大肠杆菌PTS系统改造及重组菌生长性能测定

利用Red重组系统对野生大肠杆菌Escherichia coli磷酸烯醇式丙酮酸-糖磷酸转移酶系统(Phosphoenolpyruvate:carbohydrate phosphotransferase system,PTS)进行修饰改造,敲除PTS系统中关键组分EⅡCBGlc的编码基因(ptsG),磷酸组氨酸搬运蛋白HPr的编码基因(ptsI),同时敲入来源于运动发酵单胞菌Zymomonas mobilis的葡萄糖易化体(Glucose facilitator)编码基因(glf),构建重组大肠杆菌,比较测定并系统评价了基因敲除和敲入对细胞的生长、葡萄糖代谢和乙酸积累的影响。敲除基因ptsG和ptsI造成大肠杆菌PTS系统部分功能缺失,细胞生长受到一定限制,敲入glf基因后,重组大肠杆菌能够利用Glf-Glk(葡萄糖易化体-葡萄糖激酶)途径,消耗ATP将葡萄糖进行磷酸化并转运进入细胞。通过该途径转运葡萄糖能够提高葡萄糖利用效率,降低副产物乙酸生成,同时能够使更多的碳代谢流进入后续相关合成途径,预期能够提高相关产物产量。     

理性设计和构建过量合成莽草酸的大肠杆菌代谢工程菌

利用代谢工程手段理性改造野生大肠杆菌的莽草酸(Shikimic acid,SA)合成途径及相关代谢节点,以构建高产莽草酸的工程菌株.根据细胞代谢网络分析,利用Red-Xer重组系统连续删除了野生型大肠杆菌CICIMB0013的莽草酸激酶基因(aroL、aroK),葡萄糖磷酸转移酶系统(PTS)的关键组分EIICBglc的编码基因(ptsG)以及奎宁酸/莽草酸脱氢酶基因(ydiB)并系统评价了基因删除对细胞的生长、葡萄糖代谢和莽草酸积累的影响.aroL、aroK的删除阻断了莽草酸进一步转化成为莽草酸-3-磷酸,初步提高莽草酸的累积.删除ptsG基因使大肠杆菌PTS系统部分缺失,细胞通过GalP-glk(半乳糖透性酶-葡萄糖激酶)途径,利用ATP将葡萄糖磷酸化后进入细胞.利用该途径运输葡萄糖能够减少PEP的消耗,使得更多的碳代谢流进入莽草酸合成途径,从而显著提高了莽草酸的产量.在此基础上删除ydiB基因,阻止了莽草酸合成的前体物质3-脱氢奎宁酸转化为副产物奎宁酸(Quinic acid,QA),进一步提高了莽草酸的累积.初步发酵显示4个基因缺失的大肠杆菌代谢工程菌生产莽草酸的能力比原始菌提高了90多倍.     

大肠杆菌DH5α及其耐乙酸突变株DA19在氮源限制下的代谢和关键酶特性研究

在氮源限制的基本培养基中对大肠杆菌DH5α及其耐乙酸突变株DA19进行连续培养,通过测定中心代谢途径关键酶的活性分析二者代谢差异。结果表明,DA19的6-磷酸葡萄糖脱氢酶(G6PDH)和异柠檬酸脱氢酶(ICDH)活性高于DH5α,而磷酸果糖激酶(PFK)和乙酸激酶(ACK)活性低于DH5α,反映了DA19进入磷酸戊糖途径(PPP)的碳流增加,而进入酵解和乙酸产生(Ack-Pta)途径的碳流减少。因此,关键酶活差异与DA19菌体关于葡萄糖得率提高、副产物乙酸和丙酮酸的生成减少相一致。添加腺嘌呤后,DH5α的G6PDH和ICDH活性增加,PFK和ACK活性降低,而DA19各酶活变化不明显。乙酸钠的添加导致除PFK外的其他酶活性均降低,尤其是DH5α的ICDH明显降低,这些结果反映的中心代谢途径流量变化也与二者生长和代谢副产物积累的变化一致。     

重组大肠杆菌的发酵与代谢工程

在工程菌的发酵中,外源基因高表达条件下的高密度发酵对于提高生产效率、降低生产成本、简化产品纯化工艺以及降低扩大生产投资都具有非常重要的意义．然而在大肠杆菌的高密度发酵中遇到了许多困难［‘］,其中主要包括有机酸类代谢副产物累积并抑制细菌自身生长、供氧限制和外源基因高表达引起宿主细胞生理负担过重等问题。对此,国内外学者作了许多有意义的探索。应用代谢工程技术对工程菌进行改良,使之有利于外源基因的高表达及高密度发酵,是一项很有应用前景的研究课题,引起了广泛关注。本文拟对近年来有关应用代谢工程改良重组大肠…     

