Engineering in Vivo Production of α-Branched Polyesters

Abstract

Polymers are an important class of materials that are used for a broad range of applications, from drug delivery to packaging. Given their widespread use, a major challenge in this area is the development of technology for their production from renewable sources and efforts to promote their efficient recycling and biodegradation. In this regard, the synthesis of polyesters based on the natural polyhydroxy­alkanoate (PHA) pathway offers an attractive route for producing sustainable polymers. However, monomer diversity in naturally occurring polyesters can be limited with respect to the design of polymers with material properties suitable for various applications. In this work, we have engineered a pathway to produce α-methyl-branched PHA. In the course of this work, we have also identified a PHA polymerase (CapPhaEC) from activated sludge from wastewater treatment that demonstrates a higher capacity for incorporation of α-branched monomer units than those previously identified or engineered. Production in Escherichia coli allows the construction of microbial strains that produce the copolyesters with 21–36% branched monomers using glucose and propionate as carbon sources. These polymers have typical weight-average molar masses (M w) in the range (1.7–2.0) × 105 g mol–1 and display no observable melting transition, only relatively low glass transition temperatures from −13 to −20 °C. The lack of a melting transition indicates that these polymers are amorphous materials with no crystallinity, which is in contrast to the natural poly­(hydroxybutyrate) homopolymer. Our results expand the utility of PHA-based pathways and provide biosynthetic access to α-branched polyesters to enrich the properties of bio-based sustainable polymers.

Reverse β-oxidation pathways for efficient chemical production

“…One example of using rBOX products as building blocks is the synthesis of bioderived polymers such as PHAs. Dong et al ( 2019 ) used acetyl-CoA and propionyl-CoA to produce (3 R )-hydroxyacyl-CoA which can be copolymerized by a PHA polymerase to ultimately form α-branched polyesters (Dong et al, 2019 ). In this study, the substrate selectivity of thiolase (AsAcat3) and HACD (RePhaB) plays a key role in controlling monomer availability for incorporation into the final copolymer by the PHA polymerase.…”

Structural Basis for Branched Substrate Selectivity in a Ketoreductase from Ascaris suum

“…Recognition of the acyl group is similar between the AsHadh2 and RePhaB 23 with the thioester carbonyl making a hydrogen bond to Q164, which had been identified to be important for substrate recognition in biochemical studies 14 (Figure 4). The C3 keto group interacts with two residues, S154 and Y167, of the conserved SYK motif, which places them in the correct position for their proposed roles in catalysis (Figure 1B).…”

“…This pathway bookends the AsHad2 pocket variants with a thiolase from A. suum, AsAcat3, to provide branched and unbranched ketoacyl-CoA intermediates to AsHadh2 and a thioesterase from E. coli, TesB, to release free 3-hydroxy acids for analysis (Figure 5B). 14 Twenty-four colonies for each position were picked for a total of 336 total variants screened as single replicates to assess their differential production of branched (HMB) and linear (HB) 3hydroxy acid compared to the wild-type AsHadh2 control. In this primary screen, we discovered that the I155A, M205V, P209G, and R213L mutations appeared to increase selectivity toward HMB whereas the R213S and R213T mutations generated a defect that increased HB production (Figure S4).…”

“…While the stereochemistry of methyl substituents controlled by the ketoreductase (KR) domains of modular polyketide synthases has been extensively studied, , the structural factors that mediate direct recognition of the methyl group remain incompletely determined . Given the role of ketoreductases in controlling the distribution of branched products in natural and engineered pathways, , we were interested in elucidating the molecular basis for methyl group recognition in AsHadh2. Toward this goal, we have solved the crystal structure of AsHadh2, which shows that a hydrophobic pocket helps to stabilize the substrate via interaction with the α-methyl group.…”

Respirometry and Cell Viability Studies for Sustainable Polyesters and Their Hydrolysis Products

“…Promising new candidates to address the challenges in sustainability are other related synthetic polyesters. − Specifically, we have focused research efforts toward polymers based on γ-methyl-ε-caprolactone (MCL). ,,− MCL-based polymers have potential to be both bioderived and biodegradable/compostable . While poly­(γ-methyl-ε-caprolactone) (PMCL) as a homopolymer has a low T g and limited applications, unlike PLA and or PHA homopolymers, its use in multicomponent materials is well established.…”