A phosphoglucose isomerase gene is involved in the Rag phenotype of the yeast Kluyveromyces lactis

Summary The rag2 mutant of Kluyveromyces lactis cannot grow on glucose when mitochondrial functions are blocked by various mitochondrial inhibitors, suggesting the presence of a defect in the fermentation pathway. The RAG2 gene has been cloned from a K. lactis genomic library by complementation of the rag2 mutation. The amino acid sequence of the RAG2 protein deduced from the nucleotide sequence of the cloned RAG2 gene shows homology to the sequences of known phosphoglucose isomerases (PGI and PHI). In vivo complementation of the pgi1 mutation in Saccharomyces cerevisiae by the cloned RAG2 gene, together with measurements of specific PGI activities and the detection of PGI proteins, confirm that the RAG2 gene of K. lactis codes for the phosphoglucose isomerase enzyme. Complete loss of PGI activity observed when the coding sequence of RAG2 was disrupted leads us to conclude that RAG2 is the only gene that codes for phosphoglucose isomerase in K. lactis. The RAG2 gene of K. lactis is expressed constitutively, independently of the growth substrates (glycolytic or gluconeogenic). Unlike the pgi1 mutants of S. cerevisiae, the K. lactis rag2 mutants can still grow on glucose, however they do not produce ethanol.     

Nisin-selectable food-grade secretion vector for Lactococcus lactis

A nisin-resistant Lactococcus lactis strain TML01 was isolated from crude milk. A gene with 99% homology to the nisin-resistance gene, nsr, was identified. The food-grade secretion plasmid, pLEB690 (3746 bp), was constructed based on this novel nsr gene enabling primary selection with up to 5 μg nisin/ml. The functionality of pLEB690 as a secretion vector was shown by expressing and secreting the pediocin AcH gene papA in L. lactis. pLEB690 is therefore, a functional food-grade secretion vector potentially useful for the food industry.     

Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector

Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined. One suppressor is an ochre suppressor with an altered tRNA_gin and the other an amber suppressor with an altered tRNA_ser. The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food-grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following etectroporation of a suppressible purine auxotroph, can be done on purine-free medium. The pepN gene from L. lactis Wg2 was subcloned resulting in a food-grade plasmid giving a four- to fivefold increase in lysine aminopeptidase activity.     

RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis

The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3–1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.     

The yeastKluyveromyces lactis as an efficient host for heterologous gene expression

Several different yeast species have been developed into systems for efficient heterologous gene expression. In this paper we review foreign gene expression in the dairy yeastKluyveromyces lactis. This yeast presents several advantageous properties in comparison to other yeast species. These include its impressive secretory capacities, its excellent fermentation characteristics on large scale, its food grade status and the availability of both episomal and integrative expression vectors. Moreover, in contrast to the methylotrophic yeasts that are frequently used for the expression of foreign genes,K. lactis does not require explosion-proof fermentation equipment. Here, we present an overview of the available tools for heterologous gene expression inK. lactis (available promoters, vector systems, etc). Also, the production of prochymosin, human serum albumin and pancreatic phospholipase byK. lactis is discussed in more detail, and used to rate the achievements ofK. lactis with respect to other micro-organisms in which these proteins have been produced.     

Sugar metabolism, redox balance and oxidative stress response in the respiratory yeast Kluyveromyces lactis

A lot of studies have been carried out on Saccharomyces cerevisiae, an yeast with a predominant fermentative metabolism under aerobic conditions, which allows exploring the complex response induced by oxidative stress. S. cerevisiae is considered a eukaryote model for these studies. We propose Kluyveromyces lactis as a good alternative model to analyse variants in the oxidative stress response, since the respiratory metabolism in this yeast is predominant under aerobic conditions and it shows other important differences with S. cerevisiae in catabolic repression and carbohydrate utilization. The knowledge of oxidative stress response in K. lactis is still a developing field. In this article, we summarize the state of the art derived from experimental approaches and we provide a global vision on the characteristics of the putative K. lactis components of the oxidative stress response pathway, inferred from their sequence homology with the S. cerevisiae counterparts. Since K. lactis is also a well-established alternative host for industrial production of native enzymes and heterologous proteins, relevant differences in the oxidative stress response pathway and their potential in biotechnological uses of this yeast are also reviewed.     

Cloning and Production of a Novel Bacteriocin, Lactococcin K, from Lactococcus lactis Subsp. lactis MY23

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml⁻¹ in 12 h. Revisions requested 3 November 2005; Revisions received 7 December 2005     

Loss of IrpT Function in Lactococcus lactis subsp. lactis N8 Results in Increased Nisin Resistance

The antibiotic nisin, produced by Lactococcus lactis subsp. lactis N8, offers an extensive commercial prospect as natural food preservatives. The nisin immunity of the L. lactis strains is regulated by a variety of mechanisms. In this study, we isolated a L. lactis L31 strain with increased nisin resistance from a mini-Mu transposon mutant pool of strain N8. The single Mu insertion in strain L31 was in the irpT gene with unknown function. By comparing the proteomic profiles of L. lactis L31 and its parental strain, we found that changes occurred in the synthesis of a protein involved in cell wall biosynthesis (RmlD). Strain L31 had 13.7% higher content of rhamnose in the cell wall than the N8 strain. Overexpression of RmlD involved in the synthesis of dTDP-l-rhamnose in the nisin-sensitive MG1363 strain increased nisin resistance of the strain. The results indicate that these cellular proteins effected nisin resistance in L. lactis N8.     

Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice

To develop an oral vaccine against Helicobacter pylori infection, we have expressed the H. pylori cag12 (HP0532) gene, encoding the outer membrane protein Cag12 (31 kDa), in a live delivery vehicle Lactococcus lactis. The cag12 gene was amplified by polymerase chain reaction (PCR) using the genomic DNA of H. pylori K51 isolated from Korean patients. DNA sequence analysis revealed that the cag12 gene of H. pylori K51 has 98.1 and 97.4% identity with individual cag12 genes of the H. pylori 26695 and J99, respectively. The GST–Cag12 fusion protein, produced using the Escherichia coli expression system, was used to raise a rat polyclonal anti-Cag12 antibody. The PCR-amplified cag12 gene of H. pylori K51 was cloned in the E. coli–L. lactis shuttle vector (pMG36e) and transformed into L. lactis. Western blot analysis demonstrated that the Cag12 protein was expressed in the L. lactis transformant, with a maximum level at the log phase without extracelluar secretion. The oral administration of the transformant into mice resulted in the generation of the anti-Cag12 antibody in serum in two out of five cases. These results suggest that the recombinant L. lactis, which expresses Cag12, may be applicable as an oral vaccine to induce protective immunity against H. pylori.     

A one step genotypic identification of Lactococcus lactis subspecies at the species/strain levels

A Lactococcus lactis subspecies-specific primer was designed based on their repetitive genome sequences. This primer enabled L. lactis subspecies to be identified simultaneously at both the species level and also the strain level. Based on studies using 70 strains of L. lactis and 60 strains of other non-target bacteria, the identification completely matched that obtained by the sequence of the 16S rRNA gene. However, inconsistency between phenotypic and genotypic characteristics was observed in some strains isolated from milk.     

An autoselection system in recombinant Kluyveromyces lactis enhances cloned gene stability and provides freedom in medium selection

An autoselection system for increasing plasmid stability in Kluyveromyces lactis, based on the blockage of the pyrimidine de novo and salvage pathways, was investigated. In a manner analogous to that used in Saccharomyces cerevisiae, a putative “fur1” mutation was selected in a uraA K. lactis strain using 5-fluorouracil and 5-fluorocytosine plates. Survival of the mutant required expression of a plasmid-borne URA3 gene regardless of the culture medium employed, verifying the efficacy of this autoselection system in K. lactis. The expression of heterologous invertase, encoded by the S. cerevisiae SUC2 gene, was studied during long-term sequential batch cultures (70 generations) in complex yeast/peptone/glucose medium. The fur1 mutant successfully retained the plasmid; invertase specific activity remained above 90% of the initial level. Furthermore, no mutation reversion was observed. In contrast, for the control non-fur1 strain, only 4% of the cells retained the plasmid after 70 generations, and invertase specific activity dropped to less than 10% of the initial level. Experiments comparing growth and activity in different media indicated the potential for improving productivity through medium enrichment using this autoselection system. Received: 1 April 1997 / Received revision: 16 August 1997 / Accepted: 11 September 1997     

Evolution of the alcohol dehydrogenase (ADH) genes in yeast: characterization of a fourth ADH in Kluyveromyces lactis

Summary Three alcohol dehydrogenase (ADH) genes have recently been characterized in the yeast Kluyveromyces lactis. We report on a fourth ADH in K. lactis (KADH II: KADH2 gene) which is highly similar to other ADHs in K. lactis and Saccharomyces cerevisiae. KADH II appears to be a cytoplasmic enzyme, and after expression of KADH2 in S. cerevisiae enzyme activity comigrated with a K. lactis ADH present in cells grown in glucose or in ethanol. KADH I was also expressed in S. cerevisiae and it comigrated with a major ADH species expressed under glucose growth conditions in K. lactis. The substrate specificities for KADH I and KADH II were shown to be more similar to that of SADH II than to SADH I. SADH I cannot efficiently utilize long chain alcohols, in contrast to other cytoplasmic yeast ADHs, presumably because of the presence of a methionine (residue 271) in its substrate binding cleft. A comparison of the DNA sequences of ADHs among K. lactis, S. cerevisiae and Schizosaccharomyces pombe suggests that the ancestral yeast species contained one cytoplasmic ADH. After divergence from S. pombe, the ADH in the ancestor to K. lactis and S. cerevisiae was duplicated, and one ADH became localized to the mitochondrion, presumably for the oxidative use of ethanol. Following the speciation of S. cerevisiae and K. lactis, the gene encoding the cytoplasmic ADH in S. cerevisiae duplicated, which resulted in the development of the SADH II protein as the primary oxidative enzyme in place of SADH III. In contrast, the K. lactis mitochondrial ADH duplicated to give rise to the highly expressed KADH3 and KADH4 genes, both of which may still play primary roles in oxidative metabolism. These data suggest that K. lactis and S. cerevisiae use different compartments for their metabolism of ethanol. Our results also indicate that the complex regulatory circuits controlling the glucose-repressible SADH2 in S. cerevisiae are a recent acquisition from regulatory networks used for the control of genes other than SADH2.

