Yeast sequencing reports. Isolation and sequence analysis of a gene from the linear DNA plasmid pPAC1–2 of Pichia acaciae that shows similarity to a killer toxin gene of Kluyveromyces lactis

The toxin-encoding linear plasmid systems found in Pichia acaciae and Kluyveromyces lactis yeasts appear to be quite similar, both in function and structural organization. By Southern hybridization, a linear plasmid of P. acaciae, pPac1–2, was found to hybridize to the second open reading frame (ORF2) of K. lactis plasmid pGKL1, known to encode the α and β subunits of the K. lactis toxin. A 1·7 kbp segment of pPac1–2 DNA was cloned, sequenced and shown to contain four regions of strong homology to four similarly oriented regions of K. lactis ORF2. This 1·7 kbp fragment also contained an ORF of 1473 bp that could encode a protein of ～ 55·8 kDa. Like the α subunit gene of K. lactis ORF2, a very hydrophobic region occurs at the N-terminus, perhaps representing a signal sequence for transport out of the cell. Unlike K. lactis ORF2, however, the encoded polypeptide is much smaller and lacks a recognizable domain common to chitinases. The structure of a toxin that includes the translation product of this P. acaciae ORF would likely be quite different from that of the K. lactis toxin. Analysis of the upstream region of the P. acaciae ORF revealed an upstream conserved sequence identical to that found before ORFs 8 and 9 of pGKL2. A possible hairpin loop structure, as has been described for each of the four K. lactis pGKL1 ORFs, was found just upstream of the presumed start codon. The similarity of the promoter-like elements found in the linear plasmid genes of these diverse yeasts reinforces the idea of the existence of a unique, but highly conserved, expression system for these novel plasmids. The sequence has been deposited in the GenBank data library under Accession Number U02596.     

Enhancement of nisin production in milk by conjugal transfer of the protease‐lactose plasmid pLP712 to the wild strain Lactococcus lactis UQ2

Lactococcus lactis UQ2 is a wild nisin A producer isolated from a Mexican cheese that grows poorly in milk. Conjugal matings with L. lactis NCDO712 to transfer the Lac+ Prt+ plasmid pLP712 and selection with nisin and lactose yielded L. lactis NCDO712 NisA+. Naturally rifampicin resistant L. lactis UQ2Rif was isolated to provide an additional selective marker. The identity of a transconjugant L. lactis UQ2Rif Lac+ was confirmed by RAPD‐PCR fingerprinting, nisA PCR amplification, nisin production, presence of pLP712 and phospho‐β‐galactosidase activity. This strain performed well in milk and synthesised 200 IU/mL nisin, 40 times more than the original strain.     

The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase

The 36K protein attached at the 5′ end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0·8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.     

Secretion of Mouse α-Amylase from Kluyveromyces lactis

We constructed two mouse α-amylase secretion vectors for Kluyveromyces lactis using the well-characterized signal sequence of the pGKL 128 kDa killer precursor protein. Both PHO5 and PGK expression cassettes from Saccharomyces cerevisiae directed the expression of mouse α-amylase in YPD medium at a similar level of efficiency. K. lactis transformants secreted glycosylated and non-glycosylated α-amylase into the culture medium and both species were enzymatically active. The K. lactis/S. cerevisiae shuttle secretion vector pMI6 was constructed, and K. lactis MD2/1(pMI6) secreted about four-fold more α-amylase than S. cerevisiae YNN27 harboring the same plasmid, indicating that K. lactis is an efficient host cell for the secretion and production of recombinant proteins. © 1997 John Wiley & Sons, Ltd.     

Sequence analysis of a 5·6 kb fragment of chromosome II from Saccharomyces cerevisiae reveals two new open reading frames next to CDC28

The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28. Gene disruption reveals both putative genes as non-essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Sur1 protein of S. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis-secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product. The total sequence of the fragment has been submitted to the EMBL databank (accession number X80224).     

The Linear Plasmid pDHL1 from Debaryomyces hansenii Encodes a Protein Highly Homologous to the pGKL1-Plasmid DNA Polymerase

Both the linear plasmids, pDHL1 (8·4 kb) and pDHL2 (9·2 kb), of Debaryomyces hansenii TK require the presence of a third linear plasmid pDHL3 (15·0 kb) in the same host cell for their replication. A 3·5 kb Bam HI-PstI fragment of pDHL1 strongly hybridized by Southern analysis to the 3·5 kb NcoI-AccI fragment of pDHL2, suggesting the importance of this conserved region in the replication of the two smaller pDHL plasmids. The 4·2 kb pDHL1 fragment containing the above hybridized region was cloned and sequenced. The results showed that the cloned pDHL1 fragment encodes a protein of 1000 amino acid residues, having a strong similarity to the DNA polymerase coded for by ORF1 of the killer plasmid pGKL1 from Kluyveromyces lactis. The catalytic and proof-reading exonuclease domains as well as terminal protein motif were well conserved as in DNA polymerases of pGKL1 and other yeast linear plasmids. Analysis of the cloned fragment further showed that pDHL1 encodes a protein partly similar to the α subunit of the K. lactis killer toxin, although killer activity was not known in the DHL system. Analysis of the 5′ non-coding region of the two above pDHL1-ORFs reveal the presence of the upstream conserved sequence similar to that found upstream of pGKL1-ORFs. The possible hairpin loop structure was also found just in front of the ATG start codon of the pDHL1-ORFs like pGKL1-ORFs. Thus the cytoplasmic pDHL plasmids were suggested to possess a gene expression system comparable to that of K. lactis plasmids. © John Wiley & Sons, Ltd.     

