Higher titer hyaluronic acid production in recombinant Lactococcus lactis

Abstract

Hyaluronic acid (HA) is a natural biopolymer and has long been attracting the attention of biotechnology industry due to its various biological functions. HA production with natural producer Streptococcus equi subsp. zooepidemicus has not been preferred because it has many drawbacks due to its pathogenicity. Therefore, in the present study, Streptococcal hyaluronan synthase gene (hasA) was introduced and expressed in Lactococcus lactis, through the auto inducible NICE system and the effect of nisin amount on the production of HA was examined. Newly constructed plasmid was transformed into L. lactis CES15, produced 6.09 g/l HA in static flask culture after three hours of induction period with initial 7.5 ng/ml nisin concentration within total six hours of incubation. The highest HA titer value ever was reported for recombinant HA-producing L. lactis by examining the effect of initial nisin concentration. We have shown that initial nisin concentration, which used to initiate the auto-inducing mechanism of NICE system and consequently hyaluronan synthase expression, has a direct and significant effect on the produced HA amount. Recently constructed recombinant L. lactis CES15 strain provide significant advantages for industrial HA production than those in literature in terms of production time, energy demand, carbon usage, and safety status.     

乳酸乳球菌NZ9000基因组规模代谢网络模型的构建与验证

随着后基因组时代的到来,工业微生物的代谢工程改造在工业生产上发挥着越来越重要的作用。而基因组规模代谢网络模型(Genome-scalemetabolicmodel,GSMM)将生物体体内所有已知代谢信息进行整合,为全局理解生物体的代谢状态、理性指导代谢工程改造提供了最佳的平台。乳酸乳球菌NZ9000(Lactococcuslactis NZ9000)作为工业发酵领域的重要菌株之一,由于其遗传背景清晰且几乎不分泌蛋白,是基因工程改造和外源蛋白表达的理想模式菌株。文中基于基因组功能注释和比较基因组学构建了L.lactisNZ9000的首个基因组规模代谢网络模型iWK557,包含557个基因、668个代谢物、840个反应,并进一步在定性和定量两个层次验证了iWK557的准确性,以期为理性指导L. lactis NZ9000代谢工程改造提供良好工具。     

Physical analysis of in vivo pCI301 fusion plasmids in Lactococcus lactis subsp. lactis

Abstract In vivo fusion plasmids identified following conjugative mobilization of pCI301, the 75-kilobase (kb) lactose-proteinase plasmid of Lactococcus lactis subsp. lactis UC317, were characterized. These plasmids (95 kb) were generated from fusion-deletion events involving pCI301 and the 38-kb UC317-derived cryptic plasmid, pCI303. Recombinant plasmids were separable into distinct classes based on their associated phenotypes and restriction maps. The formation of pCI301: : pCI303 composite plasmids within strain UC317 was also demonstrated.     

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。     

Time dependent responses of glycolytic intermediates in a detailed glycolytic model of Lactococcus lactis during glucose run-out experiments

Glucose addition and subsequent run-out experiments were compared to simulations with a detailed glycolytic model of Lactococcus lactis. The model was constructed largely on bases of enzyme kinetic data taken from literature and not adjusted for the specific simulations shown here. Upon glucose depletion a rapid increase in PEP, inorganic phosphate and a gradual decrease in fructose 1,6-bisphosphate (FBP) were measured and predicted by simulation. The dynamic changes in these and other intermediate concentrations as measured in the experiments were well predicted by the kinetic model.     

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.     

Overview on sugar metabolism and its control in Lactococcus lactis - the input from in vivo NMR

The wide application of lactic acid bacteria in the production of fermented foods depends to a great extent on the unique features of sugar metabolism in these organisms. The relative metabolic simplicity and the availability of genetic tools made Lactococcus lactis the organism of choice to gain insight into metabolic and regulatory networks. In vivo nuclear magnetic resonance has proven a very useful technique to monitor non-invasively the dynamics of intracellular metabolite and co-factor pools following a glucose pulse. Examples of the application of this methodology to identify metabolic bottlenecks and regulatory sites are presented. The use of this information to direct metabolic engineering strategies is illustrated.     

