简介:β-丙氨酸是自然界中唯一的β-氨基酸,被广泛应用于食品添加剂中,药物,健康产品,和表面活性剂。为避免传统生产方式造成的污染,β-丙氨酸的合成逐渐被微生物发酵和酶催化所取代,这是一个绿色的,温和,和高产率生物合成方法。方法:在本研究中,以葡萄糖为原料构建了高效生产β-丙氨酸的大肠杆菌重组菌株。L-赖氨酸产生菌的微生物合成途径,大肠杆菌CGMCC1.366,使用基因编辑通过敲除天冬氨酸激酶基因进行修饰,lysC.通过将关键酶与纤维素体组装,提高了催化效率和产物合成效率。结果:通过阻断L-赖氨酸生产途径减少了副产物的积累,从而提高β-丙氨酸的产率。此外,二酶法提高了催化效率,进一步提高了β-丙氨酸含量。关键的纤维素元素,Dockerin(docA)和cohesin(cohA),与来自枯草芽孢杆菌的L-天冬氨酸-α-脱羧酶(bspanD)和来自大肠杆菌的天冬氨酸氨基转移酶(aspC)联合使用,以提高该酶的催化效率和表达水平。两个工程菌株的β-丙氨酸产量分别达到7.439mg/L和25.87mg/L。在5L发酵罐中,β-丙氨酸含量达到755.465mg/L。讨论:构建的β-丙氨酸工程菌株合成的β-丙氨酸含量分别比没有组装纤维素体的工程菌株高10.47倍和36.42倍,分别。该研究为使用纤维素体多酶自组装系统酶法生产β-丙氨酸奠定了基础。
Introduction: β-Alanine is the only β-amino acid in nature; it is widely used in food additives, medicines, health products, and surfactants. To avoid pollution caused by traditional production methods, the synthesis of β-alanine has been gradually replaced by microbial fermentation and enzyme catalysis, which is a green, mild, and high-yield biosynthesis method. Methods: In this
study, we constructed an Escherichia coli recombinant strain for efficient β-alanine production using glucose as the raw material. The microbial synthesis pathway of L-lysine-producing strain, Escherichia coli CGMCC 1.366, was modified using gene editing by knocking out the aspartate kinase gene, lysC. The catalytic efficiency and product synthesis efficiency were improved by assembling key enzymes with cellulosome. Results: By-product accumulation was reduced by blocking the L-lysine production pathway, thereby increasing the yield of β-alanine. In addition, catalytic efficiency was improved by the two-enzyme method to further increase the β-alanine content. The key cellulosome elements, dockerin (docA) and
cohesin (cohA), were combined with L-aspartate-α-decarboxylase (bspanD) from Bacillus subtilis and aspartate aminotransferase (aspC) from E.coli to improve the catalytic efficiency and expression level of the enzyme. β-alanine production reached 7.439 mg/L and 25.87 mg/L in the two engineered strains. The β-alanine content reached 755.465 mg/L in a 5 L fermenter. Discussion: The content of β-alanine synthesized by constructed β-alanine engineering strains were 10.47 times and 36.42 times higher than the engineered strain without assembled cellulosomes, respectively. This research lays the foundation for the enzymatic production of β-alanine using a cellulosome multi-enzyme self-assembly system.