L-精氨酸生物合成的代谢流量分析

建立并完善了谷氨酸棒杆菌GWY020及其2个逐步叠加不同遗传标记的突变株HUI821和GUI089合成L-精氨酸的中心代谢网络。分别测定了它们在特定培养时段(50 h~52 h)L-精氨酸等代谢物的胞外浓度, 由此计算这一时段这些代谢物在发酵液中积累(或消耗)的速率, 分别作出这3株菌在拟稳态下的代谢流量分布图, 进而研究育种过程中不同遗传标记的叠加对代谢网络中L-精氨酸合成流量分布的影响。结果表明遗传标记的引入使流量分配发生了重大变化, 节点处的流量分配朝着有利于L-精氨酸合成的方向改变。从代谢流量分析角度上, 证明结构类似物抗性和敏感性突变是代谢流导向和设计育种的有效手段, 代谢流量分析将成为设计育种的提供新思路。     

L-缬氨酸合成的代谢流量分析

分别测定谷氨酸棒杆菌（Corynebacterium glutamicum）AS1-495及其3个逐个叠加不同遗传标记的突变株AA361、AAT231和AATV341在特定培养时段（26～28h）L缬氨酸等代谢物的胞外浓度,由此计算这一时段这些代谢物在发酵液中积累（或消耗）的速率,分别做出这4株菌在拟稳态下的代谢流量分布图,进而研究育种过程中不同遗传标记的叠加对代谢网络中L-缬氨酸合成流量分布的影响。结果表明遗传标记的引入使流量分配发生了重大变化,节点处的流量分配朝着有利于L缬氨酸合成的方向改变。6-磷酸葡萄糖节点处流入EMP途径和HMP途径的流量分配由17.0∶83.0变为24.3∶75.7;丙酮酸节点处流入L-缬氨酸合成途径和其他途径的流量分配由15.8∶842变为76.7∶23.3/L-缬氨酸合成的分支途径上的流量由最初的5.37增大为37.3,乳酸合成途径的流量从11.1最后降为1.16,L-缬氨酸产量由4g/L提高到24.5 g/L。代谢流量分布的变化趋势与L缬氨酸产量的变化趋势是互相吻合的。以2-噻唑丙氨酸抗性突变（2TAr）和L天冬氨酸氧肟酸盐超敏性突变（LAAHss）有效地进行代谢流遗传导向的事实,在代谢流量分析的层面上,证明结构类似物抗性突变和结构类似物超敏性突变是代谢流导向和设计育种的十分有效的手段,代谢流量分析会成为设计育种的校正方法。     

L-谷氨酰胺产生菌的选育和代谢流量分析

目的:建立并完善嗜乙酰乙酸棒杆菌YL012及其突变株LCHA0082合成L-谷氨酰胺的中心代谢网络.方法:分别测定了它们在特定培养时段(48h～50h)L-谷氨酰胺等代谢物的胞外浓度,由此计算这一时段这些代谢物在发酵液中积累(或消耗)的速率,分别作出这两株菌在拟稳态下的代谢流量分布图,进而研究诱变育种过程中不同诱变标记对代谢网络中L-谷氨酰胺合成流量分布的影响.结果:育种操作使流量分配朝着有利于L-谷氨酰胺合成的方向改变,流入谷氨酸节点的流量由29.198mmol/L·h上升到44.854mmol/L·h,提高到原来的1.5倍左右,合成L-谷氨酰胺的流量由18.138mmol/L·h上升至31.065mmol/L·h,效果明显.结论:从代谢流量分析角度上,证明诱变育种对代谢流量的改变起到明显的作用,代谢流量分析也为新的设计育种提供了思路.     

