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。     

丙酸的合成生物制造

丙酸是一种重要的C3平台化合物,在食品、药品和化工等领域应用广泛。以石油等化工产品为原料通过化学途径合成,环境污染严重且不可持续。近些年,利用微生物转化可再生资源生产丙酸受到了广泛关注。本文聚焦丙酸生物制造技术,首先综述了传统丙酸杆菌代谢工程改造和在大肠杆菌及酿酒酵母等异源宿主中重构丙酸合成途径的研究;其次重点讨论了基于合成生物学技术,通过对恶臭假单胞菌KT2440的途径设计和改造,实现其利用L-苏氨酸或生物基1,2-丙二醇为原料的高效生物催化合成高纯度丙酸的最新进展。     

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).     

产抑菌素菌株SM—A的分离和鉴定

自市售酸乳酪中分离到一株乳球菌SM-A菌株。该菌株产生的抑菌素能抑制或杀死芽孢杆菌、葡萄球菌、微球菌、链球菌、棒杆菌和梭菌等革兰氏阳性细菌,但对革兰氏阴性细菌、霉菌和酵母无效。SM-A菌株多为链球状,也有成对存在。革兰氏染色阳性,抗酸染色阴性,兼性厌氧生长,最适生长温度32℃,不形成芽孢,无荚膜和鞭毛,不运动;可从多种糖类产酸,但不产气;接触酶、苯丙氨酸脱氨酶和酪氨酸脱羧酶均为阴性,精氨酸双水解酶阳性;不液化明胶,还原石蕊牛奶并胨化,生长温度范围10～43℃,DNA中G Cmol为36.4％。经鉴定,SM-A菌株为乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.lactis)。     

Alternatives for biosurfactants and bacteriocins extraction from Lactococcus lactis cultures produced under different pH conditions

Aims: Study of the potential of Lactococcus lactis CECT‐4434 as a biosurfactants and nisin (the only bacteriocin allowed to be used in the food industry) producer for industrial applications, exploiting the possibility of recovering separately both metabolites, taking into account that L. lactis is an interesting micro‐organism with several applications in the food industry because it is recognized as GRAS. Methods and Results: The results showed the ability of this strain to produce cell‐bound biosurfactants, under controlled pH, and cell‐bound biosurfactants and bacteriocins, when pH was not controlled. Three extraction procedures were designed to separately recover these substances. Conclusions: The strain L. lactis CECT‐4434 showed to be a cell‐bound biosurfactants and bacterocins producer when fermentations were carried out under uncontrolled pH. Both products can be recovered separately. Significance and Impact of the Study: Development of a convenient tool for the extraction of cell‐bound biosurfactants and bacteriocins from the fermentation broth.     

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。     

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.     

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.     

PURIFICATION AND CHARACTERIZATION OF PULLULANASE FROM Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co²⁺ and completely inhibited by Hg²⁺. The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.     

Effect of bacteriocin-induced cell damage on the branched-chain amino acid transamination by Lactococcus lactis

The effect of the bacteriocin lacticin 3147 on the branched-chain amino acid transamination by Lactococcus lactis IFPL359 was investigated. The bacteriocin provokes membrane permeabilisation of the cells, rendering them non-viable but metabolically active. Free diffusion of amino acids into the cell was facilitated. In addition, membrane permeabilisation promotes further cell lysis. Both facts render the enzymes more accessible to their substrates and hence increase branched-chain amino acid transamination. This research broadens the spectrum of technological applications of lacticin 3147 in the development of cheese flavour.     

In silico analysis of lactate producing metabolic network in Lactococcus lactis

Today the importance of in silico experiment grows bigger than before by the advance of computing power. More detailed mathematical modeling handled by simulation can produce more reasonable and meaningful results. In this research, we suggest the metabolic network of Lactococcus lactis for aerobic condition. Using a mathematical model, we observed the effect of enzymes on lactate production using flux distribution analysis, metabolic control analysis, and in silico experiment by biochemical simulation software. Each analysis showed some different results because of their characteristics but some key enzymes for lactate production were found from them.