Disruption of PMR1 in Kluyveromyces lactis improves secretion of calf prochymosin

BACKGROUND: Chymosin is an important industrial enzyme widely used in cheese manufacturing. Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, only low yields of chymosin (80 U mL^?1 in shake flask culture) have been obtained using K. lactis GG799. The aim of this study was to increase the amount of recombinant calf chymosin secreted by K. lactis GG799 by disrupting the PMR1 gene. RESULTS: Kluyveromyces lactis GG799 harbouring the disrupted PMR1 gene showed reduced growth in ethylene glycol tetraacetic acid‐containing and Ca²⁺‐deficient medium, but Ca²⁺ supplementation eliminated the growth problem. The calf chymosin gene was ligated into the K. lactis GG799 expression vector, generating the plasmid pKLAC1‐N‐prochymosin. The linearised plasmid was homologously integrated into the genome of K. lactis GG799. In shake flask culture, chymosin activity was 496 U mL^?1 in the K. lactis PMR1‐deficient mutant, sixfold higher than that in wild‐type K. lactis GG799. CONCLUSION: Disrupting the PMR1 gene improved chymosin production in K. lactis GG799 sixfold. This knowledge could be applied to industrial chymosin production. Copyright © 2010 Society of Chemical Industry     

The NUM1 yeast gene: Length polymorphism and physiological aspects of mutant phenotype

We have isolated a mutant (rvs272) of the yeast (Saccharomyces cerevisiae) that displays an altered phenotype in stationary phase. It shows a high proportion of large-budded cells with two non-segregated nuclei staying in the mother cell. This phenotype is also expressed in various conditions when cells are synchronized, energy depleted or treated with the antimitotic drug benomyl. The RVS272 gene has been identified as the NUM1 gene already described. This gene presents a 192 bp tandemly repeated motif and we show that the number of repeats can vary from 1 to about 24 among different strains, without apparently affecting the function of the encoded protein. We suggest that this protein could be involved in polymerization catalysis and/or stabilization of microtubules.     

Isolation of a gene encoding a G protein α subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0·5 kb (P-1) and 0·8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the Gα subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein α subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this Gα subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian Gαs family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L45105).     

A nuclear gene required for the expression of the linear DNA-asociated killer system in the yeast Kluyveromyces lactis

The killer system of Kluveromyces lactis is associated with two linear DNA plasmids, pGKL1 and pGKL2. The killer toxin and the immunity determinant are coded for by pGKL1. Mutations which we have named KEX1. The KEX1 gene of K. lactis has been cloned by complementation of kex1 mutations by using a recombinant plasmid pool containing the entire Kluyveromyces lactis genome, on a multicopy plasmid KEp6, which contains the Saccharomyces cerevisiae URA3 gene as a marker. Genetic analyses of strains carrying a distrupted kex1 allele demonstrated that the cloned DNA corresponded to the KEX1 gene. The cloned KEX1 gene of K. lactis has low but significant sequence homology with the KEX2 gene of Saccharomyces cerevisiae. In vivo complementation of the kex1 mutations of K. lactis by the KEX2 gene of S. cerevisiae, and complementation of the kex2 mutations of S. Cerevisiae by the KEX1 gene of K. lactis, demonstrated that KEX1 of K. Lactis is functionally related to the KEX2 gene of S. cerevisiae. K. lactis diploids homozygous for kex1 are deficient for sporulation.     

Cloning and sequence of a 3·835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans

We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3′-terminal half of a gene encoding a PEX5-like protein. The EMBL/DDJB/GenBank Accession Number of this sequence is AJ003115. © 1998 John Wiley & Sons, Ltd.     

