Towards the production of bioactive short-chain-length poly-(3-hydroxyalkanoates)

Abstract

Poly-(3-hydroxyalkanoates) (PHAs) have gained significant attention as alternatives to fossil-fuel based polymers due to their renewability and biodegradability. The main objective of this thesis is to produce bioactive short-chain-length PHA (scl-PHA) with vinyl groups for potential biomedical applications. To evaluate the bioactivity of such materials, a microplate reader methodology was developed to monitor aerobic microbial growth data. That study emphasized optimization of mixing conditions to obtain maximum oxygen transfer without cross-contamination due to spillover. The production of scl-PHA with vinyl groups was studied using wild type as well as recombinant Cuprovidus necator expressing (R)-specific enoyl-CoA hydratase from Pseudonomas putida KT2440. Short-chain-length-co-medium-chain-length PHA (scl-co-mcl PHA) containing 1.7 mol% vinyl subunits was produced by the recombinant strain, co-fed with fructose, decanoic acid, and 10-undecenoic acid. Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was produced by wild type C. necator DSM545 under the same feed condition or when fed with fructose and 4-pentenoic acid. These findings suggest that both the native (R)-specific enoyl-CoA hydratase of C. necator and its PHA synthase have limited ability for processing subunits with vinyl groups. To increase the vinyl content, future work should focus on engineering a recombinant C. necator strain expressing (R)-specific enoyl-CoA hydratase from P. putida and PHA synthase from Aeromonas caviae. To further study the substrate specificity of native PHA synthase and (R)-specific enoyl-CoA hydratase of wild type C. necator, a series of pulse experiments in a nitrogen-limited chemostat were conducted. Pulses of fatty acids with different chain lengths were injected, including propionic acid, butyric acid, valeric acid, caproic acid, hexanoic acid, octanoic acid, nonanoic acid, as well as the vinyl substrates 3-butenoic acid, 4-pentenoic acid, and 10-undecenoic acid. While poly-(3-hydroxybutyrate) (PHB) and PHBV were produced, no vinyl groups were detected. It was found that the longer the fatty acid chain length, the slower the substrate consumption, and the longer the adaptation time.