Acetyl-DL-proline

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.

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 ~ 21-S9 harboring a codon-optimized proline 4-hydroxylase-encoding gene (P4H) were grown and fermented. A titer of 4.82 g/L of t4HYP production in 21-S6 overexpressing 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.