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.     

Conjugative plasmid pIP501 undergoes specific deletions after transfer from<Emphasis Type="Italic"> Lactococcus lactis</Emphasis> to<Emphasis Type="Italic"> Oenococcus oeni</Emphasis>

Conjugal transfer of plasmids pIP501 and its derivative pVA797 from Lactococcus lactis to Oenococcus oeni was assayed by filter mating. Plasmid pIP501 was transferred to a number of O. oeni strains whereas a single transconjugant of O. oeni M42 was recovered when pVA797 was used. Physical analysis of the transconjugant plasmids revealed that pIP501 and pVA797 underwent extensive deletions in O. oeni that affected the tra region (conjugal transfer) and SegB region (stability). All derivatives showed segregational instability in O. oeni, but were stably maintained in L. lactis. These differences correlated with the different plasmid copy numbers and the extent of deletions within the SegB region.Abbreviations CAT Chloramphenicol acetyltransferase - MLS Macrolides-lincosamides-streptogramin B resistance     

Genomic organization of lactic acid bacteria

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.     

Contrasting breeding systems in twoEriotheca (Bombacaceae) species of the Brazilian cerrados

The pollination biology and breeding systems ofEriotheca pubescens andE. gracilipes have been studied. These two species occur as trees in cerrado vegetation, the neotropical savannas of Central Brazil, with partially sympatric distributions. They have similar phenology and floral structure, although the flowers ofE. pubescens are larger. Both species have nectar flowers pollinated by largeAnthophoridae bees but the main pollinators of each species differ in size. The species have markedly different breeding systems: late-acting self-incompatibility inE. gracilipes and apomixis stimulated by pollination inE. pubescens.     

Loss of Plasmid-Mediated Oligopeptide Transport System in Lactococci: Another Reason for Slow Milk Coagulation

Fast milk-coagulating (Fmc⁺) strains of lactococci are known to segregate slow milk-coagulating (Fmc⁻) variants, which has been attributed to loss of proteinase (Prt) activity encoded by plasmid DNA. It was found that the Fmc⁻phenotype could also be due to loss of a plasmid encoding an oligopeptide permease (Opp) system. InLactococcus lactissubsp.lactis(L. lactis) C2O, lactose metabolism (Lac) and Prt were linked to pJK550 and the Opp system to pJK430. InLactococcus lactissubsp.cremorisSK11, known to possess Prt on a 78-kb plasmid, DNA sequence analysis of a 7.4-kb region from the Lac plasmid, pSK11L, revealed that it possessed the Opp system. The Lac plasmid inL. lactisC2 encoded both the Prt and Opp systems. Fmc⁻derivatives ofL. lactisC2 were missing theprtgenes and had Opp integrated into the chromosome, possibly due to transposition events. Growth studies showed the Opp systems were functional and, in combination with Prt, produced the Fmc⁺phenotype.     

Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora

Strains of the yeast Pichia inositovora that carry the linear plasmids pPin1-1 (18 kb) and pPin1-3 (10 kb) display a killer activity towards Saccharomyces cerevisiae. Cloning and sequencing of the smaller plasmid, pPin1-3, revealed that it is 9683 bp long and has 154-bp terminal inverted repeats. Comparison of pPin1-3 with the only other completely sequenced killer plasmid, pGKL1 of Kluyveromyces lactis, revealed differences in genome organization. The Pichia element has four ORFs that account for 95% of the sequence. ORF1 is homologous to the putative immunity gene of the K. lactis system. A viral B-type DNA polymerase is encoded by ORF2. The predicted product of ORF3 displays similarities to the - and -subunits of the heterotrimeric K. lactis killer toxin, also known as zymocin. A cysteine-rich chitin-binding site and a chitinase signature, characteristic for the -subunit of zymocin were identified in Orf3p. Chitin affinity chromatography and Western analysis confirmed the plasmid specific expression and secretion of a protein that cross-reacts with an antibody raised against the -subunit of K. lactis zymocin. Disruption of the major chitin synthase-gene ( CHS3) renders S. cerevisiae resistant to the toxin, providing further evidence that chitin is the cellular receptor for the P. inositovora toxin. Orf4p of pPin1-3 displays only weak similarities to the -subunit of zymocin, which causes a G1 cell-cycle arrest in S. cerevisiae. However, disruption of the S. cerevisiae gene ELP3/TOT3, which encodes a histone-acetyltransferase that is essential for zymocin action, resulted in reduced sensitivity to the P. inositovora toxin also. Thus, despite obvious differences in genome organization and protein architecture, both killer systems very probably have similar modes of action.Communicated by C. P. Hollenberg     

A food-grade delivery system for<Emphasis Type="Italic"> Lactococcus lactis</Emphasis> and evaluation of inducible gene expression

The genetic improvement of Lactococcus lactis is a matter of biotechnological interest in the food industry and in the pharmaceutical and medical fields. However, to construct a food-grade delivery system, both the presence of antibiotic markers or plasmid sequences should be avoided and the maintenance and expression of the cloned gene should be guaranteed. The objective of this work was to produce crossover mutants of L. lactis with a reporter gene under the control of an inducible promoter in order to evaluate the level of gene expression. We utilized a nuclease gene of Staphylococcus aureus as a reporter gene, P _nisA as the nisin-inducible promoter, a non-essential gene involved in histidine biosynthesis of L. lactis as the site for homologous recombination, and pRV300 as a suicide vector for the genomic integration in L. lactis NZ9000. Single- and double-crossover mutants were identified by genotype and phenotype. Relative to episomal transformants of L. lactis, the level of expression of the heterologous protein after nisin induction was similar in the crossover mutants, suggesting that a single copy of the heterologous gene can be used to produce the protein of interest.     

