WO1999028481A1

WO1999028481A1 - Microbial production of hydroxyacetone and 1,2-propanediol

Info

Publication number: WO1999028481A1
Application number: PCT/US1998/025318
Authority: WO
Inventors: Douglas C. Cameron; Anita J. Shaw; Michael L. Hoffman
Original assignee: Wisconsin Alumni Research Foundation
Current assignee: Wisconsin Alumni Research Foundation
Priority date: 1997-02-19
Filing date: 1998-11-30
Publication date: 1999-06-10
Anticipated expiration: 2000-06-03

Abstract

Genetically-engineered yeast which ferment utilizable carbon sources into acetol and/or 1,2-propanediol, methods to produce acetol and 1,2-propanediol by fermentation using the transformed yeast, and synthetic operons to effect the transformation are disclosed.

Description

MICROB IAL PRODUCTION OF HYDROXYACETONE AND 1 , 2-P OPANEDIOL

This is a Continuation-In-Part of co-pending application Serial No. 08/801,344, filed February 19, 1997.

FIELD OF THE INVENTION

The invention is drawn to genetically-engineered yeast and their use in the production of acetol and 1,2-propanediol. More specifically, the present invention is drawn to genetically-engineered yeast having recombinant enzymatic activity which enables the yeast to ferment sugars into acetol and/or 1,2-propanediol in isolatable quantities.

BIBLIOGRAPHY Complete bibliographic citations to the references mentioned below are included in the Bibliography section, immediately preceding the Sequence Listing. Each of the references cited below is incorporated herein by reference in its entirety.

DESCRIPTION OF THE PRIOR ART 1,2-Propanediol (1,2-PD; also known as propylene glycol) is a major commodity chemical with an annual production greater than one billion pounds in the United States. The major utilization of 1,2-PD is in unsaturated polyester resins, liquid laundry detergents, pharmaceuticals, cosmetics, antifreeze and de-icing formulations.

1,2-PD is conventionally produced from petrochemicals. Unfortunately, several toxic chemicals, such as chlorine, propylene oxide, and propylene chlorohydrin are either required or are produced as by-products in the conventional synthesis. In the conventional route, 1,2-PD is produced by the hydration of propylene oxide, which is obtained from propylene. The synthetic process produces racemic 1,2-PD, an equimolar mixture of the two enantiomers. This chemical process has a number of disadvantages.

The major problem with the conventional synthetic route to 1,2-PD arises in the production of its intermediate, propylene oxide.

Propylene oxide is manufactured by one of two standard commercial processes: the chlorohydrin process or the hydroperoxide process. The chlorohydrin process involves toxic chlorinated intermediates and the use of caustic or lime. Additionally, this process may result in air emissions of propylene chlorohydrin and chlorine. (Franklin

Associates, Ltd. (1994).) The hydroperoxide process involves oxidation of propylene by an organic hydroperoxide and results in the stoichiometric co-production of either tert- butanol or 1-phenyl ethanol. This make the economics of the production of propylene oxide via the hydroperoxide route directly related to the market for the co-produced byproducts. (Gait (1973).)

It is known that 1,2-PD is produced by several organisms when grown on exotic sugars. As early as 1937, the fermentation of L-rhamnose to 1,2-PD (later shown to be the S enantiomer) was described by Kluyver and Schnellen (1937). In E. coli and a variety of other microorganisms, L-rhamnose and L-fucose are metabolized to L- lactaldehyde and dihydroxyacetone phosphate. (Sawada and Takagi (1964) and

Ghalambor and Heath (1962), respectively.) Under aerobic conditions, L-lactaldehyde is oxidized in two steps to pyruvate (Sridhara and Wu (1969)). Under anaerobic conditions, however, L-lactaldehyde is reduced to S-1,2-PD by a nicotinamide adenine nucleotide (NAD)-linked 1,2-propanediol oxidoreductase (EC 1.1.1.77). The S-1,2-PD produced diffuses into the extra-cellular medium.

Although a variety of microorganisms, including E. coli, produce S-1,2-PD from

6-deoxyhexose sugars, Obradors et al. (1988), this route is not commercially feasible because these sugars are extremely expensive. The least expensive of these 6- deoxyhexose sugars, L-rhamnose, currently sells for approximately $325 per kilogram

(Pfanstiehl Laboratories, Chicago, Illinois).

