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 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.     

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes.

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase. Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis beta-galactosidase was overproduced in E. coli by using a lambda pL expression system. Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.     

Molecular cloning and expression in E. coli of a yeast gene coding for beta-galactosidase.

The yeast Kluyveromyces lactis synthesizes a beta-galactosidase (EC 3.2.1.32) which is inducible by lactose. We have isolated the gene that codes for this enzyme using recombinant DNA techniques. K. lactis DNA was partially digested with the restriction endonuclease Eco R1 and joined to Eco R1-digested pBR322 plasmid DNA using DNA ligase. ligase. A lac-mutant of Escherichia coli lacking the structural gene for beta-galactosidase was transformed with ligated DNA. Three lac+ transformants containing recombinant plasmids were selected. Two of the plasmids (pK15 and pK17) contain four Eco R1-K. lactis DNA fragments having molecular weights of 2.2, 1.4, 0.55 and 0.5 x 10(6) daltons. The other plasmid (pK16) lacks the smallest fragment. E. coli carrying any of these plasmids produce beta-galactosidase activity that has a sedimentation coefficient and immunological determinants that are nearly identical to K. lactis beta-galactosidase and distinctly different from E. coli beta-galactosidase. DNA-DNA hybridization studies show that the four Eco R1 fragments in pK15 hybridize to K. lactis but not to E. coli DNA.     

Structure and expression of the Lactococcus lactis gene for phospho-beta-galactosidase (lacG) in Escherichia coli and L. lactis

The Lactococcus lactis subsp. lactis 712 lacG gene encoding phospho-beta-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in Escherichia coli and L. lactis. The low phospho-beta-galactosidase activity in L. lactis transformed with high-copy-number plasmids containing the lacG gene contrasted with the high activity found in L. lactis containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In E. coli the phospho-beta-galactosidase could be overproduced using the strong inducible lambda PL promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the L. lactis lacG gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-beta-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the lacG gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the L. lactis lacG gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the lacG gene. The organization and sequence of the L. lactis lacG gene were compared with those of the highly homologous lacG gene from Staphylococcus aureus. A remarkable bias for leucine codons was observed in the lacG genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the L. lactis phospho-beta-galactosidase, that of the functionally analogous E. coli phospho-beta-glucosidase, and that of an Agrobacterium beta-glucosidase (cellobiase).     

Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N.

The chromosomal pepN gene encoding lysyl-aminopeptidase activity in Lactococcus lactis has been identified in a lambda EMBL3 library in Escherichia coli by using an immunological screening with antiserum against a purified aminopeptidase fraction. The pepN gene was localized and subcloned in E. coli on the basis of its expression and hybridization to a mixed-oligonucleotide probe for the previously determine N-terminal amino acid sequence of lysyl-aminopeptidase (P. S. T. Tan and W. N. Konings, Appl. Environ. Microbiol. 56:526-532, 1990). The L. lactis pepN gene appeared to complement an E. coli strain carrying a mutation in its pepN gene. High-level expression of the pepN gene in E. coli was obtained by using the T7 system. The overproduction of the 95-kDa aminopeptidase N could be visualized on sodium dodecyl sulfate-polyacrylamide gels and immunoblots. Cloning of the pepN gene on a multicopy plasmid in L. lactis resulted in a 20-fold increase in lysyl-aminopeptidase activity that corresponded to several percent of total protein. Nucleotide sequence analysis of the 5' region of the pepN gene allowed a comparison between the deduced and determined amino-terminal primary sequences of aminopeptidase N. The results show that the amino terminus of PepN is not processed and does not possess the characteristics of consensus signal sequences, indicating that aminopeptidase N is probably an intracellular protein. The intracellular location of aminopeptidase N in L. lactis was confirmed by immunogold labeling of lactococcal cells.     

Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N.

The chromosomal pepN gene encoding lysyl-aminopeptidase activity in Lactococcus lactis has been identified in a lambda EMBL3 library in Escherichia coli by using an immunological screening with antiserum against a purified aminopeptidase fraction. The pepN gene was localized and subcloned in E. coli on the basis of its expression and hybridization to a mixed-oligonucleotide probe for the previously determine N-terminal amino acid sequence of lysyl-aminopeptidase (P. S. T. Tan and W. N. Konings, Appl. Environ. Microbiol. 56:526-532, 1990). The L. lactis pepN gene appeared to complement an E. coli strain carrying a mutation in its pepN gene. High-level expression of the pepN gene in E. coli was obtained by using the T7 system. The overproduction of the 95-kDa aminopeptidase N could be visualized on sodium dodecyl sulfate-polyacrylamide gels and immunoblots. Cloning of the pepN gene on a multicopy plasmid in L. lactis resulted in a 20-fold increase in lysyl-aminopeptidase activity that corresponded to several percent of total protein. Nucleotide sequence analysis of the 5' region of the pepN gene allowed a comparison between the deduced and determined amino-terminal primary sequences of aminopeptidase N. The results show that the amino terminus of PepN is not processed and does not possess the characteristics of consensus signal sequences, indicating that aminopeptidase N is probably an intracellular protein. The intracellular location of aminopeptidase N in L. lactis was confirmed by immunogold labeling of lactococcal cells.     

