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  • 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
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
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  • 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
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
  • C12P7/20—Glycerol
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/42—Hydroxy-carboxylic acids

Definitions

• the invention provides a process for making glycerol, 1 ,3-propanediol and/or 3-hydroxypropionic acid from sucrose comprising:
• SEQ ID NO:73 is the nucleotide sequence of plasmid pSYCO103.
• SEQ ID NO:74 is the nucleotide sequence of plasmid pSYCOI 06.
• SEQ ID NO:77 is the nucleotide sequence of plasmid pScrl described in Example 1 herein.
• PCR Polymerase chain reaction
• polypeptide or polypeptide complex having sucrose transporter activity refers to a polypeptide or polypeptide complex that is capable of mediating the transport of sucrose into microbial cells.
• polypeptides having sucrose transporter activity include, but are not limited to, sucrose:H+ symporters.
• polypeptide complexes having sucrose transporter activity include, but are not limited to, ABC-type transporters.
• Sucrose:H+ symporters are encoded by, for example, the cscB gene found in E. coli strains such as EC3132 (Robeis et al., J. Bacteriol. 184:5307-5316, 2002) or ATCC13281 (Olson et al., Appl. Microbiol. Biotechnol.
• An example of an ABC-type transporter with activity towards sucrose is the complex encoded by the genes susT1, susT2 and susX in Streptococcus pneumoniae strain TIGR4 (Iyer and Camilli, Molecular Microbiology 66:1 -13, 2007).
• Polypeptides or polypeptide complexes having sucrose transporter activity may also have activity towards other saccharides.
• An example is the maltose transporter complex of
• Streptococcus mutans encoded by malEFGK (Kilic et al., FEMS Microbiol Lett. 266:218, 2007).
• a polypeptide having sucrose hydrolase activity may also have sucrose phosphate hydrolase activity.
• An example of such a peptide is encoded by scrB in Corynebacterium glutamicum (Engels et al., FEMS Microbiol Lett. 289:80-89, 2008).
• a polypeptide having sucrose hydrolase activity may also have sucrose phosphorylase activity. Typical of such an enzyme is EC 2.4.1 .7. Examples of genes encoding sucrose
• glycol derivative and “glycerol-derived products” are used interchangeably herein and refer to a compound that is synthesized from glycerol or in a pathway that includes glycerol.
• PTS system phosphoenolpyruvate-sugar phosphotransferase system
• PTS phosphoenolpyruvate-dependent sugar uptake system
• phosphoenolpyruvate-protein phosphotransferase and “Ptsl” refer to the phosphotransferase, EC 2.7.3.9, encoded by ptsl in E. coli.
• PtsH, Ptsl, and Crr comprise the PTS system.
• NAD-dependent and NADP-dependent glycerol-3-phosphate dehydrogenases are able to use NAD and NADP interchangeably (for example by the enzyme encoded by gpsA), the terms NAD-dependent and NADP-dependent glycerol-3-phosphate dehydrogenase will be used
• Genes encoding the large or "a" (alpha) subunit of glycerol dehydratase include dhaB1 (coding sequence set forth in SEQ ID NO:9, encoded protein sequence set forth in SEQ ID NO:10), gldA and dhaB; genes encoding the medium or " ⁇ " (beta) subunit include dhaB2 (coding sequence set forth in SEQ ID NO:1 1 , encoded protein sequence set forth in SEQ ID NO:12), gldB, and dhaC; genes encoding the small or " ⁇ " (gamma) subunit include dhaB3 (coding sequence set forth in SEQ ID NO:13, encoded protein sequence set forth in SEQ ID NO:14), gldC, and dhaE. Other genes encoding the large or
• Genes encoding one of the proteins include, for example, orfZ, dhaB4, gdrA, pduG and ddrA.
• Genes encoding the second of the two proteins include, for example, orfX, orf2b, gdrB, pduH and ddrB.
• aldehyde dehydrogenase and "Aid” refer to a
• Aldehyde dehydrogenases may use a redox cofactor such as NAD, NADP, FAD, or PQQ.