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Inulinase-hyperproducing strains of Kluyveromyces sp. isolated from aguamiel (Agave sap) and pulque

Summary Two strains of Kluyveromyces marxianus (A1 and A2) isolated from ‘aguamiel’ (agave sap) and one strain of K. lactis var. lactis (P7) isolated from ‘pulque’ (its fermented product), were studied to make a survey of inulinase production. The strains of K. marxianus A1 and A2 were the best producers of inulinase, giving up to 2.5 times more enzyme than the control hyperproducing strain K. marxianus CDBB-L-278, and showed lower catabolic repression than this. One strain isolated from pulque was identified as K. lactis var. lactis and was also an excellent inulinase producer, being the first strain of this species reported as such. These strains were very good inulinase producers and they had low susceptibility to catabolic repression probably because the source from which they were isolated was rich in sucrose and oligofructans. They can be used in the transformation of inulin to produce fructose and/or oligofructans.     

Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis

Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding α1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative α1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM–CSF in Kloch1 mutant, contained oligosaccharide Man_13–14GlcNAc₂, and in Kloch1 mnn1 mutant, contained oligosaccharide Man_9–11GlcNAc₂, whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man_>30GlcNAc₂. Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes α1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.     

The Saccharomyces cerevisiaeSOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane

The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28° C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane. Received: 22 July 1997 / Accepted: 27 October 1997     

Flavour formation in fungi: characterisation of KlAtf,the <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> orthologue of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferases Atf1 and Atf2

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. In the brewers’ yeast Saccharomyces cerevisiae, the major part of these esters is formed by two alcohol acetyltransferases, Atf1 and Atf2. In this paper, the existence of orthologues of these S. cerevisiae alcohol acetyltransferases in several ascomycetous fungi was investigated. Bioinformatic analysis of sequenced fungal genomes revealed the presence of multiple orthologues. The Saccharomyces sensu stricto yeasts all have two genes coding for orthologues. More distantly related fungi like Saccharomyces castelii, Candida glabrata, Kluyveromyces waltii and Kluyveromyces lactis have only one orthologue in their genome. The homology between the identified proteins and the S. cerevisiae alcohol acetyltransferases suggests a role for these orthologues in the aroma-active ester formation. To verify this, the K. lactis orthologue KlAtf was cloned and expressed in S. cerevisiae. Gas chromatographic analysis of small-scale fermentations with the transformant strains showed that, while S. cerevisiae ATF1 overexpression resulted in a substantial increase in acetate ester levels, S. cerevisiae ATF2 and K. lactis ATF overexpression only caused a moderate increase in acetate esters. This study is the first report of the presence of an ester synthesis gene in K. lactis.     

Enhanced expression of heterologous inulinase in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> by disruption of <Emphasis Type="Italic">hap1</Emphasis> gene

Inulinase gene (Kcinu) derived from Kluyveromyces cicerisporus was expressed extracellularly in Kluyveromyces lactis using an episomal vector directed by Kcinu promoter. The influence of hap1 gene disruption on the expression of inulinase was studied. Inulinase activity in the supernatant of the recombinant Klhap1Δ strain was 391 U ml⁻¹ after cultured 120 h, which was 2.2-fold that of the wild type host. The relative inulinase mRNA level of the Klhap1Δ strain was 11.3-fold that of the wild type strain, and the expression plasmid was more stable in the mutant host. Based on these results, the disruption of hap1 facilitated the high and stable expression of inulinase controlled by Kcinu promoter in K. lactis.     

The gene (lacA) encoding galactoside acetyltransferase fromLactococcus lactis

Summary This paper reports the cloning and characterization of a gene encoding galactoside acetyltransferase from a strain ofLactococcus lactis. AP stI library ofL. lactis strain ATCC7962 DNA was constructed in plasmid pUC18. A clone harbouring a 10 kbp DNA fragment containing part of thelac operon was isolated using a labelled probe generated by PCR. DNA sequence analysis revealed the presence of a gene encoding a protein with 64.5% similarity to the galactoside acetyltransferase fromEscherichia coli. The codon usage pattern of this gene was not typical of lactococcal genes. The lactococcallac operon organization appears to be different to that of other organisms.