Controlled Production of Stable Heterologous Proteins in Lactococcus lactis

The use of Lactococcus lactis (the most extensively characterized lactic acid bacterium) as a delivery organism for heterologous proteins is, in some cases, limited by low production levels and poor-quality products due to surface proteolysis. In this study, we combined in one L. lactis strain use of the nisin-inducible promoter P_nisA and inactivation of the extracellular housekeeping protease HtrA. The ability of the mutant strain, designated htrA-NZ9000, to produce high levels of stable proteins was confirmed by using the staphylococcal nuclease (Nuc) and the following four heterologous proteins fused or not fused to Nuc that were initially unstable in wild-type L. lactis strains: (i) Staphylococcus hyicus lipase, (ii) the bovine rotavirus antigen nonstructural protein 4, (iii) human papillomavirus antigen E7, and (iv) Brucella abortus antigen L7/L12. In all cases, protein degradation was significantly lower in strain htrA-NZ9000, demonstrating the usefulness of this strain for stable heterologous protein production.     

A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in Lactococcus lactis

Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of L. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only ~60% of total Nuc was present in a secreted form at ~5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to ~30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to ~80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to ~20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the α-amylase of Bacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.     

Heterologous gene expression of bovine plasmin in Lactococcus lactis

Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin produced was shown to be biologically active. Received: 2 December 1996 / Received revision: 17 March 1997 / Accepted: 27 April 1997     

Host specificity of nisin production by Lactococcus lactis

Kinetics of nisin production have been investigated in terms of endogenous features of the producer organism, Lactococcus lactis. Nisin-producing transposons (Tn Nip) were transferred to different hosts by conjugation. Constructs were cultivated in batch cultures and nisin produced was measured. The proteinase function of C2Prt (Tn Nip)-1 was eliminated by plasmid curing, resulting in the construct C2Prt - (Tn Nip)-1. C2Prt - (Tn Nip)-1 produced nisin to a higher concentration compared to C2Prt (Tn Nip)-1 and was able to maintain the maximum concentration till the end of cultivation. The final concentration of nisin produced was host-specific, because when different constructs carrying the same Tn Nip were cultivated they produced nisin to different concentrations. However, when the same host carried Tn Nip transposons derived from different donors the concentration of nisin produced was similar, suggesting that the two Tn Nip transposons may be similar.     

Heterologous expression of the plant coumarate: CoA ligase in Lactococcus lactis

AIMS: To demonstrate the expression of coumarate : CoA ligase of Arabidopsis thaliana in Lactococcus lactis as a first step of cloning the vanillin pathway. METHODS AND RESULTS: The 4CL gene was amplified from a cDNA library of A. thaliana by PCR and subcloned into a multicopy lactococcal vector where the expression is under the nisA promoter. The maximum yield of the protein in the recombinant strain of L. lactis was obtained 3 h after induction with 10 ng ml(-1) of nisin. However, these levels were only fraction of those detected in cell extracts of Pseudomonas fluorescens AN103 strain which naturally expresses its own enzyme when grown in the presence of ferulic acid as a carbon source. Among different substrates examined, the enzyme was most active against coumaric acid. CONCLUSIONS: The gene encoding coumarate : CoA ligase in A. thaliana was isolated, sequenced, cloned and expressed in L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first of the two steps for genetic engineering of the vanillin pathway in the GRAS (generally recognized as safe) organism L. lactis.     

Heterologous expression of Brucella abortus GroEL heat-shock protein in Lactococcus lactis

Background  

Brucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans. Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and thus are not totally safe for use in humans. Therefore, the development of mucosal live vaccines using the food-grade lactic acid bacterium, Lactococcus lactis, as an antigen delivery vector, is an attractive alternative and a safer vaccination strategy against B. abortus. Here, we report the construction of L. lactis strains genetically modified to produce B. abortus GroEL heat-shock protein, a candidate antigen, in two cellular locations, intracellular or secreted.     

硬脂酰-辅酶A脱氢酶基因在食品级乳酸菌中的表达

为了实现硬脂酰-辅酶A脱氢酶1编码基因在乳酸乳球菌中的表达,采用PCR技术扩增获得人类scd1的编码序列。Nco I和Xba I双酶切后定向插入到食品级表达载体pNZ8149中,构建表达载体pNZ8149-scd1。电转化乳酸乳球菌NZ3900,经菌落PCR和测序鉴定scd1基因成功插入到乳酸乳球菌中。在乳链菌肽诱导下进行scd1的表达,转化株提取脂肪酸,进行脂肪酸含量的气相色谱分析。结果显示,SCD1转化菌株中的C16∶1n-7和C18∶1n-7脂肪酸组分比转化pNZ8149的对照组乳酸菌分别提高了92%~169%和53%~127%。文中以scd1基因为例,尝试并证明了脂肪酸脱氢酶类基因能够在食品级乳酸菌中有效表达,为后续研究奠定了基础。     

Heterologous expression of azurin from Pseudomonas aeruginosa in food-grade Lactococcus lactis

Abstract

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3?h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10?mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.     

