• the strain WS001 (Table 1) was first constructed based on the Trp producer S028kC (Chen and Zeng, 2017) after replacement of the trpR gene with the Trp biosensor using the CRISP/Cas9 system.
• the activity of the wild-type AroG is seriously inhibited by Phe and it could not support the growth of cells. It could be, however, also possible that the recombineering efficiency is too low.
• the recombinants with the aroG WT gene were also grown on LB-agar medium with IPTG. From the complex medium, we obtained many colonies (Fig. 4), which were positively confirmed by colony PCR and designated as WS003.
• the strains WS003 and WS004 were re-checked on M9-agar medium with and without 25 mM Phe (Fig. 4). The strain WS003 was found to grow on the medium without Phe but no growth was observed on the medium with Phe (Fig. 4).
• CGSSC is an approach useful to facilitate engineering of enzymes with desired performance such as higher activity and higher inhibitor tolerance. It is used in the following to obtain AroG variants with further improved tolerance against Phe.
• AroG D6P D71 , AroG D6F D7V , AroG D6V D7C , and AroG D6F D7L with the number of occurring being 7, 6, 4, 1, 1, and 1, respectively (Table 3).
• a Number of variants refers to all the examined 20 candidates; N. D., not detected.
• the average value ⁇ standard deviation is based on three independent experiments.
• the strain S028GM1 produced a significant higher amount of Trp than the reference strain after the lag phase (about 10 h) till the end of the fermentation.
• the strain S028GM1 produced 24.03 ⁇ 1.02 g/L, which is 38.50 % higher than that (17.35 ⁇ 1.16 g/L) of the strain S028 (Fig. 7c).
• the concentrations of glucose were controlled at nearly the same level for both strains during the whole fermentations. It was found that the strain S028GM1 had a slightly faster growth rate (0.211 h 1 ) than the strain S028 (0.184 h 1 , Fig. 7a) at the beginning of the exponential growth phase.
• the enhanced DAHP synthase activity has obviously contributed to the higher growth rate to certain extent.
• a higher biomass formation rate could reasonably result in a higher productivity for the strain S028GM1 (Fig. 7d).
• its higher Trp production can be considered to be mainly due to the enhanced DAHP synthase activity directly, because the specific Trp formation rate of the strain S028GM1 was remarkably higher than that of the strain S028 (Fig. 7e) during the whole fermentation.
• the strain S028GM1 accumulated higher amounts of the intermediates SA and DSA (Fig. 7f) of the chorismate pathway than the reference strain S028.
• Both of the intermediates accumulated by the strain S028GM1 are about 36% higher than those produced by S028 at the end of fermentation. It suggests that more metabolic flux was redirected into the chorismate pathway in the strain S028GM1 than in the reference strain due to difference between the variants AroG D6G D7A and AroG sl80F . These results clearly showed that the variant AroG D6G D7A is more efficient for the chorismate pathway towards bio-production of aromatic amino acids and their derivatives.
• AroG-encoding gene variants as described above, into the chromosome it can also be simultaneously used to optimize the expression level of the engineered enzyme in the strain, i.e. by constructing the corresponding gene using different promoters and/or ribosome binding sites. It is to mention that the high efficiency of CRISPR/Cas9 technology allows multiplex genome editing.
• the method of the invention can thus be used to address a wide range of targets that require a simultaneous modulation of multiple genes, e.g. for the synthesis of metabolites like Trp requiring multiple precursors.
• Cas-OFFinder a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 30, 1473- 1475.
• Directed evolution mimics allosteric activation by stepwise tuning of the conformational ensemble. J. Am. Chem. Soc. 140, 7256-7266.
• tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNAPro. Proc. Natl. Acad. Sci. USA 98, 8997-9001.
• Vargas-Tah A., Martinez, L. M., Hemandez-Chavez, G., Rocha, M., Martinez, A., Bolivar, F., Gosset, G., 2015. Production of cinnamic and p-hydroxy cinnamic acid from sugar mixtures with engineered Escherichia coli. Microbial cell factories. 14, 6.

Landscapes

• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• General Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• Biotechnology (AREA)
• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=70166045

Family Applications (1)

Country Status (3)

Family Cites Families (5)

• 2020
  • 2020-04-09 CN CN202080027938.6A patent/CN113677795B/zh active Active
  • 2020-04-09 EP EP20716828.7A patent/EP3953465A1/en not_active Withdrawn
  • 2020-04-09 WO PCT/EP2020/060260 patent/WO2020208191A1/en unknown

Also Published As

Similar Documents

Legal Events