Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province, 214122, P. R. China.
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province, 214122, P. R. China.
J Ind Microbiol Biotechnol. 2019 Aug;46(8):1047-1059. doi: 10.1007/s10295-019-02213-2. Epub 2019 Jul 11.
L-Tyrosine serves as a common precursor for multiple valuable secondary metabolites. Synthesis of this aromatic amino acid in Bacillus licheniformis occurs via the shikimate pathway, but the underlying mechanisms involving metabolic regulation remain unclear. In this work, improved L-tyrosine accumulation was achieved in B. licheniformis via co-overexpression of aroG and tyrA from Escherichia coli to yield strain 45A12, and the L-tyrosine titer increased to 1005 mg/L with controlled glucose feeding. Quantitative RT-PCR results indicated that aroA, encoding DAHP synthase, and aroK, encoding shikimate kinase, were feedback-repressed by the end product L-tyrosine in the modified strain. Therefore, the native aroK was first expressed with multiple copies to yield strain 45A13, which could accumulate 1201 mg/L L-tyrosine. Compared with strain 45A12, the expression of aroB and aroF in strain 45A13 was upregulated by 21% and 27%, respectively, which may also have resulted in the improvement of L-tyrosine production. Furthermore, supplementation with 5 g/L shikimate enhanced the L-tyrosine titers of 45A12 and 45A13 by 29.1% and 24.0%, respectively. However, the yield of L-tyrosine per unit of shikimate decreased from 0.365 to 0.198 mol/mol after aroK overexpression in strain 45A12, which suggested that the gene product was also involved in uncharacterized pathways. This study provides a good starting point for further modification to achieve industrial-scale production of L-tyrosine using B. licheniformis, a generally recognized as safe workhorse.
L-酪氨酸是多种有价值的次生代谢物的常见前体。地衣芽孢杆菌中芳香族氨基酸的合成通过莽草酸途径进行，但涉及代谢调控的潜在机制仍不清楚。在这项工作中，通过共表达大肠杆菌的 aroG 和 tyrA ，在枯草芽孢杆菌中实现了 L-酪氨酸的积累，在控制葡萄糖补料的情况下，L-酪氨酸的产量增加到 1005mg/L。定量 RT-PCR 结果表明，修饰菌株中终产物 L-酪氨酸对编码 DAHP 合酶的 aroA 和编码莽草酸激酶的 aroK 进行了反馈抑制。因此，首先用多个拷贝表达天然的 aroK ，得到可以积累 1201mg/L L-酪氨酸的 45A13 菌株。与 45A12 菌株相比，45A13 菌株中 aroB 和 aroF 的表达分别上调了 21%和 27%，这也可能导致 L-酪氨酸产量的提高。此外，补充 5g/L 莽草酸可分别将 45A12 和 45A13 的 L-酪氨酸产量提高 29.1%和 24.0%。然而，在 45A12 菌株中过表达 aroK 后，L-酪氨酸的单位莽草酸得率从 0.365 降低到 0.198mol/mol，这表明该基因产物也参与了未明的途径。这项研究为进一步修饰提供了良好的起点，以实现使用安全的枯草芽孢杆菌进行工业规模的 L-酪氨酸生产。
