AU2007249075B2 - Bioconversion of fermentable carbon to 1,3-propanediol in a single micro-organism using dehydratases - Google Patents

Description

EDITORIAL NOTE 2007249075 Pages 40 to 59 are Gene Sequence Page 60 is part of the Description AUSTRALIA Patents Act 1990 ORIGINAL COMPLETE SPECIFICATION INVENTION TITLE: BIOCONVERSION OF FERMENTABLE CARBON TO 1,3-PROPANEDIOL IN A SINGLE MICROORGANISM USING DEHYDRATASES The following statement is a full description of this invention, including the best method of performing it known to us:- BIOCONVERSION OF FERMENTABLE CARBON TO 1,3-PROPANEDIOL IN A SINGLE MICROORGANISM USING DEHYDRATASES FELD OF INENT= O 5 This invention comprises a process for the bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism. BACKGROUND 1,3-Propanediol is a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds. 10 A variety of chemical routes to 1,3-propanediol are known. For example ethylene oxide may be converted to 1,3-propanediol over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid, by the catalytic solution phase hydration of acrolein followed by reduction, or from hydrocarbons such as glycerol, reacted in the presence of carbon monoxide and 15 hydrogen over catalysts having atoms from group VIII of the periodic table. Although it Is possible to generate 1,3-propanediol by these methods, they are expensive and generate waste streams containing environmental pollutants. It has been known for over a century that 1,3-propanediol can be produced from the fermentation of glycerol. Bacterial strains able to produce 1,3-propane 20 diol have been found, for example, in the groups Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. In each case studied, glycerol is converted to 1,3-propanediol in a two step, enzyme catalyzed reaction sequence. In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HP) and water, Equation 1. In the 25 second step, 3-HP is reduced to 1,3-propanediol by a NAD+-linked oxidoreductase, Equation 2. The 1,3-propanediol is not metabolized further and, as a result, Glycerol - 3-HP + H 2 0 (Equation 1) 30 3-HP + NADH + H+ - 1,3-Propanediol + NAD+ (Equation 2) accumulates in high concentration in the media. The overall reaction consumes a reducing equivalent in the form of a cofactor, reduced p-nicotinamnide adenine dinucleotide (NADH), which is oxidized to nicotinamide adenine dinucleotide 35 (NAD+). The production of 1,3-propanediol from glycerol is generally performed under anaerobic conditions using glycerol as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors. Under these conditions, in e.g., strains of Cirobacter, Clostridlum, and Klebsiella, a parallel

