Yang Y T, Aristidou A A, San K Y, Bennett G N
Department of Bioengineering and Chemical Engineering, Institute of Biosciences and Bioengineering, Rice University, Houston, Texas 77005-1892, USA.
Metab Eng. 1999 Jan;1(1):26-34. doi: 10.1006/mben.1998.0103.
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.
最近有报道称有几种方法可减少大肠杆菌培养物中乙酸盐的积累。这种减少随后导致重组蛋白产量显著提高。在这些研究中，通过改变葡萄糖摄取速率、消除参与乙酸盐形成途径的关键酶以及将碳通量重定向至抑制性较小的副产物，构建了乙酸盐合成速率降低的代谢工程大肠杆菌菌株。特别是，已表明携带枯草芽孢杆菌乙酰乳酸合酶（ALS）基因的菌株不仅产生较少的乙酸盐，而且具有更高的ATP产量。对中心代谢途径和丙酮酸分支点处碳通量分布的代谢通量分析表明，该菌株有能力将过量的丙酮酸导向毒性小得多的化合物乙偶姻。本研究的主要重点是系统分析宿主现有途径中的小扰动对碳通量重新分布的影响。具体而言，构建并研究了缺失乙酸激酶（ACK）和乙酰磷酸转移酶（PTA）的突变体。碳重新分布的代谢分析结果表明，ackA - pta突变将以生长速率为代价降低乙酸盐水平。此外，在ackA - pta缺陷菌株中发现乳酸形成速率高得多，同时甲酸盐和乙醇合成速率较低。在ackA - pta突变体中表达枯草芽孢杆菌ALS进一步降低了乙酸盐水平，同时达到了与亲本菌株相似的细胞密度。
