L-2-氨基丁酸作为新型药物的关键手性前体,在化工和制药行业应用广泛。该文以1株生产L-苏氨酸的大肠杆菌(Escherichia coli)THRD为出发菌株,逐步延伸代谢途径,构建了L-2-氨基丁酸高产菌株。首先,分别把苏氨酸脱水酶编码基因ilvA2和ilvA4在THRD中过表达,菌株THRD/pTrc99a-ilvA2在5 L发酵罐中分批补料发酵,2-酮基丁酸积累量达到18 g/L。然后,分别与ilvA2串联表达酪氨酸转氨酶、谷氨酸脱氢酶和亮氨酸脱氢酶编码基因tyrB、gdh和bcdBS,将L-2-酮基丁酸转化为L-2-氨基丁酸,菌株THRD/pTrc99a-bcdBS-ilvA2的L-2-氨基丁酸产量达到19 g/L。最后,研究了阻断L-苏氨酸输出途径对发酵的影响,菌株THRDΔrhtC/pTrc99a-bcdBS-ilvA2的L-2-氨基丁酸产量提升至22 g/L。因此,通过代谢途径延伸可以有效地将L-苏氨酸生产菌株转变为L-2-氨基丁酸生产菌株。该研究为L-2-氨基丁酸高产菌株的构建奠定了基础,且对其他延伸代谢途径获得新产品的代谢工程研究提供了参考。
L-2-aminobutyrate is a critical chiral precursor of new drugs, which has been widely used in chemical and pharmaceutical industries. In this study, the metabolic pathway of L-threonine in Escherichia coli THRD was extended to obtain an L-2-aminobutyrate producing strain. The threonine dehydratase encoding genes ilvA2 and ilvA4 were firstly individually overexpressed in E. coli THRD. The resulting strain THRD/pTrc99a-ilvA2 produced 18 g/L L-2-ketobutyrate in a 5 L fermenter by fed-batch fermentation. Subsequently, encoding genes of tyrosine aminotransferase, glutamate dehydrogenase, and leucine dehydrogenase, tyrB, gdh, and bcdBS, respectively, were overexpressed together with ilvA2 to catalyze the conversion of L-2-ketobutyrate to L-2-aminobutyrate. The strain THRD/pTrc99a-bcdBS-ilvA2 produced 19 g/L L-2-aminobutyrate. The effects of disrupting L-threonine exporters on L-2-aminobutyrate fermentation were investigated, and the production of L-2-aminobutyrate in strain THRDΔrhtC/pTrc99a-bcdBS-ilvA2 increased to 22 g/L. Taken together, the results clearly indicated that the L-threonine producing strain could be effectively transformed into an L-2-aminobutyrate producer by extending its downstream metabolic pathway. This study lays a solid basis for constructing L-2-aminobutyrate high producing strains. This study can also be referred in other metabolic engineering studies to biosynthesize new products by pathway extension.
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