Pseudomonas putida is a promising bacterial host for producing natural products, such as polyketides and nonribosomal peptides. In these types of projects, researchers need a genetic toolbox consisting of plasmids, characterized promoters, and techniques for rapidly editing the genome. Past reports described constitutive promoter libraries, a suite of broad host range plasmids that replicate in P. putida, and genome-editing methods. To augment those tools, we have characterized a set of inducible promoters and discovered that IPTG-inducible promoter systems have poor dynamic range due to overexpression of the LacI repressor. By replacing the promoter driving lacI expression with weaker promoters, we increased the fold induction of an IPTG-inducible promoter in P. putida KT2440 to 80-fold. Upon discovering that gene expression from a plasmid was unpredictable when using a high-copy mutant of the BBR1 origin, we determined the copy numbers of several broad host range origins and found that plasmid copy numbers are significantly higher in P. putida KT2440 than in the synthetic biology workhorse, Escherichia coli. Lastly, we developed a λRed/Cas9 recombineering method in P. putida KT2440 using the genetic tools that we characterized. This method enabled the creation of scarless mutations without the need for performing classic two-step integration and marker removal protocols that depend on selection and counterselection genes. With the method, we generated four scarless deletions, three of which we were unable to create using a previously established genome-editing technique.
Genome-scale metabolic models have been utilized extensively in the study and engineering of the organisms they describe. Here we present the analysis of a published dataset from pooled transposon mutant fitness experiments as an approach for improving the accuracy and gene-reaction associations of a metabolic model for Zymomonas mobilis ZM4, an industrially relevant ethanologenic organism with extremely high glycolytic flux and low biomass yield. Gene essentiality predictions made by the draft model were compared to data from individual pooled mutant experiments to identify areas of the model requiring deeper validation. Subsequent experiments showed that some of the discrepancies between the model and dataset were caused by polar effects, mis-mapped barcodes, or mutants carrying both wild-type and transposon disrupted gene copies—highlighting potential limitations inherent to data from individual mutants in these high-throughput datasets. Therefore, we analyzed correlations in fitness scores across all 492 experiments in the dataset in the context of functionally related metabolic reaction modules identified within the model via flux coupling analysis. These correlations were used to identify candidate genes for a reaction in histidine biosynthesis lacking an annotated gene and highlight metabolic modules with poorly correlated gene fitness scores. Additional genes for reactions involved in biotin, ubiquinone, and pyridoxine biosynthesis in Z . mobilis were identified and confirmed using mutant complementation experiments. These discovered genes, were incorporated into the final model, i ZM4_478, which contains 747 metabolic and transport reactions (of which 612 have gene-protein-reaction associations), 478 genes, and 616 unique metabolites, making it one of the most complete models of Z . mobilis ZM4 to date. The methods of analysis that we applied here with the Z . mobilis transposon mutant dataset, could easily be utilized to improve future genome-scale metabolic reconstructions for organisms where these, or similar, high-throughput datasets are available.
Microbial lipid metabolism is an attractive route for producing aliphatic chemicals, commonly referred to as oleochemicals. The predominant metabolic engineering strategy centers on heterologous thioesterases capable of producing fatty acids of desired size. To convert acids to desired oleochemicals (e.g. fatty alcohols, ketones), metabolic engineers modify cells to block beta-oxidation, reactivate fatty acids as coenzyme-A thioesters, and redirect flux towards termination enzymes with broad substrate utilization ability. These genetic modifications narrow the substrate pool available for the termination enzyme but cost one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. In this work, we demonstrate an alternative acyl-transferase strategy for producing medium-chain oleochemicals. Through bioprospecting, mutagenesis, and metabolic engineering, we developed strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones using the transacylase strategy.
Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.
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