Metabolic engineering of Komagataella phaffii for enhanced 3-hydroxypropionic acid (3-HP) production from methanol

Journal of Biological Engineering, Journal Year: 2025, Volume and Issue: 19(1)

Published: Feb. 20, 2025

Bioconversion of methanol derived from CO2 reduction into value-added chemicals provides a unique approach for mitigating global warming and reducing fossil fuels dependence. Production 3-hydroxypropionic acid (3-HP), key building block the development biobased products such as acrylates 1,3-propanediol, has been successfully achieved using sole carbon energy source in methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris). However, challenges remain meeting commercially relevant concentrations, yields productivities 3-HP, prompting further strain optimization. In present study, we have combined metabolic engineering strategies aiming at increasing precursors supply redirecting flux towards 3-HP production. A combinatorial strategy targeting export was applied to original producing K. harboring synthetic β-alanine pathway mutated NADP-dependent formate dehydrogenase Pseudomonas sp. 101 (PseFDH(V9)). To do so, several genes encoding enzymes catalyzing reactions immediately upstream were overexpressed enhance availability. only overexpression pyruvate carboxylase PYC2 gene significantly increased yield on biomass (YP/X) small-scale cultivations. Co-overexpression lactate permeases ESBP6 JEN1 led 55% improvement titer product deep-well plate cultures compared reference strain, mostly due Esbp6 activity, proving its effectiveness transporter. Deletion native FDH1 did not increase entering assimilatory pathway. Instead, knockout strains showed severe growth defects toxic intermediates accumulation. Co-expression PseFDH(V9) these failed compensate loss FDH. The combining PYC2, ESBP6, tested fed-batch pH 5, achieving concentration 27.0 g l- 1, with 0.19 g- volumetric productivity 0.56 1 h- feeding phase These results represent 42% final over 20% 3-HP-producing strain. Furthermore, bioreactor-scale cultivations 3.5 revealed robustness overexpressing monocarboxylate transporters. Our point out potential transporters efficiently drive phaffii, leading higher titers, yields, productivities, even lower conditions.

Language: Английский

Acetate metabolism during xylose fermentation enhances 3-hydroxypropionic acid production in engineered acid-tolerantIssatchenkia orientalis

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: April 17, 2025

Abstract Efficient bioconversion of acetate-rich lignocellulosic biomass into value-added chemicals remains a major challenge due to the toxicity acetic acid. In this study, we engineered an acid-tolerant Issatchenkia orientalis strain (IoDY01H) capable producing 3-hydroxypropionic acid (3-HP), key bioplastic precursor, from glucose, xylose, and acetate. Using Cas9-based genome editing system with hygromycin B resistance marker, introduced heterologous genes encoding xylose utilization β-alanine-based 3-HP biosynthetic pathways I. genome. Metabolomic analysis revealed that acetate supplementation redirected metabolic flux toward amino lipid metabolism while reducing TCA cycle intermediates. Acetate enhanced production by promoting accumulation β-alanine, but also β-alanine–pyruvate aminotransferase as bottleneck under acidic conditions. pretreated hemp stalk hydrolysate feedstock, achieved titer 8.7 g/L via separate hydrolysis fermentation (SHF), outperforming simultaneous saccharification (SSF). These findings demonstrate feasibility using non-conventional yeast highlight promising microbial chassis for industrial bioconversion. Graphical abstract Highlights Engineered co-utilized produce 3-HP. addition during fermentation. biosynthesis pathways. SHF.

Language: Английский

Transforming non-conventional yeasts into key players in biotechnology: advances in synthetic biology applications

Frontiers in Microbiology, Journal Year: 2025, Volume and Issue: 16

Published: May 2, 2025

Non-conventional yeasts exhibit exceptional genetic and functional diversity, serving as a largely untapped repertoire for biotechnological applications. Beyond the conventional yeast Saccharomyces cerevisiae , non-conventional are naturally more multifaceted, possessing ability to utilize renewable low-cost carbon sources while exhibiting robust physiology under challenging conditions. However, their vast potential remains unexplored, encompassing both challenges opportunities advancements. Over past decade, technological advancements in synthetic biology have unlocked new harness overcome inherent limitations, enabling full exploitation of advantages across broad spectrum In this review, we highlight recent advances yeasts, focusing on development building blocks (e.g., promoters terminators), genome editing tools, metabolic pathway engineering. Through these technologies, poised emerge pivotal next-generation workhorses tailored specific applications sustainable biomanufacturing, accelerating transition bio-based economy.

Language: Английский