In the mid-1980's, organisms capable of fermenting sugars such as glucose and xylose to R-1,2-PD were discovered. See, for instance, Tran-Din and Gottschalk (1985). Clostridium sphenoides produces R-1,2-PD via a methylglyoxal intermediate. In this pathway, dihydroxyacetone phosphate (DHAP) is converted to methylglyoxal (MG) by the action of methylglyoxal synthase. The MG is reduced stereospecifically to give D- lactaldehyde. The D-lactaldehyde is then further reduced to give R-1,2-PD. The commercial production of 1,2-PD by C. sphenoides is limited, however, by the fact it is only produced under phosphate limitation; it is both difficult and expensive to obtain commercial-grade medium components which are free of phosphate. Additionally, only low titers of 1,2-PD are achieved.

Thermoanaerobacteriwn thermosaccharolyticum HG-8 (formerly Clostridium thermosaccharolyticum, ATCC 31960) also produces R-1,2-PD via methylglyoxal.

Cameron and Cooney (1986). As with C. sphenoides, DHAP is converted to MG. The MG is then reduced at the aldehyde group to yield acetol (i.e., hydroxyacetone). The acetol is then further reduced at the ketone group to give R-1,2-PD. For both C. sphenoides and T. thermosaccharolyticum HG-8, the enzymes responsible for the production of 1,2-PD have not been isolated or cloned.

SUMMARY OF THE INVENTION

A first embodiment of the invention is a method of producing acetol and 1,2-PD by yeast fermentation. The method comprises culturing a genetically-engineered yeast which expresses a recombinant enzyme which enables the yeast to produce acetol, 1,2-

PD, or both, in a medium containing a suitable carbon source. By doing so, acetol and

1,2-PD are produced by the yeast and secreted into the extracellular environment.

More specifically, the preferred first embodiment of the invention is drawn to a method of producing acetol and 1,2-PD by fermentation using genetically-engineered yeast which comprises culturing a genetically-engineered yeast in a medium containing a suitable carbon source such as arabinose, glucose, galactose, lactose, xylose, sucrose, starch, and the like, wherein the yeast expresses a recombinant gene encoding methylglyoxal synthase. By doing so, the carbon source in the medium is fermented by the yeast into acetol and 1,2-PD, which can be isolated from the medium. A second embodiment of the invention is drawn to genetically-engineered yeast which ferment a suitable carbon source into acetol and 1,2-PD. The genetically- engineered yeast expresses one or more recombinant enzymes which enable the genetically-engineered yeast to produce acetol and/or 1,2-propanediol in isolatable quantities. In the preferred second embodiment, the genetically-engineered yeast express recombinant methylglyoxal synthase activity.

A third embodiment of the invention is drawn to a synthetic operon which enables the production of acetol and 1,2-PD in yeast. The operon comprises one or more genes whose encoded gene products catalyze the formation of methylglyoxal in yeast and a promoter sequence functional in yeast operationally linked to the one or more genes.

The preferred synthetic operon comprises, in 5' to 3' order, a CUP1 promoter, operationally linked to a gene encoding methylglyoxal synthase, operationally linked to a CYC1 terminator.

Overall, the preferred embodiment of the invention is drawn to the use of genetically-engineered yeast, preferably genetically-engineered S. cerevisiae, which express recombinant methylglyoxal synthase activity to produce acetol and 1,2-PD. The invention utilizes genetically-engineered yeast which express enzymes which enable the production of acetol and 1,2-PD from the fermentation of carbon sources utilizable by the yeast. As used herein, the term "suitable" or "utilizable" carbon sources refers to carbon sources utilizable by conventional and genetically-engineered yeast including, but not limited to, L-arabinose, D-glucose, D-galactose, D-xylose, lactose, sucrose, starch, and the like. With particular reference to D-xylose as a utilizable carbon source, there is known genetically-engineered yeast which utilize xylose as a carbon source. See, for instance, Ho et al. (1993).

