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随着检测手段的丰富和测序手段的日渐发达,生物过程表现出数据量大、种类多、时变性和相关耦合性的特征,反映了过程中基因、细胞、反应器不同尺度特性的混杂性。提出了基于系统生物学的生物过程全局优化方法,利用基因组学、数学模型、转录组学、蛋白组学、代谢物组学和代谢流组学数据,结合反应器流场特性和细胞生理特性的关系,对生产菌株的限制性瓶颈问题进行了探索和解决。以广泛应用于合成生物学的宿主大肠杆菌和酿酒酵母、产抗生素的放线菌属和顶头孢霉,以及用于有机酸和异源蛋白表达的黑曲霉为主要案例,对生物过程的研究进展进行综述,并为生物过程的全局优化提供了新的思路。 相似文献
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L-异亮氨酸发酵代谢分析 总被引:7,自引:0,他引:7
通过在5L自控发酵罐上对L-异亮氨酸的发酵过程进行研究,分析了发酵基本特征,并结合菌体形态及发酵控制参数的变化,指出发酵过程中代谢流流向及代谢平衡和可能存在的代谢流迁移,为进一步发酵条件优化和分阶段控制发酵研究奠定基础。 相似文献
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当前,生物制造技术和产业是世界关注的热点。然而,生物过程优化与放大过程中普遍面临以下几个难题,包括:过程检测手段缺乏,难以满足关键指标参数的监控;细胞代谢认知匮乏,无法理性实现过程最优化调控;反应器环境差异大,导致逐级放大效率低下。文中针对以上亟待解决的关键问题,通过案例分析介绍发酵过程实时检测-动态调控-理性放大全链条关键技术创新。在未来,生物过程设计将以集成细胞生理学(时空多尺度细胞代谢模型)和流体动力学(CFD模型)的全生命周期模型为指导,推进计算机辅助设计与开发,加速生物过程实现大规模智能化生产,开启绿色生物制造新时代。 相似文献
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工业生物技术是以微生物细胞工厂利用可再生的生物原料来生产能源、材料与化学品等的生物技术,在解决资源、能源与环境等问题方面起着越来越重要的作用。系统生物学是全面解析微生物细胞工厂及其发酵过程从"黑箱"到"白箱"的重要研究方法。系统生物学借助基因组、转录组、蛋白质组、代谢组以及代谢流组等多组学数据,可解析微生物细胞工厂在RNA、蛋白与代谢物等不同水平上的变化规律与调控机制。目前,系统生物学在微生物细胞工厂的设计创建与发酵工艺优化中起着越来越重要的指导作用,许多成功应用实例不断涌现,推动着工业生物技术的快速发展。文中重点综述基因组、转录组、蛋白质组、代谢组与代谢流组以及基因组规模的网络模型等各组学技术的最新发展及其在工业生物技术尤其是菌株改造与发酵优化中的应用,并就工业生物技术中系统生物学的未来发展方向进行展望。 相似文献
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【目的】通过系统研究一个、两个及多个非氧化磷酸戊糖(PP)途径基因组合过表达对酿酒酵母木糖代谢的影响,以优化重组菌株的构建过程,构建高效的木糖代谢酿酒酵母菌株。【方法】在酿酒酵母中双拷贝过表达上游代谢途径的关键酶(木糖还原酶XR,木糖醇脱氢酶XDH,木酮糖激酶XKS),在此基础上构建了一系列PP途径基因过表达菌株,并对其木糖发酵性能进行比较研究。【结果】木糖发酵结果显示,不同组合过表达PP途径基因能不同程度改善重组菌株的木糖发酵性能。其中,过表达PP途径全部基因(RKI1,RPE1,TAL1和TKL1)使菌株的发酵性能最优,其乙醇产率和产量较对照菌株分别提高了39.25%和12.57%,同时较其他基因组合过表达菌株也有不同程度的改善。【结论】通过构建PP途径基因不同组合过表达酿酒酵母菌株,首次对PP途径基因对酿酒酵母木糖代谢的影响进行了系统研究,结果表明,不同组合强化PP途径基因对重组菌株木糖代谢的影响存在差异,相对于其他基因过表达组合,同步过表达PP途径全部基因最有利于碳通量流向乙醇。 相似文献
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肌苷和鸟苷生产菌中嘌呤核苷合成途径三段基因序列的分析 总被引:3,自引:0,他引:3
为了研究肌苷和鸟苷生产菌中与产苷有关的嘌呤核苷合成途径的遗传背景,选择了pur操纵子的启动子序列、编码SAMP合成酶的purA基因和编码GMP合成酶的guaA基因,设计合适的引物,分别从野生菌、一株肌苷低产菌和肌苷鸟苷高产菌中扩增出相应片段,经克隆和测序后,对它们进行比较和分析。分析结果表明两株生产菌的purA基因发生了1个碱基缺失,导致阅读框发生移码突变;而鸟苷高产菌在pur操纵子的启动子部分和操纵子抑制蛋白结合区域发生了近10%的突变,可能影响整个操纵子的表达调控。 相似文献
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强化表达SAM合成酶促进SAM在毕赤酵母中累积 总被引:14,自引:0,他引:14
S 腺苷甲硫氨酸 (S adenosyl L methionine ,SAM)是生物体硫代谢的重要中间代谢物质 ,在体内起着转甲基、转硫基、转氨丙基的作用 ,具有重要的药用和保健价值。将酿酒酵母来源的SAM合成酶 2基因置于GAP启动子调控下 ,构建胞内组成型表达质粒 ,并电转化至毕赤酵母菌株GS115。经Zeocin抗性和培养筛选到一株高产SAM的重组菌。对重组菌表达工艺的研究表明 ,碳源、氮源、pH和溶解氧对SAM的累积有较大影响。在优化条件下 ,重组细胞培养 3天 ,SAM累积量可达 2 .49g/L。 相似文献
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Dicarboxylic acids (DCAs) can be obtained by oxidizing alkanes by Candida tropicalis. Through alpha-monocarboxylic acids (MCAs), alpha- and omega-oxidation yield alpha- or omega-DCAs, respectively. However, both MCAs and DCAs may be degraded to acetyl-CoA by beta-oxidation, resulting in a limited DCA yield. Acetyl-CoA can be transported into the mitochondrion for the TCA cycle by carnitine