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pathway for glycerol operates which first involves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD+- (or NADP-) linked glycerol dehydrogenase, Equation 3. The DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase (Equation 4), 5 Glycerol +NAD+ DRA + NADH + H+ (Equation 3) DHA + ATP -+ DHAP + ADP (Equation 4) becomes available for biosynthesis and for supporting ATP generation via e.g., 10 glycolysis. In contrast to the 1,3-propanediol pathway, this pathway may provide carbon and energy to the cell and produces rather than consumes NADH. In Klebsiella pneumoniae and Citrobacrerfreundii, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone 15 kinase (dhaK) are encompassed by the dha regulon. The dha regulons from Citrobacter and Klebsiella have been expressed in Escherichia coliand have been shown to convert glycerol to 1,3-propanediol. Biological processes for-the preparation of glycerol are known. The overwhelming majority of glycerol producers are yeasts but some bacteria, other 20 fungi and algae are also known. Both bacteria and yeasts produce glycerol by converting glucose or other carbohydrates through the fructose-1,6-bisphosphate pathway in glycolysis or the Embden Meyerhof Parnas pathway, whereas, certain algac convert dissolved carbon dioxide or bicarbonate in the chloroplasts Into the 3-carbon intermediates of the Calvin cycle. In a series of steps, the 3-carbon 25 intermediate, phosphoglyceric acid, is converted to glyceraldehyde 3-phosphate which can be readily interconverted to its keto isomer dihydroxyacetone phosphate and ultimately to glycerol. Although biological methods of both glycerol and 1,3-propanediol production are known, it has never been demonstrated that the entire process can be accomplished by a single organism. 30 Neither the chemical nor biological methods described above for the production of 1,3-propanediol is well suited for industrial scale production since the chemical processes are energy intensive and the biological processes require the expensive starting material, glycerol. A method requiring low energy input and an inexpensive starting material is needed. A more desirable process would 35 incorporate a microorganism that would have the ability to convert basic carbon sources such as carbohydrates or sugars to the desired 1,3-propanediol end product. Although a single organism conversion of feunentable carbon source other than glycerol or dihydroxyacetone to 1,3-propanediol would be desirable, it has 2 been documented that there are significant difficulties to overcome in such an endeavor. For example, Gottschalk et al. (EP 373 230) teach that the growthxif most strains useful for the production of 1,3-propanedio, including Citrobacter freundii, Clostridium autobutylicum, Clostridium butylicum, and Klebsiella 5 pneumoniae, is disturbed by the presence of a hydrogen donor such as fructose or glucose. Strains of Lactobacillus breWs and Lactobacillus buchner, which produce 1,3-propanediol in co-fermentations of glycerol and fructose or glucose, do not grow when glycerol is provided as the sole carbon source, and, although it has been shown that resting cells can metabolize glucose or fructose, they do not 10 produce 1,3-propanediol. (Veiga DA Cunha et al., J. Bacteriol. 174, 1013 (1992)). Similarly, it has been shown that a strain of Ilyobacter polytropus, which produces 1,3-propanediol when glycerol and acetate are provided, will not produce 1,3-propanediol from carbon substrates other than glycerol, including fructose and glucose. (Steib ct al., Arch. Microbiol. 140, 139 (1984)). Finally 15 Tong et al. (AppL. Biochem. Biotech. 34, 149 (1992)) has taught that recombinant Escherichia coli transformed with the dha regulon encoding glycerol dehydratase does not produce 1,3-propanediol from either glucose or xylose in the absence of exogenous glycerol. Attempts to improve the yield of 1,3-propanediol from glycerol have been 20 reported where co-substrates capable of providing reducing equivalents, typically fermentable sugars, are included in the process. Improvements in yield have been claimed for resting cells of Citrobacterfreundii and Kiebsiela pneumoniae DSM 4270 cofermenting glycerol and glucose (Gottschalk et al., supra.; and Tran-Dinh et al., DE 3734 764); but not for growing cells of Klebsiella pneumoniae 25 ATCC 25955 cofermenting glycerol and glucose, which produced no 1,3-propanediol (I-T. Tong, Ph.D. Thesis, University of Wisconsin-Madison (1992)). Increased yields have been reported for the cofermentation of glycerol and glucose or fructose by a recombinant Escherichia coli; however, no 1,3-propanediol is produced in the absence of glycerol (Tong et al, supra.). In 30 these systems, single organisms use the carbohydrate as a source of generating NADH while providing energy and carbon for cell maintenance or growth. These disclosures suggest that sugars do not enter the carbon stream that produces 1,3-propanediol. In no case is 1,3-propanediol produced in the absence of an exogenous source of glycerol. Thus the weight of literature clearly suggests that 35 the production of 1,3-propanediol from a carbohydrate source by a single organism is not possible. The problem to be solved by the present invention is the biological production of 1,3-propanediol by a single organism from an inexpensive carbon 3 substrate such as glucose or other sugars. The biological production of 1,3-propanediol requires glycerol as a substrate for a two step sequential reaction in which a dehydratase enzyme (typically a coenzyme B1 2 -dependent dehydratase) converts glycerol to an intermediate, 3-hydroxypropionaldehyde, which is then 5 reduced to 1,3-propanediol by a NADH- (or NADPH) dependent oxidoreductase. The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process which utilizes this reaction sequence for the production of 1,3-propanediol. Furthermore, in order to make the process economically viable, a less expensive feedstock than glycerol or dihydroxyacetone is 10 needed. Glucose and other carbohydrates are suitable substrates, but, as discussed above, are known to interfere with 1,3-propanediol production. As a result no single organism has been shown to convert glucose to 1,3-propanediol. Applicants have solved the stated problem and the present invention provides for bioconverting a fermentable carbon source directly to 1,3-propanediol using a 15 single organism. Glucose is used as a model substrate and the bioconversion is applicable to any existing microorganism. Microorganisms harboring the gene for a dehydratase are able to convert glucose and other sugars through the glycerol degradation pathway to 1,3-propanediol with good yields and selectivities. Furthermore, the present invention may be generally applied to include any carbon 20 substrate that is readily converted to 1) glycerol, 2) dihydroxyacetone, or 3) C 3 compounds at the oxidation state of glycerol (e.g., glycerol 3-phosphate) or 4) C 3 compounds at the oxidation state of dihydroxyacetone (e.g., dihydroxyacetone phosphate or glyceraldehyde 3-phosphate). SUMMARY OF THE INVENTION 25 The present invention is a divisional patent application of Australian Patent Application No. 2003266472, the specification of which is herein incorporated by reference (referred to as the "parent specification"). The parent application is a divisional application of 71565/00 which is itself a divisional application of 56789/96. 30 The present invention provides a recombinant microorganism comprising a host cell selected from the group consisting of yeast and filamentous fungi and expressing a diol dehydratase or a glycerol dehydratase. 12/12/07,at13772.div spec pgs, 4 4 The present invention provides a bioconversion process to produce 1,3-propanediol comprising contacting, under suitable conditions, glycerol or dihydroxyacetone with a single recombinant microorganism being a yeast or a filamentous fungus expressing an exogenous diol dehydratase or a glycerol 5 dehydratase enzyme from Klebsiella or Citrobacter. Preferably the yeast or filamentous fungi is selected from the group consisting of members of the genera Aspergillus, Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansemula, Debaryomyces, Mucor and Torulopsis. The present invention further comprises the product of the above process. 10 The present invention further comprises a cosmid comprising a DNA fragment of about 35 kb isolated from Klebsiella pneumoniae wherein said fragment encodes an active glycerol dehydratase enzyme having the restriction digest in Figure 1, columns I and 2. The present invention further comprises a transformed microorganism 15 comprising a host microorganism and the above cosmid. The present invention also comprises a transformed microorganism comprising a host microorganism and a first DNA fragment isolated from Klebsiella pneumoniae, the first DNA fragment encoding an active glycerol dehydratase enzyme having the restriction enzyme digest in Figure 1, columns I and 2, and at 20 least one second DNA fragment isolated from Kebsiella pneumoniae, the second DNA fragment encoding an active functional protein other than a glycerol dehydratsae enzyme. Recombinant microorganisms embodying the invention are set forth in the BRIEF DESCRIPTION OF THE BIOLOGICAL DEPOSITS. 25 BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows restriction digests (EcoR 1, BamH 1, EcoR V and Notl) of cosmids pKP1, pKP2 and pKP4 labeled as columns 1, 2 and 4 respectively, and separation on a 0.8% agarose gel electrophoresis. Molecular size markers were 13/12/07,at13772.div spec pgs,5 5 loaded on the lanes in the end. Columns labeled as numbers I .aM 2 reprCsAZ cosids containing a glycerol dehydratase enzyme. Figure 2 shows a partial physical map of pKP1 and the position of the genes based on DNA sequence. The genes were identified based on comparison 5 of deduced open reading franies with the G3enbank data base using the Tfasta program provided by a sequence analysis software of the University of Wisconsin [Genetics Computer Group, Verison 7. April, 1991,575 Science Drive, Madison, WI 537111. BRIEF DESCRP110N F BIIOL 10 DEPOSITS AND SEOUYENCE LITING The transformed E. coli DHSa containing cosrnid pKP1 containing a portion of the Klebsiella genone encoding the glycerol dehydratase enzyme was deposited on 18 April 1995 with the A1CC under the tem of the Budapest Treaty and was designated ATCC 69789. The transformed E. coilDH5g 15 containing cosmid pKP4 containing a portion of the Klebsiella genome encoding a diol dehydratase enzyme was deposited on 18 April- 1995 with the ATCC under the terms of the Budapest Treaty and was designated ATCC 69790. The Pseudomonas aeruginosa strain PAO 2845.pD'9, transformed with a plasmid containing the dhaB operon was deposited on 11 April 1996 with the ATCC 20 under the terms of the Budapest Treaty and was designated ATCC 55760. The Pichia pastors strain MSP42.81, transformed with non-replicativeplasmids containing expression cassettes for the dhaB1, dhaB2, dhaB3 and dhaT genes, was deposited on 11 April 1996 with the ATCC under the terms of the Budapest Treaty and was designated ATCC 74363. The Saccharomyces cerevistae, strain 25 pMCK1/10/17(HM)#A, transformed with a plasmid containing the dhaB1, dhaB2, dhaB3, and dhaT operon, was deposited before the filing of the instant international application, on May 9,1996, with the ATCC under the terms of the Budapest Treaty and was designated ATCC 74370. The Streptomyces lividans strain S./14.2, transformed with a plasnid containing the dhaB1, dhaB2, dhaB3, 30 and dhaT operon, was deposited before the filing of the instant international application, on May 9, 1996, with the ATCC under the tens of the Budapest Treaty and was designated ATCC 98052. The Bacillus lichenzformks strain BG188/pM26 (Clone #8), transformed with a plasnid containing the dhaB 1, dhaB2 and dhaB3 operon. was deposited before the filing of the instant 35 international application, on May 9, 1996, with the ATCC under the terms of the Budapest Treaty and was designated ATCC 98051. The Bacillus subtilis strain BG2864/pM27 (Clone #1), transformed with a plasmid containing the dhaBI, dhaB2, dhaB3 and dhaT operon. was deposited before the filing of the instant 6 international application, on May 9, 1996, with the ATCC under the terms of the Budapest Treaty and was designated ATCC 980505. The Aspergillus niger strain TGR40-13, transformed with a plasmid containing the dhaB1, dhaB2, dhaB3 and dhaT operon, was deposited before the filing of the instant international application, 5 on May 9, 1996, with the ATCC under the terms of the Budapest Treaty and was designated ATCC 74369. "ATCC" refers to the American Type Culture Collection international depository located at 12301 Parklawn Drive, Rockville, MD 20852, U.S.A. The designations refer to the accession number of the deposited material. Applicants have provided forty-six sequences in conformity with "Rules for 10 the Standard Representation of Nucleotide and Amino Acid Sequences in Patent Applications" (Annexes I and II to the Decision of the President of the EPO, published in Supplement No. 2 to OJ EPO, 12/1992) and with 37 C.F.R. 1.821-1.825 and Appendices A and B ("Requirements for Application Disclosures Containing Nucleotides and/or Amino Acid Sequences"). 15 DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for a biological production of 1,3 propanediol from a fermentable carbon source in a single organism. The method incorporates a microorganism containing a dehydratase enzyme which is contacted 20 with a carbon substrate and 1,3-propanediol is isolated from the growth media. The single organism may be a wild type organism or may be a genetically altered organism harbouring a gene encoding a dehydratase enzyme. The present method provides a rapid, inexpensive and environmentally responsible source of 1,3-propanediol monomer useful in the production of 25 polyesters and other polymers. As used herein the following terms have the same meaning as in the parent specification: "nucleic acid", "essentially similar", "gene", "genetically altered or genetically altered microorganism", "construct", "transformation", "transfection", "transformant", "genetically altered", "expression", "plasmid", "vector", "cosmid", 30 "dehydratase enzyme" and "carbon substrate". 