Method and nucleotide sequence for transforming microorganisms
a technology of microorganisms and nucleotides, applied in the field of methods and nucleotide sequence for transforming microorganisms, can solve the problems of malolactic fermentation and subsequent bottle fermentation, difficult control of malolactic fermentation, and inability to metabolize l-malic add in the presence of asimilable carbon sources, etc., to achieve efficient transport of malate, efficient degradation of malate, succinate or malonate
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example 1
Cloning and Characterization of Mae1
[0142] Strains and Growth Conditions:
[0143] Escherichia coli strain HB101 (hsd20 leuB supE44 ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 mtl-1 recA13 mcrB) was used. Procedures for manipulating Escherichia coli cells and DNA were based on Sambrook et al. (1989). Furthermore, a haploid strain of Schizosaccharomyces pombe 972 leu 1-32 h-(wild-type), and a haploid mae1-mutant S. pombe leu 1-32 T-h mae1-(Osothsilp and Subden, 1986b) were also used in this study. The yeast cells were grown in YE (2% glucose, 0.5% yeast extract), MM (Alfa et al., 1993) plus leucine and YEPD medium (1% yeast extract, 2% Bactopeptone, 2% glucose), supplemented with 0.8% L-malic acid (Sigma, St. Louis, Mo.) if required. Transformants were selected on YNB (0.17% yeast nitrogen base without amino acids and (NH.sub.4) SO.sub.4, [Difco Laboratories, Detroit, Mich.], 0.5% (NH.sub.4).sub.2SO.sub.4 2% glucose, 1.7% bacto-agar [Difco Laboratories, Detroit, Mich.] and malate-glucose indi...
example 2
Functional Expression of S. pombe Mae1 and Mae2 Genes in S. cerevisiae
[0177] S. cerevisiae cannot degrade malate efficiently due to the absence of a malate transporter, and a malic enzyme with low substrate affinity. In contrast, S. pombe degrades malate actively as the yeast contains a permease for malate and a malic enzyme with high substrate affinity (FIG. 8). lacZ fusions demonstrated that the promoters of the mae1 (SEQ ID NO: 1) and mae2 (SEQ ID NO: 3) genes of S. pombe are not functional in S. cerevisiae. To express these genes in S. cerevisiae, mae1 and mae2 open reading frames (ORFs) of S. pombe were subcloned into expression cassettes containing the S. cerevisiae alcohol dehydrogenase (ADH1) and 3-phosphoglycerate kinase (PGK1) promoter and terminator sequences. The different constructs employed in this study are listed in Table 1.
[0178] All plasmids listed in Table 1 were transformed into laboratory strain S. cerevisiae YPH259 (Sikorski, 1989) The recombinant S. cerevisiae...
example 9
Malolactic Fermentation in Grape Musts by a Genetically Engineered Strain of S. cerevisiae
[0183] The following materials and methods were used in the study outlined in this example:
[0184] Strains and Plasmids:
[0185] The different strains and plasmids employed are listed in Table 2.
[0186] Subcloning of the Mae1 and MleS Genes:
[0187] DNA manipulations were performed in the yeast-E. coli shuttle vector YEplac181 (Gietz and Sugino, 1988). The expression vector pHVX2 (Table 2) was obtained by subdoning a HindIII fragment from plasmid pJC (Crous et al., 1995), containing the PGK1 promoter and terminator sequences into the HindIII site of Yeplac181 (FIG. 15). The mae1 ORF was isolated as a BalI-NdeI fragment from plasmid pJGl (Grobler et al., 1996) and subdoned into YEplac181 containing a multiple cloning site with EcoRI, BaII, NdeI and BglII restriction sites. The mae1 ORF was reisolated as an EcoRI-BglII fragment and subdoned into the EcoRIlBglII site of pHVX2 to yield plasmid pHV.sup.3 ...
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