GB2178431A - Genetically engineered lactose utilizing yeast strains - Google Patents

Abstract

Yeast cells are transformed with DNA which confers on said yeast cell the ability to utilize lactose as a carbon source. Preferably the genes are those which confer the ability to utilize lactose which are present in Kluyveromyces lactis, K. fagilis and Candida pseudotropicalis. Plasmids embodying the DNA can be inserted into yeasts strains, such as k. lactis, which can naturally use lactose as a carbon source, to increase gene copy number. Transformed yeast can be grown on lactose bearing waste such as whey. <IMAGE>

Description

SPECIFICATION Genetically engineered yeast strains This invention relates to genetically engineered yeast strains.

Cheese whey is a byproduct of the cheese industry which normally contains about 5% lactose. Some of this whey is recycled back into various animal and human food products, but over half of this byproduct finds no profitable use and is dumped as waste. Because whey has a considerable biological oxygen demand, it imposes a major burden on sewage treatment plants in areas of heavy dairying. Rather than disposing of the whey byproduct, some of the larger whey producers ferment whey to ethanol using lactose-utilizing yeasts such as Kluyveromyces lactis, K. fragilis, and Candida pseudotropicalis.

According to the first aspect of the present invention, we provide a yeast cell characterised in that it is transformed with DNA which confers on said yeast cell the ability to utilize lactose as a carbon source.

Preferably, the yeast cells are of the genus Saccharomyces, e.g., S. cerevisiae or S. uvarum which, unlike many naturally lactose-utilizing strains, are able to survive or proliferate in culture media containing high (e.g., 10%, and more preferably 13%, by volume) levels of ethanol. These new strains can thus render whey fermentation to lactose commercially viable.

In preferred embodiments, the transforming DNA conferring on the host yeast cells the ability to utilize lactose in whey as a carbon source (preferably, as the only carbon source) is derived from or substantially identical to DNA of an untransformed lactose-utilizing strain such as a yeast strain of the genus Kluyveromyces, e.g., K.

lactis. This DNA, preferably carried on a plasmid vector (which can be integrating or autonomously replicating), preferably includes DNA encoding a protein exhibiting beta-galactosidase activity (i.e., a protein which cleaves lactose into glucose plus galactose); DNA encoding a protein exhibiting lactose permease activity (i.e., a protein which facilitates entry of lactose into the yeast cell); and DNA comprising one or more positive activator sequences (e.g., a gene encoding a positive activator protein) which enchance the expression of one or both of the DNA regions encoding beta-galactosidase and lactose permease activity. Preferably, the vector is free of non-yeast DNA, e.g., E. coli DNA which could otherwise introduce potentially pathogenic DNA into the yeast cells or, in the case of replicating plasmids, decrease copy number.

The invention, in a second and alternative aspect thereof, provides a plasmid containing DNA encoding one or more proteins capable of conferring on a host yeast cell transformed with said plasmid the ability to utilize lactose as its sole carbon source.

In addition to enabling commercially viable whey fermentation to ethanol, the new plasmids can be used to convert bread and dough leavening yeast strains into strains capable of growing on whey, which is an inexpensive, consistent medium, so that the strains can be propagated for distribution, e.g., to bakers, efficiently and cheaply. Any other processes utilizing stains of Saccharomyces, e.g., the production of single cell protein, particular amino acids, or high-value proteins, can be facilitated by converting the strain to one capable of using whey as a nutrient source using an embodiment of plasmid in accordance with this invention.

The plasmids; in addition to being useful for conferring on yeast cells such as S. cerevisiae the ability to utilize lactose, can also be used to transform cells naturally capable of utilizing lactose, e.g., Kluyveromyces species such as K. lactis or K. fragilis, to enchance lactose utilization in those cells.

The invention is hereinafter more particularly described by way of example only with reference to the accompanying drawings, in which: Figure 1 is a diagrammatic representation of a first embodiment of plasmid in accordance with the invention; Figure 2 is a series of restriction maps of regions of intermediate vectors used in the construction of said plasmid; Figure 3 is a diagrammatic representation of a second embodiment of plasmid also in accordance with the invention; Figure 4 is a restriction map of the 7 kb insert of K. lactis DNA, in an intermediate plasmid, which contains the LAC9 gene, located between the Bglll site on the left and the Sall site on the right; and Figure 5 is a diagrammatic representation of intermediate plasmid pGM130, an autonomously replicating plasmid capable of existing in high copy number in yeast cells.

