Development of a new strategy for production of medium-chain-length polyhydroxyalkanoates by recombinant Escherichia coli via inexpensive non-fatty acid feedstocks

Abstract

Pseudomonas putida KT2440 is capable of producing medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when grown on unrelated carbon sources during nutrient limitation. Transcription levels of genes putatively involved in PHA biosynthesis were assessed by quantitative real-time PCR (qRT-PCR) in P. putida grown on glycerol as a sole carbon source. The results showed that two genes, phaG and the PP0763 gene, were highly upregulated among genes potentially involved in the biosynthesis of MCL-PHAs from unrelated carbon sources. Previous studies have described phaG as a 3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA) transferase, and based on homology, the PP0763 gene was predicted to encode a medium-chain-fatty-acid CoA ligase. High expression levels of these genes during PHA production in P. putida led to the hypothesis that these two genes are involved in PHA biosynthesis from non-fatty acid carbon sources, such as glucose and glycerol. The phaG(pp) and PP0763 genes from P. putida were cloned and coexpressed with the engineered Pseudomonas sp. 61-3 PHA synthase gene phaCl (STQK)(ps) in recombinant Escherichia coli. Up to 400 mg liter(-1) MCL-PHAs was successfully produced from glucose. This study has produced the largest amount of MCL-PHAs reported from non-fatty acid carbon sources in recombinant E. coli to date and opens up the possibility of using inexpensive feedstocks to produce MCL-PHA polymers.

Fig 1

Proposed metabolic pathways for PHA biosynthesis. (A) Fatty acid biosynthesis; (B) PHA biosynthesis via fatty acid biosynthesis; (C) β-oxidation; (D) PHA biosynthesis via β-oxidation.

Fig 2

Differences in growth rates of P. putida KT2440 grown on mineral salt (MS) medium with different nitrogen concentrations. Growth curves of P. putida KT2440 grown on MS medium with either high N (0.072 g liter⁻¹), represented by squares, or low N (0.072 g liter⁻¹), represented by diamonds. Points represent the average absorbance of three independent data points (OD₅₄₀) ± standard deviation.

Fig 3

NMR spectrum of polymers isolated from E. coli LS5218 harboring the pTrcGK and pBBRSTQK plasmids grown in the presence of glucose. (A) ¹H NMR spectrum. Protons in the polymers are numbered and assigned to the peaks in the spectrum. (B) ¹³C NMR spectrum. Carbon atoms in polymers are numbered and assigned to peaks in the spectrum.