12/12/07,at13772.div spcc pgs,7 7 For the purposes of the present invention the dehydratase enzymes include a glycerol dehydratase and a diol dehydratase having preferred substrates of glycerol and 1,2-propanediol, respectively. Construction of Recombinant Organisms: 5 Recombinant organisms were constructed as described in the parent specification. Cosmid vectors and cosmid transformation methods were used within the context of the present invention to clone large segments of genomic DNA from bacterial genera known to possess genes capable of processing glycerol to 1,3 10 propanediol. Specifically, genomic DNA from K. pneumoniae was isolated by methods well known in the art and digested with the restriction enzyme Sau3A for insertion into a cosmid vector Supercos 1 Tm and packaged using GigapackII packaging extracts. Following construction of the vector E coli XLI-Blue MR cells were transformed with the cosmid DNA. Transformants were screened for the 15 ability to convert glycerol to 1,3-propanediol by growing the cells in the presence of glycerol and analyzing the media for 1,3-propanediol formation. Two of the 1,3-propanediol positive transformants were analyzed and the cosmids were named pKPI and pKP2. DNA sequencing revealed extensive homology to the glycerol dehydratase gene from C. freundii, demonstrating that 20 these transformants contained DNA encoding the glycerol dehydratase gene. Other 1,3-propanediol positive transformants were analyzed and the cosmids were named pKP4 and pKP5. DNA sequencing revealed that these cosmids carried DNA encoding a diol dehydratase gene. Although the instant invention utilizes the isolated genes from within a 25 Klebsiella cosmid, alternate sources of dehydratase genes include, but are not limited to, Citrobacter, Clostridia, and Salmonella. Other genes that will positively affect the production of 1,3-propanediol may be expressed in suitable hosts. For example, it may be highly desirable to over express certain enzymes in the glycerol degradation pathway and/or other pathways 30 at levels far higher than currently found in wild type oats. This may be accomplished by the selective cloning of the genes encoding those enzymes into multicopy plasmids or placing those genes under a strong inducible or constitutive promoter. 13/12/07,at13772.div spec pgs,8 8 Methods for over-expressing desired proteins are common and well known in the art of molecular biology and examples may be found in Sambrook, supra. Furthermore, specific deletion of certain genes by methods known to those skilled in the art will positively affect the production of 1,3-propanediol. Examples of such methods can 5 be found in Methods in Enzymology, Volume 217, R. Wu editor, Academic Press: San Diego (1993). Mutants: In addition to the cells exemplified, it is contemplated that the present method will be able to make use of cells having single or multiple mutations specifically 10 designed to enhance the production of 1,3-propanediol as described in the parent specification. Mutations and transformations in the 1,3-propanediol production pathway: Representative enzyme pathway. The production of 1,3-propanediol from glucose can be accomplished by the following series of steps. This series is 15 representative of a number of pathways known to those skilled in the art. Glucose is converted in a series of steps by enzymes of the glycolytic pathway to dihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG). Glycerol is then formed by either hydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction, or reduction of DHAP to glycerol 3-phosphate (G3P) 20 followed by hydrolysis. The hydrolysis step can be catalyzed by any number of cellular phosphatases which are known to be non-specific with respect to their substrates or the activity can be introduced into the host by recombination. The reduction step can be catalyzed by a NAD+ (or NADP*) linked host enzyme or the activity can be introduced into the host by recombination. It is notable that 25 13/12/07,ati3772.div spec pgs, 9 9 the dha regulon contains a glycerol dehydrogenase (E.C. 1.1.1.6) which catalyzes the reversible reaction of Equation 3. Glycerol -+ 3-HP + H20 (Equation 1) 5 3-HP + NADH + H+ -+ 1,3-Propanediol + NAD+ (Equation 2) Glycerol + NAD+ -+ DHA + NADH + H+ (Equation 3) Glycerol is converted to 1,3-propanediol via the intermediate 3-hydroxy propionaldehye (3-HP) as has been described in detail above. The intermediate 10 3-HP is produced from glycerol, Equation 1, by a dehydratase enzyme which can be encoded by the host or can introduced into the host by combination. This dehydratase can be glycerol dehydratase (E.C. 4.2.1.30), dio] dehydratase (E.C. 4.2.1.28) or any other enzyme able to catalyze this transformation. Glycerol dehydratase, but not diol dehydratase, is encoded by the dha regulon. 15 1,3-Propanediol is produced from 3-HP, Equation 2, by a NAD+- (or NADP+) linked host enzyme or the activity can introduced into the host by combination. This final reaction in the production of 1,3-propanediol can be catalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or other alcohol dehydrogenases. Mutations and transformations that affect carbon channelig. A variety of 20 mutant organisms comprising variations in the 1,3-propanediol production pathway will be useful in the present invention. For example the introduction of a triosephosphate isomerase mutation (rpl-) into the microorganism of the present invention is an example of the use of a mutation to improve the performance by carbon channeling. The mutation can be directed toward a structural gene so as to 25 impair or improve the activity of an enzymatic activity or can be directed toward a regulatory gene so as to modulate the expression level of an enzymatic activity. Alternatively, transformations and mutations can be combined so as to control particular enzyme activities for the enhancement of 1,3-propanediol production. Ihus it is within the scope of the present invention to anticipate 30 modifications of a whole cell catalyst which lead to an increased production of 1,3-propanediol. Mdia -and Carbon Substrates: Fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include but are not limited to 35 monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate may also be one-carbon substrates such as carbon dioxide, or medianol 10 for which metabolic conversion into key biochemical intennediates has bcen demonstrated. Glycerol production from single carbon sources (e.g., methanol formaldehyde or formate) has been reported in methylotrophic yeasts (K. Yamada et al., Agric. Biol. Chem., 53(2)541-543, (1989)) and in bacteria (Hunter et.al., 5 Biochemistry, 24,4148-4155, (1985)). 'hese organisms can assimilate single carbon compounds, ranging in oxidation state from methane to formate, and produce glycerol. 'he pathway of carbon assimilation can be through ribulose monophosphate, through seine, or through xylulose-momophosphate (Gottschalk, Bacterial Metab . Second Bdition, Springer-Velag: New York (1986)). The 10 ribulose monophosphate pathway involves the condensation of formate with ribulose-5-phosphate to form a 6 carbon sugar that becomes fuctose and eventually the three carbon product glyceraldchydc-3-phosphatc. Likewise, the serine pathway assimilates the one-carbon compound into the glycolytic pathway via methylenetetrahydrofolate. In addition to one and two carbon substrates methylotrophic organisms are also known to utilize a number of other carbon containing compounds *uch as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1 20 Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. (1990), 153(5), 485-9). Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon 25 containing substrates and will only be limited by the choice of organism. Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, sucrose or methanol. In addition to an appropriate carbon source, fermentation media must 30 contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for 1,3-propanediol production. Particular attention is given to Co(II) salts and/or vitamin B 12 or precursors thereof. 11 Culture Conditions: Typically cells are grown at 30*C in appropriate media as described in the parent specification. Batch and Continuous Fermentations: 5 The present process employs a batch method of fermentation as described in the parent specification. Identification and purification of 1,3-propanediol: Methods for the purification of 1,3-propanediol from fermentation media are known in the art and are as described in the parent specification. 10 Cells: Cells suitable in the present invention comprise those that harbour a dehydratase enzyme and are as described in the parent specification. On the basis of applicants' experimental work, it is contemplated that a wide variety of cells may be used in the present invention. Applicants have demonstrated 15 for example that cells varying widely in genetic and phenotypic composition are able to bioconvert a suitable carbon substrate to 1,3-propanediol. Cells exemplififed include: a K pneumoniae mutant strain constitutive for the dha genes, recombinant E. coli strains comprising elements of the Klebsiella genome containing genes encoding either glycerol or diol dehydratase, and recombinant E. coli (tpi~) strains 20 also transfected with elements of the Klebsiella genomes and harbouring a mutation in the gene encoding the triosephosphate isomerase enzyme. Although E. coli transformants containing the dha regulon from Klebsiella pneumonia were able to convert glycerol to 1,3-propanediol even in the presence of glucose or xylose (Tong et al., Appl. Biochem. Biotech., 34, 149 (1992)), no 1,3 25 propanediol was detected by these organisms in the presence of glucose alone. In direct contrast to this disclosure, applicants have discovered that three strains of E. coli, containing either of two independently isolated cosmids comprising the dha regulon from Klebsiella pneumonia, produced 1,3-propanediol from a feed of glucose with no exogenously added glycerol present. E. coli strain ECL707, 12112/07,at13772.div spec pgs,7 12 containing cosmid vectors pKP-1 or pKP-2 comprising the K. pneumoniae dha regulon, showed detectable though modest production of 1,3-propanediol-from glucose in the absense of exogenously added glycerol, (Example 4). Recombinant E. coli strains constructed from an alternate host organism, DH5 r., also containing 5 cosmid vectors pKP-1 or pKP-2, were found to be more effective than the ECL707 recombinants in producing 1,3-propanediol from glucose under the appropriate conditions, (Example 3). Most effective in producing 1,3-propanediol from glucose under the conditions of Example 4 were the recombinant E. coli strains AA200 containing cosmid vectors pKP-l or pKP-2, Example 2. E. coil 10 strain AA200 contains a defective triosephosphate isomerase enzyme (tpi-). A strain of AA200-pKP1, selected for further study from a pool of independent isolates from the transformation reaction, converted glucose to 1,3-propanediol in a two stage reaction. In the first stage, the strain AA200-pKP1-5 was grown to high cell density in the absence of glucose and 15 glycerol. In the second stage, the grown cells, suspended in a medium containing glucose but no glycerol, converted glucose to 1,3-propanediol with high conversion and selectivity, Example 5. Although differing immumochemically, chromatographically, and genetically, the coenzyme B 12 -dependent enzymes glycerol dehydratase (E.C. 4.2.1.30) and diol dehydratase (E.C. 4.2.1.28) catalyze 20 the conversion of glycerol to 3-hydroxypropionaldehyde. Glycerol dehydratase, but not diol dehydratase, is encompassed by the dha regulon. K. pneumoniae ATCC 8724, containing a diol dehydratase but not a glycerol dehydratase converts glycerol to 1,3-propanediol (Forage et al., J. Bacterial., 149, 413, (1982)). Recombinant E. coUl strains ECL707 and AA200, containing cosmid 25 vector pKF4 encoding genes for a diol dehydratase, converted glucose to 1,3-propanediol, Example 2 and Example 4. K. pneumoniae ECL2106, prepared by mutagenesis from a naturally occurring strain (Ruch et al., J. Bacteriol. 124, 348 (1975)), exhibits constitutive expression of the dha regulon (Ruch et al., supra; Johnson et al., J. Bacterial. 30 164,479 (1985)). A strain derived from K. pneumoniae ATCC 25955, displaying the same phenotype, has been simlary prepared (Forage ct al., J. Bacteriol. 149, 413 (1982)). Expression of the Klebsiella dha structural genes is, in part, controlled by a repressor (product of dha R) (Sprenger et a., J. Gen Microbiol. 135, 1255 (19g9)). Applicants have shown that ECL2106, which is constitutive 35 for the dha structural genes, produced 1,3-propanediol from a feed of glucose in the absence of exogenously added glycerol, Example 6. This is in contrast to wild type K. pneumoniae ATCC 25955 which did not produce detectable levels of 1,3-propanediol under the same conditions, Example 6. 13 The expression of the dha structural genes in ECL2106 is further controlled by catabolite expression (Sprenger et al., J. Gen Microbiol. 135, 1255 (1989)). Elimination of catabolite repression can be achieved by placing the necessary structural genes under the control of alternate promoters as has been demonstrated 5 for 1,3-propanediol oxidoreductase (dhaT) from C. freundii and diol dehydratase from K. oxytoca ATCC 8724 (Daniel et al., J. Bacteriol. 177, 2151 (1995) and Tobimatsu et al., J. Biol. Chem. 270, 7142 (1995)). By eliminating catabolite repression from ECL2106 in this manner, an improvement in the production of 1,3 propanediol from glucose in the absence of an exogenous source of glycerol is 10 achieved. An even further improvement is obtained by appropriate carbon channelling as is described, by example, with the tpi~ mutation. As the dha regulons of Citrobacter and Klebsiella sp. are strikingly similar, one of skill in the art will appreciate that teachings that involve the production of 1,3 propanediol from glucose in the absence of an exogenous source of glycerol for 15 Klebsiella sp. applies to Citrobacter sp. as well. Furthermore, as the metabolism of glycerol by C. butyricum is comparable to that of K pneumoniae [Zeng et al., Biotechnol. and Bioeng. 44, 902 (1994)], teachings will extend to Clostridia sp. as well. 20 EXAMPLES GENERAL METHODS Procedures for phosphorylations, ligations and transformations are well known in the art and are described in the parent specification. 12/12107,at13772.div spec pgs,7 14 EXAMPLE I ClQoninr and transformation of E. coli host cells with cosmid DNA for the 15 expression of I.3-propanediol Media Synthetic S12 medium was used in the screening of bacterial transformants for the ability to make 1,3-propanediol. S12 medium contains: 10 mM ammonium sulfate, 50 mM potassium phosphate buffer, pH 7.0,2 mM 20 MgC1 2 , 0.7 mM Ca 2 , 50 gM MnQ 2 , I pM FeC3, 1 pM ZnoC, 1.7 FM CuSO 4 , 2.5 pM Cod 2 , 2.4 pM Na 2 MoO 4 , and 2 pM thiamine hydrochloride. Medium A used for growth and fermentation consisted of: 10 mM annonium sulfate; 50 mM MOPS/KOH buffer, pH 7.5; 5 mM potassium phosphate buffer, pH 7.5; 2 mM MgQ 2 ; 0.7 mM CaCI 2 ; 50 FM MnC1 2 ; I PM 25 Pca 3 ; 1 pM ZnQ; 1.72 pM CuSO 4 ; 2.53 pM CoO 2 ; 2.42 pM Na 2 MoO 4 ; 2 pM thiamine hydrochloride; 0.01% yeast extract; 0.01% casamino acids; 0.8 pg/mL vitamin B 12 ; and 50 amp. Medium A was supplemented with either 0.2% glycerol or 0.2% glycerol plus 0.2% D-glucose as required. 