Plasmid structure Figures 1 and 3 illustrate pSH096 and pSH207, plasmids of the invention useful for transforming ethanol-tolerant yeast strains, e.g., Saccharomyces strains, to render them capable of utilizing lactose. The following abbreviations are used in Figures 1-5 to denote restriction sites: A, Xbal; B, BamHI; Be, BstEll; E, EcoRI; PI, Pvul; Bx, BstXI; PIl, Pvull; G, Bill; TIl, Sstll; TI, Sacl; H, Hindlll; V, EcoRV; X, Xhol; N, Notl; M, Smal. (S/X) represents the junction of a former Sall site and a former Xhol site. Genes and gene fusions are indicated by boxes. The concentric segments of the inner circle indicate segments of DNA having origins as indicated.

Structure of pSH096 Referring to Figure 1, pSH096 contains (moving clockwise) the lactose utilization genes LAC4, Lay12, and LAC13 from K. lactis; a region of the HO (homothallism) gene, which allows the plasmid to stably integrate into the homologous HO region of the host chromosome; a region containing the yeast CYC1 (iso-l-cytochrome c) promoter; the G418' gene for resistance to the antibiotic G41 8 (for selection of transformants in yeast) from the bacterial transposon Tn903; an ori (origin of replication) region from the E. coli plasmid pBR322; the pBR322-derived amp' (ampicillin resistance) gene for selecting transormants in E. coli; and a second HO region.

Construction of pSH096 Plasmid pSH096 was constructed by isolating lactose utilization genes from K. lactis and then inserting that DNA into a yeast integrating vector. The integrating vector employed, pRY296, was derived from integrating vector pRY253, described in our European Patent Application 85303625.9 (Publication No: EP 0163491) the disclosure of which is to be regarded as hereby incorporated by reference.

Isolation of the lactose utilization genes from K. lactis Standard methods were used for the construction of a K. lactis gene bank, subsequent plasmid construction, and transformation and growth of E. coli and Saccharomyces strains (Maniatis et al. (1980) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Sherman et al. (1981) Methods in Yeast Genetics; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Webster and Dickson (1983) Gene 26, 243).

A Sau3Al partial K. lactis genomic bank was constructed in E. coli from DNA of strain 71 -59 (obtained from Herman Phaff, University of California at Davis). The bank contained about 100,000 independent clones with K.

lactis inserts ranging from about 5 to 15 kilobases.

Two clones containing the K. lactis p-galactosidase gene (LAC4) were isolated from the bank by complementation of a lacZ deletion in E. coli strain ATCC 33927 (lac A 169, thi, endA1, hsdR17, supE44). A standard colorimetric plate assay was employed in which LAC4 clones give rise to blue colonies on X-gal (5'-brom-4'-chloro-3'-indoyl-,B-galactoside) indicator plates. The two clones, named pSHCBB1 and pSHCBB2 (Figure 2), contained 7 kilobase and 10.5 kilobase inserts, respectively. Restriction maps of the two inserts contained an overlap of about 6 kilobases. The EcoRI restriction map of this 6 kilobase overlapping region matched a published map of the LAC4 gene (Breunig et al. (1984) Nucleic Acids Res. 12, 2327). Appropriate Southern blots (Southern, (1975) J. Mol.Bio. 98, 503) showed that both pSHCBB1 and pSHCBB2 contained contiguous stretches of K. lactic genomic DNA.

The two LAC4 clones both gave expression of ss-galactosidase activity when transformed into S. cerevisiae strain DBY745 (x adel- 100, leu2-3, leu2- 112, ura3-52). The assay was as described by Yocum et al. (1984) Molecular and Cellular Biol. 4, 1985. However, pSHCBB1 and pSHCBB2 showed important differences in their patterns of regulation, summarized in the table below.

TABLE p-galactosidase activity of LAC4 clones in S. cerevisiae strains" grown on the carbon sources indicated Glucose + Galactose + Glucose Galactose Glycerol no plasmid/GAL44 0.1 0.1 0.1 no plasmid/gal4- 0.1 0.1 0.1 pSHCBB1/GAL4+ 0.60 0.53 13 pSHCBB1/gal4 0.35 1.9 1.6 pSHCBB2/GAL4' 15 38 34 pSHCBB2/gal4- 18 34 1.2 K. lactis 200 600 600 "YM262=z ura3-52, his3 A 200, ade2-101, lys2-801, tyrl-501.

YM335=a gal4 A 537, ura3-52, his3 A 200, ade2-101, lys2-801, met.

