人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达

以lacF基因为食品级选择标记,构建了乳酸乳球菌食品级基因表达系统,并进而实现了人铜锌超氧化物歧化酶基因在乳酸乳球菌中的食品级表达。首先构建了含有lacF基因两侧同源DNA序列(0.5kb)的整合型质粒pUCEmDE,通过pUCEmDE与乳酸乳球菌MG5267染色体上单拷贝的乳糖操纵子之间的同源双交换,构建了lacF基因缺失突变的食品级受体菌WZ103 (Lac-),并经PCR及Lac表型检测所验证。然后构建了互补质粒pMG36eF,其lacF基因的表达受组成型的强启动子P32的控制。将其电转化导入WZ103后,Lac+表型得到恢复,表明WZ103中lacF基因的功能可被互补质粒pMG36eF上的lacF基因互补。随后,以互补质粒pMG36eF为基础,构建了不含任何抗生素抗性选择标记的人铜锌超氧化物歧化酶基因的食品级表达质粒pWZ104。通过非变性聚丙烯酰胺凝胶电泳和SOD活性凝胶染色分析,检测到WZ103(pWZ104)中Cu/Zn SOD的表达,并且具有生物活性。     

乳酸乳球菌食品级诱导表达系统的构建及异源蛋白的表达

以α-aga基因为食品级选择标记构建了乳酸乳球菌食品级高效诱导细胞内和细胞壁锚定表达系统,并用这一表达系统表达了铜绿假单胞菌融合外膜蛋白基因OprF/H。首先以pRAF800和pNZ8048构建了含有α-aga、PnisA-MCS-TpepN和θ复制子的乳酸乳球菌食品级细胞内诱导表达载体pRNA48,再以pRNA48和pVE5524为出发载体构建了含有α-aga、PnisA-SPUsp45-nucA-CWAM6-t1t2和θ复制子的乳酸乳球菌细胞壁锚定诱导表达载体pRNV48。然后以食品级载体pRNA48和pRNV48为基础,构建了不含抗生素抗性选择标记的铜绿假单胞菌融合外膜蛋白基因的表达质粒pRNA48-OprF/H和pRNV48-OprF/H。利用nisin进行重组乳酸乳球菌菌株的诱导表达,通过SDS-PAGE和Western blot分析,检测到表达蛋白分别占细胞内可溶蛋白的9.6%和细胞壁锚定蛋白的9.8%,表达产物具有免疫原性,可与含OprF/H的乳球菌以及铜绿假单胞菌发生特异性的凝集反应。     

A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB590, was constructed entirely of lactococcal DNA: the pSH71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG1614 with 60 international units (IU) nisin/ml selection yielded approximately 10⁵ transformants/μg DNA. MG1614 carrying pLEB590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB590 was succesfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications. Electronic Publication     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis.

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.     

Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast.

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called Pk1) containing most of the terminal repeat sequence was isolated and examined for promoter activity. Pk1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative (lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of Pk1. The promoter activity of Pk1 fragment was independent of the presence or absence of killer plasmids. It is not known whether Pk1 can also function bidirectionally on the natural k1 plasmid. The possible functions of Pk1 for killer plasmid gene expression and plasmid replication are discussed.     

Food-grade host/vector expression system for <Emphasis Type="Italic">Lactobacillus casei</Emphasis> based on complementation of plasmid-associated phospho-β-galactosidase gene <Emphasis Type="Italic">lacG</Emphasis>

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.     

Development of an antibiotic-free plasmid selection system based on thymine auxotrophy in Lactococcus lactis

Lactococcus lactis has become the best studied species of the lactic acid bacteria (LAB) clade and an ideal cell factory for heterogenous proteins. We have constructed an antibiotic-free expression vector, pMG-thyA, using thymidine synthase gene thyA as the selection marker. The thyA gene was cloned from the food industry strain Streptococcus thermophilus St-JY and was used to replace the erythromycin resistance genes on L. lactis expression vector pMG36e in order to construct pMG-thyA. The construction of the new vector and thyA-null host L. lactis MG1363-TT yielded an antibiotic-free expression system. The α-amylase gene (amy) was cloned onto the multiple cloning site of the vector pMG-thyA as a reporter gene, yielding the recombinant plasmid pMGthyA-amy. This plasmid was electroporated into L. lactis MG1363-TT, and the recombinant strain grown on SA plates containing 0.5 % (w/v) soluble starch formed distinct bacterial colonies and clear zones (halo) around the colonies following the addition of iodine solution. These research findings lay the foundation for food-grade expression in L. lactis.     

