Abstract
While poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is a biodegradable commodity plastic with broad applications, its microbial synthesis is hindered by high production costs primarily associated with the supplementation of related carbon substrates (e.g. propionate or valerate). Here we report construction of engineered Escherichia coli strains for direct synthesis of P(3HB-co-3HV) from an unrelated carbon source (e.g. glucose or glycerol). First, an E. coli strain with an activated sleeping beauty mutase (Sbm) operon was used to generate propionyl-CoA as a precursor. Next, two acetyl-CoA moieties or acetyl-CoA and propionyl-CoA were condensed to form acetoacetyl-CoA and 3-ketovaleryl-CoA, respectively, by functional expression of β-ketothiolases from Cupriavidus necator (i.e. PhaA and BktB). The resulting thioester intermediates were channeled into the polyhydroxyalkanoate (PHA) biosynthetic pathway through functional expression of acetoacetyl-CoA reductase (PhaB) for thioester reduction and PHA synthase (PhaC) for subsequent polymerization. Metabolic engineering of E. coli host strains was further conducted to enhance total PHA content and the 3-hydroxyvaleryl (3HV) monomer fraction in the copolymer. Using a selection of engineered E. coli strains for batch cultivation with an unrelated carbon source, we achieved high-level P(3HB-co-3HV) production with the 3HV monomer fraction ranging from 3 to 19 mol%, demonstrating the potential industrial applicability of these whole-cell biocatalysts.
Highlights
Since the 1970s, petroleum-derived plastics have been deemed as the most used material in the world with a myriad of domestic, medical, and commercial applications[1,2]
We report direct and propionate-independent biosynthesis of P(3HB-co-3HV) using engineered Escherichia coli strains with an unrelated carbon source
We reported construction of engineered E. coli strains for heterologous production of 1-propanol[17,18] and propionate[16] by activating the inherently silent sleeping beauty mutase (Sbm) operon in the host genome
Summary
Since the 1970s, petroleum-derived (i.e. traditional) plastics have been deemed as the most used material in the world with a myriad of domestic, medical, and commercial applications[1,2]. Natural biodegradable polymers, such as polyhydroxyalkanoates (PHAs), have attracted considerable interest as promising candidates to replace petroleum-based plastics. PHAs are polyesters with various (R)-hydroxycarboxylic acids as monomers and their physical and mechanical properties are highly dependent on the monomeric composition[3,4]. The most well-characterized and naturally abundant member of PHAs is poly(3-hydroxybutyrate) (PHB)[4,7]. PHB has a limited range of applicability as an industrial plastic material since it is too brittle and stiff to be processed[5,8]. Numerous attempts have been made to develop copolymers with modulated properties (e.g. increased toughness, ductility, and impact strength as well as lower stiffness and crystallinity) by structurally incorporating longer-chain (R)-hydroxycarboxylic acid monomers into PHB. While Monsanto had several P(3HB-co-3HV)-based biodegradable products in the pipeline such as molded bottles, films, coatings, and even biochemical devices, the company
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