Production of Recombinant Peanut Allergen Ara h 2 using <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis.     

乳酸乳球菌双组分系统调控有氧呼吸的研究

【背景】乳酸乳球菌作为食品行业的代表性菌株,如何通过双组分系统响应环境因子与代谢调控的分子机制研究,对发酵食品产业和益生菌制剂行业有着重要的意义。【目的】探究乳酸乳球菌双组分系统对有氧呼吸代谢调控的相关网络,为乳酸菌适应性代谢研究提供新思路。【方法】采用生物信息学方法,系统性地分析乳酸乳球菌双组分系统组氨酸激酶和反应调节因子的结构域组成及预测双组分系统功能,筛选出与有氧呼吸有潜在联系的双组分,并进一步通过基因转录表达和非靶向代谢组学验证。【结果】以乳酸乳球菌的代表菌株NZ9000为例构建相互作用蛋白网络,显示双组分系统与丙酮酸代谢网络关键连接点为丙酮酸铁氧还蛋白氧化还原酶(nifJ)。在不同的生长时期,Lactococcus lactis NZ9000双组分转录表达在延滞期变化显著。与厌氧培养相比,有氧培养和有氧呼吸培养的菌体双组分呈现下调趋势。双组分系统参与乳酸菌氧化应激和血红素胁迫过程。【结论】明确乳酸乳球菌参与有氧呼吸的双组分系统以及代谢通路,有助于提高发酵剂、益生菌剂的存活率和竞争力。     

Heat shock-induced protein synthesis inLactococcus lactis subsp.lactis

Heat shock inLactococcus lactis subsp.lactis may induce as many as 16 proteins after a temperature shift from 30° to 40°C. Five induced proteins were found to be immunologically related to theEscherichia coli GroEL, DnaK, DnaJ, and GrpE proteins, and to theBacillus subtilis ⁴³ factor. From these initial studies we conclude that, inL. lactis subsp.lactis, a heat shock response similar to that known to occur in other prokaryotes might exist.     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ Nucleotide Sequence Databanks and have been assigned the accession number X78413     

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> for oral vaccination and its protective immune response in mice

K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C₈₃₅₄₉ challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice. An erratum to this article can be found at     

Expression from symbiotic promoters ofRhizobium meliloti inAzotobacter vinelandii andAzospirillum brasilense

InRhizobium meliloti, the promoter P1 of thenif HDK operon, and also the promoter P2, have earlier been shown to be active in the bacteria present in alfalfa root nodules, but not in the bacteria grown aerobically in culture. Here we have looked at the expression from P1 and P2 in two non-symbiotic nitrogen-fixing bacteria,Azotobacter vinelandii andAzospirillum brasilense, using constructions in which the promoters are fused upstream of theβ-galactosidase gene. The promoter P1, but not P2, is active inA. vinelandii, while neither P1 nor P2 is active inAzospirillum brasilense.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

荧光素酶mRNA的构建及电穿孔介导的mRNA体内表达特性北大核心CSCD

为构建一种非复制型mRNA平台并探究电穿孔介导的mRNA对小鼠健康状况的影响及蛋白的表达情况,以荧光素酶作为靶标基因,用T7 RNA聚合酶体外转录及酶法加帽加尾的策略制备mRNA,用活体基因导入仪通过电穿孔的方式体内递送mRNA,借助小动物活体成像系统观测荧光素酶蛋白在小鼠体内的表达强度和持续时间。结果表明,使用该非复制型mRNA平台得到的mRNA成功在体内外表达,电穿孔介导的mRNA对小鼠健康体征无明显影响,所有的小鼠均成功表达了荧光素酶蛋白,蛋白表达在电穿孔后第1天达到峰值,在第4天迅速下降,但蛋白表达强度和持续时间存在较大的小鼠个体间差异。研究对非复制型mRNA的构建及其应用于疫苗或肿瘤药物研发具有重要参考价值。     

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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Heterologous expression of glucose oxidase in the yeast <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Background  

In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties.     

Liquid holding recovery and photoreactivation of UV-induced damage inStreptococcus lactis

Summary Repair of ultraviolet-light-induced DNA damage inStreptococcus lactis has been examined. The wild-type strain and its derivative Lac^– possess a dark repair system (maximal increase in survival of 4-fold). Enzymatic photoreactivation exists in the two strains but a weaker photoreactivability was found in the Lac^– derivative (4 and 2-fold, respectively). Concomitant reduction of UV-induced mutagenesis (Rif^r marker) was also studied during these two repair phenomena. The absence of dark repair after saturation of photoreactivation suggests that photoreactivation is much more efficient with pyrimidine dimers as substrate.