acetyltransferase (CAT), by which the energy generation and beta-oxidation are connected. In this paper, we present a method to reconstruct the metabolic pathway by inhibiting the acetyl-CoA transportation system. Metabolic engineering is applied on the acetyl-CoA transportation system, but not the key enzymes in beta-oxidation. Starting with the original strain W10-1, cat heterozygote CZ-15 and cat homozygote CKC-11 were obtained by gene knockout. The CAT specific activity in CZ-15 was about 50% lower than that in W10-1, resulting in a 21.0% increase of the DCA concentration, and a 12% increase of the molar conversion of alkane, reaching 61.6%. However, no CAT activity was detected in CKC-11, and CKC-11 could not grow on alkane. These results indicate that inhibition of beta-oxidation via reconstruction of the transportation process between organelles can facilitate DCA production, but that totally blocking the & betagr;-oxidation would be harmful for energy supply. We thus provide a novel insight into regulation of the beta-oxidation system and metabolic flux. Further understanding of beta-oxidation and the acetyl-CoA transportation system in Candida tropicalis is reached through examination of fermentation data by metabolic flux analysis. 相似文献
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基因的表达受不同的转录调节因子调节。大肠杆菌中的异柠檬酸裂解酶调节因子(IclR)能够抑制编码乙醛酸支路酶的aceBAK操纵子的表达。本研究基于代谢物的13C同位体物质分布来定量解析代谢反应,主要研究了iclR基因在大肠杆菌生理和代谢中的作用。大肠杆菌iclR基因缺失突变株的生长速率、糖耗速率和乙酸的产量相对于原始菌株都有所降低,但菌体得率略有增加。通过代谢途径的流量比率分析发现基因缺失株的乙醛酸支路得到了激活,33%的异柠檬酸流经了乙醛酸支路;戊糖磷酸途径的流量变小,使得CO2的生成量减少。同时,乙醛酸支路激活,但草酰乙酸形成磷酸烯醇式丙酮酸的流量基本不变,说明磷酸烯醇式丙酮酸-乙醛酸循环没有激活,没有过多的碳原子在磷酸烯醇式丙酮酸羧化激酶反应中以CO2形式排出,从而确保了菌体得率。葡萄糖利用速率的降低、乙酰辅酶A的代谢效率提高等使得iclR基因敲除菌的乙酸分泌较原始菌株有所降低。 相似文献
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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. 相似文献
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Hüser AT Chassagnole C Lindley ND Merkamm M Guyonvarch A Elisáková V Pátek M Kalinowski J Brune I Pühler A Tauch A 《Applied and environmental microbiology》2005,71(6):3255-3268
A "second-generation" production strain was derived from a Corynebacterium glutamicum pantothenate producer by rational design to assess its potential to synthesize and accumulate the vitamin pantothenate by batch cultivation. The new pantothenate production strain carries a deletion of the ilvA gene to abolish isoleucine synthesis, the promoter down-mutation P-ilvEM3 to attenuate ilvE gene expression and thereby increase ketoisovalerate availability, and two compatible plasmids to overexpress the ilvBNCD genes and duplicated copies of the panBC operon. Production assays in shake flasks revealed that the P-ilvEM3 mutation and the duplication of the panBC operon had cumulative effects on pantothenate production. During pH-regulated batch cultivation, accumulation of 8 mM pantothenate was achieved, which is the highest value reported for