30 Klebsiella pneumoniae ECL2106 (Ruch et al., J. Bacteriol., 124,348 (1975)). also known in the literature as K. aerogenes or Aerobacter aerogenes. was obtained from E. C. C. Lin (Harvard Medical School, Cambridge, MA) and was maintained as a laboratory culture. Klebsiella pneumonlae ATCC 25955 was purchased from American Type 35 Culture Collection (Rockville, MD). E. coli DH15 was purchased from Gibco/BRL and was transformed with the cosmid DNA isolated from Klebsiellapneunoniae ATCC 25955 containing a gene coding for either a glycerol or diol dehydratase enzyme. Cosmids 15 containing the glycerol dehydratase were identified as pKPI and pKP2 and cosmid containing the diol dehydratase enzyme were identified as pKP4. Transformed DH5z cells were identified as DH5n-pKPI. DH5a-pKP2, and DH5r,-pKP4. 5 E. coll ECL707 (Sprenger et al., J. Gen. Microbiol., 135, 1255 (1989)) was obtained from E. C. C. Lin (Harvard Medical School, Cambridge, MA) and was similarly transformed with cosmid DNA from Klebstella pnernoniae. These transformants were identified as ECL707-pKPI and ECL707-pKP2, containing the glycerol dehydratase gene and BCL707-pKP4 containing the diol dehydratase 1.0 gene. E. coli AA200 containing a mutation in the rpi gene (Anderson et al., J. Gen Microbiol., 62, 329 (1970)) was purchased from the E. coli Genetic Stock Center, Yale University (New Haven. CT) and was transformed with Klebsiella cosmid DNA to give the recombinant organisms AA200-pKPI and AA200-pKP2, 15 containing the glycerol dehydratase gene, and AA200-pKP4, containing the diol dehydratase gene. DH5 Six transformation plates containing approximately 1,000 colonies of E. coli )L1-Blue MR transfected with K. pneunoniae DNA were washed with 20 5 mL LB medium and centrifuged. The bacteria were pelleted and suspended in 5 nL LB medium + glycerol. An aliquot (50 pL) was inoculated into a 15 mL tube containing S12 synthetic medium with 0.2% glycerol + 400 ng per mL of vitamin B 12 + 0.001% yeast extract + 50amp. The tube was filled with the medium to the top and wrapped with parafilm and incubated at 30*C. A slight 25 turbidity was observed after 48 h. Aliquots, analyzed for product distribution as described above at 78 I and 132 h, were positive for 1,3-propanediol, the later time points containing increased amounts of 1,3-propanediol. The bacteria, testing positive for 1,3-propanediol production, were serially diluted and plated onto LB-50amp plates in order to isolate single colonies. Forty 30 eight single colonies were isolated and checked again for the production of 1,3-propanediol. Cosmid DNA was isolated from 6 independent clones and transformed into E. coli strain DH5. The transformants wer again checked for the production of 1,3-propanediol. Two transformants were characterized further and designated as DH50t-pKP1 and DH5cr-pKP2. 35 A 12.1 kb EcoRI-SalI fragment from pKPI, subcloned into ptBI31 (IBI Biosystem, New Haven, CN), was sequenced and termed pHK28-26 (SEQ ID NO: 1). Sequencing revealed the loci of the relevant open reading frames of the dha operon encoding glycerol dehydratase and genes necessary for regulation. 16 Referring to SEQ ID NO:1, a fragment of the open reading frame for dhaK encoding dihydroxyacetone kinase is found at bases 1-399; the open reading frame dhaD encoding glycerol dehydrogenase is found at bases 983-2107; the open reading frame dhaR encoding the repressor is found at bases 2209-4134; the 5 open reading frame dhaTencoding 1,3-propanadiol oxidoreductase Is found at bases 5017-6180; the open reading fraum dhaBI encoding the alpha subunit glycerol dehydratase is found at bases 7044-8711; the open reading frame dhaB2 encoding the beta subunit glycerol dehydratase is found at bases 8724-9308; the open reading frame dhaB3 encoding the gamma subunit glycerol dehydratase is 10 found at bases 9311-9736; and the open reading frame dhaBX, encoding a protein of unknown function is found at bavs 9749-11572. Single colonies of E. coli XLl-Blue MR transfected v. ith packaged cosnid DNA from K. pneumoniae were inoculated into microtiter wells containing 200 uL of S15 medium (ammonium sulfate, 10 mM; potassium phosphate buffer, 15 pH 7.0, 1 mM; MOPS/KOH buffer, pH 7.0, 50 mM; MgC 2 , 2 mM; CaC12, 0.7 mM; MnC1 2 , 50 uM; FeC1 3 , I uM; ZnC, I uM; CuSO 4 , 1.72 uM; CoCl 2 , 2.53 uM; Na 2 MoO 4 , 2.42 uM and thiamine hydrochloride, 2 uM) + 0.2% glycerol + 400 ng/mL of vitami B 12+0.001% yeast extract + 50 ug/mL ampicillin. In addition to the microtiter wells, a master plate containing LB-50 20 amp was also inoculated. After 96 h, 100 uL was withdrawn and centrifuged in a Rainin microfuge tube containing a 0.2 micron nylon membrane filter. Bacteria were retained and the filtrate was processed for HPLC analysis. Positive clones demonstrating 1,3-propanediol production were identified after screening approximately 240 colonies. Three positive clones were identified, two of which 25 had grown on LB-50 amp and one of which had not. A single colony, isolated from one of the two positive clones grown on LB-50 amp and verified for the production of 1,3-propanediol, was designated as pKP4. Cosmid DNA was isolated from E. coUl strains containing pKP4 and E. coli strain DH5r was transformed. An independent transfonnant, designated as DH5or-pKP4, was 30 verified for the production of 1,3-propanediol. BCL707: E. coli strain ECL707 was transfonned with cosmid K. pneumoniae DNA corresponding to pKP1, pKP2, pKP4 and the Supercos vector alone and named ECL707-pKPI, ECL707-pKP2, ECL707-pKP4, and ECL707-sc, respectively. 35 ECL707 is defective in glpK, gld, and ptsD which encode the ATP-dependent glycerol kinase, NAD+-linked glycerol dehydrogenase, and enzyme II for dihydroxyacetone of the phosphoenolpyruvate dependent phosphotransferase system, respectively. 17 Twenty single colonies of each cosmid transfonnation and five of the Supercos vector alone (negative control) transformation, isolated from LB-50aap plates, were transferred to a master LB-50anp plate. These isolates were also tested for their ability to convert glycerol to 1,3-propanediol in order to determine 5 if they contained dehydratase activity. The transformants were transferd with a sterile toothpick to microtiter plates containing 200 pL of Medium A supplemented with either 0.2% glycerol or 0.2% glycerol plus 0.2% D-glucose. After incubation for 48 hr at 30*C, the contents of the microtiter plate wells were filtered through an 0A5 IL nylon filter and chromatographed by HPLC. The 10 results of these tests are given in Table 1. Table 1 Conversion of glycerol to 1,3-propanediol by transformed ECL707: number of positive isolates/number of isolates tested iansformant GIycoI Glycerol plus Glucose ECL707-pKPI 19/20 19/20 ECL707-pKP2 18/20 20/20 ECL707-pKP4 0/20 20/20 ECL707-sc 0/5 0/5 E. coli strain AA200 was transformed with cosmid K. pneumoniae DNA corresponding to pKP1, pKP2, pKP4 and the Supercos vector alone and named 15 AA200-pKP1, AA200-pKP2, AA200-pKP4, and AA200-sc, respectively. Strain AA200 is defective in triosephosphate isomerase, (pi). Twenty single colonies of each cosmid transformation and five of the empty vector transformation were isolated and tested for their ability to convert glycerol to 1,3-propanediol as described for E. coli strain ECL707. The results of 20 these tests are given in Table 2. Table 2 Conversion of glycerol to 1,3-propanediol by transformed AA200: Number of positive isolatesfnumber of isolates tested Trnsformant Glycrol Glycerol plus Glucose AA200-pKPI 17/20 17/20 AA200-pKP2 17/20 17/20 AA200-pKP4 2/20 16/20 AA200-sc 0/5 015 18 EXAMPLE Conversion of D-glucose to 1.3.propanediol by E. col strain AA200. tnsformed with Kiebsiellia pneumoniae DNA containing dehydratse activity 5 Glass serum bottles, filled to capacity with media (ca. 14 mL of Medium A as defined in Example 1 supplemented with 10 tg/mL kanamycin and 0.2% D-glucose, plus or minus 0.5-1.0 mM cyclic adenosine 2':3'-monophosphate (cAMP)), were innoculated with selected single colony isolates of E. coli strain AA200 containing the K. pneumoniae dha regulon cosmids pKPI or pKP2, the 10 K. pneumoniae pdu operon pKP4, or the Supercos vector alone. In order to avoid contact with glycerol, the innoculation was performed from either an agar platc of LB-50amp or from a liquid culture of the samc medium. The reactions wcrc incubated for ca. 72 hr at 30"C while shaking at 250 rpm. Growth was determined by the change in absorbance at 600 nm where the initial OD600 was 15 0.020 AU. The extent of glucose depletion and product distribution were determined by HPLC. Single colony isolates are identified by a numbered suffix --x", e.g., AA200-pKP1-x. Cumulative results are presented in Table 3 and Table 4. Table 3 Conversion of 0.2% D-glucose to 1,3-propanediol by transformed E. co/i strain AA200: without cAMP [1,3-propane Transformant O3n dioll (mM) Con. (%) Sel. (%) AA200-pKP1-3 0.056 0.05 17 1 AA200-pKP1-5 0.115 nd 0 0.007 nd 0 0.076 0.2 14 5 AA200-pKP1-20 0.116 nd 27 0 0.205 0.3 17 8 AA200-pKP2-10 0.098 .0.2 13 7 AA200-pKP2-14 0.117 0.5 17 14 0.129 0.2 19 5 AA200-pKP2-20 0.094 nd I1 0 AA200-pKP4-4 0.198 0.1 28 2 AA200-pKP4-19 0.197 0.2 34 3 AA200-pKP4-20 0.206 0.1 38 1 AA200-sc-1 0.097 nd 22 0 0.176 nd 46 0 19 Table 4 Conversion of 0.2% D-glucose to 1,3-propanediol by trnsfonned E. coi strain AA200: with cAMP [1,3-propane Transfonnant ODen diollmM) % Con. % Sel. AA200-pKP1-3 0.102 0.5 19 12 AA200-pKP1-5 0.088 1.5 21 37 0.236 1.4 24 28 0.071 0.8 15 23 AA200-pKPI-20 0.153 nd 40 0 0.185 0.9 27 16 AA200-pKP2-10 0.098 0.2 13 7 AA200-pKP2-14 0.213 2.0 26 27 0.155 0.6 25 12 AA200-pKP2-20 0.198 1.2 40 14 AA200-pKP4-4 0.218 0.1 31 2 AA200-pKP4-19 0.223 0.2 37 3 AA200-pKP4-20 0.221 0.2 35 3 AA200-sc-1 0.111 nd 23 0 0.199 nd 49 0 0.122 ad 25 0 3 The identity of 1.3-propanediol was verified by GC/MS as described in the GENERAL METHODS. EXAMPLES 3 Conversion of D-glucose to 1.3-propanediol by E. coli strain DH5;. transfored with Klebsiellia pneumoniae DNA 5 containing dehvdratase activity E. coli strain DH5cc, containing the K. pneumoniae dha rgulon cosnids pKP1 or pKP2, were tested for their ability to convert D-glucose to 1,3-propanediol as described in Examnple 2. The results are presented in Table 5. 20 Table 5 Conversion of 0.2% D-glucose to 1,3-propanediol by transformed E. coli strain DH5cc plus (+) and minus (-) cAMP [1,3-propane Transforant ODrm dioll (mM) % Con. % Sel. DH5c:-pKP1 (-) 0.630 0.5 100 2 DH5ct-pKP1 (+) 0.774 0.6 100 3 DH5cr-pKP2 (-) 0.584 0.6 100 3 DH5a-pKP2 (+) 0.699 0.7 100 3 EXAMPLE Conversion of D-glucose to 1.3-propanediol by E, coli strain ECL707. transformed with Kebsiellia pneumoniae DNA 5 contaiing dehydratase activity E. coli strain ECL707, containing the K. pneumoniae dha regulon cosmids pKP1 or pKP2, the K. pneumoniae pdu operon pKP4, or the Supercos vector alone, were tested for their ability to convert D-glucose to 1,3-propanediol as described in Example 2. In each case, conversion was quantitative. The results 10 are presented in Table 6. Table 6 Conversion of D-glucose to 1.3-propanediol by transformed E. coli strain ECL707: with and without cAMP [1,3-propane- [1,3-propane Transformant ODKn dioll (mM) ODrmnn dioll (mM) (without cAM (with cAM ECL707-pKP1-1 0.607 0.1 0.475 0.1 ECL707-pKP1-3 0.619 0.1 0.422 0.1 ECL707-pKP1-7 0.582 0.2 0.522 0.2 ECL707-pKP1-10 0.593 0.1 0.408 0.1 ECL707-pKPI-18 0.584 0.1 0.433 0.1 ECL707-pKP2-4 0.588 0.1 0.408 0.1 ECL707-pKP2-5 0.630 0.1 0.516 0.2 ECL707-pKP2-8 0.542 0.1 0.486 0.1 ECL707-pKP2-15 0.589 0.1 0.485 0.1 ECL707-pKP2-19 0.577 0.1 0.504 0.1 BCL707-pKP4-8 0.499 nd 0.361 <0.1 ECL707-pKP4-9 0.544 nd 0.354 nd 21 ECL707-pKP4-10 0.515 nd 0.265 <0.1 ECL707-pKP4-14 0.512 nd 0.318 <0.1 BCL707-pKP4-17 0.545 nd 0.388 <0.1 ECL707-sc-1 0.592 nd 0.385 nd EXAMPLE Two state conversion of D-glucose to 1.3-proanediol by Escherichia coli AA200-pKPI-5 5 Baffled flasks (250 mL) containing 50 mL LB-amp medium were inoculated with single colonies of AA200-pKPI-5. The cells were grown, in duplicate, overnight at 30 or 37*C with shaking (250 rpm). Grown cultures were spun (10 minutes, 10,000 rpm, 4*C) and resuspended in production medium without glucose (10 mM (NH4) 2 S0 4 ; 5 mM potassium 10 phosphate buffer, pH 7.5; 50 mM MOPS, pH 7.5; 0.01% yeast extract; 0.01% casamino acids; 0.8 pg/mL vitamin B,,; and 50 pg/mL ampicillin) containing either trace metals A: (0.08 pM CoC 2 , 0.06 pM CuCI 2 , 7 pM FeSO 4 , 2 pM HB0 4 , 0.2 pM MnC1 2 , 0.1 pM Na 2 MoO 4 , 0.08 pM NiCI 2 , 0.3 pM ZnSO 4 , and 0.03 mM thiamine) or trace metals B: (0.7 mM Caa 2 , 2.53 FM Coa 2 , 1.72 sM 15 CuSO 4 , 1.0 pM FeCI 3 , 2 mM MgC2, 0.05 mM MnC 2 , 2.42 pM Na 2 MoO 4 , 1.0 pM ZnC1 2 , and 0.03 mM thiamine). The cells were spun a second time, resuspended in 50 mL fresh production medium containing D-glucose and dispensed into 60 mL serum bottles which were capped and sealed with butyl rubber septa. The bottles were shaken (250 rpm) and samples withdrawn with a 20 syringe through the septum and filtered through a 0.2 p filter before analysis. Results are shown in Table 7 and Table 8; residual glucose was measured by enzymatic analysis (Biochemistry Analyzer, Yellow Springs Instruments Co., Inc.) and 1,3-propanediol was analyzed by HPLC. Table 7 Conversion of 0.2% D-glucose to 1,3-propanediol by Escherichia coli AA200-pKPI-5. Durlicate reactions were yerformeda Time [Glucose] [1,3-propane- Con. Sel. Experiment (days) (mM) diol] (mM) (%) #1 1 0.1 2.3 99 10 #1 4 0.1 2.3 99 10 #2 1 2.8 2.3 75 14 #2 4 0.1 2.4 99 11 aMe reactions mixtures, containing trace metals A, were incubated at 37*C. 25 22 Table 8 Conversion of 1% D-glucose to 13-oranediol by cherihdia coli AA200-kPl-5a time [glucose] [1,3-propane- Con. Sel. (days) (mM) dioll (mM) (%) (%) 0 53 0 0 0 1 39 5.6 26 20 2 35 8.3 34 23 3 33 8.4 38 21b aThe reactions mixtures, containing trace metals B, were incubated at 30 0 C bAt the end of the reaction, the presence of 1,3-propanediol was confirmed by GC/MS and 13 C-NMR (300 MHz). EXAMPLE 6 Conversion of D-glucose to 1.39topanediol by Klebsiella yneumpniae EL2 106 but not by 5 Klebsiella pneurnoniae ATCC 25955 Glass serum bottles, filled to capacity (ca 14 mL) with media, were lightly innoculated from a LB agar plate containing K. pneumoniae ECL2106 or K. pneumoniae ATCC 25955. The media contained 50 mM glucose, 3 mM