As noted above, while not being limited to a particular cellular mode of action, it is thought that by increasing the activity of enzymes which catalyze the formation of MG, the intracellular pool of MG is increased. The increased amount of MG then triggers cellular mechanisms which enzymatically reduce the MG to acetol and 1,2-PD. The acetol and 1,2-PD are then secreted into the extracellular environment. A major advantage of the present invention is that microbial fermentation provides a clean and "environmentally friendly" synthetic route to acetol and 1,2-PD. The microbial process can use as a substrate a renewable sugar such as glucose or xylose

(found in agricultural crops) or lactose (found in dairy industry wastes). Suitable carbon sources are also produced in commodity amounts from corn and sugar cane and from lignocellulosic biomass.

Also, the microbial process produces no toxic wastes. The byproducts of fermentation are carbon dioxide, alcohols, and organic acids, all of which can be purified as valuable co-products or used as animal feed. Another distinct advantage of the invention is that it provides a unique route to acetol and 1,2-PD from readily-available sugars or starch. These carbon sources are cheap, renewable, and readily available.

A further advantage of the present invention is that microbial processes are straightforward to operate and do not involve high temperatures and pressures. Large fermentation facilities such as those used for the production of ethanol can be readily adapted to the production of acetol and 1,2-PD.

The maximum theoretical yield of acetol and/or 1,2-PD from a sugar carbon source is favorable: on the order of about 1.0 moles or more of acetol or 1,2-PD per mole sugar. And, unlike n-butanol, 1,2-PD has very low toxicity to microorganisms. This allows for good cellular growth and viability at high final product titers. Cellular growth in the presence of 100 g/L 1,2-PD has been obtained.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic representation of the production of 1,2-PD according to the present invention.

Fig. 2 is an HPLC elution profile of a standard solution of glucose, glycerol, 1,2- PD, 2,3-butandiol, and ethanol. The HPLC protocol used was the same as that described in Examples 4-6. Fig. 3 is an HPLC elution profile of a standard solution of glucose, succinate, acetate, acetol, and ethanol. The HPLC protocol used was the same as that described in Examples 4-6.

Fig. 4 is an HPLC elution profile of culture medium taken from genetically- engineered yeast transformed and cultured as described in Example 4. In this graph, acetol and 1,2-PD display a retention times of 21.517 minutes and 20.417 minutes, respectively.

Fig. 5 is an HPLC elution profile of culture medium from the negative control yeast described in Example 3. Fig. 6 is an ultraviolet spectrum of a standard acetol solution. The spectrum displays a characteristic absorption at 263.8 nm.

Fig. 7 is an ultraviolet spectrum of the isolated peak labelled "acetol" in Fig. 4. This ultraviolet spectrum confirms the identity of the 21.517 minute peak in the HPLC elution profile of Fig. 4 as being acetol. Fig. 8 is a graph depicting the level of glucose (o), 1,2-PD (-), biomass (dry cell weight, DCW) (•) and acetol (x) over time in a culture of yeast transformed according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations:

The following abbreviations are used consistently throughout:

O OH acetol = hydroxyacetone - H₃C— C— CH₂

DHAP = dihydroxyacetone phosphate -

G-3-P = glyceraldehyde-3-phosphate -

O O

MG = methylglyoxal - ._. _c_ _Jl_Lj MGS = methylglyoxal synthase enzyme mgs = a gene encoding methylglyoxal synthase activity

OH OH 1,2-PD = 1,2-ρroρanediol - I I

H₃C CH CH2

TPI = triose phosphate isomerase

As used herein, the term "yeast" explicitly encompasses, but is not limted to, microorganisms of the genus Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluveromyces, Candida, Hansenula, Debaryomyces, and Torulopsis. Examples include Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Overview:

An abbreviated schematic diagram of sugar metabolism resulting in the production of acetol and 1,2-PD according to the present invention is shown in Fig. 1. It is hypothesized that in yeast transformed according to the present invention, sugar is metabolized into DHAP and G-3-P by glycolytic enzymes common to most organisms. Together, DHAP and G-3-P are referred to as triose phosphates. Under normal conditions, the triose phosphates are interconverted by the activity of TPI.