While pSHCBB1 encoded galactose-inducible, glucose-repressible p-galactosidase activity, pSHCBB2 gave a significantly higher level of galactose-inducible activity, some of which was not glucose repressed. Since pSHCBB1 contains all regulatory sequences upstream of LAC4 required for proper regulation in K. lactis (Das et al. (1985) EMBO J. 4, 793), and since pSHCBB2 contains sequences not contained in pSHCBB1, pSHCBB2 must contain a second gene coding for a function that positively activates expression of LAC4 in S.

cerevisiae. This expression occurs in the presence of glucose and allows for higher levels of induction of LAC4 in galactose; this gene has been designated LAC12. It is believed that LAC12 might also positively regulate the gene encoding lactose permease. This information regarding LAC12, combined with the knowledge that S.

cerevisiae galactose regulatory genes regulate K lactis LAC4 expression (see the table, above), permitting a scheme to be devised for cloning the lactose permease gene.

A 7 kilobase Xbal fragment from pSHCBB2 was subcloned into the unique EcoR1 site of the integrating vector Ylp1 (Botstein et al. (1979) Gene 8, 17) to give plasmid pSHE29. pSHE29 was transformed into S.

cerevisiae strain SH33-5dR (x gal80, his3- 1, ura3-52, leu2-3, leu2- 112, adel- 100) by cutting with Xho1 to direct integration at the his3 locus (Orr-Weaver et al. (1981) PNAS USA 78, 634). Transformants were assayed for p-galactosidase activity after growth in various carbon sources to demonstrate that the glucose-resistant regulatory activity ascribed to LAC12 was still contained on the subcloned Xbal fragment.

The integrated transformant, SH33-5dR/pSHE29,expressed ss-galactosidase but did not grow on lactose as a sole carbon source, presumably because it lacked the lactose permease gene. SH33-5dR/pSHE29 was transformed with the K. lactis gene bank, and a pool containing about 100,000 independent transformants was plated on a minimal selective medium containing lactose as the sole carbon source at a density of 2.5 x 106 colonies per 100 mm petri plate. Several doubly transformed yeast colonies appeared out of the background after 5 to 7 days. These colonies were purified and circular plasmid DNA was recovered from the isolates by standard methods.

One plasmid, designated pSHL6, was further characterized. Retransformation of the naive SH33-5dR/pSHE29 with pSH L6 showed that the ability to grow on a minimal selective lactose medium was conferred by pSHL6.

That SH33-5dR/pSHE29/pSHL6 contained lactose permease activity was demonstrated by uptake of '4C-thiomethyl-/X-D-galactoside (Dickson petal. (1983) Cell 15,123); SH33-5dR/pSHE29 did not take up the lactose analog.

Restriction analysis of pSHL6 showed that it contained a 13 kilobase insert that included the LAC4gene. It was subsequently shown that pSHL6 alone conferred a p-galactosidase activity on SH33-5dR, with a pattern of regulation similar to that of pSHCBB2. Finally, it was shown that both pSHL6 and pSHCBB2 conferred the ability to grow on a minimal lactose medium on both SH33-5dR and DBY745.

Thus pSHL6 and pSHCBB2, but not pSHCBB1 or pSHE29, contained a gene coding for lactose permease, which we have designated LAC13. A summary of the K. lactis inserts in the plasmids described and the genes contained therein is given in Figure 2. The arrow in the upper left of Figure 2 indicates the approximate location and direction of transcription of the LAC4 gene.

Given that pSHCBB2 contained LAC4, LAC12, and LAC13, it was deduced that the 10.5 kilobase Xho1 fragment of pSHL6 must also contain all three of these LAC genes.

Installing the LAC genes in industrial yeasts The 10.5 kilobaseXhol fragment containing LEA CM LAC12, and LAC13 from pSHL6 was ligated into the unique Sall site of pRY296 to give pSH096 (Figure 1). pRY296 is a vector designed to integrate at the HO (homothallism) locus of wild type Saccharomyces strains, using antibiotic G41 8 resistance for selection of transformants. pRY296 was derived from pRY253 (described in Yocum, id) by 1) deleting a 2.7 kilobase Sall fragment that contained the URA3 gene, 2) converting the remaining Sall site to an EcoRV site with synthetic linkers, and 3) converting the unique Kpnl site in the HO gene to a unique Sall site using synthetic linkers.

pSH096, linearized at a unique Sacl site, was used to transform several strains of S. cerevisiae and S. uvarum to G41 8 resistance by the method of Webster et al. (1983) Gene 26, 243. The strains transformed were DBY745, ATCC 24858, ATCC 26602, and 42133, a distiller's active dry yeast obtained from Universal Foods (Milwaukee). Transformants of all four strains grew weakly on minimal lactose medium; the untransformed strains were unable to grow on lactose.

