Parimi Naga Sirisha, Durie Ian A, Wu Xianghao, Niyas Afaq M M, Eiteman Mark A
School of Chemical, Materials and Biomedical Engineering, Driftmier Engineering Center, University of Georgia, Athens, GA, 30602, USA.
Microb Cell Fact. 2017 Jun 21;16(1):114. doi: 10.1186/s12934-017-0729-2.
Citramalate, a chemical precursor to the industrially important methacrylic acid (MAA), can be synthesized using Escherichia coli overexpressing citramalate synthase (cimA gene). Deletion of gltA encoding citrate synthase and leuC encoding 3-isopropylmalate dehydratase were critical to achieving high citramalate yields. Acetate is an undesirable by-product potentially formed from pyruvate and acetyl-CoA, the precursors of citramalate during aerobic growth of E. coli. This study investigated strategies to minimize acetate and maximize citramalate production in E. coli mutants expressing the cimA gene.
Key knockouts that minimized acetate formation included acetate kinase (ackA), phosphotransacetylase (pta), and in particular pyruvate oxidase (poxB). Deletion of glucose 6-phosphate dehydrogenase (zwf) and ATP synthase (atpFH) aimed at improving glycolytic flux negatively impacted cell growth and citramalate accumulation in shake flasks. In a repetitive fed-batch process, E. coli gltA leuC ackA-pta poxB overexpressing cimA generated 54.1 g/L citramalate with a yield of 0.64 g/g glucose (78% of theoretical maximum yield), and only 1.4 g/L acetate in 87 h.
This study identified the gene deletions critical to reducing acetate accumulation during aerobic growth and citramalate production in metabolically engineered E. coli strains. The citramalate yield and final titer relative to acetate at the end of the fed-batch process are the highest reported to date (a mass ratio of citramalate to acetate of nearly 40) without being detrimental to citramalate productivity, significantly improving a potential process for the production of this five-carbon chemical.
柠苹酸是工业上重要的甲基丙烯酸（MAA）的化学前体，可以通过过表达柠苹酸合酶（cimA基因）的大肠杆菌来合成。删除编码柠檬酸合酶的gltA和编码3-异丙基苹果酸脱水酶的leuC对于实现高柠苹酸产量至关重要。乙酸是在大肠杆菌有氧生长过程中由柠苹酸的前体丙酮酸和乙酰辅酶A潜在形成的不良副产物。本研究调查了在表达cimA基因的大肠杆菌突变体中使乙酸最小化并使柠苹酸产量最大化的策略。
使乙酸形成最小化的关键基因敲除包括乙酸激酶（ackA）、磷酸转乙酰酶（pta），特别是丙酮酸氧化酶（poxB）。旨在改善糖酵解通量而删除葡萄糖6-磷酸脱氢酶（zwf）和ATP合酶（atpFH）对摇瓶中的细胞生长和柠苹酸积累产生了负面影响。在重复补料分批过程中，过表达cimA的大肠杆菌gltA leuC ackA-pta poxB在87小时内产生了54.1 g/L的柠苹酸，产率为0.64 g/g葡萄糖（理论最大产率的78%），且仅产生1.4 g/L的乙酸。
本研究确定了在代谢工程改造的大肠杆菌菌株有氧生长和柠苹酸生产过程中对减少乙酸积累至关重要的基因缺失。在补料分批过程结束时，相对于乙酸的柠苹酸产量和最终滴度是迄今为止报道的最高值（柠苹酸与乙酸的质量比接近40），且对柠苹酸生产力没有不利影响，显著改进了生产这种五碳化学品的潜在工艺。