Food-grade gene expression in lactic acid bacteria

In the 1990s, significant efforts were invested in the research and development of food-grade expression systems in lactic acid bacteria (LAB). At this time, Lactococcus lactis in particular was demonstrated to be an ideal cell factory for the food-grade production of recombinant proteins. Steady progress has since been made in research on LAB, including Lactococcus, Lactobacillus and Streptococcus, in the areas of recombinant enzyme production, industrial food fermentation, and gene and metabolic pathway regulation. Over the past decade, this work has also led to new approaches on chromosomal integration vectors and host/vector systems. These newly constructed food-grade gene expression systems were designed with specific attention to self-cloning strategies, food-grade selection markers, plasmid replication and chromosomal gene replacements. In this review, we discuss some well-characterized chromosomal integration and food-grade host/vector systems used in LAB, with a special focus on sustainability, stability and overall safety, and give some attractive examples of protein expression that are based on these systems.     

Construction of a new food-grade expression system for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> based on theta replication plasmids and auxotrophic complementation

A new food-grade expression system was constructed for Bacillus subtilis based on replicative food-grade expression plasmids and auxotrophic complementation. The food-grade B. subtilis host FG01 was created by knockout of the dal locus from the chromosome of B. subtilis 168. Two food-grade expression plasmids pXFGT03 and pXFGT05 were constructed by combining a novel theta-type Bacillus replicon with the B. subtilis endogenous gene dal and P43 promoter; while pXFGT05 was derived from pXFGT03 by deletion of two open reading frames (ORFs) from the original replicon. Upon transformation of FG01 with pXFGT03 or pXFGT05, the host phenotype was complemented on Luria–Bertani agar plates by the plasmid-coded dal gene, which served as a food-grade selection marker for recombinants. Results showed that deletion of the two ORFs had no impact on plasmid replication. A reporter gene bgaB was cloned into pXFGT03 and pXFGT05, respectively, under control of the P43 promoter, and it was successfully expressed in this food-grade expression system. Segregational stabilities of two recombinant plasmids were investigated, and they were fully stable.     

Heterologous leaky production of transglutaminase in Lactococcus lactis significantly enhances the growth performance of the host

This study describes a novel strategy to improve the growth performance of Lactococcus lactis by heterologous production of food-grade transglutaminase. The mtg gene from Streptoverticillium mobaraense that encodes the transglutaminase mature protein was cloned into a nisin-inducible expression vector and transformed into L. lactis subsp. cremoris NZ9000. The leaky expression of the mtg gene from the nisA promoter resulted in ammonia formation and carbon flux redistribution at the pyruvate branch. As a consequence, medium acidification was lessened and energy utilization was improved. This led to significantly higher biomass production under aerobic conditions and particularly under non-pH-controlled conditions (up to a 12-fold increase). The results presented here provide a novel way to enhance the growth yield of L. lactis, which is an important step for the purposes of producing proteins of commercial interest using L. lactis as a host.     

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.     

南极假丝酵母脂肪酶B(CALB)基因在乳酸乳球菌MG1363中的整合及表达

根据南极假丝酵母脂肪酶B (CALB)的基因序列将CALB基因进行TA克隆、酶切鉴定及测序后,亚克隆至大肠杆菌-乳酸乳球菌穿梭表达栽体pMG36e-Nisl中,构建重组表达栽体pMG36e-Nisl-CALB.设计特异性引物P3和P4,对重组质粒pMG36e-NisI-CALB进行红霉素抗性基因的敲除,以构建食品级表达载体pMG36N-CALB,后再将两种重组质粒分别电转化入乳酸乳球菌MG1363,以Nisin为选择压力,考察CALB在MG1363中的表达情况.结果显示,成功构建了表达载体pMG36e-NisI-CALB及pMG36N-CALB,两株重组菌在含有20 IU Nisir/mL的培养基中均生长情况良好,遗传性能稳定,且经水解圈鉴定,CALB能够进行活性表达.进一步研究发现,CALB基因整合到乳酸乳球菌MG1363染色体中.     

