Cloning and expression of an alpha-acetolactate decarboxylase gene from Streptococcus lactis subsp. diacetylactis in Escherichia coli

The Streptococcus lactis gene coding for alpha-acetolactate decarboxylase (ADC) was cloned in Escherichia coli. Subsequent subcloning in E. coli showed that the ADC gene was located within a 1.3-kilobase DNA fragment. The ADC gene was controlled by its own promoter. Gas chromatography showed that S. lactis and the transformed E. coli strains produced the two optical isomers of acetoin in different ratios.     

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Cloning of the citrate permease gene of Lactococcus lactis subsp. lactis biovar diacetylactis and expression in Escherichia coli.

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.

The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the alpha-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.     

Diacetyl and alpha-acetolactate overproduction by Lactococcus lactis subsp. lactis biovar diacetylactis mutants that are deficient in alpha-acetolactate decarboxylase and have a low lactate dehydrogenase activity

Lactococcus lactis subsp. lactis biovar diacetylactis strains are utilized in several industrial processes for producing the flavoring compound diacetyl or its precursor alpha-acetolactate. Using random mutagenesis with nitrosoguanidine, we selected mutants that were deficient in alpha-acetolactate decarboxylase and had low lactate dehydrogenase activity. The mutants produced large amounts of alpha-acetolactate in anaerobic milk cultures but not in aerobic cultures, except when the medium was supplemented with catalase, yeast extract, or hemoglobin.     

Structure of the beta-galactosidase gene from Streptococcus diacetylactis 144 and its expression in Escherichia coli and Streptococcus diacetylactis cells

The ability of industrial strains of mesophylic Streptococcus diacetylactis to synthesize the enzyme beta-galactosidase has been studied. Among the 22 studied strains 8 were found to synthesize the enzyme. Plasmid DNA was isolated from the Streptococcus diacetylactis strain 144 possessing the highest level of beta-galactosidase activity. The cells of the strain harbour the 35, 40 and 60 kb plasmids. The alpha-galactosidase genes from this strain was cloned in Escherichia coli cells. The gene is located on the BglIII DNA fragment of the total plasmid DNA from Streptococcus diacetylactis the size of 2.8 kb. Following the Sau3A restriction endonuclease digestion the gene was subcloned on a birepliconed vector plasmid pCB20. The latter is capable of replication in the Gram-negative as well as Gram-positive microorganisms. The pCB20 derivatives carrying the different length fragments with the beta-galactosidase gene were isolated. DNA of an obtained plasmid was used for transformation of Streptococcus diacetylactis cells. The presence of the recombinant plasmid in streptococcus strain 144 results in the 1.8 fold increase in beta-galactosidase production.     

Cloning and expression of an amylase gene from Streptococcus bovis in Escherichia coli

An amylase gene was identified in a Streptococcus bovis 033 gtWESB genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.     

Improved Medium for Detection of Citrate-Fermenting Streptococcus lactis subsp. diacetylactis

A modified medium which distinguished between citrate-fermenting and non-citrate-fermenting species of lactic streptococci within 48 h was developed. In addition, the occurrence of citrate-negative variants in citrate-positive populations of Streptococcus lactis subsp. diacetylactis could be detected.     

Mutants of Streptococcus lactis subsp. diacetylactis lacking diacetyl reductase activity.

Three strains of Streptococcus lactis subsp. diacetylactis, namely DRC-1, DRC-2 and DRC-3 which produced diacetyl up to 120 h of incubation were exposed to the ultraviolet irradiation as well as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) to isolate mutants lacking diacetyl reductase activity. UV irradiation did not produce any isolate completely devoid of diacetyl reductase activity, though, 99.5% loss in activity could be achieved. NTG treatment proved to be more effective and seven survivors exhibiting complete loss of diacetyl reductase activity were recovered. These altered characteristics were retained on repeated subculturing.     

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli.

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.     