大肠杆菌乙酸耐受性菌株的构建及其耐受机制研究进展

乙酸是微生物发酵生产常见的副产物,也可作为碳源存在于木质纤维素水解液等非粮原料发酵培养基中。培养基中含有高浓度的乙酸/乙酸盐时会抑制细胞生长、降低生物量,影响目标产品的产量和产率。研究乙酸耐受性机制,改进菌株的乙酸耐受性,构建具有高乙酸耐受性工程菌株,对于以乙酸为碳源或利用含乙酸的原料进行高附加值产品发酵生产具有重要意义。本文综述了通过代谢工程、实验室适应性进化、全局转录机器工程和基于CRISPR可追踪基因组工程等方法构建大肠杆菌乙酸耐受性菌株的研究进展,进一步从乙酸同化代谢、氨基酸依赖型代谢、离子转运系统调节和细胞膜成分修饰等4个方面阐述了大肠杆菌乙酸耐受性菌株的耐受性应答机制,总结了大肠杆菌乙酸耐受菌株的生产应用,展望了提高大肠杆菌乙酸耐受方法和大肠杆菌乙酸耐受机制的研究方向。     

重组大肠杆菌高密度发酵研究进展

重组大肠杆菌的高密度发酵是提高基因工程产品产量的一个非常有效的手段,是现代发酵工程研究的一个热点。本文就高密度发酵中影响重组大肠杆菌发酵产率的几个因素,包括宿主菌、培养基、培养条件、补料方法以及高密度发酵过程中存在的问题和对策加以讨论,着重探讨了高密度下大肠杆菌产生的有害代谢副产物———乙酸的产生机制、抑制作用机理,以及控制乙酸积累的技术方法 。     

不同溶氧条件下L-苏氨酸生物合成菌株的代谢流量分析

[目的]探索L-苏氨酸生物合成机理及影响因素.[方法]建立了大肠杆菌L-苏氨酸的代谢流平衡模型,应用MATLAB软件计算出不同溶氧条件下发酵中后期代谢网络的代谢流分布及理想代谢流分布.[结果]5%溶氧条件下,25.5%碳架进入HMP途径,74.5%碳架进入糖酵解途径,获得33.9%质量转化率;20%溶氧条件下,58.08%碳架进入HMP途径,41.92%碳架进入糖酵解途径,获得46.5%质量转化率;[结论]与理想代谢流(88.23%质量转化率)相比,应从菌种改造和发酵控制方面通过改变6-磷酸葡萄糖异构酶借以增加HMP途径代谢流量,通过增加磷酸烯醇式丙酮酸羧化反应代谢流提高天冬氨酸族合成代谢流,减少TCA循环代谢流量,从而达到减少副产物生成,增加L-苏氨酸生物合成的目的.     

植物挥发物代谢工程在改良香气品质和植物防御中的应用

挥发物次生代谢在植物繁殖、植物防御和改良食物品质方面发挥着重要作用。近年来,随着参与挥发物生物合成的基因和酶类的鉴定以及代谢途径和调控机理等研究的不断发展和深入,挥发物代谢工程已经具备较高的可行性。应用代谢工程改良花、果实的香气品质以及提高植物防御能力的研究成效显著。主要介绍了这些方面的最新进展,同时也讨论了植物挥发物代谢工程应用存在的问题和挑战以及研究思路。     

从碳代谢流角度新解代谢工程策略

代谢工程通过改造微生物代谢过程,进而利用微生物生产各种有用的医药、化学产品及工业原料.本文从细胞代谢中碳代谢流的角度入手,将代谢工程的传统与新型策略进行分类解析.其中,传统代谢工程手段主要对目标代谢路径的关键酶进行改造,通过过表达或基因敲除增大目的代谢路径碳代谢流.然而,在代谢路径改造需要进行多基因表达的情况下,传统手段在如何最佳表达多种酶使碳流通畅上会受到很大限制.本文提出利用高碳流路径,通过简单基因改造以获得高效目标产物生产的新策略.同时,随着合成生物学与系统生物学的发展,精细调控多基因表达成为可能.本文进一步举例讨论了代谢工程中粗略与精细调控基因表达水平对碳流的影响,以期对教学与前沿科研有助.     

