Metabolic analyses of Yarrowia lipolytica for biopolymer production reveals roadblocks and strategies for microbial utilizing volatile fatty acids as sustainable feedstocks

Abstract

This study quantifies metabolic features of engineered Yarrowia lipolytica strains for converting volatile fatty acids (VFAs) into poly-3-hydroxybutyrate (PHB) via ¹³C-metabolic flux analysis and RNA-Seq. Yarrowia lipolytica is unable to grow with C4 ∼ C6 VFAs due to substrate toxicity, while propionate (C3) metabolism leads to slow growth and minimal PHB production due to enzymatic limitations in substrate assimilation pathways. Acetate is a viable but challenging VFA feedstock. Comparing to glucose, acetate catabolism results in low ATP/ADP ratios, high enzyme usage, substantial CO₂ release (>50 % of input carbon), and limited NADPH. Several strategies may overcome these roadblocks: 1) glucose-VFA co-catabolism improves energy charge, alleviates metabolic imbalances, reduces flux rigidity, and lowers the enzyme expression burden; 2) overexpressing acetyl-CoA synthetase and nitrogen limitation increase acetate uptake and PHB synthesis during glucose-acetate co-utilization; and 3) repression of oxidase facilitates fluxes towards PHB synthesis. The results provide insights into efficient utilization of acetate as feedstock.

Keywords: ATP; Co-catabolism; Metabolic flux analysis; PHB; VFA.