This thesis presents an integrated approach to microbial strain engineering and
process optimization for the sustainable production of polyhydroxyalkanoates (PHAs),
a family of biodegradable and biocompatible polymers with significant potential to
replace conventional plastics. The research focuses on enhancing PHA production,
tailoring polymer composition, and evaluating the use of renewable feedstocks in
industrially relevant microbial hosts.
A key outcome of this work is the successful construction of genetically engineered
Pseudomonas putida capable of synthesizing short-chain-length (scl-) and medium
chain-length (mcl-) PHA copolymers through the heterologous expression of the
phaCAB operon from Cupriavidus necator. By systematically evaluating promoter
strength, PHA monomer composition was modulated from 17.9 mol% to 99.6 mol% 3
hydroxybutyrate (3HB), with polymer titers reaching up to 1.48 g/L and intracellular
PHA content exceeding 42 wt% under glucose-supplemented conditions.
Physicochemical characterization using gas chromatography–mass spectrometry
(GC-MS), gel permeation chromatography (GPC), nuclear magnetic resonance (NMR),
and differential scanning calorimetry (DSC) confirmed the production of structurally
distinct polymer blends, with tunable molecular weights and thermal properties aligned
with target applications.
In parallel, this thesis explored the integration of synthetic mcl-PHA biosynthetic
pathways into native scl-PHA producers, including Rhodococcus and C. necator.
Although functional expression was achieved in E. coli, no detectable mcl-PHA
production was observed in the native hosts, revealing critical limitations in carbon flux
and pathway compatibility. These findings highlight the importance of host-specific
engineering strategies and underscore the metabolic rigidity of non-model organisms.
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Furthermore, the potential of low-cost, renewable substrates was assessed using
sugarcane molasses and potato peel hydrolysate (PPH). Burkholderia BCC 59163
achieved PHA accumulation up to 57.01 wt% from molasses, while the engineered P.
putida demonstrated the ability to produce PHA blends from PPH. However, both
substrates presented technical challenges related to nutrient variability, low titers, or
process consistency, emphasizing the need for further feedstock optimization.
Overall, this thesis advances the field of sustainable biopolymer production by
demonstrating how genetic control, host selection, and feedstock integration
collectively determine polymer output and quality. The findings lay a foundation for the
development of next-generation microbial platforms capable of producing application
specific PHAs with reduced environmental and economic costs.
