A metabolic-engineering framework approach via fed-batch fermentation for enhancing glucaric acid production in Komagataella phaffii.

Glucaric acid (D-saccharic acid) is an organic compound belonging to glucuronic acid derivatives, whose commercial synthesis involves the use of hazardous solvents. Biosynthetic production in Saccharomyces cerevisiae has limitations, such as ethanolic fermentation, redox strategy limitations, and low pH toxicity. Komagataella phaffii (K. phaffii) formly known Pichia pastoris, an alternative and robust engineerable organism, is a promising biotransformation agent for glucaric acid production. However, K. phaffii lacks native biosynthetic pathways for glucaric acid synthesis at the industrial scale. There is no proof-of-concept glucaric acid production system. Therefore, gene expression profiling-based metabolic engineering of glucaric acid producing gene cassette was performed using in-fusion cloning. Product production was enhanced using fed-batch fermentation of the key metabolite, myo-inositol; this improved the yield of glucaric acid. The expression was optimized through cofactor recycling and codon optimization for the UDH gene. Fed-batch fermentation with mixed supplementation (Myo-inositol + Monosodium glutamate) as substrate in engineered K. phaffii (X33-GA) enhanced glucaric acid synthesis to 17.6 g/L. In addition, we present simple HPLC and LC-MS techniques for quantifying glucaric acid and its precursors in the fermentation samples. The proof-of-concept results from both shake flask and bioreactor studies provide a unique perspective on sustainable, cost-effective, and green technological alternatives for glucaric acid synthesis.