In the preferred embodiment, DHAP is the initial intermediate in the acetol/ 1,2- PD pathway. By transforming yeast to express a recombinant MGS activity, DHAP is converted to MG. While not being limited to a particular cellular mechanism, it is believed that in response to the increased intracellular level of MG, the transformed yeast detoxify the MG by a reductive pathway leading to acetol and then to 1 ,2-PD, which then diffuses or is actively secreted from the cell. Regardless of the actual mechanism, the present inventors have shown that by expressing recombinant MGS activity in yeast, the yeast will produce both acetol and 1,2-PD in isolatable quantities.

The crux of the invention, therefore, is a method to produce acetol and/or 1,2-PD using genetically-engineered yeast which express recombinant enzyme activities whereby the yeast produce intracellular MG (or whereby the yeast produce increased amounts of intracellular MG as compared to non-transformed yeast). The MG is then converted into acetol and 1,2-PD. The acetol and 1,2-PD so formed may then be harvested from the cell culture medium.

The first step of the process is to identify and/or obtain the DNA sequences which encode the desired enzyme activities (i.e., activities leading to increased production of MG) and to insert them into the yeast. This can be accomplished by any means known to the art.

The preferred enzyme for the production of 1,2-PD by yeast is methylglyoxal synthase (MGS). The preferred form of MGS is that from E. coli. It must be noted, however, that because MGS is highly conserved, the source of the mgs gene is not critical to the present invention.

The gene which encodes the enzyme having the required activity is then incorporated into a suitable vector which is then used to transform a yeast host. The preferred host is S. cerevisiae (baker's yeast).

Incorporation of the gene into a plasmid vector is accomplished by digesting the plasmid with suitable restriction endonucleases, followed by annealing the gene insert to the plasmid "sticky ends," and then ligating the construct with suitable ligation enzymes to re-circulize the plasmid. Each of these steps is well known to those skilled in the art. (See, for instance, Sambrook, Fritsch, and Maniatis (1989), Molecular Cloning, A Laboratory Manual, incorporated herein by reference for its teaching of vector construction, and Kaiser et al. (1994) for transformation of yeast.)

Once successfully transformed with the required gene(s), the genetically- engineered yeast produce acetol and 1,2-PD from the fermentation of commonly utilized carbon sources, including arabinose, galactose, glucose, lactose, sucrose, xylose, and starch. Careful selection of mutant hosts can also be used to increase the yield of acetol and 1 ,2-PD. A TPI knockout mutant or a mutant having altered TPI activity can be used as the host cell to increase the intracellular levels of MG, thereby increasing acetol and 1,2-PD production. See, for instance, Pompliano et al. (1990) for a discussion on altering the specificity of TPI. Similarly, glyoxalase knockout mutants can also be used as host cells, thereby increasing the intracellular level of MG for conversion to acetol and

1,2-PD.

Appropriate host selection (using other mutants) also allows the conditions under which 1,2-PD is produced to be varied and/or optimized, e.g., aerobic or anaerobic production, different substrates as a carbon source, etc.

Isolation of the acetol and 1,2-PD formed from the cell medium can be accomplished by any means known in the separation art. The preferred method is to filter the culture medium to separate cells and cellular debris, and then to isolate the acetol and 1,2-PD from the medium by vacuum distillation. (See, for instance, Simon et al. (1987).) If so desired, the yeast may be completely lysed by any known means prior to isolation of the products.

Negative Controls

As noted in Example 3 below, yeast transformed to contain the preferred vector which contains an insert encoding green fluorescence protein rather than MGS do not produce acetol or 1,2-PD. See Example 3 and Fig. 5.

Vectors and Yeast Hosts:

In yeast, the preferred mgs gene is first inserted into an appropriate vector. YpJ66 is constructed from YEp352, whose oligonucleotide sequence is shown in SEQ.

ID. NO: 5., and can be constructed according to the method of Hill et al. (1986). In short, this is accomplished by inserting the CUP1 promoter, (galK) and CYC1 terminator sequence into the Xbal site of Yep352.