• Typical of aldehyde dehydrogenases is EC 1 .2.1 .3 (NAD-dependent); EC 1 .2.1 .4 (NADP-dependent); EC 1 .2.99.3 (PQQ- dependent); or EC 1 .2.99.7 (FAD-dependent).
• An example of an NADP- dependent aldehyde dehydrogenase is AldB (SEQ ID NO:16), encoded by the E. coli gene aldB (coding sequence set forth in SEQ ID NO:15).
• carbon sources comprising fructose and glucose.
• the carbon source may further comprise other monosaccharides; disaccharides, such as sucrose; oligosaccharides; or polysaccharides.
• production microorganism refers to a microorganism, including, but not limited to, those that are recombinant, used to make a specific product such as 1 ,3-propanediol, glycerol, 3- hydroxypropionic acid, polyunsaturated fatty acids, and the like.
• Foreign genes can comprise genes inserted into a non-native organism, genes introduced into a new location within the native host, or chimeric genes.
• non-native polypeptide refers to a polypeptide that is not normally found in the host microorganism.
• target sequences can be identified which are 100% complementary to the probe (homologous probing).
• stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
• Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl).
• sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified.
• default values will mean any set of values or parameters that originally load with the software when first initialized.
• BLASTN method of alignment is an algorithm provided by the National Center for Biotechnology Information (NCBI) to compare nucleotide sequences using default parameters.
• the invention encompasses more than the specific exemplary nucleotide sequences disclosed herein.
• alterations in the gene sequence which reflect the degeneracy of the genetic code are contemplated.
• alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded protein are common. Substitutions are defined for the discussion herein as exchanges within one of the following five groups:
• a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine).
• a codon encoding another less hydrophobic residue such as glycine
• a more hydrophobic residue such as valine, leucine, or isoleucine
• changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid
• one positively charged residue for another such as lysine for arginine
• nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.
• polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)).
• BLAST Basic Local Alignment Search Tool
• Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993) In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
• oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
• nucleotide bases that are capable of Watson-Crick base- pairing when aligned in an anti-parallel orientation.
• adenosine is capable of base-pairing with thymine
• cytosine is capable of base-pairing with guanine. Accordingly, the instant invention may make use of isolated nucleic acid molecules that are complementary to the complete sequences as reported in the
• isolated refers to a polypeptide or nucleotide sequence that is removed from at least one component with which it is naturally associated.
• promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters”.
• 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., the coding sequence is under the transcriptional control of the promoter).
• Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.
• the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA.
• sequences, phage or nucleotide sequences linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing an expression cassette(s) into a cell.
• regenerant refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. "Recombinant” also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation, natural transduction, natural transposition) such as those occurring without deliberate human intervention.
• naturally occurring events e.g., spontaneous mutation, natural transformation, natural transduction, natural transposition
• chimeric construct “construct”, and “recombinant DNA construct”, are used interchangeably herein.
• a recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature.
• a recombinant construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory
• Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.
• expression refers to the production of a functional end-product (e.g., an mRNA or a protein [either precursor or mature]).
• Suitable host bacteria for use in the construction of the recombinant bacteria disclosed herein include, but are not limited to organisms of the genera: Escherichia, Streptococcus, Agrobacterium, Bacillus,
• the host bacterium is selected from the genera: Escherichia, Klebsiella, Citrobacter, and Aerobacter.
• the host bacterium is PTS minus.
• the host bacterium is PTS minus in its native state, or may be rendered PTS minus through inactivation of a PTS gene as described below.
• Sucrose transporter polypeptides or polypeptide complexes are polypeptides or polypeptide complexes that are capable of mediating the transport of sucrose into microbial cells.
• Sucrose transport polypeptides and polypeptide complexes are known, as described above.
• Examples of polypeptides having sucrose transporter activity include, but are not limited to, CscB from E. coli wild-type strain EC3132 (set forth in SEQ ID NO:24), encoded by gene cscB (coding sequence set forth in SEQ ID NO:23); CscB from E.