Characterization of the genetic element coding for lactose metabolism in Lactococcus lactis subsp. lactis KP3

The Lactococcus lactis subsp. lactis KP3 Lac genetic element was investigated. KP3 is a lactose-positive (Lac+) transconjugant which contains no detectable plasmid DNA. The KP3 Lac genetic element was self-transmissible (Tra+) and encoded a reduced bacteriophage sensitivity (Rbs+) phenotype. Matings of KP3 with a recombination-deficient (Rec-) recipient resulted in Lac+ transconjugants which were phenotypically indistinguishable from KP3 and contained a 96-MDa plasmid (pJS96). Phenotypic and physical analyses of pJS96 indicated that it was a deletion derivative of a putative pKB32::pJS88 Lac+ Tra+ cointegrate. pKB32 is the Lac plasmid and pJS88 is the Tra+ Rbs+ plasmid in L. lactis subsp. lactis 11007, the donor used in obtaining KP3. The results presented suggest that pJS96 is an episome, since it appeared to replicate both as a plasmid and as an integrated part of the chromosome. Conjugal transfer of chromosomal DNA mediated by pJS96 was not observed. Conjugal transfer of pJS96 resulted in Lac+ transconjugants containing plasmids ranging in size from 21 to 90 MDa. Only in Rec+ recipients were transconjugants isolated which appeared to contain pJS96 integrated into the host chromosome. Restriction analysis of several plasmids in the 21 to 90 MDa range suggested the deletions were due to intramolecular transposition of a transposable element on pJS96. This report suggests that a self-transmissible episome exists in KP3 and provides an explanation of how plasmids which vary in size yet encode similar phenotypes may be formed and disseminated.     

Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis

Cloning vector plasmids have been constructed on the basis of the broad host range plasmid pAM beta 1 and used for the cloning of a nisin resistance determinant in Streptococcus lactis. They incorporate several desirable features for gene cloning in S. lactis and other transformable Gram-positive bacteria. They carry an easily selectable erythromycin resistance marker, are present at low (6-9) or high (45-85) copy number in S. lactis and possess a convenient polyrestriction site sequence. A significant advantage of these plasmids is their capability to carry and stably maintain very large cloned DNA fragments (up to 30 kilobases).     

A potential food-grade cloning vector for Streptococcus thermophilus that uses cadmium resistance as the selectable marker

A potential food-grade cloning vector, pND919, was constructed and transformed into S. thermophilus ST3-1, a plasmid-free strain. The vector contains DNAs from two different food-approved organisms, Streptococcus thermophilus and Lactococcus lactis. The 5.0-kb pND919 is a derivative of the cloning vector pND918 (9.3 kb) and was constructed by deletion of the 4.3-kb region of pND918 which contained DNA from non-food-approved organisms. pND919 carries a heterologous native cadmium resistance selectable marker from L. lactis M71 and expresses the Cd(r) phenotype in S. thermophilus transformants. With the S. thermophilus replicon derived from the shuttle vector pND913, pND919 is able to replicate in the two S. thermophilus industrial strains tested, ST3-1 and ST4-1. Its relatively high retention rate in S. thermophilus further indicates its usefulness as a potential food-grade cloning vector. To our knowledge, this is the first report of a replicative potential food-grade vector for the industrially important organism S. thermophilus.     

牛凝乳酶原基因在乳酸乳球菌中的表达

【目的】利用乳酸乳球菌nisin诱导基因表达系统(the NIsin Controlled gene Expression system,NICE)表达牛凝乳酶原。【方法】从克隆载体pS19-PPC中获得牛凝乳酶原基因,将该基因与表达载体pNZ8148连接并电转化乳酸乳球菌NZ9000,转化子经酶切、PCR和测序鉴定后,用nisin进行诱导表达,表达产物利用SDS-PAGE和Western blot鉴定,表达产物纯化后检测凝乳活性。【结果】重组牛凝乳酶原与天然牛凝乳酶原比较,其分子量大小、免疫性质、生物活性和抑制剂敏感性没有发现显著差异,其凝乳活性可达2×103IMCU/mL。【结论】在乳酸乳球菌中表达了具有凝乳活性的牛凝乳酶原,同时乳酸乳球菌作为发酵剂和凝乳酶产生菌双重角色的实现,为奶酪加工提供了新思路和新方法。     

Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis

pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putative cis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-acting ori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is a repB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis if trans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactis and two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.