Genetic Evidence for Plasmid-Linked Lactose Metabolism in Streptococcus lactis subsp. diacetylactis

It has been previously observed that loss of plasmid pGK4101 occurred concomitantly with loss of lactose-fermenting ability in Streptococcus lactis subsp. diacetylactis 18-16. Transfer of this 41-megadalton plasmid to LM0230, a lactosenegative (Lac⁻) strain of S. lactis, required cell-to-cell contact and resulted in a conversion of LM0230 to the Lac⁺ phenotype. This confirms the linkage of lactose-fermenting ability to the 41-megadalton plasmid in S. lactis subsp. diacetylactis and, in addition, demonstrates transfer by a process resembling conjugation in the group N streptococci.     

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.     

Cloning of a chromosomal fragment from Lactococcus lactis subsp. lactis partially complementing Escherichia coli recA functions

A recA-like gene was isolated from a gene library of Lactococcus lactis subsp. lactis by intergeneric complementation of an E. coli recA mutant. A plasmid was obtained which fully complemented the RecA response to DNA damaging agents and UV inducibility of prophage, but not P1 plating efficiency in an E. coli recA mutant. The cloned DNA fragment also partially complemented the rec mutation in Lc. lactis MMS36. Hybridization studies showed that there was no detectable sequence homology between the recA gene of E. coli and Lc. lactis subsp. lactis chromosomal DNA.     

Plasmid linkage of a bacteriocin-like substance in Streptococcus lactis subsp. diacetylactis strain WM4: transferability to Streptococcus lactis.

Streptococcus lactis subsp. diacetylactis strain WM4 transferred lactose-fermenting and bacteriocin-producing (Bac+) abilities to S. lactis LM2301, a lactose-negative, streptomycin-resistant (Lac- Strr), plasmid-cured derivative of S. lactis C2. Three types of transconjugants were obtained: Lac+ Bac+, Lac+ Bac-, and Lac-Bac+.S. diacetylactis WM4 possessed plasmids of 88, 33, 30, 5.5, 4.8, and 3.8 megadaltons (Mdal). In Lac+ Bac+ transconjugants, lactose-fermenting ability was linked to the 33-Mdal plasmid and bacteriocin-producing ability to the 88-Mdal plasmid. Curing the 33-Mdal plasmid from Lac+ Bac+ transconjugants resulted in loss of lactose-fermenting ability but not bacteriocin-producing ability (Lac- Bac+). These strains retained the 88-Mdal plasmid. Curing of both plasmids resulted in a Lac- Bac- phenotype. The Lac+ Bac- transconjugant phenotype was associated with a recombinant plasmid of 55 or 65 Mdal. When these transconjugants were used as donors in subsequent matings, the frequency of Lac transfer was about 2.0 X 10(-2) per recipient plated, whereas when Lac+ Bac+ transconjugants served as donors, the frequency of Lac transfer was about 2.0 X 10(-5) per recipient plated. Also, Lac- Bac+ transconjugants were found to contain the 88-Mdal plasmid. The data indicate that the ability of WM4 to produce bacteriocin is linked to an 88-Mdal conjugative plasmid and that lactose-fermenting ability resides on a 33-Mdal plasmid.     

Citrate utilization by free and immobilized Streptococcus lactis subsp. diacetylactis in continuous culture

Summary The effects of pH, temperature and concentration of citrate were investigated to achieve an optimal production of diacetyl, acetoin and C₂ compounds such as acetaldehyde, acetate and ethanol for free and immobilized cells. The critical conditions of culture, 22°C, pH 4.8, increased the production of C₄ compounds (diacetyl, acetoin, 2, 3 butylene glycol), C₂ compounds (acetaldehyde, ethanol, acetate) and formate. A higher yield of C₂ and C₄ compounds was observed for the immobilized cells than for the free cells in continuous culture. At 75 mMol/l of citrate, the citrate bioconversion yield was 42.8% and 80% for free and immobilized cells, respectively. This paper discusses citrate and lactose utilization and NADH₂ part on diacetyl reduction.     

Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis.

The pyruvate dehydrogenase complex of Lactococcus lactis subsp. lactis bv. diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate. The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively. The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa). The L. lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruvate. Thiamine pyrophosphate (Km = 3.2 microM) and divalent cations are essential for activity. The native enzyme measures 172 kDa and consists of 62-kDa monomers. The role of both enzymes in product formation is discussed in view of NADH inhibition and competition for pyruvate.