L-缬氨酸产生菌的选育及其代谢流分析

以谷氨酸棒杆菌(Corynebacterium glutamicum)AS1.495(Leu^-)为出发菌株,通过多次亚硝基胍(NTG)诱变,给AS1．495(Leu^-)依次叠加L-AAH^as,2-TA^r,Vd^-的遗传标记,得到突变株AATV341(Leu^-,L-AAH^as,2-TA^r,Vd^-),可在8％的葡萄糖培养基积累L-缬氨酸24．5g／L,比出发菌株提高了5．13倍。同时运用代谢流量分析理论,测定出发菌株AS1．495及其突变株AATV341在L-缬氨酸合成阶段的代谢流量,并初步进行比较和分析,发现遗传标记的引入使流量分配发生了重大变化,流量分配朝着有利于L-缬氨酸合成的方向改变。     

L-精氨酸高产菌的选育及基于代谢流量分布的育种机制

从分析钝齿棒杆菌(Corynebacterium crenatum)的精氨酸合成途径入手,提出了一种通过选育脯氨酸结构类似物抗性突变株以提高其精氨酸合成能力的育种思路。采用亚硝基胍(NTG)诱变处理出发菌株YD8(His-,SGr1.2 mg/mL,D-Argr15 mg/mL),经含15 mg/mL的脯氨酸结构类似物S-甲基半胱氨酸(S-MC)的抗性筛选获得精氨酸高产突变株YDM403(His-,SGr1.2 mg/mL,D-Argr15 mg/mL,S-MCr15 mg/mL),产酸水平可达29.4 g/L,较出发菌株YD8的产酸高出55.0%。通过代谢流量分布分析了菌株YD8和YDM403代谢网络的变化,结果表明,菌株YD8可能存在顺序反馈抑制作用,出发株YD8解除了Arg对Glu到Arg的反馈抑制,而YDM403又解除了Pro对Glu到Pro的反馈抑制,从而使中间物Glu累积量下降而对-αKG到Glu不再有反馈抑制,其通量提高,与此同时从Glu向Arg的代谢通量也相应增加。     

须糖多孢菌Saccharopolyspora pogona的核糖体工程改造对丁烯基多杀菌素合成的影响

核糖体工程是以微生物的各类抗生素抗性突变为筛选标记,高效获得次生代谢产物合成能力提高的突变株的一种育种新方法。通过核糖体工程技术,使用链霉素对须糖多孢菌Saccharopolyspora pogona进行抗性选育,以获得高产丁烯基多杀菌素突变菌株。对原始菌株和所获得的突变菌株代谢产物的研究发现,相对于原始菌株,其中突变株S13的丁烯基多杀菌素产量提高幅度最大,相比原始菌株提高了1.79倍。经质谱测定表明,其代谢物中比原始菌株多了一种丁烯基多杀菌素组分Spinosynα1。对抗性突变株S13的DNA序列进行分析,发现在编码核糖体S12蛋白的rps L基因保守区域中出现点突变,第314位和第320位的胞嘧啶(C)分别突变为腺嘌呤(A)和胸腺嘧啶(T),对应的氨基酸残基分别由脯氨酸突变为谷氨酰胺,丙氨酸突变为缬氨酸。研究显示,突变株S13遗传稳定性良好。     

代谢流量比率分析及其在代谢工程中的应用

细胞内代谢反应流量在系统理解细胞代谢特性和指导代谢工程改造等方面都起着重要的作用。由于代谢流量难以直接测量得到,在很多情况下通过跟踪稳定同位素在代谢网络中的转移并进行相应的模型计算能有效地定量代谢流量。代谢流量比率分析法能够高度体现系统的生物化学真实性、辨别细胞代谢网络的拓扑结构,并且能够相对简单快速地定量反应速率等,因此受到代谢工程研究者越来越多的重视。以下着重介绍并讨论了利用代谢物同位体分布信息分析关键代谢节点合成途径的流量比率、基于流量比率的代谢流量解析、以及应用于代谢工程等的相关原理、实验测量、数据分析、使用条件等,以期充分发挥代谢流量比率分析法的优势,并将其拓展推广至更多细胞体系的代谢特性阐明和代谢工程改造中去。     