(NH

4

)

2

SO

4 , 0.9 MM CaC 2 , 4 pM CoO 2 , 0.06 pM CuC2, 7 pM PeSO 4 , 2 pM 10 H 3

BO

4 , 0.8 mM MgSO 4 , 0.2 pM MnC 2 , 0.1 pM Na 2 MoO 4 , 0.08 pM NiC 2 , 0.3 pM ZnSO 4 , 0.1 mg/mL DL-cysteine, 10 pM ethylenedianinetetrancetic acid, 0.8 Rg/mL vitamin B 12 , potassium phosphate as indicated in Table 9, and either 50 mM HEPES or 50 mM MOPS buffer, pH 7.5. The reactions were incubated for 47 hr at 30 0 C while shaking at 250 rpm. Otherwise, the reaction was 15 performed as described in Example 2. The results are given in Table 9. Table Conversion of D-glucose to 1,3-propanediol by Klebsiella pneumoniae ECL2106 but not by Klebsiella pneumoniae ATCC 25955 Strain Buffer Pi [Glucose] (1,3-Propane (mM) (mM) dioll (mM) 2106 MOPS 5.0 11.4 0.2 2106 MOPS 2.5 13.9 0.2 2106 MOPS 1.3 14.8 0.1 2106 MOPS 0.6 15.8 0.1 2106 HEPES 5.0 21.1 0.1 2106 HEPES 2.5 23.4 0.1 23 2106 HEPES 1.3 26.4 0.1 2106 HEPES 0.6 27.5 0.1 25955 MOPS 5.0 4.4 nd 25955 MOPS 2.5 5.4 nd 25955 MOPS 1.3 2.8 nd 25955 MOPS 0.6 7.8 nd 25955 HEPES 5.0 7.0 nd 25955 HEPES 2.5 13.5 nd 25955 HEPES 1.3 10.2 nd 25955 HEPES 0.6 17.8 nd EXAMPLE 7 Production of I.3-propanediol by recombinant Pichia pastoris Construction of general purpose expression n'lasmid 5 The 0.9 kb EcoR 1/Xbal fragment in pHIL-D4 (Phillips Petroleum, Bartlesville, OK) was replaced by the 0.9 kb EcoRl/Xbal fragment fro-m pAO815 (Invitrogen, San Diego, CA) to generate the plasmid pHIL-D4B2 which contains the following elements: S'AOX1, P. pastoris methanol inducible alcohol oxidase I (AOX1) promoter, AOX1 term, P. pastoris AOX I transcriptional termination 10 region; HIS4, P. pastoris histidinol dehydrogenase-encoding gene for selection in his4 hosts; kan, sequence derived from transposon Tn9O3 encoding aminoglycoside X-phosphotransferase, conferring kanamycin, neomycin and G418 resistance in a wide variety of hosts, and useful as an indicator of cassette copy number; 3AOX1, P. pastoris sequence downstream from AOX1, used in 15 conjunction with 5AOXJ for site-directed vector integration; ori, pBR322 origin of DNA replication allowing plasmid manipulations in E. coli; and amp, $-lactamase gene from pBR322 conferring resistance to ampicillin. An additional feature of pHIL-D4B2 is that multiple expression cassettes (5AOXI - gene AOXJterm) can easily be placed onto one plasmid by subcloning cassettes on 20 Bgl2/Xbal fragments into BamH1/Xbal sites. Construction of plasmid for co-expssion of dhaB1_and dhaB2 The open reading frames for dhaB) and dhaB2 were amplified from cosmid pKPI by PCR using primers (SEQ ID NO:2 with SEQ ID NO:3 and SEQ ID NO:4 with SEQ ID NO:5 for dhaB and dhaB2, respectively) incorporating 25 EcoRI sites at the 5' ends (10 mM Tris pH 8. 50 mM KCl, 1.5 MM MgC1 2 , 0.0001% gelatin, 200 pM dATP, 200 pM dCMP, 200 IM dGTP, 200 pM d'TP, I pM each primer, 1-10 ng target DNA, 25 units/mL AmplitaqO DNA polymerase Perkin Elmer Cetus, Norwalk CT). PCR parameters were I min at 94 0 C, 1 min at 55 0 C, 1 min at 72 0 C, 35 cycles. The products were subcloned into the EcoRI site 24 of pHIL-D4B2 to generate the expression plasmids pMP19 and pMP20 containing dhaBl and dhaB2, respectively. The dhaBl expression cassette on a Bgl2/Xbal fragment from pMP19 was subcloned into the BamH1/Xbal site of pMP20 to generate pMP21. Plasmid 5 pMP21 contains expression cassettes for both dhaB2 and dhaB), and the HIS4 selectable maker. Construction of plasmid for co-expression of dhaB3 and dhaT The open reading frames for dhaT and dhaB3 were amplified by PCR from cosmid pKP1 using primers (SEQ ID NO:6 with SEQ ID NO:7 and SEQ ID 10 NO:8 with SEQ ID NO:9 for dhaT and dhaB3, respectively) incorporating EcoRI sites at the 5' ends. The products were subcloned into the EcoRI site of pHIL-D4B2 to generate the expression plasmids pMP17 and pMP18 containing dhaT and dhaB3, respectively. The dhaT expression cassette on aBgl2/Xbal fragment from pMP17 was 15 subcloned into the BamHl/Xbal site of pMP18 to generate pMP22 which contains expression cassettes for both dhaT and dhaB3. The 4.1 kb EcoRI fragment containing SUC2 was deleted from pRK20 (Phillips Petroleum, Bartlesville, OK) to generate pMP2. SUC2 encodes for an invertase which may be used a second selectable marker in Pichia. The 4.0 kb 20 Hind3 fragment containing lacZ was deleted form pMP2 to generate pMP3. The 0.4 kb Hind3 fragment containing AOXI term from pHIL-D4 was subcloned into the Hind3 site of pMP3 to generate pMP1O. The 2.0 kb Bgl2/Xbal fragment in pMP10 was replaced with the 5.0 kb Bgl2/Xbal fragment containing the dhaB3 and dhaT expression cassettes from 25 pMP22 to generate pMP23. The 5.4 kb Pstl/Bgl2 fragment containing SUC2 from pRK20 was subcloned into the Pstl/Bg12 sites of pSP73 (Promega, Madison, WI) to generate pMPIla. Plasmid pMP11a was cut with BooR1, filled with T4 DNA polymerase and religated to generate pMP1 lb. The 1.1 kb Pst/Bgl2 fragment in pMP1O was replaced with the 5.4 kb Bg12/Pstl fragment containing 30 SUC2 from pMP11b to generate pMP12. The 1.0 kb Scal/Bg12 fragment in pMP23 was replaced with the 5.2 kb Scal/Bgl2 fragment containing SUC2 firom pMP12 to generate pMP24. Plasmid pMP24 contains expression cassettes for both dhaT and dhaB3, and the SUC2 selectable marker. 35 Transformation of P. pastors with dhaBlldhaB2 expression plasmid pMP2I P. pastors strain GTS 1 15(his4) (Phillips Petroleum, Bartlesville, OK) was transformed with 1-2 ug of Bg12-linearized plasmid pMP21 using the spheroplast transformation method described by Cregg et al., (Mol. Cell. Biol. 5,3376, 25 (1985)). Cells were regenerated on plates without histidine for 3-4 days at 30*C. All transfonnants arise after integration of plasmid DNA into the chromos-om Transfonnmants were patched onto a YPD (1% Bacto yeast extract, 2% peptone, 2% glucose) master plate. 5 Screening of P. astois transformnants for dhaBI and dhaB2 Chromosomal DNA was prepared from his+ transfornants described above and subjected to PCR analysis with primers specific for dhaB) and dhaB2. High copy number strains were selected from transformants containing both dhaB1 and dhaB2 by growth in YPD media supplemented with increasing levels 10 of G418 (Sigma, St. Louis, MO) up to 2000 pghnL. Resistance to a high level of G418 suggests significant duplication of expression cassettes. Secondary transformation of F. pasroris with dhaB3/dhaT expression plasmid Transformants with resistance to a high level of 0418 as described above 15 were re-transformed with plasmid pMP24 using the spheroplast transformation method. Cells were first regenerated on non-selective plates for 2 days at 30*C, after which top agar containing the regenerated cells was scraped from the plate and vortexed extensively in 20 mL water. After passing through 4 folds of cheesecloth, the cells were pelleted by centrifugation and resuspended in 10 mL 20 water. Aliquots of 200 uL were plated onto sucrose plates, and incubated for 2 days at 30 0 C. All transformants arise after integration of plasmid DNA into the chromosome. Transformants appear as large colonies in a background of small colonies, and require isolation. After 24 h growth with shaking at 30"C in Msu media (per L, 13.4 g yeast nitrogen base w/o amino acids, 10 g sucrose, 0.4 g 25 biotin), transformants were streaked onto Msu plates (Msu media plus 15 g/L agar) and grown for 2 days at 30'C. Large isolated colonies were patched onto a YPD master plate. Screening of P. pasroris double transfornants for dhaBI. dhaB2. dhaB3.and dhaT and their corsndi enz ivte 30 . Chromosomal DNA was prepared from suc+ double transfomants described above and subjected to PCR analysis with primers specific for dhaB1, dhaB2, dhaB3, and dhaT. Thus, the presence of all four open reading frames was confirmed. The presence of active glycerol dehydratase (dhaB) and 1,3-propanediol 35 oxido-reductase (dhaT) was demonstrated using in vitro enzyme assays. Additionally, westem blot analysis confirmed protein expression from all four open reading frames. Cell free extracts for these protein characterizations were prepared as follows. Double transforants containing dhaBl, dhaB2, dhaB3, and 26 dhaT were grown aerobically with shaking at 30*C in MGY (Per L, 13.4 g yeast .nitrogen base w/o amino acids, 0.4 mg biotin, 10 mL glycerol) for 2 days. The: cells were pelleted by centrifugation, resuspended in MM (Per L, 13.4 g yeast nitrogen base w/o amino acids, 0.4 mg biotin, 5 mL methanol) and incubated as 5 above. After approximately 24 h, the cells were harvested, resuspended in buffer (0.1 M tricene/KOH buffer, pH 8.2,50 mM KCl, and 2% 1,2-propanediol), mechanically disrupted (using a glass rod while vortexing in the presence of glass beads), and centrifuged. One strain that showed positive for the presence of all four open reading 10 frames (dhaBl, dhaB2, dhaB3, and dhaT) and their corresponding activities was designated MSP42.81 and was selected for further study. In yivo production of 1.3-propanediol using recombinant Pichia p2astoris P. pastoris MSP42.81 (ATCC 74363) were grown in a BiostatB fermenter (B Braun Biotech, Inc.) in 1.5 L minimal medium containing 8.5 g/L KH 2