In order to select a mutant that was capable of more rapid aerobic growth on lactose, the ethanol-tolerant distillery strain 42133, transformed with pSH096, was continuously cultured aerobically in a New Brunswick Model C30 C32 fermenter with a working volume of 1.3 litres. The minimal lactose medium consited of 0.7% Difco Yeast Nitrogen Base (without amino acids) and 0.44% lactose. The flow rate was 4.8 ml/min and aeration was 1 v/v/min, which resulted in a steady state culture density of 2.4 OD600 units. During three weeks of continuous culture, a spontaneous mutant arose which exhibited an increased aerobic growth rate on lactose as a sole carbon source. This mutant manifested itself by an increase in the steady state cell density, which rose to 3.3 OD600 units. A sample was taken from the culture and plated onto a minimal lactose plate containing 2% lactose.Colonies of various sizes arose on the plate; one of the larger colonies was chosen and subcultured. The aerobic doubling time for this selected mutant yeast strain, 421 33/pSH096-21 c, in minimal 2% lactose medium, was about 2 hours. This strain was capable of anaerobically producing 11.5% ethanol (v/v) in 84 hours from a medium consisting of 25% lactose, 1.4% Difco Yeast Nitrogen Base (without amino acids), 0.002% ergosterol, and 0.1% Tween 80.

421 33/pSH096-21 C had an anaerobic doubling time of 12 hours, which is too slow for optimal commercial use. In order to further select a mutant that was capable of more rapid anaerobic growth on lactose, strain 421 33/pSH096-21 c was continuously cultured anaerobically in the Model C30 C32 fermenter, with a working volume of 1:3 litres. The minimal lactose medium consisted of 1.4% Difco Yeast Nitrogen Base (without amino acids), 0.002% ergosterol, 0.1% Tween 80, and 1% lactose. The initial flow rate was 0.8 ml/min, which resulted in a steady state culture density of 1.0 OD600 unit. During three months of continuous culture, a spontaneous mutant arose which exhibited an increased anaerobic growth rate on lactose as a sole carbon source. This mutant manifested itself by an increase in the steady state cell density, which which rose to 2.4 OD600. The final flow rate was 3.5 ml/min. A sample was taken from the culture and plated onto a minimal lactose plate containing 2% lactose. Colonies of various sizes arose on the plate; one of the larger colonies was chosen and subcultured. The anaerobic doubling time for this selected mutant yeast strain, which we have designated 421 33/pSH096-1 43c, was 4.5 hours, and it retained the original aerobic doubling time of about 2 hours. This This strain was capable of anaerobically producing ethanol concentrations comparable to 42133/pSH096-21 c, but in only 35 hours. In addition, strain 421 33/pSH096-1 43c is capable of growing on and fermenting 25% solids whey permeate concentrate.

The K. lactis LAC genes transformed into the above-described yeast strains are mitotically stable: the lac+ phenotype was not lost after many generations of growth in a non-selective medium. This suggests that the LAC genes have been integrated into a chromosome of the transformed S. cerevisiae strains. However, since the original plasmid pSH096 carries an ars (autonomously replicating sequence), another possibility is that the ars enables the genes to be maintained in the strains in an autonomously replicating form.

In either case, the K. lactis LAC genes can be recovered from transformed yeast strains such as 421 33/pSH096-21 C or 42133/pSH096-143c, in order to retransform these genes into other yeast strains.

Genomic DNA from the transformed yeast strain can be digested with Sacl, religated, and the mixture transformed into E. coli. Plasmids similar or identical to pSH096 can be recovered from E. coli transformants, since it has an E. coli replicon. Purified plasmid can then be used to transform yeast strains of interest. For example, K. lactis 71-59 was transformed with pSH096-21 c. DNA of pSH096-21 c (which is a plasmid similar or identical to pSH096, that was recovered from 421 33/pSH096-21 c by the above-described procedure) was digested with Xbal to facilitate integration at the LAC4 locus of K. lactis. The plasmid was transformed into K.

lactis by the lithium acetate transformation procedure (Ito et al. (1983) J. Bacteriol 153, 163) and stable G418R transformants were obtained.

Structure of pSH207 Referring to Figure 3, pSH207 contains regions of the naturally occuring yeast 2,u circle, including REP 1, FLP, REP2, and a yeast origin of replication. REP1 and REP2 are thought to encode different subunits of a replication enzyme complex, while the FLP gene encodes a site-specific recombinase that catalyzes recombination between the two copies of a 600 basepair inverted repeat (IR) carried on the plasmid. It is thought that his recombination, or "flipping", aids in the maintenance of the relatively high copy number of the 2y plasmid, about 50 per cell (Sutton & Broach (1985) Mol. Cell. Biol. 5, 2770-2780).

