Zhang Chuanzhi, Zhang Junli, Kang Zhen, Du Guocheng, Chen Jian
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
J Ind Microbiol Biotechnol. 2015 May;42(5):787-97. doi: 10.1007/s10295-015-1593-x. Epub 2015 Feb 10.
Microbial production of L-phenylalanine (L-Phe) from renewable sources has attracted much attention recently. In the present study, Corynebacterium glutamicum 13032 was rationally engineered to produce L-Phe from inexpensive glucose. First, all the L-Phe biosynthesis pathway genes were investigated and the results demonstrated that in addition to AroF and PheA, the native PpsA, TktA, AroE and AroA, and the heterologous AroL and TyrB were also the key enzymes for L-Phe biosynthesis. Through combinational expression of these key enzymes, the L-Phe production was increased to 6.33 ± 0.13 g l(-1) which was about 1.48-fold of that of the parent strain C. glutamicum (pXM-pheA (fbr)-aroF (fbr)) (fbr, feedback-inhibition resistance). Furthermore, the production of L-Phe was improved to 9.14 ± 0.21 g l(-1) by modifying the glucose and L-Phe transport systems and blocking the acetate and lactate biosynthesis pathways. Eventually, the titer of L-Phe was enhanced to 15.76 ± 0.23 g l(-1) with a fed-batch fermentation strategy. To the best of our knowledge, this was the highest value reported in rationally engineered C. glutamicum 13032 strains. The results obtained will also contribute to rational engineering of C. glutamicum for production of other valuable aromatic compounds.
近年来，利用可再生资源通过微生物生产L-苯丙氨酸（L-Phe）备受关注。在本研究中，对谷氨酸棒杆菌13032进行了合理改造，使其能从廉价的葡萄糖生产L-Phe。首先，对所有L-Phe生物合成途径基因进行了研究，结果表明，除了AroF和PheA外，天然的PpsA、TktA、AroE和AroA，以及异源的AroL和TyrB也是L-Phe生物合成的关键酶。通过这些关键酶的组合表达，L-Phe产量提高到6.33±0.13 g l(-1)，约为亲本菌株谷氨酸棒杆菌（pXM-pheA (fbr)-aroF (fbr)）（fbr，反馈抑制抗性）的1.48倍。此外，通过改造葡萄糖和L-Phe转运系统并阻断乙酸盐和乳酸盐生物合成途径，L-Phe产量提高到9.14±0.21 g l(-1)。最终，采用分批补料发酵策略，L-Phe的滴度提高到15.76±0.23 g l(-1)。据我们所知，这是在经过合理改造的谷氨酸棒杆菌13032菌株中报道的最高值。所获得的结果也将有助于对谷氨酸棒杆菌进行合理改造，以生产其他有价值的芳香族化合物。
