Osaka Municipal Technical Research Institute, Osaka, Japan.
Appl Environ Microbiol. 2012 Sep;78(17):6203-16. doi: 10.1128/AEM.01148-12. Epub 2012 Jun 29.
Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.
大肠杆菌通过扩展莽草酸途径进行代谢工程改造，生成能够生产 6 种芳香族化合物的菌株，包括苯乳酸、4-羟基苯乳酸、苯乙酸、4-羟基苯乙酸、2-苯乙醇和 2-(4-羟基苯基)乙醇，这些化合物被广泛应用于医药、农药、抗生素、香料等多个行业。为了生成生产苯乳酸和 4-羟基苯乳酸的菌株，分别将铜绿假单胞菌的乳酸脱氢酶基因（ldhA）引入到苯丙氨酸和酪氨酸高产菌的染色体中。将来自巴西固氮螺菌的苯丙酮酸脱羧酶基因（ipdC）和大肠杆菌的苯乙醛脱氢酶基因（feaB）分别引入到苯丙氨酸和酪氨酸高产菌的染色体中，以生成苯乙酸和 4-羟基苯乙酸的生产者，而 ipdC 和短乳杆菌的醇脱氢酶基因（adhC）则被引入以分别生成 2-苯乙醇和 2-(4-羟基苯基)乙醇的生产者。各自引入的基因的表达由 T7 启动子控制。在生成 2-苯乙醇和 2-(4-羟基苯基)乙醇的生产者时，我们发现大肠杆菌中由 yqhD、yjgB 和 yahK 基因编码的内源性醛还原酶会自动将生成的苯乙醛和 4-羟基苯乙醛还原为 2-苯乙醇和 2-(4-羟基苯基)乙醇。在苯丙氨酸和酪氨酸高产菌中，与 ipdC 共同引入和共表达每个基因都增强了葡萄糖生产 2-苯乙醇和 2-(4-羟基苯基)乙醇的能力。引入 yahK 基因可使两种芳香醇的产量达到最高。在生产 2-苯乙醇、2-(4-羟基苯基)乙醇、苯乙酸和 4-羟基苯乙酸时，观察到一些副产物的积累。从构建的菌株的染色体中删除 feaB、pheA 和/或 tyrA 基因导致所需芳香族化合物的产量增加，而副产物的产量减少。最后，构建的 6 株工程菌均能够成功生产出不同的芳香族化合物作为主要产物。我们在这里表明，可以从可再生资源中生产 6 种芳香族化合物，而无需补充昂贵的前体。