• polypeptide having fructokinase activity corresponds substantially to the sequence set forth in SEQ ID NO:48
• polypeptide having sucrose hydrolase activity corresponds substantially to the amino acid sequence set forth in SEQ ID NO:58.
• polypeptides may be effected using one of many methods known to one skilled in the art.
• the nucleotide sequences encoding the polypeptides described above may be introduced into the bacterium on at least one multicopy plasmid, or by integrating one or more copies of the coding sequences into the host genome.
• the nucleotide sequences encoding the polypeptides may be introduced into the host bacterium separately (e.g., on separate plasmids) or in any combination (e.g., on a single plasmid, as described in the Examples herein).
• the host bacterium contains a gene encoding one of the polynucleotides, then only the remaining nucleotide sequences need to be introduced into the bacterium. For example, if the host bacterium contains a nucleotide sequence encoding a polypeptide having fructokinase activity, only a nucleotide sequence encoding a polypeptide having sucrose transporter activity and a nucleotide sequence encoding a polypeptide having sucrose hydrolase activity need to be introduced into the bacterium to enable sucrose utilization.
• the introduced coding regions that are either on a plasmid(s) or in the genome may be expressed from at least one highly active promoter.
• Increased production of glycerol may be attained through reducing expression of target endogenous genes. Down-regulation of endogenous genes encoding glycerol kinase and glycerol dehydrogenase activities further enhance glycerol production as described in U.S. Patent No.
• Down-regulation may be accomplished by using any method known in the art, for example, the methods described above for down-regulation of genes of the PTS system.
• Glycerol provides a substrate for microbial production of useful products.
• examples of such products, i.e., glycerol derivatives include, but are not limited to, 3-hydroxypropionic acid, methylglyoxal, 1 ,2-propanediol, and 1 ,3-propanediol.
• 1 ,3-propanediol is a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds.
• 1 ,3-Propanediol can be produced by a single microorganism by bioconversion of a carbon substrate other than glycerol or
• glycerol is produced from the carbon substrate, as described above.
• Glycerol is converted to the intermediate 3- hydroxypropionaldehyde by a dehydratase enzyme, which can be encoded by the host bacterium or can be introduced into the host by recombination.
• the dehydratase can be glycerol dehydratase (E.C.
• Bacteria can be recombinantly engineered to provide more efficient production of glycerol and the glycerol derivative 1 ,3-propanediol.
• U.S. Patent No. 7,005,291 discloses transformed
• U.S. Patent No. 6,013,494 describes a process for the production of 1 ,3-propanediol using a single microorganism comprising exogenous glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate phosphatase, dehydratase, and 1 ,3-propanediol oxidoreductase (e.g., dhaT).
• 6,136,576 discloses a method for the production of 1 ,3- propanediol comprising a recombinant microorganism further comprising a dehydratase and protein X (later identified as being a dehydratase reactivation factor peptide).
• Increased production of 1 ,3-propanediol may be achieved by further modifications to a host bacterium, including down-regulating expression of some target genes and up-regulating, expression of other target genes, as described in U.S. Patent No. 7,371 ,558.
• expression of glucokinase activity may be increased.
• the recombinant bacteria disclosed herein are capable of producing 3-hydroxypropionic acid.
• 3-Hydroxypropionic acid has utility for specialty synthesis and can be converted to
• Mutations can be directed toward a structural gene so as to impair or improve the activity of an enzymatic activity or can be directed toward a regulatory gene, including promoter regions and ribosome binding sites, so as to modulate the expression level of an enzymatic activity.
• genes involved in an enzyme pathway may be up- regulated to increase the activity of their encoded function(s). For example, additional copies of selected genes may be introduced into the host cell on multicopy plasmids such as pBR322. Such genes may also be integrated into the chromosome with appropriate regulatory
• target genes may be modified so as to be under the control of non- native promoters or altered native promoters. Endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution.