Preferably, the vector is then transformed into YPH500 (ATCC 76626) (leu , trp^', urd, lys^', ode^', his^') by standard methods and fed the required amino acids for growth, except uracil, which is used as the marker to maintain the plasmid in yeast. Yeast transformed to contain the mgs insert produce acetol and 1,2-PD in isolatable quantities when fermented on a wide variety of substrates. Any vector capable of successfully transforming yeast to express the required enzyme activity can be used in the present invention. The vector may be an integrating plasmid, in which case the recombinant gene is incorporated into the genome of the yeast host, or the vector may be replicating, in which case the recombinant gene might be present only on one or more copies of a self-replicating plasmid, or the required genetic elements may be placed on a yeast artificial chromosome (YAC). A large number of suitable vectors, including shuttle vectors, are known to the art. For standard techniques to integrate heterologous genes into yeast, see, for instance, Lee and Da Silva (1996).

Any number of yeast auxotrophic markers can be used, such as HIS3 (imidazole glycerolphosphate dehydratase), LEU2 (θ-isopropylmalate dehydrogenase), LYS2 (α- aminoadipate reductase), TRP1 (N-(5'-phosphoribosyl)-anthranilate isomerase), and URA3 (orotidine-5'-phosphate decarboxylase). Selection is accomplished by culturing the yeast in a suitable media lacking the required nutrient.

Replicating vectors include an autonomously replicating sequence (ARS) or a 2μ sequence to allow multiple copies of the plasmid to be replicated within each yeast cell.

The vector may also include a centromere sequence (CEN), which will generally limit the copy number of the plasmid vector to one or two per cell. (Again, see Lee and Da Silva (1996) for conventional integration methodology.) YAC's, which further include telomere sequences, may also be used to carry the recombinant genes necessary to induce the production of acetol and 1,2-PD by the transformed yeast.

Selection of suitable restriction endonucleases and ligases and appropriate laboratory procedures to insert the required gene(s) into the shuttle vector and to mobilize the vector into yeast are all well known to the art. See the Examples for illustrative protocols.

EXAMPLES The following Examples are included solely for illustrative purposes to provide a more complete understanding of the invention. The Examples do not limit the scope of the invention disclosed or claimed herein in any fashion. Example 1 - Plasmid Construction:

The DNA coding region for E. coli. MGS, shown in SEQ. ID. NO: 1, was amplififed by the polymerase chain reaction (PCR) using conventional and well known techniques. The PCR primers used are listed in SEQ. ID. NO: 2 (a Kpnl restriction site is defined by nucleotides 3-8: GGTACC) and SEQ. ID. NO: 3 (a EcoRI restriction site is defined by nucleotides 3-8: GAATTC). The nucleotide base sequence of the amplified fragment was confirmed by sequencing using conventional methods.

The PCR product was inserted, using restriction enzymes, into the expression vector YpJ66. YpJ66 is based on YEp352 and contains the CUP1 promoter and the CYC1 terminator for the expression of proteins in S. cerevisiae. YpJ66 is constructed from YEp352, whose oligonucleotide sequence is shown in SEQ. ID. NO: 4, according to the method of Hill et al. (1986). This is accomplished by inserting the CUP1 promoter, (galK), and CYC1 terminator sequence into the Xbal site of Yep352. YEP352 also contains the URA3 gene wich allows selection in a URA3- host strain. In more detail, starting with YEp352 (SEQ. ID. NO: 4), the EcoRI, Kpnl, Smal, and Hindlll sites are knocked out using T4 DNA polymerase. Then, a BamHI/EcoRI fragment of the genomic CUP1 promoter is ligated at the EcoRI site to a Clal/EcoRI fragment of the CYC1 terminator. This fragement is blunt-ended (BamHI and Clal blunt-ended with T4 polymerase) into the blunt-ended (T4 polyerase) Xbal site in YEp352, with the filled-in BamHI site of the insert furthest from the BamHI site in

YEp352 to yield YpJ66.

To insert the E. coli mgs gene into YpJ66, E. coli DNA was subjected to PCR using the primers depicted in SEQ. ID NOS: 2 and 3. The PCR amplification was digested with EcoRI/KpnI. The fragments were separated on an agarose gel. YpJ66 was also digested with EcoRI/KpnI and the fragments separated on an agarose gel. The digested PCR fragment was then ligated to the large fragment from YpJ66. This plasmid was named pMH36.

