Dataset Information

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol.

ABSTRACT: Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific phaB gene from Cupriavidus necator. The heterologous pathway was optimised in three ways: at the transcriptional level choosing strongly expressed promoters and comparing plasmid borne with chromosomal gene expression, at the translational level by optimising the codon usage of the gene to fit the inherent codon adaptation index of C. saccharoperbutylacetonicum, and at the enzyme level by introducing point mutations which led to increased enzymatic activity. The resulting whole cell catalyst produced up to 20 mM (1.8 g/l) (R)-1,3-BDO in non-optimised batch fermentation which is a promising starting position for economical production of this chiral chemical.

SUBMITTER: Grosse-Honebrink A

PROVIDER: S-EPMC8155681 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol.

Grosse-Honebrink Alexander A Little Gareth T GT Bean Zak Z Heldt Dana D Cornock Ruth H M RHM Winzer Klaus K Minton Nigel P NP Green Edward E Zhang Ying Y

Frontiers in bioengineering and biotechnology 20210513

Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific pha ...[more]

PMID: 34055760

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Project description:The carboxylic acid propionate is a valuable platform chemical with applications in various fields. The biological production of this acid has become of great interest as it can be considered a sustainable alternative to petrochemical synthesis. In this work, Clostridium saccharoperbutylacetonicum was metabolically engineered to produce propionate via the acrylate pathway. In total, the established synthetic pathway comprised eight genes encoding the enzymes catalyzing the conversion of pyruvate to propionate. These included the propionate CoA-transferase, the lactoyl-CoA dehydratase, and the acryloyl-CoA reductase from Anaerotignum neopropionicum as well as a D-lactate dehydrogenase from Leuconostoc mesenteroides subsp. mesenteroides. Due to difficulties in assembling all genes on one plasmid under the control of standard promoters, the PtcdB-tcdR promoter system from Clostridium difficile was integrated into a two-plasmid system carrying the acrylate pathway genes. Several promoters were analyzed for their activity in C. saccharoperbutylacetonicum using the fluorescence-activating and absorption-shifting tag (FAST) as a fluorescent reporter to identify suitable candidates to drive tcdR expression. After selecting the lactose-inducible PbgaL promoter, engineered C. saccharoperbutylacetonicum strains produced 0.7 mM propionate upon induction of gene expression. The low productivity was suspected to be a consequence of a metabolic imbalance leading to acryloyl-CoA accumulation in the cells. To even out the proposed imbalance, the propionate-synthesis operons were rearranged, thereby increasing the propionate concentration by almost four-fold. This study is the first one to report recombinant propionate production using a clostridial host strain that has opened a new path towards bio-based propionate to be improved further in subsequent work. KEY POINTS: • Determination of promoter activities in C. saccharoperbutylacetonicum using FAST. • Implementation of propionate production in C. saccharoperbutylacetonicum. • Elevation of propionate production by 375% to a concentration of 3 mM.

Project description:Butyrate is produced by chemical synthesis based on crude oil, produced by microbial fermentation, or extracted from animal fats (M. Dwidar, J.-Y. Park, R. J. Mitchell, and B.-I. Sang, The Scientific World Journal, 2012:471417, 2012, https://doi.org/10.1100/2012/471417). Butyrate production by anaerobic bacteria is highly favorable since waste or sustainable resources can be used as the substrates. For this purpose, the native hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was used as a chassis strain due to its broad substrate spectrum. BLASTp analysis of the predicted proteome of C. saccharoperbutylacetonicum N1-4(HMT) resulted in the identification of gene products potentially involved in acetone-butanol-ethanol (ABE) fermentation. Their participation in ABE fermentation was either confirmed or disproven by the parallel production of acids or solvents and the respective transcript levels obtained by transcriptome analysis of this strain. The genes encoding phosphotransacetylase (pta) and butyraldehyde dehydrogenase (bld) were deleted to reduce acetate and alcohol formation. The genes located in the butyryl-CoA synthesis (bcs) operon encoding crotonase, butyryl-CoA dehydrogenase with electron-transferring protein subunits α and β, and 3-hydroxybutyryl-CoA dehydrogenase were overexpressed to channel the flux further towards butyrate formation. Thereby, the native hyper-butanol producer C. saccharoperbutylacetonicum N1-4(HMT) was converted into the hyper-butyrate producer C. saccharoperbutylacetonicum ΔbldΔpta [pMTL83151_BCS_PbgaL]. The transcription pattern following deletion and overexpression was characterized by a second transcriptomic study, revealing partial compensation for the deletion. Furthermore, this strain was characterized in pH-controlled fermentations with either glucose or Excello, a substrate yielded from spruce biomass. Butyrate was the main product, with maximum butyrate concentrations of 11.7 g·L-1 and 14.3 g·L-1, respectively. Minimal amounts of by-products were detected. IMPORTANCE Platform chemicals such as butyrate are usually produced chemically from crude oil, resulting in the carry-over of harmful compounds. The selective production of butyrate using sustainable resources or waste without harmful by-products can be achieved by bacteria such as clostridia. The hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was converted into a hyper-butyrate producer. Butyrate production with very small amounts of by-products was established with glucose and the sustainable lignocellulosic sugar substrate Excello extracted from spruce biomass by the biorefinery Borregaard (Sarpsborg, Norway).

Project description:Gas fermentation provides a promising platform to turn low-cost and readily available single-carbon waste gases into commodity chemicals, such as 2,3-butanediol. Clostridium autoethanogenum is usually used as a robust and flexible chassis for gas fermentation. Here, we leveraged constraint-based stoichiometric modeling and kinetic ensemble modeling of the C. autoethanogenum metabolic network to provide a systematic in silico analysis of metabolic engineering interventions for 2,3-butanediol overproduction and low carbon substrate loss in dissipated CO2. Our analysis allowed us to identify and to assess comparatively the expected performances for a wide range of single, double, and triple interventions. Our analysis managed to individuate bottleneck reactions in relevant metabolic pathways when suggesting intervening strategies. Besides recapitulating intuitive and/or previously attempted genetic modifications, our analysis neatly outlined that interventions-at least partially-impinging on by-products branching from acetyl coenzyme A (acetyl-CoA) and pyruvate (acetate, ethanol, amino acids) offer valuable alternatives to the interventions focusing directly on the specific branch from pyruvate to 2,3-butanediol. IMPORTANCE Envisioning value chains inspired by environmental sustainability and circularity in economic models is essential to counteract the alterations in the global natural carbon cycle induced by humans. Recycling carbon-based waste gas streams into chemicals by devising gas fermentation bioprocesses mediated by acetogens of the genus Clostridium is one component of the solution. Carbon monoxide originates from multiple biogenic and abiogenic sources and bears a significant environmental impact. This study aims at identifying metabolic engineering interventions for increasing 2,3-butanediol production and avoiding carbon loss in CO2 dissipation via C. autoethanogenum fermenting a substrate comprising CO and H2. 2,3-Butanediol is a valuable biochemical by-product since, due to its versatility, can be transformed quite easily into chemical compounds such as butadiene, diacetyl, acetoin, and methyl ethyl ketone. These compounds are usable as building blocks to manufacture a vast range of industrially produced chemicals.

Dataset Information

Development of Clostridium saccharoperbutylacetonicum as a Whole Cell Biocatalyst for Production of Chirally Pure (R)-1,3-Butanediol.

Publications

Development of <i>Clostridium saccharoperbutylacetonicum</i> as a Whole Cell Biocatalyst for Production of Chirally Pure (<i>R</i>)-1,3-Butanediol.

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