PO

4 , 15 2.1 g/L (NH 4

)

2

SO

4 , 10 g/L glycerol, 2.3 g/L MgSO 4 7H 2 0, 0.18 g/L CaSO 4 '2H 2 0, and 0.29 ml/L PTMI. PTMI is a stock mineral solution containing 24 mM CUSO 4 , 4.8 mM a1, 18 mM MnSO 4 , 0.8 mM Na 2 MoQ 4 , 0.3 MM H 3 B0 3 , 2.1 mM Co2,70 mM ZnSO 4 , 26 mM H 2

SO

4 , 234mM FeSO 4 , and 0.8 mM biotin. The fermenter was controlled at pH 5.0 with addition of 2 M NH40H, 20 30 0 C, and 30% dissolved oxygen tension through agitation control. A culture of P. pastoris MSP42.81 grown in YM broth at 30*C was used as an inoculum; 20 mL of the culture was used to inoculate the fermenter. When glycerol was shown to be depleted (24 h after inoculation), induction of the AOX promoters was initiated by the addition of a methanol feed. 25 The feed contained I liter of methanol, 5 mL PMl and 5 mL of a stock biotin solution prepared as 0.2 g/L in water. The methanol solution was added manually to maintain an average concentration of 0.5% as determined by HPLC analysis. Fifty mL of cells ODw0 = 20 AU) were removed from the reactor after 15 h of induction. 30 The 50 mL cell suspension was pelleted and resuspended in 12.5 nIL nitrogen sparged base medium (6.7 giL yeast nitrogen base, 3.0 g/L yeast extract, 1.0 g/L K 2

HPO

4 , 1.0 g/L KH 2

PO

4 , 3.0 g/L (NH 4

)

2

SO

4 , titrated to pH 7.2 and filter sterilized). Coenzyme B12, prepared as a stock solution at 2 mg/nL in nitrogen sparged water, was added to the cell suspension to give a final 35 concentration of 20 pg/mL. Three media stock solutions were prepared in base medium containing 1% glucose, 0.5% glucose and 0.5% glycerol (w/v), and 1.0% glycerol (w/v). A stock solution of chloroquine (1.06 g/50 mL, pH 7.2) was also prepared. 27 Two nL of the media stock solutions and 1 mL mixtures of chloroquine and water to give the final concentrations listed in Table 10 were placed in10 .mC crimp sealed serum bottles and sparged with nitrogen before adding I mL of cells with coenzyme B 12 mixture. The serum bottles were incubated at 30*C with 5 shaking. Samples taken immediately after the addition of cells and after 24 h incubation were analyzed by HPLC. The results are shown in Table 10. Ible 10 In vivo production of 1,3-propanediol using recombinant Pichia nastoris chloroquine 1,3-propanediol reaction medium' (mM) (mM) I glub 0 0.04 2 glu 2.5 0.2 .3 glu 5.0 0.1 4 glu 10.0 0.1 5 glub/gly 0 0.2 6 glu/gly 2.5 0.4 7 glu/gly 5.0 0.4 8 glu/gly 10.0 1.2c 9 gly 0 0.2 10 gly 2.5 0.3 11 gly 5.0 0.3 12 gly 10.0 1.4c aLoss than 10% of each substrate was used in 24 h unless noted. bNo glucose remained after 24 h. CThe presence of 1,3-propanediol was confined by GC/MS as described in GENERAL METHODS. EXAMPLE 10 Use of a Pichia pastoris double transformant for production of 1.3-propanedig from D-glucose P pastoris MSP42.81 were grown in a BiostatB fermenter (B Braun Biotech, Inc.) in 1.5 L minimal medium containing 8.5 g/L KH 2 P0 4 , 2.1 g/L

(NH

4

)

2

SO

4 , 10 g/L glucose, 2.3 g/L MgSO 4 7H 2 0, 0.18 g/L CaSO 4 2H 2 O, and 15 0.29 miL PTM[. Otherwise, fermentation and induction conditions were identical to those described in Example 7. Fifty niL of cells were removed from the reactor after 15 h of induction. The cell suspension was handled as described in Example 7, with the exception that a modified base medium (6.7 g/L yeast nitrogen base, 1.0 g/L 28

KH

2

PO

4 , 1 g/L K 2

HFO

4 , 3 g/L (NH 4

)

