Multiplex Amino Acid Metabolism Engineering for Increased Production of L-Ornithine in Yeast

Recent advances in metabolic engineering and synthetic biology enable the rational engineering of microbial amino acid metabolism for efficient production of valuable amino acid-derived molecules. Several unique superiorities of Baker’s yeast Saccharomyces cerevisiae make it an alternative host for production of valuable chemicals. However, its complex regulation of metabolism and compartmentalization of specific metabolic functions imposes additional challenges for engineering its metabolism compared with bacteria. Here, we demonstrate a multiplex and systematic engineering approach to thoroughly engineer the amino acid metabolism of yeast. Our multiple engineering strategies includes, besides traditional over-expression of pathway enzymes, recruitment of subcellular trafficking and its endogenous regulation, and we demonstrate that it is hereby possible to overproduce L-ornithine, an intermediate of the arginine biosynthetic pathway. The final engineered strain could produce 1.46 g l^-1 of L-ornithine with a specific yield of 78.27 mg (g DCW)^-1 during a fed-batch fermentation, which is the highest reported production of any amino acid by S. cerevisiae. Our multiple engineering strategy demonstrate the importance of systematic pathway optimization and show how recruitment of endogenous regulation can possible be used as a generalized approach for overproducing amino acids by S. cerevisiae and possible even other eukaryotic microorganisms.