One of the optimization strategies of an artificial biosynthetic metabolic flux with a multienzyme pathway is when the enzyme concentrations are present at the appropriate ratios rather than at their maximum expression. Thus, many recent research efforts have focused on the development of tools that fine-tune the enzyme expression, and these research efforts have facilitated the search for the optimum balance between pathway expression and cell viability. However, the rational approach has some limitations in finding the most optimized expression ratio in in vivo systems. In our study, we focused on fine-tuning the expression level of a six-enzyme reaction for the artificial biosynthesis of curcumin by screening a library of 5′-untranslational region (UTR) sequence mutants made by a multiplex automatic genome engineering (MAGE) tool. From the screening results, a variant (6M08rv) showed about a 38.2-fold improvement in the production of curcumin compared to the parent strain, in which the calculated expression levels of 4-coumarate:CoA ligase (4CL) and phenyldiketide-CoA synthase (DCS), two of the six enzymes, were much lower than those of the parent strain.
Bibliographical noteFunding Information:
This work was supported in part by the KRIBB Research Initiative Program and by the Next-Generation BioGreen 21 Program (SSAC, PJ001108401) funded by the RDA, Republic of Korea. We would like to express our gratitude to Prof. S.K. Lee (UNIST) for kindly providing us with pRED1 vector. We thank to Mr. B.S. Lee (KRIBB) for his help with processing LC−MS/ MS data.
- artificial biosynthesis
- multienzyme pathway
- multiplex automatic genome engineering