The CUP1 promoter is induced by copper and other heavy metals in fermentation media (Etcheverry, 1990). The pMH36 construct was designed so that the coding region of MGS is downstream from the CUPl promoter (i.e., in the 3' direction from CUPl) and upstream from the CYCl terminator (i.e., in the 5' direction from CYCl). The identity of the pMH36 construct was confirmed by restriction enzyme analysis on agarose gel. The complete nucleotide sequence of pMH36 is provided in SEQ. ID. NO: 5. All plasmid construction steps were performed in E. coli strain AG1 (Stratagene,

La Jolla, California). All molecular biology steps were performed using standard protocols (Sambrook, Fritsch, and Maniatis, 1989).

Example 2 - Yeast Transformation: Yeast strain YPH500 (ATCC 76626) (MATΆ ura3 lys2 ade2 trpl his3 leu2) was used for all of the Examples (Sikorski & Hieter, 1989). Yeast were transformed according to the lithium acetate method of Ito as modified by Kaiser et al. , 1994:

Yeast cultures were grown overnight in the medium defined below to an OD 600 of from about 0.3 to about 0.5. The cultures were then centrifuged at 4000 x g for 5 minutes and the cells resuspended in 10 mL sterile water. The cells were then centrifuged at 5000 x g for 5 minutes and the cells resuspended in 1.5 mL of a buffered lithium acetate solution (1 vol. lOx Tris/EDTA buffer, pH 7.5; 1 vol. lOx LiAc (1 M), pH 7.5; 8 vols. sterile water) and incubated for 1 hour at 30°C.

After incubation, 100 μL of the resuspended cells were mixed with 10 μL of calf thymus DNA (10 mg/mL) and about 5 μg of the DNA to be transformed in 10 μL sterile water. The mixture was incubated for 30 minutes at 30 °C. To the mixture was then added 0.7 mL PEG/TE/LiAc (1 vol. lOx Tris/EDTA buffer, pH 7.5; 1 vol. lOx LiAc (1 M), pH 7.5; 8 vols. 50% PEG 2000). This mixture was then incubated at room temperature overnight. The cells were then heat-shocked at 42°C for 5 minutes. The cells were then centrifuged and washed with 1 mL of Tris/EDTA, pH 7.5. The cells were again centrifuged and resuspended in 20 μL Tris/EDTA, pH 7.5. The cells were then plated out onto selective media and examined after 1 to 2 days for transformants. Example 3 - Negative controls:

As a negative control, yeast strain YPH500 was transformed with a plasmid designated pMHl. pMHl encodes (and expresses) green fluoresence protein in the same cassette as pMH36 (CUPl promoter and CYCl terminator). YPH500 strains transformed with pMHl and cultured under a variety of aerobic and anaerobic conditions showed no acetol or 1,2-PD production.

Fig. 5 shows an HPLC elution profile of culture medium from a culture of yeast transformed with pMHl. No acetol or 1,2-PD is evident in the elution profile. Yeast were cultured and HPLC analyses were performed as detailed in Examples 4-6.

Examples 4 through 6 - Production of Acetol and 1,2-PD: Standard Conditions:

All fermentations were performed in synthetic defined medium (SDM) containing: glucose, 20 g/L; (NH₄)₂SO₄, 5 g/L; KH₂PO₄, 1 g/L; MgSO₄, 0.5 g/L; NaCl, 0.1 g/L; CaCl₂, 0.1 g/L; BH₃O₃, 0.5 mg/L; CuCl₂, 0.005 mg/L; KI, 0.2 mg/L; FeCl₃, 0.2 mg/L;

MnSO₄, 0.4 mg/L; Na₂MoO₄, 0.2 mg/L; ZnSO₄, 0.4 mg/L; biotin, 2 mg/L; calcium pantothenate, 0.4 mg/L; folic acid, 2 mg/L; nicotinic acid, 0.4 mg/L; /.-aminobenzoic acid, 0.2 mg/L; pyridoxine hydrochloride, 0.4 mg/L; riboflavin, 0.2 mg/L; and thiamine hydrochloride, 0.4 mg/L. This media was supplemented with the required nutrients (lysine, adenine, tryptophan, histidine and leucine) to support the auxotrophs and maintain the plasmid (Kaiser et al., 1994). Copper (as CuCl₂) was added to the medium to induce the expression of MGS.

