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Metabolic and genetic control of the fermentative fluxes in the cyanobacterium Synechococcus sp. PCC 7002 for biofuel production.

机译：蓝藻Syechococcus sp。中发酵通量的代谢和遗传控制。 PCC 7002用于生物燃料生产。

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摘要

Some aquatic microbial oxygenic photoautotrophs (AMOPs) excrete products such as H2, lactate, ethanol, and acetate in low yields during auto-fermentation (in dark, anoxic conditions) of intracellular carbohydrates previously stored during aerobic photosynthesis. We have utilized NMR and LC/MS metabolomics methods to characterize the marine cyanobacterium, Synechococcus sp. strain PCC 7002. In addition to identifying the WT strain, we have analyzed the phenotype of 3 mutant strains: ldhA, ldhAEx, and nifJ. In addition, we analyzed the WT strain under auto- fermentative conditions in the presence of nitrate. We have gained insight into manipulating fermentative fluxes of this bacterium, which may have future applications for fuels like bio-hydrogen.;In Chapter One, we have constructed a mutant (ldhA) of the cyanobacterium Synechococcus sp. strain PCC 7002 lacking the enzyme for the NADH-dependent reduction of pyruvate to D-lactate, the major fermentative reductant sink in this cyanobacterium. Auto-fermentation by the ldhA mutant resulted in no D-lactate production and redistributed fluxes of the excreted acetate, alanine, succinate, and hydrogen, which was 5-fold higher during auto-fermentation. The NAD(P)H/NAD(P) + ratio was also higher in the ldhA strain and this increased ratio is the source of the increased H2, which is generated via the NADH-dependent, bidirectional [NiFe]-hydrogenase.;In Chapter 2, we overexpress the ldhA gene for increased lactate production. In addition to a 2-fold increase in fermentative lactate production, the strain we have developed, LdhAEx, also excretes lactate (up to 20 mM) during photoautotrophic growth. Surprisingly, LdhAEx, which overexpresses only the lactate dehydrogenase enzyme, excretes large concentrations of acetate (up to 80 mM) during photoautotrophic growth, likely as an energy balance for the lactate production.;In Chapter 3, we have constructed a knock-out mutant (nifJ) of Synechococcus sp. strain PCC 7002 that lacks one of two enzymes for the oxidation of pyruvate to acetyl-CoA: pyruvate: ferredoxin oxidoreductase (PFOR). During auto-fermentation of nifJ the excreted carbon flux decreased by 2-fold for acetate and 1.2-fold for lactate, while reductant flux to H2 was 1.3-fold lower than WT. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production during auto-fermentation. The first phase proceeded via reduced ferredoxin, as this phase decreased 2-fold in nifJ. Measurement of the intracellular NADH/NAD + ratio revealed that the first phase of H2 production arose from a reductant source that was not in equilibrium with NADH/NAD +.;In Chapter 4, we examine the effects of nitrate during auto-fermentation in Synechococcus 7002. This cyanobacterium imports and reduces nitrate to nitrite, a majority of which is excreted rather than being further reduced and assimilated into the cells during auto-fermentation. Hydrogen production increases 11-fold, lactate increases 2.5-fold, and CO2 decreases 3-fold when nitrate is removed from fermentation media after cultures are grown in nitrate replete conditions. The cells that were fermented in the presence of nitrate seem to utilize enzymes of the oxidative pentose phosphate pathway in addition to glycolysis in order to produce more reductant equivalents per glucose equivalent catabolized as well as more CO2, while cells fermented in the absence of nitrate utilize predominantly glycolysis.
机译：某些水生微生物的氧合自养生物（AMOP）会在以前发酵有氧光合作用过程中储存的细胞内碳水化合物进行自动发酵（在黑暗，缺氧条件下）的过程中以低收率分泌H2，乳酸，乙醇和乙酸盐等产品。我们已经利用NMR和LC / MS代谢组学方法来表征海洋蓝细菌Synechococcus sp.。 PCC 7002菌株。除了鉴定WT菌株外，我们还分析了3个突变菌株的表型：ldhA，ldhAEx和nifJ。另外，我们在硝酸盐存在下在自动发酵条件下分析了野生型。我们已经掌握了操纵这种细菌的发酵通量的见解，这可能在生物氢等燃料中有未来的应用。在第一章中，我们构建了蓝藻Synechococcus sp。的突变体（ldhA）。菌株PCC 7002缺乏NADH依赖的丙酮酸还原为D-乳酸的酶，D-乳酸是该蓝细菌的主要发酵还原剂。通过ldhA突变体进行的自动发酵导致没有D-乳酸的产生，并且排泄的乙酸盐，丙氨酸，琥珀酸和氢的通量重新分配，这在自动发酵过程中提高了5倍。在ldhA菌株中，NAD（P）H / NAD（P）+的比率也更高，并且这种增加的比率是H2升高的来源，H2是通过NADH依赖的双向[NiFe]-氢化酶产生的。第2章，我们过表达ldhA基因以增加乳酸的产生。除了发酵乳酸产量增加2倍外，我们开发的菌株LdhAEx在光合养分生长过程中也会分泌乳酸（最高20 mM）。令人惊讶的是，仅过表达乳酸脱氢酶的LdhAEx在光合养分生长过程中会排泄大量乙酸盐（最高80 mM），这可能是乳酸产生的能量平衡。在第三章中，我们构建了一个敲除突变体。 （球菌）（NifJ）菌株PCC 7002，它缺乏将丙酮酸氧化为乙酰辅酶A的两种酶之一：丙酮酸：铁氧还蛋白氧化还原酶（PFOR）。在nifJ的自动发酵过程中，醋酸盐的排泄碳通量减少了2倍，乳酸盐的排泄的碳通量减少了1.2倍，而还原到H2的通量则比WT低1.3倍。连续电化学检测溶解的H2揭示了自动发酵过程中H2产生的两个时间分辨阶段。第一阶段通过还原性铁氧还蛋白进行，因为该阶段的nifJ降低了2倍。测量细胞内NADH / NAD +的比例显示，H2产生的第一阶段是由与NADH / NAD +不平衡的还原剂产生的。在第4章中，我们研究了硝酸盐在Synechococcus中自动发酵过程中的作用。 7002.这种蓝细菌输入硝酸盐并将其还原为亚硝酸盐，其中大部分会被排泄，而不是在自动发酵过程中被进一步还原并吸收到细胞中。当在硝酸盐充足的条件下培养后，从发酵培养基中除去硝酸盐时，产氢量增加11倍，乳酸增加2.5倍，二氧化碳减少3倍。在存在硝酸盐的情况下发酵的细胞似乎除了糖酵解外还利用氧化戊糖磷酸途径的酶，以便在分解的葡萄糖当量中产生更多的还原剂当量以及更多的CO2，而在没有硝酸盐的情况下发酵的细胞则利用主要是糖酵解。

著录项

  • 作者

    McNeely, Kelsey Marie.;

  • 作者单位

    Princeton University.;

  • 授予单位 Princeton University.;
  • 学科 Biology Molecular.;Biology Microbiology.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 133 p.
  • 总页数 133
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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