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

Abstract

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.

Fig. 1

Simplified representation of the conversion of glycerol to 3‐HP through the malonyl‐CoA route. The metabolic engineering targets to increase the availability of the precursors of the malonyl‐CoA to 3‐HP pathway are included. Acc, acetyl‐CoA carboxylase; cPos5, cytosolic NADH kinase; MCR (C‐ter), C‐terminal domain of malonyl‐CoA reductase; MCR (N‐ter), N‐terminal domain of malonyl‐CoA reductase.

Fig. 2

Production of 3‐HP for each strain in the screening experiments. The genes heterologously expressed in each strain are depicted in the left side of the graph. The grey bars show the average 3‐HP concentration at the end of the culture, the discontinuous line shows the standard deviation, the solid line indicates the SE and the circles show the average result for each clone. The solid circle of a PpHP5 clone shows the result of a clone which was discarded for the calculations, as it had a different behaviour from the rest of the clones of that strain. For PpHP6, only 3 clones could be screened. Despite that more than 50 transformants of PpHP6 from 2 independent transformations were checked using colony PCR, only 3 clones resulted positive.

Fig. 3

Fed‐batch culture of PpHP6. A. Biomass and metabolites concentration during cultivation and total glycerol added (per litre) into the reactor is shown. It was calculated considering the volume of feeding added to the reactor and the actual culture volume. Error bars denote SE. B. Growth rate (µ), product yield (Yield_3‐HP/Glyc), q‐rate of 3‐HP (qP 3‐HP), and q‐rate of arabitol (qP Arabitol) of the strain PpHP6 throughout the feeding phase of the fed‐batch culture at a pre‐set µ of 0.1 h⁻¹. Error bars show the SE.