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Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli.

ABSTRACT: Strain engineering and bioprocessing strategies were applied for biobased production of porphobilinogen (PBG) using Escherichia coli as the cell factory. The non-native Shemin/C4 pathway was first implemented by heterologous expression of hemA from Rhodopseudomonas spheroids to supply carbon flux from the natural tricarboxylic acid (TCA) pathways for PBG biosynthesis via succinyl-CoA. Metabolic strategies were then applied for carbon flux direction from the TCA pathways to the C4 pathway. To promote PBG stability and accumulation, Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) was applied to repress hemC expression and, therefore, reduce carbon flowthrough toward porphyrin biosynthesis with minimal impact to cell physiology. To further enhance PBG biosynthesis and accumulation under the hemC-repressed genetic background, we further heterologously expressed native E. coli hemB. Using these engineered E. coli strains for bioreactor cultivation based on ~ 30 g L^-1 glycerol, we achieved high PBG titers up to 209 mg L^-1, representing 1.73% of the theoretical PBG yield, with improved PBG stability and accumulation. Potential biochemical, genetic, and metabolic factors limiting PBG production were systematically identified for characterization.

Supplementary information

The online version contains supplementary material available at 10.1186/s40643-021-00482-3.

SUBMITTER: Lall D

PROVIDER: S-EPMC8668860 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli.

Lall Davinder D Miscevic Dragan D Bruder Mark M Westbrook Adam A Aucoin Marc M Moo-Young Murray M Perry Chou C C

Bioresources and bioprocessing 20211213 1

Strain engineering and bioprocessing strategies were applied for biobased production of porphobilinogen (PBG) using Escherichia coli as the cell factory. The non-native Shemin/C4 pathway was first implemented by heterologous expression of hemA from Rhodopseudomonas spheroids to supply carbon flux from the natural tricarboxylic acid (TCA) pathways for PBG biosynthesis via succinyl-CoA. Metabolic strategies were then applied for carbon flux direction from the TCA pathways to t ...[more]

PMID: 34970474

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Project description:BACKGROUND: The recalcitrant nature of hemicellulosic materials and the high cost in depolymerization are the primary obstacles preventing the use of xylan as feedstock for fuel and chemical production. Consolidated bioprocessing, incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single microorganism, could potentially avoid the cost of the dedicated enzyme generation in the process of xylan utilization. In this study, we engineered Escherichia coli strains capable of exporting three hemicellulases to the broth for the succinate production directly from beechwood xylan. RESULTS: Xylanases were extracellular environment-directed by fusing with OsmY. Subsequently, twelve variant OsmY fused endoxylanase-xylosidase combinations were characterized and tested. The combination of XynC-A from Fibrobacter succinogenes S85 and XyloA from Fusarium graminearum which appeared to have optimal enzymatic properties was identified as the best choice for xylan hydrolysis (0.18 ± 0.01 g/l protein in the broth with endoxylanase activity of 12.14 ± 0.34 U/mg protein and xylosidase activity of 92 ± 3 mU/mg protein at 8 h after induction). Further improvements of hemicellulases secretion were investigated by lpp deletion, dsbA overexpression and expression level optimization. With co-expression of ?-arabinofuranosidase, the engineered E. coli could hydrolyze beechwood xylan to pentose monosaccharides. The hemicellulolytic capacity was further integrated with a succinate-producing strain to demonstrate the production of succinate directly from xylan without externally supplied hydrolases and any other organic nutrient. The resulting E. coli Z6373 was able to produce 0.37 g/g succinate from xylan anaerobically equivalent to 76% of that from xylan acid hydrolysates. CONCLUSIONS: This report represents a promising step towards the goal of hemicellulosic chemical production. This engineered E. coli expressing and secreting three hemicellulases demonstrated a considerable succinate production on the released monosaccharides from xylan. The ability to use lower-cost crude feedstock will make biological succinate production more economically attractive.

Project description:BackgroundFlavonoids are a structurally diverse group of secondary metabolites, predominantly produced by plants, which include a range of compounds with pharmacological importance. Pinocembrin is a key branch point intermediate in the biosynthesis of a wide range of flavonoid subclasses. However, replicating the biosynthesis of these structurally diverse molecules in heterologous microbial cell factories has encountered challenges, in particular the modest pinocembrin titres achieved to date. In this study, we combined genome engineering and enzyme candidate screening to significantly enhance the production of pinocembrin and its derivatives, including chrysin, pinostrobin, pinobanksin, and galangin, in Escherichia coli.ResultsBy implementing a combination of established strain engineering strategies aimed at enhancing the supply of the building blocks phenylalanine and malonyl-CoA, we constructed an E. coli chassis capable of accumulating 353 ± 19 mg/L pinocembrin from glycerol, without the need for precursor supplementation or the fatty acid biosynthesis inhibitor cerulenin. This chassis was subsequently employed for the production of chrysin, pinostrobin, pinobanksin, and galangin. Through an enzyme candidate screening process involving eight type-1 and five type-2 flavone synthases (FNS), we identified Petroselinum crispum FNSI as the top candidate, producing 82 ± 5 mg/L chrysin. Similarly, from a panel of five flavonoid 7-O-methyltransferases (7-OMT), we found pinocembrin 7-OMT from Eucalyptus nitida to yield 153 ± 10 mg/L pinostrobin. To produce pinobanksin, we screened seven enzyme candidates exhibiting flavanone 3-hydroxylase (F3H) or F3H/flavonol synthase (FLS) activity, with the bifunctional F3H/FLS enzyme from Glycine max being the top performer, achieving a pinobanksin titre of 12.6 ± 1.8 mg/L. Lastly, by utilising a combinatorial library of plasmids encoding G. max F3H and Citrus unshiu FLS, we obtained a maximum galangin titre of 18.2 ± 5.3 mg/L.ConclusionThrough the integration of microbial chassis engineering and screening of enzyme candidates, we considerably increased the production levels of microbially synthesised pinocembrin, chrysin, pinostrobin, pinobanksin, and galangin. With the introduction of additional chassis modifications geared towards improving cofactor supply and regeneration, as well as alleviating potential toxic effects of intermediates and end products, we anticipate further enhancements in the yields of these pinocembrin derivatives, potentially enabling greater diversification in microbial hosts.

Dataset Information

Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli.

Supplementary information

Publications

Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in <i>Escherichia coli</i>.

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