Study of intraspecific variations of the bacterium Lactococcus lactis in adaptation to high acidity of the medium

This paper reports on the study of acid tolerance of lactic acid bacteria as a property of cells, determining their ability to divide efficiently and retain viability under conditions of increased nutrient medium acidity during bacterial growth. The bacteria of the strain TV2, isolated from a self-soured curd, similar to the bacteria of the strain STE05 (Russian National Collection of Industrial Microorganisms), were assigned to the species Lactococcus lactis according to their GC composition (36.7–36.5 mol %) and the high level of DNA-DNA hybridization (93%). However, these strains were essentially different in the number and size of the plasmids and the chromosomal DNA restriction fragments, as well as in the sensitivity to phages of lactic acid bacteria. It was found that bacteria of the strain TV2 were stable (i.e., they divided efficiently at a pH as low as 5.3) and tolerant to the lactic acid that they produced while growing (i.e., they retained viability at pH 4.4). Bacteria of the strain STE05 lacked acid tolerance (at pH below 6.5, growth was retarded, and pH 5.0 was the lowest value at which the cells remained viable). The acid tolerance and phage resistance of TV2 bacteria are likely to characterize their higher adaptive capacity in comparison with STE05 bacteria. Acid tolerance is inherited in a stable manner and retained by the segregants of TV2 strain obtained in the course of long-term storage of the bacteria. Specifically, the strains TV29, TV13, and TV229, which displayed this property, had altered physiological and biochemical characteristics (accumulation of biomass and fermentation of lactose) in spite of their genetic identity to the original strain (pulse electrophoresis of chromosomal DNA restriction fragments).     

Expression of green fluorescent protein in Lactococcus lactis

The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp. lactis biovar cremoris MG1363, upon construction and introduction of plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in L. lactis.     

Dynamics of pyruvate metabolism in Lactococcus lactis.

The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acid-product formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis in continuous cultures subjected to step changes in dilution rate. Both shift-up and shift-down experiments were carried out, and these experiments showed that the enzyme pyruvate formate-lyase (PFL) plays a key role in the regulation of the shift. Pyruvate formate-lyase in vivo activity was regulated both at the level of gene expression and by allosteric modulation of the enzyme. A simple mathematical model was proposed to estimate the relative significance of the regulatory mechanisms involved.     

Proton-dependent kinetics of citrate uptake in growing cells of Lactococcus lactis subsp. lactis bv. diacetylactis

Abstract The kinetic analysis of citrate uptake in growing cells of Lactococcus lactis subsp. lactis biovar. diacetylactis identified a proton-dependent transport and suggested the divalent anionic species as the form of citrate transported across cell membranes. The reaction followed Michaelis-Menten kinetics for a two-substrate reaction. The limiting steps were the formation of the ternary complex and the rate of transport. Temperature modified the activity of the permease, increasing the uptake rate.     

生长抑制剂所诱导的乳酸乳球菌MG1363的自溶表型及其生长阶段特异的性质

【目的】自溶是细菌在压力环境下通过自身裂解而获得的一种生理适应现象,研究的目的是全面探讨乳酸菌株在生长抑制剂条件下的自溶表型及机理。【方法】对多种来源的乳酸菌株的自溶能力进行检测,通过在不同生长条件和抑制剂压力条件下乳酸乳球菌MG1363的生长检测对其自溶表型进行分析。【结果】在葡萄糖严格受限的培养基中,氨苄青霉素的加入能够显著诱导MG1363的自溶,而且该自溶现象只发生在葡萄糖耗尽的时间点,展现出一种狭窄的生长时期依赖的特征。与此同时,因为氨苄青霉素的加入,4种主要的自溶酶的表达都发生了不同程度的显著改变。此外,所有受试的抑制剂都削弱了MG1363在非营养条件下的自溶,表明该菌株可能具有一种涉及细胞壁合成和降解酶的共同调控的模式。【结论】乳酸菌株在不同生长抑制剂条件下的自溶表型存在很大差异,且该自溶体现出营养条件和生长时期严格依赖的特征。     