In addition, pSH207 contains the following: 1) a portion of E. coli plasmid pBR322, which enables replication in E. coli; this E. coli DNA, which is not located in any region required for vector replication in yeast, is bounded by Notl sites, which allow easy removal of the DNA prior to transformation of yeast cells.The Notl sites are particularly advantageous because they are rare, recognizing DNA regions eight base pairs long, rather than the more usual six; 2) the LEU2-d gene (Beggs (1981) in Molecular Genetics in Yeast, Von Wettstein et al., eds., Alfred Benzon Symposium Vol. 16, pp 383-390), which is a defective form of the LEU2 gene which encodes an enzyme permitting growth of host yeast cells on leucine-deficient media; the LEU2-d gene also permits such growth, but is expressed poorly, so that many (about 100) copies of the vector containing the gene are required for growth on leucine-deficient media, and high copy number thus can be induced at the desired fermentation stage by growing transformed cells on medium deficient in leucine; 3) the above-described LAC4, LAC12, and LAC13 genes from K. lactis; and 4) the K. Iactis LAC9 gene, which, like LAC12, encodes a positive activator protein which induces the LAC4 and LAC13 genes. Plasmid pSH207 differs from pSH096 primarily in that pSH207 is capable of autonomously replicating in constitutively high copy number in yeast cells, and it contains the LAC9 gene.

Cloning of LA C9 Because it was believed that LAC9 was analogous to the S. cerevisiae gene, GAL4, which encodes a trans-acting positive regulator of S. cerevisiae GAL 1, GAL7 and GAL 10, as well as LAC4 which has been introduced into S. cerevisiae, the strategy for cloning LAC9 was based on the hypothesis that LAC9 could, to some extent, substitute for GAL4 for the induction of LAC4 in S. cerevisiae. A yeast strain was constructed that was mutated in the GAL4 gene, and contained LAC4 and LAC12 integrated into a chromosome. pSHE29 (see Figure 2) was integrated into the his3 locus of S. cerevisiae strain SH31 -8c (a ga14-1 ura 3-52 his3 Al leu 2-3 leu 2- 112) after linearizing the plasmid at its unique Xhol site.Since SH31 -8c is gal4, the integrated transformant expresses virtually no p-galactosidase activity, and gives white colonies on Xgal indicator plates.

The LAC9 gene was then isolated by screening the K. lactis gene bank (described above) in this strain for clones that would complement the ga/4 mutation, i.e. clones that would turn yeast colonies from white to blue on indicator plates. SH31 -8c/pSHE29 was transformed with the K. lactis gene bank and selected for URA3+ on minimal glucose medium. About 60,000 URA3+ transformants were pooled and plated on buffered selective minimal galactose medium containing Xgal indicator at a density of 105 cells per plate. Blue colonies appeared at a frequency of about 1/104. Plasmid DNA was recovered into E. coli from 9 blue isolates, and each plasmid was retransformed into naive SH31 -8c/pSHE29. One plasmid, called pSHP8b, which conferred the blue colony phenotype upon retransformation, was further characterized. A restriction map of the 7 kb K. lactis insert is shown in Figure 4.

Plasmid pSHP8b presumably contains a clone of the LAC9 gene. The insert was shown to be derived from K.

lactis DNA by Southern blotting. In order to demonstrate that the LAC9 gene stimulated expression of LAC4 in S. cerevisiae, these two genes (as well as LAC12 and LAC13) were combined on the same 2 micron replicating plasmid as follows.

The 10.5 kb Xhol fragment from pSHL6 was inserted between the Sall site near the right end of the insert in pSHP8b and a second Sall site just beyond the end of the insert in the tetr gene of the vector, to give pSHl 01.

pSH101 and pSHL6 were then independently transformed into S. cerevisiae strain YM335 (a ga/4 A537 ura3-52 ade2- 101 lys2-801 his3-200 met). Both URA3 transformants were grown in minimal selective glucose medium, and assayed for tS-galactosidase activity. YM335/pSH101 gave 9.6 units of activity while YM335/pSHL6 gave only 4.5 units, Therefore, we deduced that DNA sequences between the left hand end of the insert of pSHP8b and the Sall site of pSHP8b were capable of a two-fold stimulation of expression of the LAC4 gene in S.

cerevisiae. We believe that the positive regulatory activity on pSHP8b is due to the LAC9 gene. Our conclusion is that a combination of both LAC9 and LAC12 in S. cerevisiae gives a higher level of ss-galactosidase than either LAC9 or LAC12 alone.