α-核突触蛋白基因突变小鼠脑组织中内源性代谢产物的变化

目的:明确α-核突触蛋白与帕金森病的病理生理相关性及其临床意义。方法:采用相色谱-质谱联用(UPLC-MS)检测野生型小鼠和基因突变型小鼠脑组织中内源性代谢性产物,通过mzcloud法对小鼠脑组织中内源性代谢物质进行鉴定,将相应数据进行主成分分析(PCA)和聚类分析,分析其相关差异表达代谢物,并构建通路图和互作网络图。结果:(1)基于LC/MS法的代谢组分析结果显示两组间差异代谢物以氨基酸类及磷脂类等为主,包括β-丙氨酰-L-组氨酸、L-精氨酸、L-组氨酸、L-亮氨酸、L-苯丙氨酸、L-缬氨酸、L-天门冬氨酸、L-丙氨酸、磷脂酰胆碱等;(2)构建的代谢通路主要涉及酮体的合成和降解、牛磺酸和亚牛磺酸代谢、丙氨酸,天冬氨酸和谷氨酸代谢、精氨酸和脯氨酸代谢、组氨酸代谢、苯丙氨酸代谢、缬氨酸,亮氨酸和异亮氨酸的生物合成、甘油磷脂代谢等,从中发现18个具有标志性的代谢成分。结论:α-核突触蛋白基因突变后,酮体的合成和降解、牛磺酸和亚牛磺酸代谢、丙氨酸,天冬氨酸和谷氨酸代谢、精氨酸和脯氨酸代谢、组氨酸代谢、苯丙氨酸代谢、缬氨酸,亮氨酸和异亮氨酸的生物合成、甘油磷脂代谢等代谢通路发生了变化,涉及β-丙氨酰-L-组氨酸、L-精氨酸、L-组氨酸、L-亮氨酸、L-苯丙氨酸、L-缬氨酸、L-天门冬氨酸、L-丙氨酸、磷脂酰胆碱等的生物学标志性代谢产物变化。     

钝齿棒杆菌GlnK在氮代谢调控及L-精氨酸合成中的功能

【目的】钝齿棒杆菌是重要的氨基酸生产菌株,本研究针对氮代谢PⅡ信号转导蛋白GlnK展开相关功能研究,分析其在钝齿棒杆菌氮代谢调控及L-精氨酸合成中的作用。【方法】以GlnK蛋白为研究对象,通过基因敲除等遗传方法获得过表达、敲除及敲弱glnK的重组钝齿棒杆菌,研究GlnK对NH_4~+吸收的影响,通过RT-qPCR和酶活测定,从转录水平和蛋白水平上揭示GlnK对氮代谢和L-精氨酸合成相关基因表达水平及酶活的影响,通过5-L发酵罐发酵产L-精氨酸研究GlnK对L-精氨酸合成的影响。【结果】过表达glnK能明显促进NH_4~+的吸收,而敲除glnK后则会抑制NH_4~+的摄取;RT-qPCR和酶活测定发现,相比于野生型菌株Cc5-5,glnK过表达菌株Cc-glnK中与铵吸收相关的基因,表达量平均上调约4.58倍,L-精氨酸合成基因簇中基因的表达水平平均上调1.50倍。Cc-glnK中氮代谢相关蛋白的酶活平均提高46.97%;L-精氨酸合成途径上7个关键酶的酶活平均提高30.00%;5-L发酵罐发酵各重组菌株结果表明,Cc-glnK菌株的产量可达49.53 g/L,产率为0.516 g/(L·h),相比于出发菌株Cc5-5,其L-精氨酸产量提高了28.65%。【结论】过表达GlnK能促进NH_4~+的吸收及利用,并通过影响L-精氨酸合成途径上关键基因的表达水平,提高关键酶的酶活,最终提高L-精氨酸的产量。本研究为后续探索钝齿棒杆菌氮代谢调控机制及代谢工程改造钝齿棒杆菌生产L-精氨酸提供了一种新的策略。     

