Engineering endogenous L-proline biosynthetic pathway to boost trans-4-hydroxy-L-proline production in Escherichia coli

Non-proteinogenic trans-4-hydroxy-L-proline (t4HYP), a crucial naturally occurred amino acid, is present in most organisms. t4HYP is a regio- and stereo-selectively hydroxylated product of L-proline and a valuable building block for pharmaceutically important intermediates/ingredients synthesis. Microbial production of t4HYP has aroused extensive investigations because of its low-cost and environmentally benign features. Herein, we reported metabolic engineering of endogenous L-proline biosynthetic pathway to enhance t4HYP production in trace L-proline-producing Escherichia coli BL21(DE3) (21-S0). The genes responsible for by-product formation from L-proline, pyruvate, acetyl-CoA, and isocitrate in the biosynthetic network of 21-S0 were knocked out to channel the metabolic flux towards L-proline biosynthesis. PdhR was knocked out to remove its negative regulation and aceK was deleted to ensure isocitrate dehydrogenase's activity and to increase NADPH/NADP(+) level. The other genes for L-proline biosynthesis were enhanced by integration of strong promoters and 5'-untranslated regions. The resulting engineered E. coli strains 21-S1 similar to 21-S9 harboring a codon-optimized proline 4-hydroxy-lase-encoding gene (P4H) were grown and fermented. A titer of 4.82 g/L of t4HYP production in 21-S6 over-expressing P4H was obtained at conical flask level, comparing with the starting 21-S0 (26 mg/L). The present work paves an efficient metabolic engineering way for higher t4HYP production in E. coli.