Dissertation
Fundamentals of biological production of polyhydroxyalkanoate from biomass-derived carbon sources in Pseudomonas putida
Washington State University
Doctor of Philosophy (PhD), Washington State University
01/2022
DOI:
https://doi.org/10.7273/000004439
Handle:
https://hdl.handle.net/2376/118820
Abstract
Efficient utilization of all available carbons from lignocellulosic biomass is critical for the economic efficiency of a bioconversion process to produce renewable bioproducts. This dissertation aims to understand the fundamentals of bioconversion of biomass-derived carbons to polyhydroxyalkoate (PHA) by Pseudomonas strain. The key contributions of this work include the elucidation of two distinct kinetic patterns of PHA accumulation with benzoate under nitrogen-limited and surplus conditions (ammonium chloride form) in Pseudomonas putida KT2440. Moreover, a novel strategy—co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in P. putida KT2440—was developed for the first time, which simultaneously improved both cell biomass and PHA production. NMR metabolite analysis and global proteome profiles suggested the stimulated oxidative-stress responses altered the intracellular NAD+/NADH and NADPH/NADP+ ratios, up-regulated the proteins involved in energy generation and storage processes, including the Entner–Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid β-oxidation, and PHA biosynthesis. Furthermore, the benzoate addition inhibited cell biomass and PHA production in the glucose system, presenting a divergent effect to glycerol. Global proteomics results revealed that in both glucose and glycerol co-feeding systems, the addition of benzoate dynamically regulated the energy-related pathways such as the ED/PP pathway, trehalose, and PHA biosynthesis pathways during early and late fermentation periods. Redox proteomics analysis results further confirmed that co-feeding benzoate with glucose dynamically regulates the energy generation pathway by increasing protein thiol oxidation in the PP pathway and glyoxylate shunt while decreasing fatty acid and trehalose metabolism, resulting in a smaller cell dry weight and PHA yield. These results showed that aromatics would not only serve as the carbon source but also generate distinct redox environments and finally reflected by different PHA accumulation patterns. In addition, the mechanisms of bacterial lignin depolymerization with P. putida were investigated via secretomic analysis. Depolymerization reaction mechanisms involved in a couple of oxidoreductases such as Dyp, multicopper oxidase, NAD(P)H dehydrogenase, glutathione reductase, glutathione S-transferases, choline dehydrogenase, and FAD-binding oxidoreductase, followed by β-Ο-4, β-β, β-5, and β-1 cleavage and aromatic intermediates release (e.g., vanillin and vanillic acid).
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Details
- Title
- Fundamentals of biological production of polyhydroxyalkanoate from biomass-derived carbon sources in Pseudomonas putida
- Creators
- Zhangyang Xu
- Contributors
- Bin Yang (Advisor)John R. Cort (Committee Member)Shulin Chen (Committee Member)John H. Miller (Committee Member)Scott E. Baker (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- College of Agricultural, Human, and Natural Resource Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 284
- Identifiers
- 99900883437101842
- Language
- English
- Resource Type
- Dissertation