鸟苷产生菌的代谢途径分析

代谢工程要解决的主要问题就是改变某些途径中的碳架物质流量或改变碳架物质流在不同途径中的流量分布,其目标就是修饰初级代谢,将碳架物质流导入目的产物的载流途径以获得产物的最大转化率。利用途径分析方法对枯草芽孢杆菌生产鸟苷的途径进行了分析,建立了3种基础模型,鸟苷理论摩尔产率分别是0．625、0．75和0．667,确定了枯草芽孢杆菌生产鸟苷的最佳途径的通量分布。     

Metabolic flux analysis of Escherichia coli deficient in the acetate production pathway and expressing the Bacillus subtilis acetolactate synthase

Several approaches to reduce acetate accumulation in Escherichia coli cultures have recently been reported. This reduction subsequently led to a significant enhancement in recombinant protein production. In those studies, metabolically engineered E. coli strains with reduced acetate synthesis rates were constructed through the modification of glucose uptake rate, the elimination of critical enzymes that are involved in the acetate formation pathways, and the redirection of carbon flux toward less inhibitory byproducts. In particular, it has been shown that strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene not only produce less acetate but also have a higher ATP yield. Metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point revealed that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin. The main focus of this study is the systematic analysis of the effects of small perturbations in the host's existing pathways on the redistribution of carbon fluxes. Specifically, a mutant with deleted acetate kinase (ACK) and acetyl phosphotransferase (PTA) was constructed and studied. Results from the metabolic analysis of carbon redistribution show the ackA-pta mutation will reduce acetate level at the expense of the growth rate. In addition, in the ackA-pta deficient strain a much higher lactate formation rate with simultaneously lower formate and ethanol synthesis rates was found. Expression of the B. subtilis ALS in ackA-pta mutants further reduces acetate levels while cell density similar to that of the parent strain is attained.     

Expression of two recombinant chloramphenicol acetyltransferase variants in highly reduced genome Escherichia coli strains

Highly reduced E. coli strains, MDS40, MDS41, and MDS42, lacking approximately 15% of the genome, were grown to high cell densities to test their ability to produce a recombinant protein with high yields. These strains lack all transposons and insertion sequences, cryptic prophage and many genes of unknown function. In addition to improving genetic stability, these deletions may reduce the biosynthetic requirements of the cell potentially allowing more efficient production of recombinant protein. Basic growth parameters and the ability of the strains to produce chloramphenicol acetyltransferase (CAT) under high cell density, batch cultivation were assessed. Although growth rate and recombinant protein production of the reduced genome strains are comparable to the parental MG1655 strain, the reduced genome strains were found to accumulate significant amounts of acetate in the medium at the expense of additional biomass. A number of hypotheses were examined to explain the accumulation of acetate, including oxygen limitation, carbon flux imbalance, and metabolic activity of the recombinant protein. Use of a non-catalytic CAT variant identified the recombinant protein activity as the source of this phenomenon; implications for the metabolic efficiency of the reduced genome strains are discussed.     

Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production

The culture of Escherichia coli for the commercial production of recombinant proteins has increased significantly in recent years. The production of acetate as a byproduct retards cell growth, inhibits protein formation, and diverts carbon from biomass to protein product. Our approach to reducing acetate accumulation was to disable the phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) by deleting the ptsHI operon in the wild-type E. coli strain GJT001. The mutation caused a severe reduction in growth rate and glucose uptake rate in glucose-supplemented M9 minimal medium, which confirmed the mutation, and eliminated acetate accumulation. The mutant strain (TC110) apparently metabolized glucose by a non-PTS mechanism that we are currently investigating, followed by phosphorylation by glucokinase. In complex medium such as 2xLB broth with 2% glucose, TC110 was able to grow quickly and still retained the phenotype of significantly reduced acetate accumulation (9.1+/-6.6 vs. 90.4+/-1.6mM in GJT001, P<0.05). The reduced acetate accumulation resulted in a significant improvement in final OD (23.5+/-0.7 in TC110 vs. 8.0+/-0.1 in GJT001, P<0.05). We tested the strains for the production of model recombinant proteins such as green fluorescent protein (GFP) and beta-galactosidase. TC110 had a 385-fold improvement in final volumetric productivity of GFP over GJT001 in shake flasks with 2xLB broth with 2% glucose. The distribution of GFP fluorescence in the cell population, as determined by flow cytometry, was much broader in GJT001 (coefficient of variation=466+/-35%) than in TC110 (coefficient of variation=55+/-1%). In corn steep liquor medium with 2% glucose, we observed a 28.5-fold improvement in final volumetric production of GFP in TC110 over GJT001. TC110 had a 7.5-fold improvement in final volumetric productivity of beta-galactosidase over GJT001 in 2xLB broth with 2% glucose medium. When tested in a batch bioreactor cultures with 2xLB broth with 2% glucose medium, the volumetric production of GFP by TC110 was 25-fold higher than that of GJT001. In summary, the ptsHI mutant of GJT001 resulted in reduced acetate accumulation, which led to significant improvements in recombinant protein production in batch bioreactors.     

Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method

A new algorithm was developed for the estimation of the metabolic flux distribution based on GC-MS data of proteinogenic amino acids. By using a sensitive GC-MS protocol as well as by combining the global search algorithm such as the genetic algorithm with the local search algorithm such as the Levenberg-Marquardt algorithm, not only the distribution of the net fluxes in the entire network, but also certain exchange fluxes which contribute significantly to the isotopomer distribution could be quantified. This mass isotopomer analysis could identify the biochemical changes involved in the regulation where acetate or glucose was used as a main carbon source. The metabolic flux analysis clearly revealed that when the specific growth rate increased, only a slight change in flux distribution was observed for acetate metabolism, indicating that subtle regulation mechanism exists in certain key junctions of this network system. Different from acetate metabolism, when glucose was used as a carbon source, as the growth rate increased, a significant increase in relative pentose phosphate pathway (PPP) flux was observed for Escherichia coli K12 at the expense of the citric acid cycle, suggesting that when growing on glucose, the flux catalyzed by isocitrate dehydrogenase could not fully fulfill the NADPH demand for cell growth, causing the oxidative PPP to be utilized to a larger extent so as to complement the NADPH demand. The GC-MS protocol as well as the new algorithm demonstrated here proved to be a powerful tool for characterizing metabolic regulation and can be utilized for strain improvement and bioprocess optimization.     

Response of fluxome and metabolome to temperature-induced recombinant protein synthesis in Escherichia coli

The response of the central carbon metabolism of Escherichia coli to temperature-induced recombinant production of human fibroblast growth factor was studied on the level of metabolic fluxes and intracellular metabolite levels. During production, E. coli TG1:plambdaFGFB, carrying a plasmid encoded gene for the recombinant product, revealed stress related characteristics such as decreased growth rate and biomass yield and enhanced by-product excretion (acetate, pyruvate, lactate). With the onset of production, the adenylate energy charge dropped from 0.85 to 0.60, indicating the occurrence of a severe energy limitation. This triggered an increase of the glycolytic flux which, however, was not sufficient to compensate for the increased ATP demand. The activation of the glycolytic flux was also indicated by the readjustment of glycolytic pool sizes leading to an increased driving force for the reaction catalyzed by phosphofructokinase. Moreover, fluxes through the TCA cycle, into the pentose phosphate pathway and into anabolic pathways decreased significantly. The strong increase of flux into overflow pathways, especially towards acetate was most likely caused by a flux redirection from pyruvate dehydrogenase to pyruvate oxidase. The glyoxylate shunt, not active during growth, was the dominating anaplerotic pathway during production. Together with pyruvate oxidase and acetyl CoA synthase this pathway could function as a metabolic by-pass to overcome the limitation in the junction between glycolysis and TCA cycle and partly recycle the acetate formed back into the metabolism.     

Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering

Escherichia coli only maintains a small amount of cellular malonyl-CoA, impeding its utility for overproducing natural products such as polyketides and flavonoids. Here, we report the use of various metabolic engineering strategies to redirect the carbon flux inside E. coli to pathways responsible for the generation of malonyl-CoA. Overexpression of acetyl-CoA carboxylase (Acc) resulted in 3-fold increase in cellular malonyl-CoA concentration. More importantly, overexpression of Acc showed a synergistic effect with increased acetyl-CoA availability, which was achieved by deletion of competing pathways leading to the byproducts acetate and ethanol as well as overexpression of an acetate assimilation enzyme. These engineering efforts led to the creation of an E. coli strain with 15-fold elevated cellular malonyl-CoA level. To demonstrate its utility, this engineered E. coli strain was used to produce an important polyketide, phloroglucinol, and showed near 4-fold higher titer compared with wild-type E. coli, despite the toxicity of phloroglucinol to cell growth. This engineered E. coli strain with elevated cellular malonyl-CoA level should be highly useful for improved production of important natural products where the cellular malonyl-CoA level is rate-limiting.     

