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Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris.

ABSTRACT: Concerns over climate change have led to increased interest in renewable fuels in recent years. Microbial production of advanced fuels from renewable and readily available carbon sources has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the yeast Pichia pastoris, an industrial powerhouse in heterologous enzyme production, to produce the advanced biofuel isobutanol from sugarcane trash hydrolysates. Our strategy involved overexpressing a heterologous xylose isomerase and the endogenous xylulokinase to enable the yeast to consume both C5 and C6 sugars in biomass. To enable the yeast to produce isobutanol, we then overexpressed the endogenous amino acid biosynthetic pathway and the 2-keto acid degradation pathway. The engineered strains produced isobutanol at a titer of up to 48.2 ± 1.7 mg/L directly from a minimal medium containing sugarcane trash hydrolysates as the sole carbon source. To our knowledge, this is the first demonstration of advanced biofuel production using agricultural waste-derived hydrolysates in the yeast P. pastoris. We envision that our work will pave the way for a scalable route to this advanced biofuel and further establish P. pastoris as a versatile production platform for fuels and high-value chemicals.

SUBMITTER: Bumrungtham P

PROVIDER: S-EPMC9330720 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPolyketides are one of the most important classes of secondary metabolites and usually make good drugs. Currently, heterologous production of fungal polyketides for developing a high potential industrial application system with high production capacity and pharmaceutical feasibility was still at its infancy. Pichia pastoris is a highly successful system for the high production of a variety of heterologous proteins. In this work, we aim to develop a P. pastoris based in vivo fungal polyketide production system for first time and evaluate its feasibility for future industrial application.ResultsA recombinant P. pastoris GS115-NpgA-ATX with Aspergillus nidulans phosphopantetheinyl transferase (PPtase) gene npgA and Aspergillus terrus 6-methylsalicylic acid (6-MSA) synthase (6-MSAS) gene atX was constructed. A specific compound was isolated and identified as 6-MSA by HPLC, LC-MS and NMR. Transcription of both genes were detected. In 5-L bioreactor, the GS115-NpgA-ATX grew well and produced 6-MSA quickly until reached a high value of 2.2 g/L by methanol induction for 20 hours. Thereafter, the cells turned to death ascribing to high concentration of antimicrobial 6-MSA. The distribution of 6-MSA changed that during early and late induction phase it existed more in supernatant while during intermediate stage it mainly located intracellular. Different from 6-MSA production strain, recombinant M. purpureus pksCT expression strains for citrinin intermediate production, no matter PksCT located in cytoplasm or in peroxisomes, did not produce any specific compound. However, both npgA and pksCT transcripted effectively in cells and western blot analysis proved the expression of PPtase. Then the PPTase was expressed and purified, marked by fluorescent probes, and reacted with purified ACP domain and its mutant ACPm of PksCT. Fluoresence was only observed in ACP but not ACPm, indicating that the PPTase worked well with ACP to make it bioactive holo-ACP. Thus, some other factors may affect polyketide synthesis that include activities of the individual catalytic domains and release of the product from the synthase of PksCT.ConclusionsAn efficient P. pastoris expression system of fungal polyketides was successfully constructed. It produced a high production of 6-MSA and holds potential for future industrial application of 6-MSA and other fungal polyketides.

Project description:Background:2,3-butanediol (2,3-BD) is a bulk platform chemical with various potential applications such as aviation fuel. 2,3-BD has three optical isomers: (2R, 3R)-, (2S, 3S)- and meso-2,3-BD. Optically pure 2,3-BD is a crucial precursor for the chiral synthesis and it can also be used as anti-freeze agent due to its low freezing point. 2,3-BD has been produced in both native and non-native hosts. Several pathogenic bacteria were reported to produce 2,3-BD in mixture of its optical isomers including Klebsiella pneumoniae and Klebsiella oxytoca. Engineered hosts based on episomal plasmid expression such as Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis are not ideal for industrial fermentation due to plasmid instability. Results:Pichia pastoris is generally regarded as safe and a well-established host for high-level heterologous protein production. To produce pure (2R, 3R)-2,3-BD enantiomer, we developed a P. pastoris strain by introducing a synthetic pathway. The alsS and alsD genes from B. subtilis were codon-optimized and synthesized. The BDH1 gene from S. cerevisiae was cloned. These three pathway genes were integrated into the genome of P. pastoris and expressed under the control of GAP promoter. Production of (2R, 3R)-2,3-BD was achieved using glucose as feedstock. The optical purity of (2R, 3R)-2,3-BD was more than 99%. The titer of (2R, 3R)-2,3-BD reached 12 g/L with 40 g/L glucose as carbon source in shake flask fermentation. The fermentation conditions including pH, agitation speeds and aeration rates were optimized in batch cultivations. The highest titer of (2R, 3R)-2,3-BD achieved in fed-batch fermentation using YPD media was 45 g/L. The titer of 2,3-BD was enhanced to 74.5 g/L through statistical medium optimization. Conclusions:The potential of engineering P. pastoris into a microbial cell factory for biofuel production was evaluated in this work using (2R, 3R)-2,3-BD as an example. Engineered P. pastoris could be a promising workhorse for the production of optically pure (2R, 3R)-2,3-BD.

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Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris.

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