Location Effects of a Reporter Gene on Expression Levels and on Native Protein Synthesis in Lactococcus lactis and Saccharomyces cerevisiae

The engineering of industrially important genetically modified organisms by the integration of heterologous genes into the chromosome is often the method of choice for several reasons concerned with long-term stability, homogeneous population distribution, and the enabling of selection without the addition of antibiotics. However, integration may disrupt endogenous gene expression, giving rise to increased levels of toxic metabolic byproducts or activating otherwise silent genes. The position of integration of a foreign gene in the chromosome can also influence its expression levels, and this effect will be of relevance in terms of optimizing protein production parameters. In this study, we determine how the random integration of a foreign reporter gene might affect expression levels and assess the use of proteome analysis to investigate possible effects on synthesis of endogenous proteins in two important food-relevant microorganisms, Saccharomyces cerevisiae and Lactococcus lactis. Eleven L. lactis integrants carrying the gusA gene were analyzed, and expression levels were found to vary by a factor of threefold in contrast to expression levels of lacZ in 18 S. cerevisiae integrants, which showed a 14-fold variation. Of relevance to industry is whether any changes in expression levels might occur as a consequence of storage of the modified strains. Here it is also shown that the above differences in expression levels were not significantly affected by storage of frozen cultures over a period of several months. Analysis of the protein composition of the yeast and lactococcal integrant strains by separation on one-dimensional (1D) and 2D gels showed no significant variations in position beyond those observed in control samples.     

The effects of osmotic stress on survival and alkaline phosphatase activity of Aeromonas hydrophila

Abstract Lactococcus lactis MG5267 is a plasmid-free strain in which the lactose operon is integrated in the bacterial chromosome. The chromosomal lac G gene which encodes phospho-β-galactosidase was inactivated by a double cross-over integration event. Unexpectedly, the resultant mutant was shown to retain a Lac-positive phenotype. The lysin gene from Listeria monocytogenes bacteriophage LM-4 was subsequently integrated into the chromosome of this strain such that expression of the heterologous gene was mediated by the lactose operon promoter. Expression of the lysin gene was shown to be regulated by growth on lactose. This represents an important strategy for the controlled and stabilised expression of biotechnologically useful genes in L lactis .     

A truncated form of KlLsm4p and the absence of factors involved in mRNA decapping trigger apoptosis in yeast

The LSM4 gene of Saccharomyces cerevisiae codes for an essential protein involved in pre-mRNA splicing and also in mRNA decapping, a crucial step for mRNA degradation. We previously demonstrated that the first 72 amino acids of the Kluyveromyces lactis Lsm4p (KlLsm4p), which contain the Sm-like domains, can restore cell viability in both K. lactis and S. cerevisiae cells not expressing the endogenous protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of viability in stationary phase cells (). Herein, we demonstrate that S. cerevisiae cells expressing the truncated LSM4 protein of K. lactis showed the phenotypic markers of yeast apoptosis such as chromatin condensation, DNA fragmentation, and accumulation of reactive oxygen species. The study of deletion mutants revealed that apoptotic markers were clearly evident also in strains lacking genes involved in mRNA decapping, such as LSM1, DCP1, and DCP2, whereas a slight effect was observed in strains lacking the genes DHH1 and PAT1. This is the first time that a connection between mRNA stability and apoptosis is reported in yeast, pointing to mRNA decapping as the crucial step responsible of the observed apoptotic phenotypes.     

New tools for the physical and genetic mapping of Lactococcus strains.

Tools for the genetic and physical analysis of the Lactococcus lactis subsp. lactis genome were developed. Plasmid pRC1 does not replicate in Gram+ bacteria; it contains unique ApaI, NotI and SmaI restriction sites and an erythromycin-resistance (ErR) encoding gene, ermAM, functional in L. lactis subsp. lactis. When a chromosomal L. lactis subsp. lactis DNA fragment was cloned into this vector, the resulting plasmid became integrated, after transformation, into the bacterial chromosome by homologous recombination in a Campbell-like manner. The integration lead to the generation of new rare restriction sites near to the host fragment. This procedure allows precise mapping of cloned genes onto the chromosomal restriction map. The mapping of the his operon of L. lactis subsp. lactis provides an illustration. The cloning into pRC1 of an IS element able to transpose into the chromosome of the target cell, gave rise to an integration plasmid able to insert randomly rare restriction sites onto the bacterial chromosome. The L. lactis IS element, ISS1RS, was cloned into pRC1, yielding pRL1. Pulsed-field gel electrophoresis analysis of ErR clones obtained after transformation with pRL1, showed that this plasmid was stably integrated at a number of different sites in the L. lactis subsp. lactis chromosome, via transposition. Plasmids pRC1 and pRL1 can greatly facilitate the construction of the physical and genetic map of the chromosome of lactococcal strains.     

Gene expression in Lactococcus lactis

Abstract Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis . A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis . This feature allowed the expression of a number of L. lactis -derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.     

Gene expression in Lactococcus lactis.