基于代谢通量分析的琥珀酸放线杆菌高产选育研究

在对产琥珀酸放线杆菌代谢分析的基础上选育出高产突变株对琥珀酸的工业生物转化有重要意义.在矩阵分析代谢通量基础上,围绕柔性节点下的副产物乙酸及乙醇的降低分别实施软X诱变及定点突变选育,并对比分析了突变株与出发株相关酶活及基因序列变化.针对出发株的流量分析显示产物琥珀酸的代谢通量为1.78(mmol/g/h),主要副产物乙酸与乙醇的代谢通量分别为(0.60mmol/g/h)和(1.04 mmol/g/h),并发现乙醇代谢加剧了琥珀酸合成中的H电子供体的不足;筛选出的氟乙酸抗性突变株S.JST1的乙酸代谢通量降低了96%,为0.024(mmol/g/h),酶活检测表明磷酸乙酰转移酶(Pta)的酶比活力从602降低到74,进一步的序列对比分析发现pta突变基因中产生了一个突变位点:adh定点复合突变株S.JST2的乙醇代谢通量降低了98%,为0.020(mmol/g/h),酶活检测表明Adh的酶比活力从585降低到62.最终突变株S.JST2琥珀酸累积产量达65.7 g/L.围绕产琥珀酸放线杆菌Pta及Adh酶活的降低实施定向选育,在降低副产物流量的同时,有助于改善细胞H供体代谢平衡进而提高琥珀酸的流量.所获突变株具有工业应用潜力.     

Metabolic analysis of adaptive evolution for in silico-designed lactate-producing strains

Experimental evolution is now frequently applied to many biological systems to achieve desired objectives. To obtain optimized performance for metabolite production, a successful strategy has been recently developed that couples metabolic engineering techniques with laboratory evolution of microorganisms. Previously, we reported the growth characteristics of three lactate-producing, adaptively evolved Escherichia coli mutant strains designed by the OptKnock computational algorithm. Here, we describe the use of (13)C-labeled experiments and mass distribution measurements to study the evolutionary effects on the fluxome of these differently designed strains. Metabolic flux ratios and intracellular flux distributions as well as physiological data were used to elucidate metabolic responses over the course of adaptive evolution and metabolic differences among strains. The study of 3 unevolved and 12 evolved engineered strains as well as a wild-type strain suggests that evolution resulted in remarkable improvements in both substrate utilization rate and the proportion of glycolytic flux to total glucose utilization flux. Among three strain designs, the most significant increases in the fraction of glucose catabolized through glycolysis (>50%) and the glycolytic fluxes (>twofold) were observed in phosphotransacetylase and phosphofructokinase 1 (PFK1) double deletion (pta- pfkA) strains, which were likely attributed to the dramatic evolutionary increase in gene expression and catalytic activity of the minor PFK encoded by pfkB. These fluxomic studies also revealed the important role of acetate synthetic pathway in anaerobic lactate production. Moreover, flux analysis suggested that independent of genetic background, optimal relative flux distributions in cells could be achieved faster than physiological parameters such as nutrient utilization rate.     

Effects of the changes in enzyme activities on metabolic flux redistribution around the 2-oxoglutarate branch in glutamate production by<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

An experimental method for metabolic control analysis (MCA) was applied to the investigation of a metabolic network of glutamate production by Corynebacterium glutamicum. A metabolic reaction (MR) model was constructed and used for flux distribution analysis (MFA). The flux distribution at a key branch point, 2-oxoglutarate, was investigated in detail. Activities of isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH), and 2-oxoglutarate dehydrogenase complex (ODHC) around this the branch point were changed, using two genetically engineered strains (one with enhanced ICDH activity and the other with enhanced GDH activity) and by controlling environmental conditions (i.e. biotin-deficient conditions). The mole flux distribution was determined by an MR model, and the effects of the changes in the enzyme activities on the mole flux distribution were compared. Even though both GDH and ICDH activities were enhanced, the mole flux distribution was not significantly changed. When the ODHC activity was attenuated, the flux through ODHC decreased, and glutamate production was markedly increased. The flux control coefficients of the above three enzymes for glutamate production were determined based on changes in enzyme activities and the mole flux distributions. It was found that the factor with greatest impact on glutamate production in the metabolic network was obtained by attenuation of ODHC activity.     

Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β-alanine metabolism

Mevalonate (MVA) pathway is the core for terpene and sterol biosynthesis, whose metabolic flux influences the synthesis efficiency of such compounds. Saccharomyces cerevisiae is an attractive chassis for the native active MVA pathway. Here, the truncated form of Enterococcus faecalis MvaE with only 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was found to be the most effective enzyme for MVA pathway flux using squalene as the metabolic marker, resulting in 431-fold and 9-fold increases of squalene content in haploid and industrial yeast strains respectively. Furthermore, a positive correlation between MVA metabolic flux and β-alanine metabolic activity was found based on a metabolomic analysis. An industrial strain SQ3-4 with high MVA metabolic flux was constructed by combined engineering HMGR activity, NADPH regeneration, cytosolic acetyl-CoA supply and β-alanine metabolism. The strain was further evaluated as the chassis for terpenoids production. Strain SQ3-4-CPS generated from expressing β-caryophyllene synthase in SQ3-4 produced 11.86 ± 0.09 mg l⁻¹ β-caryophyllene, while strain SQ3-5 resulted from down-regulation of ERG1 in SQ3-4 produced 408.88 ± 0.09 mg l⁻¹ squalene in shake flask cultivations. Strain SQ3-5 produced 4.94 g l⁻¹ squalene in fed-batch fermentation in cane molasses medium, indicating the promising potential for cost-effective production of squalene.     

13C标记代谢流分析中天然稳定性同位素修正矩阵构建方法及应用

稳定性同位素¹³C标记实验是分析细胞代谢流的一种重要手段,主要通过质谱检测胞内代谢物中¹³C标记的同位素分布,并作为胞内代谢流计算时的约束条件,进而通过代谢流分析算法得到相应代谢网络中的通量分布。然而在自然界中,并非只有C元素存在天然稳定性同位素¹³C,其他元素如O元素也有其天然稳定性同位素¹⁷O、¹⁸O等,这使得质谱方法所测得的同位素分布中会夹杂除¹³C标记之外的其他元素的同位素信息,特别是分子中含有较多其他元素的分子,这将导致很大的实验误差,因此需要在进行代谢流计算前进行质谱数据的矫正。本研究提出了一种基于Python语言的天然同位素修正矩阵的构建方法,用于修正同位素分布测量值中由于天然同位素分布引起的测定误差。文中提出的基本修正矩阵幂方法用于构建各元素修正矩阵,结构简单、易于编码实现,可直接应用于¹³C代谢流分析软件数据前处理。将该修正方法应用于¹³C标记的黑曲霉（Aspergillus niger）胞内代谢流分析,结果表明本研究提出的方法准确有效,为准确获取微生物胞内代谢流分析提供了可靠的数据修正方法。     

Batch culture characterization and metabolic flux analysis of succinate-producing Escherichia coli strains

This study presents an in-depth analysis of the anaerobic metabolic fluxes of various mutant strains of Escherichia coli overexpressing the Lactococcus lactis pyruvate carboxylase (PYC) for the production of succinate. Previously, a metabolic network design that includes an active glyoxylate pathway implemented in vivo increased succinate yield from glucose in an E. coli mutant to 1.6 mol/mol under fully anaerobic conditions. The design consists of a dual succinate synthesis route, which diverts required quantities of NADH through the traditional fermentative pathway and maximizes the carbon converted to succinate by balancing the carbon flux through the fermentative pathway and the glyoxylate pathway (which has a lower NADH requirement). Mutant strains previously constructed during the development of high-yield succinate-producing strains were selected for further characterization to understand their metabolic response as a result of several genetic manipulations and to determine the significance of the fermentative and the glyoxylate pathways in the production of succinate. Measured fluxes obtained under batch cultivation conditions were used to estimate intracellular fluxes and identify critical branch point flux split ratios. The comparison of changes in branch point flux split ratios to the glyoxylate pathway and the fermentative pathway at the oxaloacetate (OAA) node as a result of different mutations revealed the sensitivity of succinate yield to these manipulations. The most favorable split ratio to obtain the highest succinate yield was the fractional partition of OAA to glyoxylate of 0.32 and 0.68 to the fermentative pathway obtained in strains SBS550MG (pHL413) and SBS990MG (pHL413). The succinate yields achieved in these two strains were 1.6 and 1.7 mol/mol, respectively. In addition, an active glyoxylate pathway in an ldhA, adhE, ack-pta mutant strain is shown to be responsible for the high succinate yields achieved anaerobically. Furthermore, in vitro activity measurements of seven crucial enzymes involved in the pathways studied and intracellular measurements of key intermediate metabolite pools provided additional insights on the physiological perturbations caused by these mutations. The characterization of these recombinant mutant strains in terms of flux distribution pattern, in vitro enzyme activity and intracellular metabolite pools provides useful information for the rational modification of metabolic fluxes to improve succinate production.