Study of alternative metabolic pathways for the production of (R)-3-hydroxybutyric acid in polyhydroxybutyrate producing bacteria
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Date
2022
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Universidad de los Andes
Abstract
Considerable rich literature has accumulated concerning biochemical, physiological, and genetic aspects of polyhydroxybutyrate (PHB) intracellular accumulation in bacteria. The costs of substrates and processing, including the extraction of the polymer accumulated in intracellular granules, still
hampers a more widespread use of this family of polymers. The PHB monomeric unit, (R)-3-hydroxybutyric acid (R3HBA) has found uses at the biomedical, chemical and supplement industries. The literature shows that two main process engineering and metabolic engineering strategies have
been identified aimed at the production of chiral R3HBA: (i) production from the accumulated polymer (polymerization and depolymerization system, PDS); (ii) by bypassing the accumulation of PHB using metabolically engineered bacteria. The later includes the use of thioesterases (thioesterase
shortcut system, TSS) that removes CoA from R3HBA-CoA, resulting in the R3HBA release to extracellular medium. This PhD thesis aims at broadening the understanding of the genetic and operational factors leading to PHB polymerization and R3HBA production in Azohydromonas lata DSM 1123, Cupriavidus necator H16 and Methylocystis parvus OBBP. Results showed that the growth associated PHB production observed in A. lata mimics an overflow metabolism, additionally, a successful PHB depolymerization in a two stage chemostat was obtained. The feasibility of producing R3HBA
through in-vivo depolymerization of the intracellularly accumulated PHB in M. parvus was investigated. A PHB to R3HBA conversion of 77.2 ± 0.9% (R3HBA titer of 0.153 ± 0.002 g L⁻¹) can be attained in a mineral medium containing 1.0 g L⁻¹ KNO₃ at 30 °C with shaking at 200 rpm and a constant pH of 11 for 72 hours. Nitrogen deprivation, oxygen limitation, the supplementation with exogenous R3HBA and neutral or acidic pHs strongly reduced the excreted R3HBA concentration and yield. The implementation of the TSS system in M. parvus and C. necator by the construction of an expression vector containing tesB was hampered by inconsistencies in the constructed plasmids pLY01 and pLY02. Finally, the production of R3HBA by redirecting fluxes in the PHB metabolic pathway was investigated in C. necator; two mutant strains were constructed using the suicide vector pT18mobsacB: C. necator ∆phaC and C. necator ∆phaC ∆hbd, both unable to polymerize PHB and the last one incapable to transform R3HBA into acetoacetate. The mutant trains released pyruvate and R3HBA, suggesting that a native thioesterase of C. necator may play a role in the release of R3HBA by removing CoA from 3HBA-CoA. A protein homology on the genome of C. necator showed an enzyme encoded as WP_037025319.1 with a percent identity of 44 % in comparison with Ycia that may trigger R3HBA release. The results obtained in this work demonstrated the feasibility of R3HBA production by reducing or eliminating the fluxes of the reactions consuming R3HBA via operational manipulation as described in M. parvus and A. lata or via gene knock outs in C. necator.
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Yáñez Meneses, L. F. (2022). Study of alternative metabolic pathways for the production of (R)-3-hydroxybutyric acid in polyhydroxybutyrate producing bacteria (Tesis doctoral, Universidad de los Andes, Chile). Santiago, Chile.