• down-regulation of gene expression may be used to either prevent expression of the protein of interest or result in the expression of a protein that is non-functional. This may be accomplished for example, by 1 ) deleting coding regions and/or regulatory (promoter) regions, 2) inserting exogenous nucleic acid sequences into coding regions and/regulatory (promoter) regions, and 3) altering coding regions and/or regulatory (promoter) regions (for example, by making DNA base pair changes).
• Specific disruptions may be obtained by random mutation followed by screening or selection, or, in cases where the gene sequences in known, specific disruptions may be obtained by direct intervention using molecular biology methods know to those skilled in the art.
• a particularly useful method is the deletion of significant amounts of coding regions and/or regulatory (promoter) regions.
• bacterium a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast or a plant. Protocols for obtaining and using such vectors are known to those skilled in the art (Sambrook et al., supra).
• Initiation control regions, or promoters, which are useful to drive expression of coding regions for the instant invention in the desired host bacterium are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving expression is suitable for use herein. For example, any of the promoters listed above may be used.
• Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.
• PSYCO109, and pSYCO400/AGRO are set forth in SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76, respectively.
• the differences between the vectors are illustrated in the chart below [the prefix "p-" indicates a promoter; the open reading frames contained within each "( )" represent the composition of an operon]: pSYCO101 (SEQ ID NO:72):
• suitable expression cassettes are constructed, they are used to transform appropriate host bacteria. Introduction of the cassette containing the coding regions into the host bacterium may be
• E. coli for example, cannot fabricate the corrin ring structure, but is able to catalyze the conversion of cobinamide to corrinoid and can introduce the
• Vitamin B12 may be added continuously to E. coli fermentations at a constant rate or staged as to coincide with the generation of cell mass, or may be added in single or multiple bolus additions.
• vitamin B12 is added to the transformed E. coli described herein, it is contemplated that other bacteria, capable of de novo vitamin B12 biosynthesis will also be suitable production cells and the addition of vitamin B12 to these bacteria will be unnecessary.
• bacterial cells are grown at 25 to 40 °C in an appropriate medium containing sucrose.
• suitable growth media for use herein are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth.
• LB Luria Bertani
• SD Sabouraud Dextrose
• YM Yeast medium
• Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular bacterium will be known by someone skilled in the art of microbiology or fermentation science.
• agents known to modulate catabolite repression directly or indirectly e.g., cyclic adenosine 2':3'-monophosphate, may also be incorporated into the reaction media.
• Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is typical as the initial condition.
• Fed-Batch fermentation processes are also suitable for use herein 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 media. Measurement of the actual substrate concentration in
• 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 log phase growth.
• Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration.
• 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.
• a number of factors affecting growth can be altered continuously while the cell concentration, measured by the turbidity of the medium, is kept constant.
• Continuous systems strive to maintain steady state growth conditions, and thus the cell loss due to medium being drawn off must be balanced against the cell growth rate in the fermentation.
• a process for making glycerol, 1 ,3-propanediol, and/or 3-hydroxypropionic acid from sucrose comprises the steps of culturing a recombinant bacterium, as described above, in the presence of sucrose, and optionally recovering the glycerol, 1 ,3-propanediol, and/or 3-hydroxypropionic acid produced.
• the product may be recovered using methods known in the art. For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the product may be isolated from the fermentation medium, which has been treated to remove solids as described above, using methods such as distillation, liquid-liquid
• the present invention is further defined in the following Examples.
• Genomic DNA was isolated from E. coli strain ATCC13281 and digested with EcoRI and BamHI. Fragments approximately 4 kbp in length were isolated by Tris-Borate-EDTA agarose gel electrophoresis and Iigated with plasmid vector pLitmus28 (New England Biolabs, Beverly, MA) that had also been digested with EcoRI and BamHI. The resulting plasmids were used to transform E. coli strain DH5alpha (Invitrogen, Carlsbad, CA), and transformants containing the genes required for sucrose utilization were identified by growth on MacConkey sucrose agar (MacConkey agar base from Difco, Sparks, MD) containing 100 g/mL ampicillin.