Lactic acid bacteria are of major economic importance, as they occupy a key position in the manufacture of fermented foods. A considerable body of research is currently being devoted to the development of lactic acid bacterial strains with improved characteristics, that may be used to make fermentations pass of more efficiently, or to make new applications possible. Therefore, and because the lactococci are designated 'GRAS' organisms ('generally recognized as safe') which may be used for safe production of foreign proteins, detailed knowledge of homologous and heterologous gene expression in these organisms is desired. An overview is given of our current knowledge concerning gene expression in Lactococcus lactis. A general picture of gene expression signals in L. lactis emerges that shows considerable similarity to those observed in Escherichia coli and Bacillus subtilis. This feature allowed the expression of a number of L. lactis-derived genes in the latter bacterial species. Several studies have indicated, however, that in spite of the similarities, the expression signals from E. coli, B. subtilis and L. lactis are not equally efficient in these three organisms.     

Disruption of the MNN10 gene enhances protein secretion in Kluyveromyces lactis and Saccharomyces cerevisiae

Screening for genes affecting super-secreting phenotype of the over-secreting mutant of Kluyveromyces lactis resulted in isolation of the gene named KlMNN10, sharing high homology with Saccharomyces cerevisiae MNN10. The disruption of the KlMNN10 in Kluyveromyces lactis, as well as of MNN10 and MNN11 in Saccharomyces cerevisiae, conferred the super-secreting phenotype. MNN10 isolated from Saccharomyces cerevisiae suppressed the super-secretion phenotype in Kluyveromyces lactis klmnn10, as did the homologous KlMNN10. The genes MNN10 and MNN11 of Saccharomyces cerevisiae encode mannosyltransferases responsible for the majority of the alpha-1,6-polymerizing activity of the mannosyltransferase complex. These data agree with the view that the structure of glycoproteins in a yeast cell wall strongly influences the release of homologous and heterologous proteins in the medium. The set of genes namely the suppressors of the over-secreting phenotype, could be attractive for further analysis of gene functions, over-secreting mechanisms and for construction of new strains optimized for heterologous protein secretion. KlMNN10 has EMBL accession no. AJ575132.     

Host-vector system for phenol-degrading Rhodococcus erythropolis based on Corynebacterium plasmids

Two integrating vectors developed for use in Saccharomyces cerevisiae were successfully employed for cloned gene integration in the yeast Kluyveromyces lactis. A delta-integrating vector carrying the dominant selection marker neo allowed tandem integrations of a CUP1p-lacZ cassette into one or two chromosomal sites. A delta/UB-integrating vector, which contains a reusable selection cassette, enabled multiple rounds of integration and the sequential insertion of stable, dispersed copies of CUP1p-lacZ. Subsequent gene expression was closely correlated with integrated copy number illustrating the promise of this method for metabolic engineering in K. lactis. While both vectors contain an S. cerevisiae delta target sequence, the presence of delta-like elements in K. lactis has not been confirmed. Given the degree of illegitimate recombination in this yeast species, the insertions likely occurred at random locations in the chromosomes.     

Modulation of gene expression made easy

A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the pfk gene was cloned upstream of a gusA gene encoding beta-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected the activities of all three enzymes to the same extent, and enzyme activities ranging from 0.5 to 3.5 times the wild-type level were obtained.     

Genetic and biochemical characterization of the galactose gene cluster in Kluyveromyces lactis.

We isolated and identified mutant strains of Kluyveromyces lactis that are defective for the Leloir pathway enzymes galactokinase, transferase, and epimerase, and we termed these loci GAL1 , GAL7 , and GAL10 , respectively. Genetic data indicate that these three genes are tightly linked, having an apparent order of GAL7 - GAL10 - GAL1 . This same gene order has been observed in Saccharomyces cerevisiae. Strains harboring gal7 mutations have elevated levels of beta-galactosidase, coded by an unlinked gene, galactokinase, and epimerase activities under uninduced conditions. We investigated the genetic basis of this constitutive gene expression and found no recombinants between the constitutive and Gal- phenotypes among 76 tetrads, suggesting that either GAL7 or a tightly linked gene codes for a regulatory function. This is the second gene that has been shown to specifically coregulate expression of the genes coding for beta-galactosidase and the Leloir pathway enzymes.     

不同宿主来源的重组酿酒酵母混合糖代谢比较

【目的】研究不同工业酿酒酵母宿主背景对重组酵母木糖利用效率的影响。【方法】将木糖利用途径的木糖还原酶(XR)、木糖醇脱氢酶(XDH)和木酮糖激酶(XK)编码基因串联后分别转入3株不同的工业酿酒酵母中,得到重组酵母ZQ1、ZQ5和ZQ7。分别对3个木糖途径代谢基因的表达水平、酶活和重组菌株的木糖发酵效率进行比较。【结果】重组菌株在木糖代谢基因转录、酶活性和木糖利用性能方面有很大差异,其中ZQ5木糖代谢能力最强,ZQ7其次,ZQ1木糖利用能力最弱。ZQ7在初始木糖浓度为20 g/L时木糖利用速率快于ZQ5,表明木糖浓度对重组菌发酵性能评价具有影响。【结论】不同菌株的遗传背景和木糖浓度对重组菌木糖利用的影响很大,评价重组酵母的木糖利用需考虑宿主的遗传背景和底物浓度的影响。     

Use of the alr gene as a food-grade selection marker in lactic acid bacteria

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.