Engineering Komagataella phaffii for citric acid production through carbon-conserving supply of acetyl-CoA

The oxidative formation of AcCoA limits the glycolytic pathway yield (Y^P_GLY) for citric acid due to the NADH overflow and carbon loss as CO₂. An interesting approach to enhance product yields is the incorporation of carbon-conserving pathways. This study assesses the potential of a carbon-conserving AcCoA pathway, the glycolysis alternative high carbon yield cycle (GATHCYC), to improve citric acid production, utilizing the non-native citric acid producer Komagataella phaffii as an orthogonal test system. The combination of different metabolic engineering strategies enabled K. phaffii to acquire the ability to produce extracellular citric acid. By constructing the GATHCYC in the cytosol and peroxisomes, the intracellular concentration of AcCoA increased. Overexpression of the genes encoding pyruvate carboxylase (PYC2), citrate synthase (CIT2) and citrate exporter protein (cexA) in the peroxisomal AcCoA strains boosted the citric acid production. Thus, the best producer strain reached a citric acid titer of 51.3 ± 0.9 g L^-1 and a yield of 0.59 ± 0.01 g g^-1 after 76 h of glucose-limited fed-batch cultivation. Our results highlight the potential of using GATHCYC to provide an efficient supply of acetyl-CoA to enhance citric acid production. This approach could be exploited for the production of other AcCoA-derived compounds of industrial relevance in different cell factories.