Benchmarking recombinant Pichiapastoris 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 3hydroxypropionic acid (3-HP) from glycerol. 3-HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleumbased 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 À1 , a product yield of 0.13 g g À1 and a volumetric productivity of 0.54 g l À1 h À1 , 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.

Combining Metabolic Engineering and Multiplexed Screening Methods for 3-Hydroxypropionic Acid Production in Pichia pastoris

“…However, this strategy resulted in rather modest improvements in terms of product yield, pointing to the delivery of malonyl-CoA as a major bottleneck to further improve 3-HP production. Notably, this strain produced up to 24.75 g L −1 of 3-HP in 45.5 h in a fed-batch culture using glycerol as a carbon source, which is, to our knowledge, the highest productivity reported so far in yeast (0.54 g L −1 h −1 ) (Fina et al, 2021).…”

“…In a recent study, we addressed the use of P. pastoris to produce 3-HP from glycerol (Fina et al, 2021). This yeast was metabolically engineered to express the malonyl-CoA reductase pathway, which consists of two consecutive steps reducing malonyl-CoA into 3-HP, while consuming two NADPH molecules (Figure 1).…”

Heterologous Production of Acrylic Acid: Current Challenges and Perspectives

“…However, even when expressing multiple copies of these genes in the genome, the concentrations obtained in S. cerevisiae and S. pombe did not surpass 9.8 g/L [92], which is significantly lower than that reached in E. coli. Notwithstanding, recently, 3-HP production in P. pastoris reached 24.75 g/L from glycerol through the overexpression of MCR, ACC and cPOS5, a cytosolic NADH kinase used to increase NADPH availability [95]. This concentration is still lower than the one obtained in E. coli, but P. pastoris is able to efficiently use crude glycerol without purification as a substrate [97], which can also be highly advantageous.…”

“…The first step is rate-limiting [91]. So far, 3-HP heterologous production using the malonyl-CoA route was reported least in E. coli, S. cerevisiae, Pichia pastoris, Schizosaccharomyces pombe and Synechococcus el gatus [42,86,[92][93][94][95]. Rathnasingh et al [86] were the first to report 3-HP production in coli using this route (Table 2).…”

“…Rathnasingh et al [86] were the first to report 3-HP production in coli using this route (Table 2). In addition to overexpressing MCR from C. aurantiacus, th overexpressed ACC (accADBC) and BirA (biotinilase) genes to increase malonyl-C availability and pyridine nucleotide transhydrogenase subunits α and β (PntAB from So far, 3-HP heterologous production using the malonyl-CoA route was reported at least in E. coli, S. cerevisiae, Pichia pastoris, Schizosaccharomyces pombe and Synechococcus elongatus [42,86,[92][93][94][95]. Rathnasingh et al [86] were the first to report 3-HP production in E. coli using this route (Table 2).…”