Benchmarking recombinant Pichia pastoris for 3‐hydroxypropionic acid production from glycerol

Summary The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3‐hydroxypropionic acid (3‐HP) from glycerol. 3‐HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum‐based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3‐HP from glycerol through the malonyl‐CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14‐fold increase in the production of 3‐HP compared to the initial strain. The best strain (PpHP6) was tested in a fed‐batch culture, achieving a final concentration of 3‐HP of 24.75 g l−1, a product yield of 0.13 g g−1 and a volumetric productivity of 0.54 g l−1 h−1, which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3‐HP. Production of 3‐HP through the Malonyl‐CoA pathway was achieved in Pichia pastoris. Expressing the two subunits of the Malonyl‐CoA reductase separately led to a substantial increase in 3‐HP production. Further metabolic engineering to increase precursors availability increased the 3‐HP titre, achieving a final product concentration of 24.75 g l−1 in fed‐batch cultures, and the highest 3‐HP volumetric productivity value reported for yeast to date (0.54 g l−1 h−1).