Dataset Information

Efficient production of itaconic acid from the single-carbon substrate methanol with engineered Komagataella phaffii.

ABSTRACT:

Background

Amidst the escalating carbon dioxide levels resulting from fossil fuel consumption, there is a pressing need for sustainable, bio-based alternatives to underpin future global economies. Single-carbon feedstocks, derived from CO₂, represent promising substrates for biotechnological applications. Especially, methanol is gaining prominence for bio-production of commodity chemicals.

Results

In this study, we show the potential of Komagataella phaffii as a production platform for itaconic acid using methanol as the carbon source. Successful integration of heterologous genes from Aspergillus terreus (cadA, mttA and mfsA) alongside fine-tuning of the mfsA gene expression, led to promising initial itaconic acid titers of 28 g·L^-1 after 5 days of fed-batch cultivation. Through the combined efforts of process optimization and strain engineering strategies, we further boosted the itaconic acid production reaching titers of 55 g·L^-1 after less than 5 days of methanol feed, while increasing the product yield on methanol from 0.06 g·g^-1 to 0.24 g·g^-1.

Conclusion

Our results highlight the potential of K. phaffii as a methanol-based platform organism for sustainable biochemical production.

SUBMITTER: Severinsen MM

PROVIDER: S-EPMC11251334 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Publications

Efficient production of itaconic acid from the single-carbon substrate methanol with engineered Komagataella phaffii.

Severinsen Manja Mølgaard MM Bachleitner Simone S Modenese Viola V Ata Özge Ö Mattanovich Diethard D

Biotechnology for biofuels and bioproducts 20240715 1

<h4>Background</h4>Amidst the escalating carbon dioxide levels resulting from fossil fuel consumption, there is a pressing need for sustainable, bio-based alternatives to underpin future global economies. Single-carbon feedstocks, derived from CO<sub>2</sub>, represent promising substrates for biotechnological applications. Especially, methanol is gaining prominence for bio-production of commodity chemicals.<h4>Results</h4>In this study, we show the potential of Komagataella phaffii as a product ...[more]

PMID: 39010147

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Project description:BackgroundBioconversion of methanol derived from CO2 reduction into value-added chemicals provides a unique approach for mitigating global warming and reducing fossil fuels dependence. Production of 3-hydroxypropionic acid (3-HP), 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.ResultsA combinatorial metabolic engineering strategy targeting precursors supply and 3-HP export was applied to the original 3-HP producing K. phaffii strain harboring the synthetic β-alanine pathway and a mutated NADP-dependent formate dehydrogenase from Pseudomonas sp. 101 (PseFDH(V9)). To do so, several genes encoding enzymes catalyzing reactions immediately upstream of the β-alanine pathway were overexpressed to enhance precursors availability. 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 3-HP titer and product yield in methanol deep-well plate cultures 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 the PseFDH(V9) encoding gene in these strains failed to compensate for the loss of the native FDH. The strain combining PYC2, ESBP6, and JEN1 overexpression was further tested in fed-batch cultures at pH 5, achieving a 3-HP concentration of 27.0 g l- 1, with a product yield of 0.19 g g- 1, and a volumetric productivity of 0.56 g l- 1 h- 1 for the methanol feeding phase of the cultivations. 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 overexpressing monocarboxylate transporters.ConclusionsOur 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.

Project description:BackgroundMethanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways.ResultsIn this study, production of 3-HP via the synthetic β-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus, and ydfG from Escherichia coli K-12. The expression of these key enzymes allowed a production of 1.0 g l-1 of 3-HP in small-scale cultivations using methanol as substrate. The addition of a second copy of the panD gene and selection of a weak promoter to drive expression of the ydfG gene in the PpCβ21 strain resulted in an additional increase in the final 3-HP titer (1.2 g l-1). The 3-HP-producing strains were further tested in fed-batch cultures. The best strain (PpCβ21) achieved a final 3-HP concentration of 21.4 g l-1 after 39 h of methanol feeding, a product yield of 0.15 g g-1, and a volumetric productivity of 0.48 g l-1 h-1. Further engineering of this strain aiming at increasing NADPH availability led to a 16% increase in the methanol consumption rate and 10% higher specific productivity compared to the reference strain PpCβ21.ConclusionsOur results show the potential of K. phaffii as platform cell factory to produce organic acids such as 3-HP from renewable one-carbon feedstocks, achieving the highest volumetric productivities reported so far for a 3-HP production process through the β-alanine pathway.

Project description:BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Dataset Information

Efficient production of itaconic acid from the single-carbon substrate methanol with engineered Komagataella phaffii.

Background

Results

Conclusion

Publications

Efficient production of itaconic acid from the single-carbon substrate methanol with engineered Komagataella phaffii.

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