丙酮酸和乙酰-CoA协同促进L-亮氨酸的合成

Pyruvate works synergistically with acetyl-CoA to boost L-leucine production

摘要:

[目的] 通过理性改造柠檬酸合酶（citrate synthase，CS）、丙酮酸脱氢酶系E1p（pyruvate dehydrogenase complex，PDHC，编码基因aceE）和ATP-柠檬酸裂解酶（ATP-Citrate lyase，ACL），有效供应胞内丙酮酸和乙酰-CoA，以提高L-亮氨酸产量。[方法] 以谷氨酸棒杆菌（Corynebacterium glutamicum）为底盘细胞，分析不同CS和PDHC酶活水平对L-亮氨酸合成的影响。随后，考查协同改造CS和PDHC或引入绿硫菌（Chlorobium tepidum）中ACL对L-亮氨酸合成的影响。[结果] 低强度的CS酶活（即重组菌XL-3 P_dapA-R2gltA）有利于L-亮氨酸的合成，L-亮氨酸产量达到17.5±0.6 g/L。而改变PDHC酶活水平不利于L-亮氨酸的合成。此外，以启动子P_dapA-R2控制CS表达，而以启动子P_gapA控制PDHC表达时（即重组菌XL-4），可实现胞内丙酮酸和乙酰-CoA的有效供给，L-亮氨酸产量达到20.2±1.7 g/L，且显著降低副产物产量。若在重组菌XL-4中引入C.tepidum，ACL会显著抑制菌体生长而不利于L-亮氨酸合成，而引入到出发菌XL-3中因胞内丙酮酸和乙酰-CoA得到有效供给，目标重组菌XL-5 L-亮氨酸产量达到18.5±1.2 g/L，比出发菌株XL-3增加了14.2%。[结论] 重组菌XL-4中因协同控制CS和PDHC酶活，从而实现胞内丙酮酸和乙酰-CoA有效供给，促进L-亮氨酸的合成。该研究结果对后续利用代谢工程技术强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。

关键词:谷氨酸棒杆菌;L-亮氨酸合成;丙酮酸;乙酰-CoA;协同作用;代谢工程

Abstract:

[Objective] Pyruvate and acetyl-CoA were efficiently supplied by modifying citrate synthase (CS), pyruvate dehydrogenase complex (PDHC) or ATP-citrate lyase (ACL), thus increasing the L-leucine production in C. glutamicum.[Methods] Firstly, the effects of CS and PDHC activity on L-leucine production were studied using C. glutamicum as chassis cells, and then the effects of co-modifying CS and PDHC as well as introduction of ACL from C. tepidum on L-leucine production were investigated.[Results] The strain with low activity of CS (i.e, C. glutamicum XL-3 P_dapA-R2gltA) was beneficial to L-leucine production, and the yield of L-leucine was 17.5±0.6 g/L. However, changing the activity of PDHC is not conducive to L-leucine production. In addition, the strain with expression of CS under the weak promoter P_dapA-R2 and expression of PDHC under the medium promoter P_gapA (i.e., XL-4) produced 20.2±1.7 g/L of L-leucine. In addition, the yield of by-products in strain XL-4 was significantly decreased. Moreover, the ACL of Chlorobium tepidum was introduced into the recombinant strain XL-4, resulting in the low cell growth and L-leucine production. However, the production of L-leucine of recombinant strain with introduction of ACL in original strain XL-3 (i.e., XL-5) was significantly increased because of the efficient supply of pyruvate and acetyl-CoA. Recombinant strain XL-5 produced 18.5±1.2 g/L of L-leucine, which was 14.2% higher than that of the original strain XL-3.[Conclusion] Pyruvate and acetyl-CoA has been effectively in strain XL-4 by modifying citrate synthase (CS) and pyruvate dehydrogenase complex (PDHC), thus increasing the L-leucine production. Therefoere, the results of this study have important reference value for the following application of metabolic engineering to improve branched-chain amino acid producing strains.

Key words:Corynebacterium glutamicum;L-leucine production;pyruvate;acetyl-CoA;synergy;metabolic engineering