Introduction of the LA C genes on a replicating plasmid Replicating plasmids often have the disadvantage of being unstable, i.e. they are readily lost from a population of cells in the absence of specific selection for the plasmid. However, growth on lactose as a carbon source can provide the specific selection necessary for maintenance of the plasmid. As is mentioned above, pSH207 is such a replicating plasmid, constructed by introducing the LAC genes, including LAC9, into pGM130 (Figure 5), which was constructed as follows.

Plasmid pJDB219 (Parent et al. (1985) Yeast 1, 83-138), cdntaining the LEU2-d gene, was cut partially with Hpal and an 8 bp Xhol linker (New England Biolabs) was inserted. An isolate containing an Xhol site at the former 2 Hpal site was named pGM1 14. pGMl 14 was cut with Ndel and Mstll and the sticky ends were filled in with reverse transcriptase. This DNA was ligated, treated at 65"C for 10 minutes, and digested with Xhol. The DNA fragment containing the LEU2-d gene was gel purified and ligated to Xhol-cut pGM111 to give pGM120; pGM111 was obtained by insertion of an 8 bp Xhol linker into the Pvull site of Ylp5.

pgml 20 was partially cut with Xhol and ligated in the presence of a synthetic poly-linker of the following sequence: 5' - TCGAACAGCTCAGATCTCCCGGGGCGGCCGCA TCTCGAGTCTAGAGGG CCCCG CCG G CGTAGCT -5' An isolate that contained one copy of the poly-linker on the LEU2-d side of the pGM1 11 insert was named pGM1 20P. The linker in pGM1 20P was oriented such that the Notl site was toward the pGMl 11 insert. The URA3 gene of pGM120P was deleted by cleaving with BamHI and Xhol, treating the Mung Bean Nuclease (P-L Biochemicals), and ligating in the presence of an 8 bp Notl linker (New England Biolabs). The resulting plasmid, which contained a Notl linker, was called pGM128.

pGM128 was cut with Xbal and treated with bacterial alkaline phosphatase (Bethesda Research Labs) and ligated to the Xbal fragment of CV21 (publicly available) that contains the FLP gene, to give pGM130.

pSH206 was then constructed as follows. First, the Smal site of pGM1 30 was converted to a Sall site by insertion of an 8 bp Sall linker (New England Biolabs) to give pSH200. Next, the 10.5 kb Xhol fragment from pSHL6 that contains LAC4, LAC12, and LAC13 was ligated into the Sall site of pSH200 to give pSH206.

pSH206 was transformed into S. cervisiae DBY745 (x adel- 100, leu2-3, leu2- 112, ura3-52), selecting for growth on minimal complete medium containing lactose as the sole carbon source. A resulting transformant was subcultured five times at 1 to 100 dilution in minimal complete lactose liquid medium, which resulted in a strain that had an aerobic doubling time of 4 hours in the aforementioned medium.

Finally, a plasmid, pSH207, was constructed in which the LAC9 gene was present, along with the LAC 4, 12, and 13 genes; the LAC9 gene, obtained as a 5.6 kb Bglll to Sall fragment from pSHP86 was inserted into the Bglll to Sall backbone of pSH200 (see above) to give pSH204. Next, the 10.5 kb Xhol fragment from pSHL6 that contained LAY4, LAC12, and LAC13 was ligated into the Sall site of the pSH204 to give pSH207. Plasmid pSH207 can be transformed into yeast strains as described above.

Overproduction of GAL4 protein Since the GAL4 gene functions in S. cerevisiae to stimulate expression of LAC4 (see the Table below), and since GAL4 protein is known to be present at very low levels in S. cerevisiae (Klar and Halvorson (1976) Mol.

Gen. Genet. 135, 203), we hypothesized that overproduction of GAL4 protein might lead to enhanced levels of LAC4 expression. Thus, the GAL4 gene was introduced on a multicopy 2 micron plasmid into a yeast strain containing LAC4, LAC12, and LAC13 integrated into the chromosome. pSH1 b-2 was constructed by subcloning the 10.5 kb Xhol fragment from pSHL6 that contains LA Cl, LAC12, and LAC13 into the unique Sall site of the integrating vector Ylpl (Botstein et al. (1979) Gene 8, 17). Thus pSH1 b-2 is similar to pSHE29, above, but contains the LAC13 gene in addition. Yeast strain SH31 -8c was integratively transformed with pSH1 b-2 after cleavage at its unique Xhol site and selecting for HIS3+. This transformant was the recipient for the GAL4 overproducing plasmid.

