| Lactoyl-CoA is an important intermediate in the metabolism of intestinal microorganism in some herbivores,which is mainly from the nonrandomizing pathway of lactate reduction.The lactate-based copolymers are widely used in the production of biodegradable plastics and biomedicine,which is important for environment protection and biomedicine production.The conversion of lactate to lactoyl-CoA catalyzed by acetyl-CoA:lactate CoA-transferase（ALCT）is the key step in the lactate-based copolymers production with biosysthesis.However,there are few alternatives for ALCTs.To exploit novel and efficient lactoyl-CoA transferases,the amino acid sequence similarity was analysised by BLAST with the well-known CoA-transferases from Clostridium propionicum and Megasphaera elsdenii as templates.Three candidate CoA-transferases were screened,which are derived from Megasphaera sp.DISK 18,Clostridium lactatifermentans An7 and Firmicutes bacteria CAG:466,respectively.Sequence analysis and phylogenetic tree analysis were performed.They were found to be consistent with the sequence characteristics of class I CoA-transferases,and the phylogenetic tree branches were distributed as expected.The genes of three candidates and two known ALCTs were integrated into the p ET28a.Then,the enzyme activity for converting lactate and acetyl-CoA into lactoyl-CoA and acetate were indentified.Then,the optimal reaction conditions,stability and kinetic properties of the five ALCTs for both D-lactate and acetyl-CoA were systematically investigated.The catalytic efficiency of ALCTs were then characterized in vivo.Among the five ALCTs,the propionyl-CoA from Megasphaera elsdenii（ME-PCT）exhibited the highest catalytic efficiency for both acetyl-CoA(264.22 s-1 m M-1)and D-lactate(84.18 s-1 m M-1)and showed a broad thermal adaptability and good stability.Moreover,it also showed the highest catalytic efficiency in vivo.We initially studied on ALCT,which can provide novel and efficient enzyme candidates for lactoyl-CoA generation.The ME-PCT with the best enzymatic properties will surely provide a huge driving force for the synthesis of products with lactoyl-CoA as an important intermediate.It can also offer valuable new tools for the engineering of pathways that rely on these enzymes. |
