Engineering Pseudomonas putida for production of 3-hydroxyacids using hybrid type I polyketide synthases

Abstract

Engineered type I polyketide synthases (T1PKSs) are a potentially transformative platform for the biosynthesis of small molecules. Due to their modular nature, T1PKSs can be rationally designed to produce a wide range of bulk or specialty chemicals. While heterologous PKS expression is best studied in microbes of the genus Streptomyces, recent studies have focused on the exploration of non-native PKS hosts. The biotechnological production of chemicals in fast growing and industrial relevant hosts has numerous economic and logistic advantages. With its native ability to utilize alternative feedstocks, Pseudomonas putida has emerged as a promising workhorse for the sustainable production of small molecules. Here, we outline the assessment of P. putida as a host for the expression of engineered T1PKSs and production of 3-hydroxyacids. After establishing the functional expression of an engineered T1PKS, we successfully expanded and increased the pool of available acyl-CoAs needed for the synthesis of polyketides using transposon sequencing and protein degradation tagging. This work demonstrates the potential of T1PKSs in P. putida as a production platform for the sustainable biosynthesis of unnatural polyketides.

Keywords: 3-Hydroxyacid production; Polyketide synthase engineering; Protease degradation tag; Pseudomonas putida; Transposon sequencing.

Fig. 1

Engineered lipomycin polyketide synthase (LipPKS) expression in Pseudomonas putida. (a) The blue-pigment synthase A (BpsA) assay (blue) in P. putida with and without the addition of sfp. (b) 3-Hydroxy-2,4-dimethylpentanoic acid (3H24DMPA) production in engineered strains (orange). (c) General design of the engineered LipPKS pathway and its required precursors. The tested methylmalonyl-CoA (mmCoA) pathways comprised of either the mmCoA mutase and epimerase (MCM/EPI) from Sorangium cellulosum or the propionyl-CoA carboxylase (PCCase) complex from Streptomyces coelicolor. The preferred loading substrates are located in P. putida's native branched-chain amino acid (BCAA) catabolism. The polyketide product is an enantiopure 3-hydroxyacid, which varies based on the substrate loaded. AT: acyl transferase; ACP: acyl carrier protein; KS: keto synthase; KR: keto reductase; TE: thioesterase; ibCoA: isobutyryl-CoA; 2mbCoA: 2-methylbutyryl-CoA; ivCoA: isovaleryl-CoA; 3H4MPA: 3-hydroxy-4-methylpentanoic acid; 3H4MHA: 3-hydroxy-4-methylhexanoic acid; (∗∗∗∗) = p < 0.0001; NS: non-significant. Error bars represent the standard deviation of n = 3.

Fig. 2

The branched-chain amino acid (BCAA) metabolism in Pseudomonas putida. (a) BarSeq analysis of the BCAA degradation pathway in P. putida. Fitness defects were considered significant when the fitness value was > |1| and the t-value was >5. Shown are the fitness values (n = 2) associated with growth on L-leucine (green), L-valine (red) and L-isoleucine (blue) as the sole source of carbon. Fitness values marked with an asterisk (∗) represent averages across multiple genes. (b) Genomic arrangement of PP_5452 and PP_0642 operon with heatmap of the associated fitness values (n = 2) for L-valine carbon source (C), 3-methyl-2-oxobutanoic acid (C) and L-valine nitrogen source (N) conditions.

Fig. 3

Protein degradation tags as a tool to control malonyl-CoA (mCoA) levels in Pseudomonas putida. (a) Engineered ssrA tags and their corresponding degradation rates in P. putida. (b) Flaviolin production by the mCoA reporter RppA in modified P. putida strains with ssrA-tagged malonyl-CoA:ACP transacylase (FabD). WT: Wild-type; LB: Luria-Bertani medium; TB: Terrific Broth; mM9: modified M9 medium. Error bars represent the standard deviation of n = 3.

Fig. 4

Optimization of polyketide titers in Pseudomonas putida. (a) Media optimization for the polyketides 3-hydroxy-4-methylpentanoic acid (3H4MPA) (left) and 3-hydroxy-4-methylhexanoic acid (3H4MHA) (right). (b) Polyketide production in combinatorial library of strains deficient for acyl-CoA dehydrogenases (ACDHs) PP_2216, PP_3492, or PP_4064. In addition, a deletion of the potential 3-methyl-2-oxobutanoic acid transporter (PP_0642) was tested in strain ΔPP_2216ΔPP_3492ΔPP_4064. LB: Luria-Bertani medium; mM9: modified M9 medium; WT: wild-type. Error bars represent the standard deviation of n = 3.