Heterologous extracellular production of enterocin P in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by a food-grade expression system

In order to develop an entirely food-grade enterocin P expression system for the food industry, the enterocin P structural gene (entP) with or without the enterocin P immunity gene (entiP) was cloned in plasmid pLEB590 under control of the lactococcal constitutive promoter P₄₅. Introduction of the recombinant vectors in L. lactis MG1614 resulted in production of biologically active enterocin P in the supernatants of recombinant L. lactis MG1614. Moreover, coexpression of the entP and entiP genes could increase the production of enterocin P in all L. lactis MG1614 hosts. Recombinant enterocin P from L. lactis MG1614 (pLEB590-entP2) was purified by a three-step procedure involving ammonium sulfate precipitation, SP-Sepharose Fast Flow cation exchange, and hydrophobic adsorption chromatography. The purified bacteriocin protein concentration from recombinant L. lactis MG1614 (pLEB590-entP2) was 3.9-fold greater than that of E. faecium LM-2, and the final recovery of enterocin P activity from the supernatant of L. lactis MG1614 (40.2%) was dramatically improved compared with that of the native host strain (19.9%). Bacteriocin activity and Tricine-SDS–PAGE analysis revealed that purified recombinant enterocin P is biologically active and has a molecular mass corresponding to the native enterocin P from E. faecium LM-2, suggesting that the synthesis, process, and secretion of enterocin P progresses efficiently in recombinant L. lactis MG1614 hosts. The enterocin P was expressed successfully in this food-grade system.     

YHp as a highly stable,hyper-copy,hyper-expression plasmid constructed using a full 2-μm circle sequence in cir0 strains of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2-μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir⁰ strain as a host for constructing an entire 2-μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2-μm plasmid contains two long inverted repeats, which is problematic for the amplification by polymerase chain reaction. Therefore, we amplified it by dividing into two fragments, each containing a single repeat together with an overlapping sequence for homologous recombination. TDH3 promoter-driven yEmRFP (TDH3p-yEmRFP) and the URA3 were used as a reporter gene and a selection marker, respectively, and inserted at the 3′ end of the RAF1 gene on the 2-μm plasmid. The three fragments were combined and used for the transformation of sake yeast cir⁰ ura3^- strain. The resulting transformant colonies showed a red or purple coloration, which was significantly stronger than that of the cells transformed with YEp-TDH3p-yEmRFP. The 2-μm transformants were cultured in YPD medium and observed by fluorescence microscopy. Almost all cells showed strong fluorescence, suggesting that the plasmid was preserved during nonselective culture conditions. The constructed plasmid maintained a high copy state similar to that of the natural 2-μm plasmid, and the red fluorescent protein expression was 54 fold compared with the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.     

The KlPMR1 gene of Kluyveromyces lactis encodes for a P‐type Ca2+‐ATPase

A novel P‐type Ca²⁺‐ATPase gene has been cloned and sequenced in the yeast Kluyveromyces lactis. The gene has been named KlPMR1 and is localized on chromosome I. The putative gene product contains 936 residues and has a calculated molecular weight of 102 437 Da. Analysis of the deduced amino acid sequence (KlPmr1p) indicated that the encoded protein retains all the highly conserved domains characterizing the P‐type ATPases. KlPmr1p shares 71% amino acid identity with Pmr1p of S. cerevisiae, 62% with HpPmr1p of Hansenula polymorpha, 56% with YlPmr1p of Yarrowia lipolytica and 52% with the Ca²⁺‐ATPase encoded for by the SPCA1 gene of Rattus norvegicus; these similarities place KlPmr1p in the SPCA group (secretory pathway Ca²⁺‐ATPase) of the P‐type ATPases. The K. lactis strain harbouring the Klpmr1 disrupted gene is not able to grow in presence of low calcium concentrations and shows hypersensitivity to high concentrations of EGTA in the medium. These defects are relieved by PMR1 of S. cerevisiae on a centromeric plasmid, demonstrating that KlPMR1 encodes for a functional Pmr1p homologue. The sequence described can be retrieved under EMBL Accession No. AJ001018. Copyright © 1999 John Wiley & Sons, Ltd.     

Expression of milk-derived angiotensin I-converting enzyme-inhibitory peptides in Lactococcus lactis

Angiotensin I-converting enzyme (ACE) plays a key role in the regulation of blood pressure. Currently, most single or tandem repeats of ACE-inhibitory (ACE-I) peptides have been expressed in Escherichia coli. However, in this study, a food-grade system was constructed using Lactococcus lactis (L. lactis) to simultaneously express four different milk-derived ACE-I peptides with antihypertensive activity. Mixed peptides (MPs) fused with green fluorescent protein (GFP) and eight histidines were synthesized. To ensure potent ACE inhibition by the MPs in human digestive juice, pepsin and trypsin cleavage sites were introduced among the four ACE-I peptides. The MP fusion gene was inserted into expression vector pSEC-E7 with nisin induction and expressed in L. lactis NZ9000, then purified by affinity chromatography. The transformants containing pSEC-MP:GFP were identified based on green fluorescence using Leica laser scanning confocal microscopy. The target proteins were detected by SDS-PAGE analysis and displayed obvious immunogenicity by western blot. After hydrolysis with digestive enzymes, the IC₅₀ of the MPs was 118.63 μM. These results suggested that multiple milk-derived ACE-I peptides with antihypertensive properties could be produced using a food-grade lactococcal expression system.     

Cloning,sequencing and disruption of the ARG8 gene encoding acetylornithine aminotransferase in the petite-negative yeast Kluyveromyces lactis

A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.