Anaerobic fermentations were performed in 15 mL anaerobic tubes; aerobic fermentations were performed in 250 mL baffled shake flasks. All fermentations were agitated by mixing at 200 rpm and maintained at 30°C.

All fermentation products were determined by HPLC according to the method of Tong et al. (1991). An organic acids column (Bio-Rad "HPX87H", Hercules, California) was used to measure acetol and 1,2-PD in the fermentation broth. The following conditions were used for the analysis: 50 mL injection size, H₂SO₄ mobile phase (pH 2), 0.5 ml/min flow rate, and 40°C column temperature. Peaks were detected by a 410 differential refractometer (Waters, Milford, Massachusetts), also at 40°C. Acetol and 1 ,2-PD produced in the fermentation broth was compared to a 1 ,2-PD standard solution (Sigma, St. Louis, Missouri).

HPLC elution profiles for standard solutions using the above protocol are depicted in Figs. 2 and 3. Fig. 2 is an HPLC elution profile of a standard solution containing glucose, glycerol, 1,2-PD, 2,3-butandiol, and ethanol. Fig. 3 is an HPLC elution profile of a standard solution containing glucose, succinate, acetate, acetol, and ethanol.

Example 4 - Anaerobic Production of Acetol and 1,2-PD:

An overnight culture of YPH500 yeast transformed with plasmid pMH36 was grown in SDM for 24 hrs. Aliquots (0.1 mL each) of this culture were then inoculated into a series of 10 mL cultures of SDM containing various levels of copper (0 to 0.6 mM) (in addition to the copper already present in the SDM) and allowed to ferment in anaerobic tubes for 60 hours at 30°C. The fermentation broth was collected and analysed using HPLC as described above. The results are shown in Table 1. The values given are the averages of three fermentations.

Table 1

Copper Concentration (mM) 1,2-PD Produced (g/L)

0 0.094

0.025 0.15

0.05 0.19

0.1 0.20

0.15 0.21

0.2 0.19

0.3 0.20

0.4 0.20

0.5 0.19

0.6 0.16 An HPLC elution profile of the culture medium from this Example is presented in Fig. 4. As shown in Fig. 4, 1,2-PD eluted from the column at 20.417 minutes, which is the same retention time displayed by the 1,2-PD standard shown in Fig. 2. In Fig. 4, acetol eluted at 21.517 minutes, which is slightly faster than the 21.650 retention time displayed by the acetol standard shown in Fig. 3.

To confirm the identity of the 21.517 minute peak in Fig. 4 as being acetol, the peak was collected and analyzed by ultraviolet spectroscopy against an acetol standard. The U.V. spectrum for the acetol standard is depicted in Fig. 6. Fig. 6 displays a very characteristic U.V. absorption at 263.8 nm. The U.V. spectrum for the 21.517 minute peak of Fig. 4 is shown in Fig. 7. These two U.V. spectra are virtually identical, indicating that the 21.517 minute peak is, in fact, acetol.

This Example clearly demonstrates that by transforming yeast to express recombinant MGS activity, the yeast will produce acetol and 1,2-PD.

Example 5 - Aerobic production of 1,2-PD:

An overnight culture of YPH500 yeast transformed with plasmid pMH36 was grown in SDM for 24 hrs. Aliquots (0.1 mL each) of this culture were inocculated into 25 mL cultures of SDM containing various levels of added copper (0 to 0.6 mM added copper) and allowed to ferment aerobically in 250 mL baffled flasks for 72 hours at 30°C. The fermentation broth was collected and analysed using HPLC as described above. The results are shown in Table 2. The values given are the averages of three fermentations

Copper Concetration (mM) 1,2-PD Produced (g/L)

0 0

0.05 0.007

0.1 0.016

0.25 0.056

0.4 0.082

0.6 0.070 Example 6 - Time Course Study of Aerobic and Anaerobic 1,2-PD production:

An overnight culture of the YPH500 yeast transformed with plasmid pMH36 was grown in SDM for 24 hrs. Fermentations were performed in a volume of 200 mL in either 300 mL anaerobic flasks or 1000 mL baffled aerobic flasks, and maintained at 30°C and 200 rpm. Optical density was measured at 600 ran using a spectrophotometer

(model 340, Sequoia-Turner, Mountain View, California) and converted to dry cell weight. Fermentation products and glucose concentrations were measured using HPLC.