The GAL4 gene was obtained on a plasmid called pG525, which contains a 3.7 kilobase BamHI fragment of yeast genomic DNA that includes the GAL4 gene, cloned into the BamHI site of pBR322 (Laughon and Gestland (1984) Mol. Cell. Biol. 4, 260). The sequence of the GAL4 gene is provided in Laughon and Gestland.

The 3.7 kilobase BamHI fragment containing GAL4 was inserted into a 2 micron, URA3 plasmid to give pRY233, which is maintained in yeast at about 7 copies per cell. pRY233 or empty vector was transformed into SH31 -8c/pSH1 b-2, and both URA3 transformants were grown in minimal selective galactose medium. The transformant containing pRY233 gave 90 units of ss-galactosidase activity while the transformant containing the empty vector gave 2 units. A GAL4+ yeast strain, YM262, (one chromosomal copy of GAL4) transformed with pSH1 b-2 gives 14 units of p-galactosidase. Thus overproduction of GAL4 from a multicopy plasmid clearly boosts ss-gaiactosidase expression from the LAC4 gene in S. cerevisiae.

The Ga14 gene, like the LAC9 gene, can also be introduced into an autonomously replicating vector, e.g., pGM1 30. The 3.7 kb BamHI fragment containing GAL4 from pG525 was ligated into the Bglll site of pGM1 30 to give pSH203. pSH208, analogous to pSH207 but containing the GAL4 gene rather than the LAC9 gene, can be constructed by cleaving pSH23 with Smal, and ligating in the 10.5 kb Xhol fragment from pSHL6 that contains LA Cl, LAC12, and LAC13, after filling in the Xhol sticky ends with Klenow fragment, and screening for isolates that give blue colonies in E. coli YMC9 on Xgal indicator plates.

If it is desired that the transformed yeast strains be free of E. coli DNA, the pBR322-derived sequences in pSH206, pSH207, or pSH208 can be removed just prior to transformation of yeast. The plasmid is cleaved with Note, and the larger of the two fragments is purified by preparative agarose gel electrophoresis. The purified fragment is circularized by ligation at a concentration of less than or equal to 10 ug/ml. The circularized plasmid is then phenol extracted, ethanol precipitated, and transformed into yeast by standard procedures.

Use Yeast strains produced in accordance with this invention can be used as the fermenting strains in any industrial applications in which a lactose-containing medium, e.g., whey, or whey derivatives, is used. Use of the transformed strains to ferment the whey which is a byproduct of the dairy industry not only reduces the burden the whey would otherwise impose on sewage treatment plants, it also results in the production of valuable products such as ethanol, single-cell protein, or bakers yeast. As mentioned above, any other industrially important strains can, by practising the teachings of this invention, be made to grow on economical lactose-containing media. Fermentation using the transformed strains produced according to our teachings is carried out using conventional fermentation methods.

Deposits Yeast strain 42133/pSH096-143c, and plasmid pSH207 in E.coliYMC9, have been deposited with the American Type Culture Collection, and have been given ATCC Accession Number 20763 and 67149, respectively, and were deposited on July 5, 1985 and July 1, 1986, respectively.

Other embodiments Other embodiments are feasible. For example, any host yeast cells can be used; they need not be polyploid or have high ethanol tolerance like S. cerevisiae strains, but can be haploid or diploid, and can have low ethanol tolerance, if ethanol production is not the primary goal of fermentation. Although in many instances, regions of homology with the host chromosome or the plasmid are desired for integration, autonomously replicating plasmids not containing such sequences can be used as well. The LAC genes can be derived from any lactose-utilizing yeast strains or other organisms, and could be synthesized in whole or in part rather than being obtained from such strains. Minor differences between synthesized DNA sequences and naturally occurring LAC genes could be acceptable if not resulting in loss of the ss-galactosidase, lactose permease, and positive activator functions. Further, the LAC genes might well be expected to vary somewhat in structure between lactose-utilizing strains. Although a mutant selection step following transformation is desirable to enhance lactose utilization, such a step will not necessarily be required in every instance.

Claims (42)

1. A yeast cell characterised in that it is transformed with DNA which confers on said yeast cell the ability to utilize lactose as a carbon source.

2. A yeast cell according to Claim 1, wherein said yeast cell has the ability to utilize lactose as its sole carbon source.

3. A yeast cell according to Claim 1 or Claim 2, wherein said yeast cell is capable of surviving or proliferating in a culture medium containing 10% ethanol by volume.

4. A yeast cell according to Claim 3, wherein said yeast cell is capable of surviving or proliferating in a culture medium containing 13% ethanol by volume.

