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

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.