Pichia pastoris growth - coupled heme biosynthesis analysis using metabolic modelling

Soy legHemoglobin is one of the most important and key ingredients in plant-based meat substitutes that can imitate the colour and flavour of the meat. In order to improve the high-yield production of legHemoglobin protein and its main component - heme in the yeast Pichia pastoris, glycerol and methanol cultivation conditions were studied. Additionally, in-silico metabolic modelling analysis of growth-coupled enzyme quantity, suggests metabolic gene up/down-regulation strategies for heme production. First, cultivations and metabolic modelling analysis of P. pastoris were performed on glycerol and methanol in different growth media. Glycerol cultivation uptake and production rates can be increased by 50 % according to metabolic modelling results, but methanol cultivation - is near the theoretical maximum. Growth-coupled metabolic optimisation results revealed the best feasible upregulation (33 reactions) (1.47 % of total reactions) and 67 downregulation/deletion (2.98 % of total) reaction suggestions. Finally, we describe reaction regulation suggestions with the highest potential to increase heme production yields.