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。     

基因组重排筛选高产谷氨酰胺转胺酶菌株

谷氨酰胺转胺酶(TGase)的产量不足的问题一直限制其工业化生产规模,故采用基因组重排的方法,筛选高产谷氨酰胺转胺酶菌株。通过对不同制备条件下原生质体纯度和形成率的考量,获得制备原生质体的最优条件为以6mg/ml的溶菌酶浓度进行酶解,酶解时间2h。再优化融合条件,以2min紫外灭活和40min热灭活结合的方法挑选出融合子。通过两轮基因组重排,经过96孔板发酵高通量筛选和摇瓶发酵复筛验证,获得了一株产酶达7.12U/ml的茂源链霉菌,相比最初选用菌株的平均酶活提高65.5%。发酵结果显示,酶活提高的原因可能是在重组后原酶成熟更快、更彻底,且得到的菌株遗传稳定性良好。证明基因组重排能够有效提高菌株的产酶水平,同时为谷氨酰胺转胺酶产量提高提供理论依据。     

构建重组乳酸乳球菌生产谷胱甘肽

以大肠杆菌染色体DNA为模板,分别扩增得到编码γ-谷氨酰半胱氨酸合成酶和谷胱甘肽合成酶的基因gsbA和gshB。将gsbA和gshB基因克隆到质粒pNZSl48中,电转化乳酸乳球菌NZ9000,获得重组菌NZ9000(pNZ3203)。在添加10mmol／L谷氨酸、半胱氨酸和甘氨酸的M17培养基中培养该重组茵,当OD600达到0、4时用乳酸链球菌素诱导4h,胞内谷胱甘肽含量达到358mmol／mg蛋白(胞内浓度相当于140mmol／L),这是在革兰氏阳性茵中生产谷胱甘肽的首例报道。     

Overproduction of Heterologous Mannitol 1-Phosphatase: a Key Factor for Engineering Mannitol Production by Lactococcus lactis

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.     

Signal Peptide and Propeptide Optimization for Heterologous Protein Secretion in Lactococcus lactis

Lactic acid bacteria are food-grade microorganisms that are potentially good candidates for production of heterologous proteins of therapeutical or technological interest. We developed a model for heterologous protein secretion in Lactococcus lactis using the staphylococcal nuclease (Nuc). The effects on protein secretion of alterations in either (i) signal peptide or (ii) propeptide sequences were examined. (i) Replacement of the native Nuc signal peptide (SP_Nuc) by that of L. lactis protein Usp45 (SP_Usp) resulted in greatly improved secretion efficiency (SE). Pulse-chase experiments showed that Nuc secretion kinetics was better when directed by SP_Usp than when directed by SP_Nuc. This SP_Usp effect on Nuc secretion is not due to a better antifolding activity, since SP_Usp:Nuc precursor proteins display enzymatic activity in vitro, while SP_Nuc:Nuc precursor proteins do not. (ii) Deletion of the native Nuc propeptide dramatically reduces Nuc SE, regardless of which SP is used. We previously reported that a synthetic propeptide, LEISSTCDA, could efficiently replace the native Nuc propeptide to promote heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895–1903, 1998). To determine whether the LEISSTCDA effect is due to its acidic residues, specific substitutions were introduced, resulting in neutral or basic propeptides. Effects of these two new propeptides and of a different acidic synthetic propeptide were tested. Acidic and neutral propeptides were equally effective in enhancing Nuc SE and also increased Nuc yields. In contrast, the basic propeptide strongly reduced both SE and the quantity of secreted Nuc. We have shown that the combination of the native SP_Usp and a neutral or acidic synthetic propeptide leads to a significant improvement in SE and in the quantity of synthesized Nuc. These observations will be valuable in the production of heterologous proteins in L. lactis.     

Production of dehydroamino acid-containing peptides by Lactococcus lactis

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.     

Production of Dehydroamino Acid-Containing Peptides by Lactococcus lactis

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.     

Minimal Requirements for Exponential Growth of Lactococcus lactis

A minimal growth medium containing glucose, acetate, vitamins, and eight amino acids allowed for growth of Lactococcus lactis subsp. lactis, with a specific growth rate in batch culture of μ = 0.3 h^-1. With 19 amino acids added, the growth rate increased to μ = 0.7 h^-1 and the exponential growth phase proceeded until high cell concentrations were reached. We show that morpholinepropanesulfonic acid (MOPS) is a suitable buffer for L. lactis and may be applied in high concentrations.     

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.     

High-Level Acetaldehyde Production in Lactococcus lactis by Metabolic Engineering

Efficient conversion of glucose to acetaldehyde is achieved by nisin-controlled overexpression of Zymomonas mobilis pyruvate decarboxylase (pdc) and Lactococcus lactis NADH oxidase (nox) in L. lactis. In resting cells, almost 50% of the glucose consumed could be redirected towards acetaldehyde by combined overexpression of pdc and nox under anaerobic conditions.