The results are shown in Fig. 8: glucose (π), 1,2-PD (-), biomass (•) and acetol (x). Each data point is the average of three experiments. Over the course of time, the concentration of acetol (x) and 1,2-PD (~) increases markedly. Concurrently, the concentration of glucose drops quickly. This graph shows that as the yeast consume the glucose, they convert this carbon source to acetol and 1,2- PD, which are then secreted into the culture medium.

The invention is not limited to the particular protocols, reagents, host strains, plasmids, and promoters described herein, but encompasses all modified forms thereof which are encompassed by the attached claims.

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Claims

CLAIMS What is claimed is:

1. A genetically-engineered yeast which expresses one or more recombinant enzymes which enable the genetically-engineered yeast to produce acetol, 1,2- propanediol, or both, in isolatable quantities.

2. The genetically-engineered yeast of Claim 1 which expresses recombinant methylglyoxal synthase.

3. The genetically-engineered yeast of Claim 2 which has been transformed to contain and express SEQ. ID. NO: 1.

4. The genetically-engineered yeast of Claim 2 which has been transformed to contain SEQ. ID. NO: 5.

5. A method of producing a compound selected from the group consisting of acetol, 1,2-propanediol, and combinations thereof comprising: culturing a genetically- engineered yeast which expresses one or more recombinant enzymes which enable the genetically-engineered yeast to produce acetol, 1,2-propanediol, or both, in a medium containing a carbon substrate utilizable by the yeast, wherein acetol, 1,2-propanediol, or both, is produced.

6. The method of Claim 5, wherein a genetically-engineered yeast which expresses recombinant methylglyoxal synthase is cultured.

7. The method of Claim 5, wherein a genetically-engineered yeast which expresses recombinant E. coli methylglyoxal synthase is cultured.

8. The method of Claim 5, wherein a genetically-engineered yeast transformed with a transformation vector containing a gene sequence as shown in SEQ. ID. NO: 1 is cultured.

9. The method of Claim 8, wherein a genetically-engineered yeast transformed with a transformation vector containing a gene sequence as shown in SEQ. ID. NO: 1 operationally linked to one or more promoter sequences whereby transcription of the gene sequence is controlled, is cultured.

10. The method of Claim 5, wherein a genetically-engineered yeast transformed with SEQ. ID. NO: 5 is cultured.

11. The method of Claim 5, wherein genetically-engineered S. cerevisiae is cultured.

12. The method of Claim 5, wherein the genetically-engineered yeast is cultured in a medium containing a carbon source selected from the group consisting of arabinose, galactose, glucose, lactose, sucrose, xylose, starch, and combinations thereof.

13. The method of Claim 5, wherein the genetically-engineered yeast is cultured aerobically.

14. The method of Claim 5, wherein the genetically-engineered yeast is cultured anaerobicallly.

15. The method of Claim 5, further comprising the step of isolating the acetol or 1,2-propanediol or both.

16. A method of producing a compound selected from the group consisting of acetol, 1,2-propanediol, or combinations thereof comprising: culturing a genetically- engineered yeast as recited in Claim 2 in a medium containing a carbon source selected from the group consisting of arabinose, galactose, glucose, lactose, sucrose, xylose, starch, and combinations thereof, wherein the carbon source is fermented into acetol and 1,2-propanediol.

17. The method of Claim 16, further comprising the step of isolating the acetol or 1,2-propanediol or both.

18. A synthetic operon which enables the production of 1,2-propanediol and acetol in yeast transformed to contain the operon, the operon comprising one or more genes whose encoded gene products catalyze the formation of methylglyoxal in yeast and a promoter sequence functional in yeast operationally linked to the one or more genes.

19. The synthetic operon of Claim 18 comprising, in 5' to 3' order, a CUPl promoter, operationally linked to a gene encoding methylglyoxal synthase, operationally linked to a CYCl terminator.

20. The synthetic operon of Claim 19 wherein the gene encoding methylglyoxal synthase is SEQ. ID. NO: 1.

21. A synthetic operon functional in yeast comprising SEQ. ID. NO: 5.