2

SO

4 , titrated to pH 7.2 and filter sterilized) was used. The three media stock solutions were prepared in this. modified base medium as well. All other solutions were the same. Reaction mixtures were prepared as described, and incubated at 30*C with shaking. 5 Samples taken immediately after the addition of cells and after 75 hours incubation were analyzed by HPLC. In a reaction containing glucose as the carbon source and 5 mM chloroquine, 0.17 mM 1,3-propanediol was produced. EXAMPLE 2 Plasmid consmiction for the transformation and expression of dha and dAT in 10 Saccharomyces cerevisiae Construction of general pupose expression plasmids Two types of expression plasmids were created, those that could integrate by recombination into chromosomes, and those that could exist as replicating episomal elements. For each type of generl expression plasmid a yeast promoter 15 was present and separated from a yeast transcription terminator by fragments of DNA containing recognition sites for one or more restriction endonuccascs. Each type of general expression plasrnid also contained the gene for -lactamase for selection in E. coHl on media containing ampicillin, an origin of replication for plasmid maintainence in E. coli, and either a 2 micron origin of replication for 20 episomal elements or sequences homologous to those found in S. cerevisiae chromosomes for recombination and integration of introduced DNA into chromosomes. The selectable nutritional markers used for yeast and present on the expression plasmids were one of the following: HIS3 gene encoding miridazoleglycerolphosphate dehydratase, URA3 gene encoding orotidine 25 5'-phosphate decarboxylase, TRP1 gene encoding N-(5'-phosphoribosyl) anthranilate isomerase and LEU2 encoding 0-isopropylmalate dehydrogenase. The yeast promoters used were ADHI or GAL1, and the transcription tenninators ADHI, CYCI or AOXl; the later from Pichia pastors. Plasmid pGADGH (Clontech, Palo Alto, CA) was digested with HindiH 30 and the single-strand ends converted to EcoRI ends by ligation with HindM-XmnI and EcoRI-XmnI adaptors (New England Biolabs, Beverly, MA). Selection for plasmids with correct EcoRI ends was achieved by ligation to a kanamycin resistance gene on an EcoRI fragment from plasmid pUC4K (Pharmacia Biotech, Uppsala), transformation into E. coli strain DH5a and 35 selection on LB plates containing 25 pg/mL kanamycin. The resulting plasmid (pGAD/KAN2) was digested with SnaBI and EcoRI and a 1.8 kb fragment with the ADHI promoter was isolated. Plasmid pGBI (Clontech, Palo Alto, CA) was digested with SnaBI and EcoRI, and the 1.5kb ADHI1GALA fragment replaced 29 by the 1.8 kb ADHI promoter fragment isolated from pGAD/KAN2 by digestion with SnaBI and EcoRI. The resulting vector (pMCKI 1) is a replicating pianid in yeast with ADHI promoter and terminator and a TRPI marker. Plasmid pGADGH was digested with SnaBI and HindIB and a 1.8 kb 5 fragment containing the ADHI promoter isolated. This fragment was ligated into the vector pRS405 (Stratagene, La Jolla, CA) previously digested with Smal and HindIII. Positive clones were identified by insertional-inactivation of the plasmid-encoded lacZ alpha peptide and the presence of the ADHI promoter fragment. The resulting plasmid (pMCK4) contained an ADHI promoter and a 10 LEU2 marker. be -0.2 kb NacI-EcoRI fragment from pGBT9 containing the ADH I terminator was ligated to EcoRI-HincII digested pRS403 (Stratagene, La Jolla, CA) to yield the -4.8 kb plasmid pRVN5. The -2.0 kb SnaBI-EcoRI fragment from pGAD/KAN2 containing the ADHI promoter was ligated to Smal-EcoRI 15 digested pRVN5 to yield the -6.8 kb plasmid pRVN6 with the ADH1 prornoter and terminator and a unique EcoRI cloning site in between. The OA kb HindMl fragment from pGADGH containing an additional XnnI site was deleted and the vector was relegated to yield the 7.0 kb vector pGAD-D3. Vector pGAD-D3 was digested with XrnnI and the -2.4 kb fragment 20 containing the ADHI promoter and terminator and an intervening HindlI cloning site was purified. The pRS404 vector (Stratagene, La Jolla, CA) was digested with Pvull and the larger 3.8 kb fragment with TRP1 was purified and ligated to the XnnI promoter and terminator fragment from pGAD-D3 to give plasmid pRVN11. 25 The open reading frames for dhaT, dhaB3, and dhaB1 were amplified from pHK28-26 (SEQ ID NO:1) by PCR using primers (SEQ ID NO:6 with SEQ ID NO:7, SEQ ID NO.8 with SEQ ID NO:9, and SEQ ID NO:2 with SEQ ID NO:3 for dhaT, dhaB3, and dhaBI, respectively) incorporating EcoRI sites at the 5' ends(10 mM Tris pH 8.3, 50 mM KCI, 1.5 mM MgC1 2 , 0.0001% gelatin, 30 200 pM dATP, 200 pM dCTP, 200 pM dGTP, 200 pM dTTP, 1 pM each primer, 1-10 ng target DNA, 25 units/niL Amplitaq DNA polymerase (Perkin Elmer Cetus, Norwalk CT)). PCR parameters were I min at 94*C, 1 min at 55"C, 1 min at 72*C, 35 cycles. The products were subcloned into the EcoR1 site of pHIL-D4 (Phillips Petroleum, Bartlesville, OK) to generate the plasmids pMP13, pMP14, 35 and pMP15 containing dhaT, dhaB3, and dhaBI, respectively. Constmction of plasmids for expression of dhaT The replicating plasmid pGAD/KAN2 was digested with EcoRI to remove the kanamycin resistance fragment, dephosphorylated, and ligated to the dhaT 30 EcoRI fragment from pMP13. The resulting plasmid (pMCK13) had dhaT correctly oriented for transcription from the ADH I promoter and contained a LEU2 marker. Plasmid pRNV6 was digested with EcoRI and ligated to the dhaT EcoRi 5 fragment from pMP13. The resulting plasnmid (pRVN6T) had dhaT correctly orientated for transcription from the ADHI promoter and contained a HIS3 marker. Contction-of plasmids for exrsionof dhaB I The replicating plasmid pGADOH was digested with Hindl, 10 dephosphorylated, and ligated to the dhaB1 HindI fragment from pMP15. The resulting plasmid (pMCKI0) had dhaBl correctly oriented for transcription from the ADH1 promoter and contained a LEU2 marker. Construction of plasmids for expression of dhaBf2 The replicating plasmid pMCK1 1 was digested with EcoRI, 15 dephosphorylated, and ligated to the dhaB2 EcoRI fragment from pMP20. The resulting plasmid (pMCK17) had dhaB2 correctly oriented for transcription from the ADHl promoter and contained a TRPI marker. Plasmid pRS403 was digested with Smal and ligated to a SnaBI/Nael dhaB2 fragment from pMCK17. The resulting plasmid (pMCK21) had dhaB2 20 correctly orientated for transcription from the ADHI promoter and contained a HIS3 marker. Construction of plasmids for expression of dhB3 The replicating plasmid pYES2 (Invitrogen, San Diego, CA) was digested with EcoRI, dephosphorylated, and ligated to the dhaB3 EcoRI fragment from 25 pMP14. The resulting plasmid (pMCK1) had dhaB3 correctly oriented for transcription from the GAL promoter and contained a URA3 marker. The replicating plasmid pGAD/KAN2 was digested with EcoRL dephosphorylated, and ligated to the dhaB3 EcoRI fragment from pMP14. The resulting plasmid (pMCK15) had dhaB3 correctly oriented for transcription from 30 the ADHI promoter and contained a LEU2 marker. Plasmid pRS404 was digested with Pst and HincII and ligated to the Psti/EcoRV dhaB3 fragment from pMCK15. The resulting plasmid (pMCK2O) had dhaB3 correctly orientated for transcription from the ADHI promoter and contained a TRP1 marker. 35 Transformation of S. cerevisiae with dha expression Diasmids S. cerevisiae strain YPH499 (ura3-52 lvs2-801 ade2-101 trpl-del63 his3-del200 leu2-dell) (Stratagene, La Jolla, CA) was transformed with 1-2 pg of plasmid DNA using a UCl/polyethylene glycol protocol published by Stratagene 31 (Catalog #217406). Alternatively, transformation was achieved using a Frozen-EZ Yeast Transformation Kit (Catalog #T2001) from Zymo Researtif (Orange, CA). Colonies were grown on Supplemented Minimal Medium (SMM 0.67% yeast nitrogen base without amino acids, 2% glucose) for 3-4 days at 29*C 5 with one or more of the following additions adenine sulfate (20 mgIL), uracil (20 mg/L), L-tryptophan (20 mg/L), L-histidine (20 mg/L), I-leucine (30 mg/L), L-lysine (30 mg/L). Colonies were streaked on selective plates and used to inoculate liquid media. Depending on the vector used, colonies arose either after integration of plasmid DNA or from replication of an episode. In addition to 10 transformations with single plasmid types, co-transformations with two or more plasmids were carried out. Expression of dhaB activity in transformed S. cerevisiae Strain YPH499 transformed with plasmids pMCKI, pMCKIO and pMCK17 was grown on Supplemented Minimal Medium containing 0.67% yeast 15 nitrogen base without amino acids, 2% galactose, 2% raffmose, 20 mg/L adenine sulfate, 30 mg/L L-lysine and 20 mg/L histidine. Cells were homogenized and extracts assayed for dhaB activity. A specific activity of 0.021 units per mg was obtained. EXAMPLE IQ 20 Construction of altemate replicating and interaction plasmids for the transformation of S. cerevisiae A general purpose expression plasmid is constructed by isolating a SnaBI/EcoRI ADH 1 promoter fragment from pGAD/KAN2 and ligating this fragment into the vector pRS406 (Stratagene, La Jolla, CA) previously digested 25 with HincH and EcoRI. Positive clones are identified by insertional-inactivation of the plasmid-encoded lacZ alpha peptide and the presence of the ADHI promoter fragment. The resulting plasmid (pMCK3) is digested with EcoRI and SmaIand ligated to the 0.2 kb ADHI terminator fragment released from plasmid pGBT9 by digestion with EcoRI and NaeI. The resulting plasmid (pMCK5) 30 contains both ADHI promoter and terminator sequences and a URA3 marker. Construction of plasmids for expression of dhaT The vector pMCK5 is digested with EcoRI and dephosphorylated. 'he dhaT gene is excised as an EcoRI fragment from plasmid pMP13 and ligated to pMCK5. The resulting plasmid (pMCK7) has dhaT correctly orientated for 35 transcription from the ADH1 promoter and contains a URA3 marker. The integration vector pRS404 is digested with KpnI and Sac. The dhaT gene with flanking promoter and terminator is excised as a KpnI/SacI fragment from plasmid pMCK7 and ligated to pRS404. The resulting plasmid has dhaT 32 correctly orientated for transcription from the ADHI promoter and contains a. TRP1 marker. Construction of plasmids for expression of dhaBI The vector pMCK5 is digested with EcoR, and dephosphorylated. The 5 dhaBl gene is excised as an EcoRI fragment from plasmid pMP15 and ligated to pMCK5. The resulting plasmid (pMCK8) has dhaB1 correctly orientated for transcription from the ADHI promoter and contains a URA3 marker. The integration vector pRS403 is digested with Cal and AatHI. The dhaBl gene with flanking promoter and terminator is excised as a CI/Aatfl 10 fragment from plasmid pMCKS and ligated to pRS403. The resulting plasmid has dhaBI correctly orientated for transcription from the ADHI promoter and contains a HIS3 marker. The replicating plasmid pYES2 is digested with HindIII and SnaBI, and the GALl promoter element is replaced by ligation with a SnaBI and HindlI 15 digested ADHI promoter fragment from pGADGH. A dhaBi HindHI and XbaI fragment from pMP19 is ligated to those sites in the modified, ADHI promoter version of pYES2. The resulting plasmid has dhaB1 correctly oriented for transcription from the ADHI promoter and contains a URA3 marker. The vector pMCK4 is digested with HindI and dephosphorylated. The 20 dhaB1 gene is excised as an Hinm fragment from plasmid pMP15 and ligated to pMCK4. The resulting plasmid has dhaB1 correctly orientated for transcription from the ADHi promoter and contains a LEU2 marker. Construction of plasmids for expression of dhaB2 The vectors pRS404, pRS405 and pRS406 are digested with SmaI. The 25 dhaB2 gene with flanking promoter and terminator is excised as a SnaBI/NaeI fragment from plasmid pMCK17 and ligated to each of the integration vectors. The resulting plasmids have dhaB2 correctly orientated for transcription from the .ADHI promoter and contain either the LEt2 TRP1, or URA3 markers. EXAMPLB11 30 Screenine of S. cerevisiae for dha transformants and conversion of D-alucose to 1.3-propanediol Screening of S. cerevisiae for dha enes Chromosomal DNA from Ura+, His+, Trp+ or Leu+ transformants, constructed as described in Examples 9 and 10, is analyzed by PCR using primers 35 specific for each gene, as described for Pichia pastoris (SEQ ID NO:2-9). 33 Production of 1,3-propanediol from D-glucose by S. cerevisiae transformed with dha genes Transformants containing dhaT, dhaBl, dhaB2 and dhaB3, constructed as described in Examples 9 and 10, are grown aerobically or anaerobically with shaking 5 at 29*C in SMM supplemented with 20 mg/L adenine sulphate, 30 mg/L L-lysine, I mg/L vitamin B 12 . Growth continues until stationary phase is reached and the presence of 1,3-propanediol is determined by HPLC. Transformant S. cerevisiae pMCKI/10/1 7(HM)#A was deposited and designated ATCC . EXAMPLES 12 TO 21 10 These examples and the tables referred to therein are described in the parent specification. EXAMPLE 22 Construction of expression cassettes for expression of dhaB1, dhaB2, dhaB3 and dhaT in Aspergillus niger 15 General expression cassette (pAEX): The 1.4 kb Spe- I -Eco RV fragment from the plasmid pGPTpyrG (Berka et al., "The development of gene expression systems for filamentous fungi", Biotechnol. Adv., 7:127-154 (1989)), containing sufficient portions for proper 12/12/07,atl3772.div spec pgs,7 34 regulation of the Aspergillus niger gla A promoter and terminator, was ligated into the Spel and EcoRV sites in the polylinke of pLITMUS39 (New Englkand Biolabs, Beverly, MA). Individual clone expression cassettes for A. niger 5 The open reading frames (ORF's) for individual Klebsiella pnewnoniae dhaB subunits and dhaT were cloned and lighted into the general expression vector (pAEX) separately, using the same cloning strategy: Primer pairs for PCR amplification of each individual dhaB ORF and the dhaT ORF were designed to match the 5' and 3'ends sequence for each ORF 10 based on known sequence of the entire gene operon (dhaB 1, dhaB2, dhaB3, dhaBX and dhaT: SEQ ID NO:38 and 12,39 and 40,41 and 42,45 and 46,43 and 44, respectively). In addition to the matching sequence, the primers for the 5' end of each ORF were designed to include an EcoRI restriction site followed by a Bgl 11 restriction site at the 5' most end of t sequence as well as the five base 15 sequence CAGCA upstream of the first ATO of each ORF. Primers designed to match the 3 ends of each ORF placed an Xbal restriction site downstream of the translation stop codon, at the 3' most end of the clone. Individual clone fragments for the dhaB and dhaT ORF's were amplified by PCR from the plasmid pHK26-28, containing the entire K. pneumoniae dha 20 operon, using the primers described above. The individual ORF clone fragments were isolated based on their respective molecular weights (dhaB 1 = 1540 bp; dhaB2 =607 bp; dhaB3 = 448 bp; dhaBX= 1846 bp; dhaT = 1187 bp). Using the unique EcoRI and Xbal restriction sites designed in the PCR primers, each individual dhaB and dhaT ORF fragment was ligated into the EcoR1 and Xbal 25 restriction sites in the polylinker of pLITMUS29 (New England Biolabs). The dhaB2 and dhaB3 clones in pLIMUS29 were confirmed to be correct by sequencing. A unique 1363 bp Ncol-EcoRV restriction fragment from the coding region of dhaBI clone in pLITMUS29 was removed and replaced with the conesponding restriction fragment from pHK26-28. A unique 783 bp Tthi 11 30 I-Mlu I restriction fragment from the coding region of dhaT clone in pLTMUS29 was replaced with the corresponding restriction fragment from pHK26-28. A unique 1626 bp EcoRV restriction fragment from the coding region of dhaBX clone in pLITMUS29 was replaced with the coresponding restriction fragment from pM7 (containing the K. pneumoniae dhaB operon). The 5' and 3'end 35 sequences of the dhaB1, dhaBX and dhaT clones, approximately 250 bp which includes some sequence from the substituted fragment, was confirmed to be correct by sequencing. 35 The unique Bgl H-Xbal restriction fragments containing the ORF's of dhaB1, dhaB2, dhaB3, dhaBX and dhdT clones in piLTMUS29 were ligatecda u the Bgl fl-Xbal restriction sites in the general expression vector pAEX separately, placing expression of each clone under the control of the A. niger glaA promoter 5 and terminator. Each resulting vector was named by the respective ORP, i.e.; pABXdhaB1, pAEXdhaB2, pAEX-dhaB3, pAEXdhaBX and pAEXdhaT. Dual expression cassette vectors for A. niger. The unique SnaB I-Stul restriction fragment containing the dhaB 1 expression cassette (consisting of the A. niger glaA promoter, the dhaB I ORF, 10 and teminator) was isolated from the vector pAEX:dhaB 1 and ligated into the unique SnaBI restriction site in the pAEXdhaB2 vector. The approx. 2.2 kb Scal-Smal restriction fragment from pBH2 (Ward et. al., Exp. Myc., 13, 289 (1989)) containing the Aspergillus nidulans pyrG auxotrophy selectable marker, was ligated into the unique Stul restriction site in the vector containing the dhaB 1 15 and dhaB2 expression cassettes. This vector was named pAEX:B l+B2. The unique Spel-Hind IM restriction fragments containing the entire expression cassettes for dhaB3 and dhaT were isolated from the respective pAEX-dhaB3 and pAEXdhaT vectors. The two expression cassette fragments were simultaneously ligated, in tandem, in the unique Hind M restriction site in 20 the vector pUCIS. This vector was named pAEXB3+T. Transformation, isolation of transformants. confirmation of intearation of expression cassettes and expession of dhaR and dhaT genes in Asperrillus Aspergillis niger strain FSI (pyrG~) was co-transformed with the two expression vectors pAEX:B 1+B2 and pABX:B3+T using the method of 25 (Campbell et al., "Improved transformation efficiency of A. niger using homologous niaD gene for nitrate reductase", Curr. Genet., 16:53-56 (1989)). Transformants were selected for by their ability to grow on selective media without uridine. Genomic DNA of transformants was digested with Hind I and Spel to liberate fragments of predicted molecular weights, demonstrating 30 integration of intact expression cassettes. Detection of each expression cassette was done by Southern analysis, probing with individual genes separately. The presence of the dhaB2 protein was detected by western analysis using anti-dhaB antibody. Expression of each ORF was tested by growing transformants, that have 35 the pAEX:BI+B2 and pAEX:B3+T vectors integrated, in 10% CSL media (corn steep liquor (50% solids), 10% (w/v); NaH 2 POiH 2 0, 1.0 g/L; MgSO 4 , 0.50 g/L; maltose, 100.0 g/L; glucose, 10.0 g/L; and Mazu Antifoam, 0.003% (v/v)) as a seed culture then transferring 1/10 volume of the seed culture to MBM carbon 36 media (NaH 2