Expression, purification, and characterization of arginine deiminase from Lactococcus lactis ssp. lactis ATCC 7962 in Escherichia coli BL21

The arcA gene that encodes arginine deiminase (ADI, EC 3.5.3.6)--a key enzyme of the ADI pathway--was cloned from Lactococcus lactis ssp. lactis ATCC 7962. The deduced amino acid sequence of the arcA gene showed high homology with the arcA gene from Lactobacillus plantarum (99%) and from Lactobacillus sakei (60%), respectively. The arcA gene from Lc. lactis spp. lactis ATCC 7962 was expressed in soluble fraction of recombinant Escherichia coli BL21. ADI produced from Lc. lactis spp. lactis ATCC 7962 (LADI) in E. coli BL21 (DE3) was purified using sequential Q-Sepharose anion exchange and Sephacryl S-200 gel filtration column chromatography. The final yield of LADI in the purification procedure was 63.5%, and the specific activity was 140.27 U/mg. The presence of purified LADI was confirmed by N-terminal sequencing and determination of the molecular mass. The LADI had a molecular mass of about 140 kDa, and comprised a homotrimer of 46 kDa in the native condition. LADI exhibited only 35% amino acid sequence homology with ADI from Mycoplasma arginini. However, LADI shared a similar three dimensional structure. The K(M) and V(max) values for arginine were 8.67+/-0.045 mM (mean+/-SD) and 344.83+/-1.79 micromol/min/mg, respectively, and the optimum temperature and pH for the production of LADI were 60 degrees C and 7.2.     

Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment

Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli, retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli, using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis, in E. coli, Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.     

Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase

The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.     

Tripeptidase gene (pepT) of Lactococcus lactis: molecular cloning and nucleotide sequencing of pepT and construction of a chromosomal deletion mutant.

The gene encoding a tripeptidase (pepT) of Lactococcus lactis subsp. cremoris (formerly subsp. lactis) MG1363 was cloned from a genomic library in pUC19 and subsequently sequenced. The tripeptidase of L. lactis was shown to be homologous to PepT of Salmonella typhimurium with 47.4% identity in the deduced amino acid sequences. L. lactis PepT was enzymatically active in Escherichia coli and allowed growth of a peptidase-negative leucine-auxotrophic E. coli strain by liberation of Leu from a tripeptide. Using a two-step integration-excision system, a pepT-negative mutant of L. lactis was constructed. No differences between the growth of the mutant and that of the wild-type strain in milk or in chemically defined medium with casein as the sole source of essential amino acids were observed.     

产异丁醇关键基因在大肠杆菌中表达的研究

[目的]改造大肠杆菌缬氨酸合成途径,使其能够代谢合成异丁醇.[方法]将乳酸乳球菌(Lactococcus lactis) 1.2829的2-酮异戊酸脱羧酶基因(kivD)和醇脱氢酶基因(adhA)串联克隆到大肠杆菌DH5α宿主中表达.[结果]经过改造的宿主菌发酵24 h后异丁醇产量为0.12 g/L.酶活测定实验发现,kivD和adhA基因在宿主菌中均得到表达,但由于KivD的低表达量导致宿主菌最终的异丁醇合成能力偏低.通过研究温度和pH对KivD和AdhA酶活的影响,最终选定二者的最适温度为30℃,最适pH为6.5. [结论]通过向宿主菌导入外源异丁醇合成基因能够改造其自身代谢途径,从而合成异丁醇.     

An origin of transfer from Lactococcus lactis subsp. lactis causes rearrangement in the temperature sensitive plasmids pUCB3522 and pJRS290

Cloning of the oriT region derived from Lactococcus lactis subsp. lactis into plasmids is expected to convert them into mobilizable plasmids in a suitable genetic background. In some cases, however, the cloning of oriT causes deleterious DNA rearrangements. In the work presented, oriT was cloned into two temperature sensitive plasmids, pJRS290 and pUCB3522. The plasmids were transformed into E. coli and electroporated into L. lactis. Restriction digestion of the plasmids derived from the cloned transformed cells showed fragment sizes different from those expected, indicating that rearrangements had occurred. Furthermore, the inability to successfully transform E. coli with one of the plasmids supports this conclusion.