5. A yeast cell according to any preceding claim, wherein the yeast cell which was transformed was of the genus Saccharomyces.

6. A yeast cell according to Claim 5, wherein the yeast cell which was transformed was of the species S.

cerevisiae.

7. A yeast cell according to Claim 5, wherein the yeast cell which was transformed was of the species S.

uvarum.

8. A yeast cell according to any preceding claim, wherein said DNA is derived from or substantially identical to DNA of an untransformed yeast cell capable of utilizing lactose as a carbon source.

9. A yeast cell according to Claim 8, wherein said untransformed yeast cell is of the genus Kluyveromyces.

10. A yeast cell according to Claim 9, wherein said untransformed yeast cell is of the species K. lactis.

11. A yeast cell according to any of Claims 1 to 7, wherein said DNA comprises DNA which encodes a protein exhibiting beta-galactosidase activity.

12. A yeast cell according to any of Claims 1 to 7 or 11, wherein said DNA comprises DNA which encodes a protein exhibiting lactose permease activity.

13. A yeast cell according to Claim 11, wherein said DNA further comprises a positive activator sequence which enhances the expression of said DNA encoding a protein exhibiting beta-galactosidase activity.

14. A yeast cell according to Claim 12, wherein said DNA further comprises a positive activator'sequence which enhances the expression of said DNA encoding a protein exhibiting lactose permease activity.

15. A yeast cell according to Claims 13 or 14, wherein said positive activator sequence is a gene encoding a positive activator protein.

1 6. A yeast cell according to Claim 14, wherein said positive activator sequence further enhances the expression of DNA encoding a protein exhibiting beta-galactosidase activity.

17. A yeast cell according to Claims 13 or 14, wherein said positive activator sequence is the K. lactis LAC12 gene.

18. A yeast cell according to Claim 15, wherein said positive activator protein is encoded by the K. lactis LAC9 gene.

19. A yeast cell according to Claim 15, wherein said positive activator protein is encoded by the S. cerevisiae GAL4 gene.

20. A yeast cell according to Claim 17, wherein said DNA further comprises the K. lactis LAC9 gene.

21. A yeast cell according to Claim 17, wherein said DNA further comprises the S. cerevisiae GAL4 gene.

22. A yeast cell according to any preceding claim, wherein said DNA is integrated into the chromosome of said yeast cell.

23. A yeast cell according to any of Claims 1 to 22, wherein said DNA is capable of automonously replicating in said yeast cell.

24. A yeast cell according to any preceding claim, wherein said yeast cell is capable of producing ethanol.

25. A yeast cell according to Claim 24, wherein said yeast cell is capable of converting lactose from whey or whey derivatives to ethanol.

26. The yeast cell having ATCC Accession No. 20763.

27. A plasmid containing DNA encoding one or more proteins capable of conferring on a host yeast cell transformed with said plasmid the ability to utilize lactose as its sole carbon source.

28. A plasmid according to Claim 27, wherein said DNA comprises DNA encoding a protein exhibiting beta-galactosidase activity; DNA encoding a protein exhibiting lactose permease activity; and DNA comprising a positive activator sequence which is capable of enhancing the expression of said DNA encoding betagalactosidase activity.

29. A plasmid according to Claim 28, wherein said positive activator sequence further enhances the expression of said DNA encoding lactose permease activity.

30. A plasmid according to any of Claims 27, 28 or 29, wherein said plasmid is free of non-yeast DNA.

31. A plasmid having ATCC Accession No. 67149.

32. A yeast cell transformed with a plasmid according to any of Claims 27 to 31.

33. A yeast cell according to Claim 32, wherein the yeast cell which was transformed was of the genus Kluyveromyces.

34. A method of producing ethanol comprising culturing a yeast cell according to any of Claims 1 to 26, or Claim 32 in a culture medium containing lactose.

35. A method according to Claim 34, wherein said lactose-containing medium comprises whey or a whey derivative.

36. Ethanol whenever produced by the method of Claims 34 or 35.

37. A method of producing single-cell protein comprising culturing a yeast cell according to Claim 1 or Claim 32 in a culture medium containing lactose.

38. Single-cell protein whenever produced by the method of Claim 37.

39. A method of producing active bakers' yeast comprising culturing a yeast cell according to Claim 1 or Claim 32 in a culture medium containing lactose.

40. Active bakers' yeast whenever produced by the method of Claim 39.

41. A method of producing a desired protein or amino acid comprising culturing a yeast cell according to Claim 1 or Claim 32 in a culture medium containing lactose.

42. Proteins and/or amino acids whenever produced by the method of Claim 41.