Metabolic engineering of Komagataella phaffii for enhanced 3-hydroxypropionic acid (3-HP) production from methanol
Year: 2025
Type: article
Abstract: Background Bioconversion of methanol derived from CO2 reduction into value-added chemicals is crucial for mitigating global warming and reducing fossil fuels dependence within a circular economy. Production of 3-hydroxypropionic (3-HP) acid, a key building block for the development of biobased products such as acrylates and 1,3-propanediol, has been successfully achieved using methanol as the sole carbon and energy source in the methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris). However, challenges remain in meeting commercially relevant concentrations, yields and productivities of 3-HP, prompting further strain optimization. In the present study, we have combined metabolic engineering strategies aiming at increasing metabolic precursors supply and redirecting carbon flux towards 3-HP production. Results A combinatorial metabolic engineering strategy targeted to increase precursor supply and 3-HP export was applied to the original 3-HP producing K. phaffii strain harboring the synthetic b-alanine pathway and a heterologous NADP-dependent formate dehydrogenase. To do so, several genes encoding for enzymes catalyzing reactions immediately upstream of the β-alanine pathway were overexpressed to enhance the pathway’s precursors supply. However, only the overexpression of the pyruvate carboxylase PYC2 gene significantly increased the 3-HP yield on biomass (YP/X) in small-scale cultivations. Co-overexpression of PYC2 and the lactate permeases ESBP6 and JEN1 genes led to a 55% improvement in titer (1.5 g l− 1) and product yield (0.13 g g− 1) compared to the reference strain, mostly due to Esbp6 activity, proving its effectiveness as a 3-HP transporter. Deletion of the native formate dehydrogenase gene FDH1 did not increase methanol flux entering the assimilatory pathway. Instead, knockout strains showed severe growth defects due to toxic intermediates accumulation. Co-expression of a gene encoding for a mutated NADP-dependent formate dehydrogenase in these strains failed to compensate for the loss of native FDH. The strain combining PYC2, ESBP6 and JEN1 overexpression was further tested in fed-batch cultures at pH 5, achieving a final 3-HP concentration of 27.0 g l− 1 in 39.3 h, with a product yield of 0.19 g g− 1 and a volumetric productivity of 0.56 g l− 1 h− 1. These results represent a 42% increase in final concentration and over 20% improvement in volumetric productivity compared to the original 3-HP producing strain. Furthermore, bioreactor-scale cultivations at pH 3.5 revealed increased robustness of the strains overproducing monocarboxylate transporters. Conclusions Our results point out the potential of lactate transporters to efficiently drive 3-HP export in K. phaffii, leading to higher titers, yields, and productivities, even at lower pH conditions.
Authors: Sílvia Àvila-Cabré, Joan Albiol, Pau Ferrer
Institution: Universitat Autònoma de Barcelona
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,890
OpenAlex ID: W4407781789