Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A

Abstract

Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) comprise biosynthetic pathways that provide access to diverse, often bioactive natural products. Metabolic engineering can improve production metrics to support characterization and drug-development studies, but often native hosts are difficult to genetically manipulate and/or culture. For this reason, heterologous expression is a common strategy for natural product discovery and characterization. Many bacteria have been developed to express heterologous biosynthetic gene clusters (BGCs) for producing polyketides and nonribosomal peptides. In this article, we describe tools for using Pseudomonas putida, a Gram-negative soil bacterium, as a heterologous host for producing natural products. Pseudomonads are known to produce many natural products, but P. putida production titers have been inconsistent in the literature and often low compared to other hosts. In recent years, synthetic biology tools for engineering P. putida have greatly improved, but their application towards production of natural products is limited. To demonstrate the potential of P. putida as a heterologous host, we introduced BGCs encoding the synthesis of prodigiosin and glidobactin A, two bioactive natural products synthesized from a combination of PKS and NRPS enzymology. Engineered strains exhibited robust production of both compounds after a single chromosomal integration of the corresponding BGC. Next, we took advantage of a set of genome-editing tools to increase titers by modifying transcription and translation of the BGCs and increasing the availability of auxiliary proteins required for PKS and NRPS activity. Lastly, we discovered genetic modifications to P. putida that affect natural product synthesis, including a strategy for removing a carbon sink that improves product titers. These efforts resulted in production strains capable of producing 1.1 g/L prodigiosin and 470 mg/L glidobactin A.

Keywords: Genome editing; Heterologous expression; Non-ribosomal peptide; Polyketide; Pseudomonas putida.

Fig. 1.. Establishing a prodigiosin production strain.

(a) Map of prodigiosin BGC from S. marcescens ATCC274. Colored arrows indicate the enzyme class according to the legend. (b) Prodigiosin biosynthesis begins with intermediates in fatty acid metabolism and L-proline. 2-octenoyl-CoA/ACP is converted to MAP by PigDEB. L-proline, serine, and malonyl-CoA are converted to MBC by PigIAJHMFN. The two intermediates are then fused by PigC to produce prodigiosin which generates a visible red color (inset) in culture. (c) Integration of BGCs into the chromosome of P. putida. A pNVLTv2 integration vector is introduced via conjugation into a strain of P. putida containing pCas9. The integration vector carries out a single-crossover recombination with the P. putida chromosome. The Cas9 counterselection is then enabled after electroporating pgRNA targeting the wild-type sequence to be replaced by the BGC. This selects for the double-crossover recombination of the integration vector and results in a markerless and stable integration of the BGC. (d) Prodigiosin production from cultures grown in glucose or glycerol-based media under control of P_rha or P_trc promoters. All cultures were supplied with dodecane as a product-sink. Error bars represent standard deviation, n = 3 biological replicates. Differences in values between all samples were found to be statistically significant (P<0.01) by the Student’s t-test.

Fig. 2.. Tn5 libraries highlight disruptions in electron transport chain.

(a) Generalized workflow for generating and screening mutant libraries for improved prodigiosin production. (b) Components of electron transport chain, highlighting role of the bo₃ oxidase, Cyo. UQ – ubiquinones, UQH₂ – ubiquinols, CIO – cyanide insensitive oxidase. Adapted from Ugidos et al. and Nikel et al. (c) Effect of deleting cyo operon on prodigiosin production on glucose and glycerol. Error bars represent standard deviation, n = 3 biological replicates. Differences between samples marked with asterisks were found to be statistically significant by the Student’s t-test (** = P<0.01).

Fig. 3.. Identifying glidobactin A as primary product from expression of glb and lum clusters.

(a) Gene maps of glidobactin BGCs from S. brevitalea DSM7029 and P. luminescens TT01. Colored arrows indicate the enzyme class according to the legend. The structure of glidobactin A is shown to the right. (b) Unique peak identified in HPLC analysis of extracts from glidobactin production strains. (c) LC-MS shows that the major peak has m/z value corresponding to glidobactin A + H⁺ (calculated m/z = 521.3334). (d) Effects of promoter and RBS strength on glidobactin production. All strains were grown in 25 mL of glycerol-based media in 250-mL non-baffled shake flasks. SD=Shine-Dalgarno sequence. Error bars represent standard deviation, n = 3 biological replicates. Differences in values between all samples were found to be statistically significant (P<0.01) by the Student’s t-test.

Fig. 4.. Improving functional expression of PKS/NRPS enzymes in glidobactin pathway.

(a) Strategies for overexpressing MLPs and PPTases in production strains. The MLP gene, glbE, was integrated in place of PP_3808, the gene encoding P. putida’s native MLP, along with a constitutive promoter from the Anderson promoter library. A constitutive promoter from the Anderson library was integrated upstream of PP_1183, the gene encoding P. putida’s native PPTase. The inducible promoter, P_tac, and sfp were integrated in place of pvdL. (b) Cas9-assisted ssDNA oligo recombination scheme for introducing ATG start codons in glbC/plu1880. To select for start codon mutations, an sgRNA targets the PAM sequence immediately downstream of the start codon. A mutagenic oligo contains mutations in the PAM and the start codon, and Cas9 activity selects against cells that don’t incorporate mutations from this oligo. PAM=protospacer adjacent motif. (c) and (d) Effects of MLP overexpression, PPtase overexpression, and start codon mutagenesis on glidobactin production strains. Charts are labeled and split into sections to emphasize genetic changes made (e.g. “PPTase” samples have modifications to PPTase expression). All cultures were in 25-mL of glycerol-based media in 250-mL non-baffled flasks. Error bars represent standard devitaion, n ≥ 3 biological replicates. Differences between samples marked with asterisks were found to be statistically significant by the Student’s t-test (n.s. = not significant, * = P<0.05, ** = P<0.01, *** = P<0.001).

Fig. 5.. Identification of fatty acyl precursors as a metabolic engineering target.

(a) Supplementing various precursors in minimal media (RK 2.5% glycerol) affects prodigiosin production. Cultures were grown in 3 mL media without dodecane overlay. (b) Metabolites from fatty acid metabolism that are incorporated into PHA, prodigiosin, and glidobactin A biosynthesis. P. putida incorporates mostly C8-C12 3-hydroxyalkanoates into PHAs. 2-octenoyl-ACP or CoA is incorporated into prodigiosin, and dodecanoyl-CoA or 2-dodecenoyl-CoA is incorporated into glidobactin A. (c) PHA composition from P. putida strains with and without ΔglpR and ΔphaC1ZC2 genotypes. Cultures were grown in RK media with glycerol and extractions were completed at 24h of growth. (d) Prodigiosin production from P. putida strains with and without ΔglpR and ΔphaC1ZC2 genotypes. Cultures were grown in 25 mL RK glycerol with dodecane overlay for 48h. (e) Effect of ΔglpR ΔphaC1ZC2 genotype on glidobactin A production. Cultures were grown in 25 mL RK glycerol for 48h. Error bars represent standard deviation, n = 3 biological replicates. Differences between samples marked with asterisks were found to be statistically significant by the Student’s t-test (n.s. = not significant, * = P<0.05, ** = P<0.01, *** = P<0.001).