C. glutamicum. Metabolic flux analysis during the fermentation demonstrated that the P-ilvEM3 mutation successfully reoriented the carbon flux towards pantothenate biosynthesis. Despite this repartition of the carbon flux, ketoisovalerate not converted to pantothenate was excreted by the cell and dissipated as by-products (ketoisocaproate, DL-2,3,-dihydroxy-isovalerate, ketopantoate, pantoate), which are indicative of saturation of the pantothenate biosynthetic pathway. Genome-wide expression analysis of the production strain during batch cultivation was performed by whole-genome DNA microarray hybridization and agglomerative hierarchical clustering, which detected the enhanced expression of genes involved in leucine biosynthesis, in serine and glycine formation, in regeneration of methylenetetrahydrofolate, in de novo synthesis of nicotinic acid mononucleotide, and in a complete pathway of acyl coenzyme A conversion. Our strategy not only successfully improved pantothenate production by genetically modified C. glutamicum strains but also revealed new constraints in attaining high productivity. 相似文献
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基于途径分析的L-异亮氨酸发酵溶氧控制研究 总被引:4,自引:0,他引:4
利用途径分析方法对黄色短杆菌(Brevibacterium flavum)TC-21 生产L-异亮氨酸的途径进行了分析,确定了黄色短杆菌TC-21生产L-异亮氨酸的最佳途径的通量分布,根据途径分析的结果,TCA循环的代谢流量对L-异亮氨酸产量有明显影响,而TCA循环与发酵过程中的溶氧密切相关,因此可以通过控制溶氧来提高L-异亮氨酸产量。在发酵过程的不同阶段,根据菌体生长和产酸的需求,改变TCA代谢流量,可以有效提高产酸率。实验证明,通过溶氧分阶段控制发酵生产L-异亮氨酸,比溶氧恒定控制方式发酵产率提高了15.77%。实验结果说明,用途径分析的结果指导发酵过程中的溶氧可以大幅度提高L-异亮氨酸的产量。 相似文献
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Depending on the availability of oxygen, Escherichia coli is able to switch between aerobic respiratory metabolism and anaerobic mixed acid fermentation. An important, yet understudied, metabolic mode is the micro-aerobic metabolism at intermediate oxygen availabilities. The relationship between oxygen input, physiology and gene expression of E. coli MG1655 and two isogenic mutants lacking succinate dehydrogenase (SDH) and fumarate reductase (FRD) activities was analyzed at different aerobiosis levels. Growth rate and cell yield were very similar to the parent strain. By-product formation was altered in the sdhC mutant to higher acetic acid and glutamate production in batch cultures. In continuous cultures with defined oxygen input gene expression analysis revealed a dependency of many catabolic genes to aerobiosis. Acetate excretion was still detectable under aerobic conditions in the sdhC mutant; the frdA mutant lacked anaerobic succinate excretion. Anaerobic repression of the sdh operon was diminished in the frdA strain, possibly to allow SDH to partially replace FRD. The experiments illustrate the remarkable adaptability of E. coli physiology—to compensate for the absence of important metabolic genes by altering carbon flux and/or gene expression such that there are only minor changes in growth capability across the aerobiosis range. 相似文献