Dataset Information

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida.

ABSTRACT: Pseudomonas putida is a saprophytic bacterium with robust metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic catabolism, confirmed known pathways, and proposed new annotations for two aromatic transporters. The 1,3,6,8-tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA hydratase-lyase (Ech), resulted in increased production of the type III polyketide bisdemethoxycurcumin.

SUBMITTER: Incha MR

PROVIDER: S-EPMC7136493 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Publications

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida.

Incha Matthew R MR Thompson Mitchell G MG Blake-Hedges Jacquelyn M JM Liu Yuzhong Y Pearson Allison N AN Schmidt Matthias M Gin Jennifer W JW Petzold Christopher J CJ Deutschbauer Adam M AM Keasling Jay D JD

Metabolic engineering communications 20191217

Pseudomonas putida is a saprophytic bacterium with robust metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PK ...[more]

PMID: 32280587

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Project description:Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.

Dataset Information

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida.

Publications

Leveraging host metabolism for bisdemethoxycurcumin production in <i>Pseudomonas putida</i>.

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