PO

4 , 0.70 g/L; K 2 1P0 4 , 0.70 g/L; KH 2 P0 4 , 0.70 g/L; MgSO 4 *7H 2 0, 1.40 g/L; (NH4) 2

SO

4 , 10.5 g/L; CaaI2H 2 0, 0.70 g/L; NH 4 NG, 3.50 g/L; sodium citrate, 14 g/L; FeCl 2 4H 2 0, 1.0 mg/L; ZnC1 2 , 5.87 mg/L; CuaQ2H 2 0, 0.42 mg/L; Mnai4H 2 0, 0.21 mg/L; Na 2

B

4 0'10H 2 0, 0.07 mg/L; 5 folic acid, 0.174 mg/L; pyridoxineliCi, 6.12 mg/L; riboflavin, 1.83 mg/L; pantothenic acid, 23.60 mg/L; nicotinic acid, 26.66 mg/L; biotin, 0.49 mg/L; thiarninelI, 1.39 mg/L; maltose, 120.0 g/L; carbenicillin, 0.035 mg/L; streptomycin, 0.035 mg/L; tween 80. 0.07% (w/v); and Mazu antifoam, 0.14% (v/v)) for induction of the glaA promoter. mRNA was isolated from transfornant 10 cultures (Fast Track 2 Kit, Invitrogen Corp.) and Northern analysis performed with chemiluminescence (Genius* System, Boehringer-Mannheim) to detect transcribed message from each gene. Co-ordinate transcription of the dhaB 1, dhaB2, dhaB3 and dhaT genes in shakeflask cultues was demonstrated by Northern hybridizaton, probing with gene fragments of each dhaB and dhaT 15 ORF. Isolated colonies shown to transcribe all of the transformed genes were chosen to be further transformed with pAEX-dhaBX. These isolates were co transformed with pAEXdhaBX and pAAIO (3.2kb. Acc1-Asp718 restricton fragment containing the Aspergillus nidulans amdS selectable marker in pUC18). 20 These newly transformed cultures were selected for on media containing acetanide as the sole carbori source. Transformant colonies able to utilize acetamide as a sole carbon source were demonstrated to have the dhaBX ORF integrated by PCR amplification of the dhaBX ORF from gcnomic DNA using primers KpdhaBX-5' and KpdhaBX-3'(SEQ ID NO:45 and 46). 25 Production of givcerol by A. Niger. strain FSI Aspergillus niger strain FSI was grown in 10% CSL media as a seed culture and transferred as a 1:10 dilution to MBM carbon media + 12% maltose. Culture supernatent was demonstrated to contain 6 g/L glycerol produced by Aspergillus. Analysis of glycerol was done by HPLC. 30 Production of 1.3-propanediol by recombinant A nier Aspergillus fermentations were carried out in 15.5 L total volume Biolafitte fermenters, working volume initially 8 L, increasing to I I L during the run. Aerobic conditions were insured by aeration with air at a rate of 10 Lmin., at an impeller speed of 700-800 rpm and a back-pressure of 1.1 bar (aerobic 35 conditions are defined by the % Dissolved Oxygen (100% DO defined at ambient pressure), measured with installed DO-probes; a minimal value of 35% DO was considered aerobic). The pH was maintained at 5.60 by automatic addition of 10% H 3

PO

4 or 28% NH 4 OH. Temperature was maintained at 32*C. 37 The following compounds were batched into the tank and sterilized at 121*C for 30 min.: 2 g/L NaH 2

PO

4

H

2 0, 17 g/L (Ni 4

)

2

SO

4 , 1 g/L MgSO 4 , 2 g/L Tween 80,45 g/L Promosoy-100 (a soy concentrate of 70% protein), 6 g/L con steep liquor (50% solids), 10 gIL maltose, and 2 g/L MAZU DF204 (a 5 custom-made antifoam). After sterilization, 500 gram of the 50% Maltrin 150 feed was added, together with carbenicillin and streptomycin (both up to a final concentration of 10 mg/L). One liter of a 45 h old Aspergillus niger strain (strain TGR40) transformed with the two expression vectors pAEX:B1+B2 and pABXB3+T, growing in a 10 shakeflask containing 10% CSL was used to inoculate the fermenter. The culture was then allowed to grow batchwise, fully aerobic, for 28 h before a feed (a 50% Maltrin 150 solution, heat sterilized) was started at a rate of 0.8-1.0 g/min. The culture was then run for another 20 h, during which the %DO dropped to virtually zero because of the 0 2 -demand of the cells (the culture remained at zero to 5% 15 throughout the rest of the run). After that (48 h after inoculation), glycerol was fed in over a period of 8 h, up to a final glycerol concentration of 163 g/L. The maltrin feed was stopped 97 h after inoculation, backpressure and aeration lowered to respectively 0.2 bar and 4 Lmin. (0.5 vvM), and co-enzyme B 12 added to a final concentration of 10 mg/L. When the culture was 122 h old, broth 20 was harvested, centrifuged, and 0.2 L of ethanol added to 1 L of supernatant. One L of cell-free fermentation broth was vacuum-distilled, yielding about 60 rnL of a dark slurry. The slurry was centrifuged, and about 40 rmL of liquid supernatant were collected. This liquid was then treated with 40 nL of ethanol in order to precipitate out residual solids, which were removed by centrifugation. A 25 small sample of the decanted liquid was analyzed by HPLC and found to contain 1,3-propanediol: the identity of the propanediol was confinned by GC/MS. Applicants have deposited a recombinant Aspergillus niger strain TGR40-13, comprising a DNA fragment encoding dhaB(1-3), dhaBx and dhaT (ATCC:74369). 30 EXAMPLE 23 Production of 1.3-provanediol from maltose using recombinant A. niger Aspergillus fermentations are carried out in 15.5 L total volume Biolafitte fermenters, working volume initially 8 L, increasing to 11 L during the run. Aerobic conditions are insured by aeration with air at a rate of 10 1./min, at an 35 impeller speed of 700-800 rpm and a back-pressure of 1.1 bar (aerobic conditions are defined by the % Dissolved Oxygen (100% DO defined at ambient pressure), measured with installed DO-probes; a minimal value of 35% DO was considered 38 aerobic). The pH is maintained at 5.60 by automatic addition of 10% H 3 PO or 28% NH40H. Temperature is maintained at 320C. The following compounds are batched into the tank and sterilized at 121"C for 30 min: 2 g/L NaH 2 POjH 2 0, 17 g/L (NH4) 2 S04, 1 g/L MgSO 4 , 2 g/L 5 Tween 80,45 g/L.Promosoy-100 (a soy concentrate of 70% protein), 6 g/L com steep liquor (50% solids), 10 g/L maltose, and 2 g/L MAZU DF204 (a custom made antifoam). After sterilization, 500 gram of the 50% Maltrin 150 feed is added, together with carbenicilin and streptomycin (both up to a final concentration of 10 mg/L). 10 One L of a 40-48 h old Aspergillus niger strain transformed with dhaB 1, dhaB2, dhaB3, dhaB4 and dhaT genes, growing in a shakeflask containing 10% CSL (defined in Example 22), is used to inoculate the fermenter. The culture is then allowed to grow for 30-35 h before the feed (a 50% Maltrin 150 solution, heat sterilized) is started, at a rate of I g/nin. The culture is then run for another 15 5 h under 02 limited conditions (%DO zero, under full aeration). After that, the Maltrin feed is stopped and when the measured glucose in the supernatart is virtually zero, the rpm is lowered to 150, the BP to 0.2, and aeration is stopped. The fermenter is flushed with an anaerobic gas-mixture (5% H 2 , 5% C02,90%

N

2 ) at a rate of 7 /min for 30 min. Gas inlet and outlet is then closed, BP is 20 maintained at 0.4 bar, and co-enzyme B 12is added to a final concentration of 5 mg/L Throughout, broth samples are centrifuged and the supernatants are prepared for HPLC and GC analysis. 1,3-propanediol is detected in the supernatant. 39 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group 5 of integers or steps. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form or suggestion that the prior art forms part of the common general knowledge in Australia. 12/12/07,at13772.div spec pgs,7 60

Claims (2)

1. A recombinant microorganism comprising a host cell selected from the group consisting of yeast and filamentous fungi and expressing a diol dehydratase or 5 a glycerol dehydratase.

2. A substantially pure culture of a microorganism in accordance with that deposited under Accession No. ATCC 69790. 02/11 .k7067elaimsdoc,61 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007 2007249075 18 Dec 2007