Quantification of proteomic and metabolic burdens predicts growth retardation and overflow metabolism in recombinant Escherichia coli

Escherichia coli has been the host organism most frequently investigated for efficient recombinant protein production. However, the production of a foreign protein in recombinant E. coli often leads to growth deterioration and elevated secretion of acetic acid. Such observed phenomena have been widely linked with cell stress responses and metabolic burdens originated particularly from the increased energy demand. In this study, flux balance analysis and dynamic flux balance analysis were applied to investigate the observed growth physiology of recombinant E. coli, incorporating the proteome allocation theory and an adjustable maintenance energy level (ATPM) to capture the proteomic and energetic burdens introduced by recombinant protein synthesis. Model predictions of biomass growth, substrate consumption, acetate excretion, and protein production with two different strains were in good agreement with the experimental data, indicating that the constraint on the available proteomic resource and the change in ATPM might be important contributors governing the growth physiology of recombinant strains. The modeling framework developed in this work, currently with several limitations to overcome, offers a starting point for the development of a practical, model-based tool to guide metabolic engineering decisions for boosting recombinant protein production.     

Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis

Several bacterial species and filamentous fungi utilize the phosphoketolase pathway (PHK) for glucose dissimilation as an alternative to the Embden-Meyerhof-Parnas pathway. In Aspergillus nidulans, the utilization of this metabolic pathway leads to increased carbon flow towards acetate and acetyl CoA. In the first step of the PHK, the pentose phosphate pathway intermediate xylulose-5-phosphate is converted into acetylphosphate and glyceraldehyde-3-phosphate through the action of xylulose-5-phosphate phosphoketolase, and successively acetylphosphate is converted into acetate by the action of acetate kinase. In the present work, we describe a metabolic engineering strategy used to express the fungal genes of the phosphoketolase pathway in Saccharomyces cerevisiae and the effects of the expression of this recombinant route in yeast. The phenotype of the engineered yeast strain MP003 was studied during batch and chemostat cultivations, showing a reduced biomass yield and an increased acetate yield during batch cultures. To establish whether the observed effects in the recombinant strain MP003 were due directly or indirectly to the expression of the phosphoketolase pathway, we resolved the intracellular flux distribution based on (13)C labeling during chemostat cultivations. From flux analysis it is possible to conclude that yeast is able to use the recombinant pathway. Our work indicates that the utilization of the phosphoketolase pathway does not interfere with glucose assimilation through the Embden-Meyerhof-Parnas pathway and that the expression of this route can contribute to increase the acetyl CoA supply, therefore holding potential for future metabolic engineering strategies having acetyl CoA as precursor for the biosynthesis of industrially relevant compounds.     

Gene modification of <Emphasis Type="Italic">Escherichia coli</Emphasis> and incorporation of process control to decrease acetate accumulation and increase ʟ-tryptophan production

Acetate is a primary inhibitory metabolite in cultures of Escherichia coli, and the production of both biomass and desired products are increased by reducing the accumulation of acetate. In this study, the accumulation of acetate during ?-tryptophan production was decreased by genetic modification of ?-tryptophan-producing strain (BCTRP) and optimization of the fermentation process. The mutant (BCTRPG), which has a deletion of the integral membrane permease IICB^Glc (ptsG), produces a higher concentration of ?-tryptophan than mutants with deletions of either phosphate acetyltransferase (pta) or pta–ptsG, due to the low accumulation of acetate and other byproducts, as well as high biomass production. The appropriate dissolved oxygen (DO) level, glucose feeding mode, and pH control strategy were applied to ?-tryptophan production using the BCTRPG mutant. The BCTRPG strain with optimized conditions resulted in a reduction in acetate accumulation (71.08% reduction to 0.72 g/L) and an increase in ?-tryptophan production (35.81% increase to 17.14 g/L) compared with the BCTRP strain in the original culture condition. Meanwhile, an analysis of the metabolic flux distribution indicated that the acetate synthesis flux decreased from 19.2% (original conditions) to 8.4% (optimized conditions), and the flux of tryptophan formation with the optimized conditions was 18.5%, which was 1.89 times higher than under the original conditions. This study provided the theoretical foundation and technical support for high-level industrialization production of ?-tryptophan.