乳链菌肽前体基因（nisZ）在乳酸乳球菌中的克隆和表达

用PCR技术从克隆有完整乳链菌肽生物合成基因簇（来自于乳链菌肽高产菌株L.lactis AL2)的重组噬菌体λHJ-3中扩增了编码乳链菌肽的前体基因,与pMG36e连接得到重组质粒pHJ201,用电击转化法将pHJ201转化到L.lactis NZ9800中,经活性测定和Tricine－SDS－PAGE电泳证实乳链菌肽前体基因获得了功能表达。DNA序列分析表明乳链菌肽高产菌株L.lactis AL2产生的是NisinZ。发现pHJ201d L.lactis NZ9800 中有良好的稳定性。     

一株能够利用血红素进行有氧呼吸的乳酸乳球菌

[目的]乳酸乳球菌在呼吸状态下,生长速度快,生物产量大,是改善发酵剂生产效率的潜在途径之一,本研究旨在观察由传统乳制品中分离得到的乳酸乳球菌在血红素存在状态下有氧呼吸情况以及代谢的变化.[方法]对本实验室保藏的12株乳酸乳球菌进行有氧培养实验,比较其生物量和代谢产物的差异.观测在4℃下储藏30 d后的活菌数差异.[结果]筛选出一株在血红素存在条件下进行有氧呼吸的菌株KLDS 4.0316,与没有添加血红素的原菌株相比生物量增长了50%,在4℃储藏30 d后,添加血红素并振荡的活菌数依然维持在100 CFU/mL,而未添加血红素未振荡检测不到活菌.在血红素存在下,KLDS 4.0316代谢产物发生了变化,与没有添加血红素的原菌株相比乳酸产量减少了48%.[结论]KLDS 4.0316在血红素存在条件下能够进行有氧呼吸,乳酸产生减少,生物量增加.     

In situ delivery of cytokines by genetically engineered Lactococcus lactis

The development of novel approaches that allow for accurate targeting of therapeutics to the bowel mucosa is a priority in the research on inflammatory bowel disease. We have engineered Lactococcus lactis to secrete soluble, fully active, correctly processed cytokines. We have used these live, recombinant strains for the in situ delivery of mouse interleukin (mIL)-2, -6 and -10 at airway mucosa or mucosa of the colon. Strains that secrete mIL-2 or mIL-6 and produce TTFC intracellular show a higher level of anti-TTFC induction in mice following intranasal inoculation. We showed that mIL-10 producing L. lactis can prevent and cure enterocolitis in mice. The daily ingestion of this strain leads to the prevention of colitis in IL-10 –/– 129 Sv/Ev mice. The repeated addition of DSS to the drinking water of Balb/c mice leads to the induction of chronic colitis with a typical mean histological score of five points. Subsequent daily treatment with 10⁸ IL-10 producing L. lactis reduced the inflammation to a score of approximately 1 in 40% of the treated mice, which is a status equal to that of healthy control mice. Most other animals from the treated group only showed minor patchy remnants of the inflammation. Killing of the IL-10 producing bacteria by UV irradiation immediately prior to inoculation abrogates this therapeutic effect. Therefore it can be attributed to the active in vivo delivery of IL-10. We have further documented this by demonstrating in situ de novo synthesis of IL-10 in the colon of IL-10 –/– mice.     

Lactic acid bacteria as a cell factory: rerouting of carbon metabolism in Lactococcus lactis by metabolic engineering

Lactic acid bacteria display a relatively simple metabolism wherein the sugar is converted mainly to lactic acid. The extensive knowledge of metabolic pathways and the increasing information of the genes involved allows for the rerouting of natural metabolic pathways by genetic and physiological engineering. We discuss several examples of metabolic engineering of Lactococcus lactis for the production of important compounds, including diacetyl, alanine and exopolysaccharides.     

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.     

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。