• MacConkey sucrose agar MacConkey agar base from Difco, Sparks, MD
• Plasmid DNA was isolated from a colony that had acquired the ability to metabolize sucrose, and the plasmid (designated pScrl ; set forth in SEQ ID NO:77) was sequenced to identify the region of DNA necessary for sucrose utilization.
• the insert was 4140 bp in length and contained putatitve open reading frames homologous to the known E. coli sucrose utilization genes cscB, cscK, and cscA (Robeis et al., J. Bacteriol.
• the esc operon was subsequently moved to plasmid pBHR1
• TT aldA described in U.S. Patent No. 7,371 ,558 (Example 17). Briefly, an aldB deletion was made by first replacing 1 .5 kbp of the coding region of aldB in E. coli strain MG1655 (available from The American Type Culture Collection as ATCC No: 700926) with the FRT-CmR-FRT cassette of the pKD3 plasmid (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA
• a replacement cassette was amplified with the primer pair SEQ ID NO:80 and SEQ ID NO:81 using pKD3 as the template.
• the primer SEQ ID NO:80 contains 80 bp of homology to the 5'- end of aldB and 20 bp of homology to pKD3.
• Primer SEQ ID NO:81 contains 80 bp of homology to the 3' end of aldB and 20 bp homology to pKD3.
• the PCR products were gel-purified and electroporated into MG1655/pKD46 competent cells (U.S. Patent No. 7,371 ,558).
• Recombinant strains were selected on LB plates with 12.5 mg/L of chloroamphenicol.
• the deletion of the aldB gene was confirmed by PCR, using the primer pair SEQ ID NO:82 and SEQ ID NO:83.
• the wild-type strain gave a 1 .5 kbp PCR product while the recombinant strain gave a characteristic 1 .1 kbp PCR product.
• a P1 lysate was prepared and used to move the mutation to the TT aldA strain to form the TT aldAAaldB::Cm strain.
• a chloramphenicol-resistant clone was checked by genomic PCR with the primer pair SEQ ID NO:82 and SEQ ID NO:83 to ensure that the mutation was present.
• the chloramphenicol resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) to create strain TTab.
• Strain TTab was then transformed with pSYCO109 (set forth in SEQ ID NO:75), described in U.S. Patent No. 7,371 ,558, to generate strain TTab pSYCOI 09.
• Plasmid pSYCO109 contains genes encoding a glycerol production pathway (DAR1 and GPP2) and genes encoding a glycerol dehydratase and associated reactivating factor (dhaB123, dhaX, orfX, orfY).
• Strain TTab/pSYCO109 was transformed with each of the two esc operon overexpression plasmids pBHRcscBKA and pBHRcscBKAmutB, described in Example 1 .
• Transformants were selected by growth on LB agar containing 50 pg/nriL of spectinomycin and 50 pg/nriL of kanamycin. Individual colonies were picked and grown overnight at 34 °C with shaking (250 rpm) in LB broth with the same antibiotics.
• TTab/pSYCO109 was grown under identical conditions with the exception of the kanamycin.
• TM3 is a minimal medium containing 13.6 g/L KH 2 PO 4 , 2.04 g/L citric acid dihydrate, 2 g/L magnesium sulfate heptahydrate, 0.33 g/L ferric ammonium citrate, 0.5 g/L yeast extract, 3 g/L ammonium sulfate, 0.2 g/L CaCI 2 -2H 2 O, 0.03 g MnSO 4 H 2 O, 0.01 g/L NaCI, 1 mg/L FeSO 4 -7H 2 O, 1 mg/L, CoCI 2 -6H 2 O, 1 mg/L ZnSO 4 -7H 2 O, 0.1 mg/L CuSO 4 -5H 2 O, 0.1 mg/L H 3 BO 4 , 0.1 mg/L NaMoO 4 -2H 2 O and sufficient NH 4 OH to provide a final

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