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

【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-R2)gltA)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。

[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-R2)gltA)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.