Modification of Glucose Metabolic Pathway to Enhance Polyhydroxyalkanoate Synthesis in <i>Pseudomonas putida</i>
Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are semi-crystalline elastomers with a low melting point and high elongation at break, allowing for a wide range of applications in domestic, agricultural, industrial, and mainly medical fields. Utilizing low-cost cellulose hydrolyzed sugar as a c...
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| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-11-01
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| Series: | Current Issues in Molecular Biology |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1467-3045/46/11/761 |
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| Summary: | Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are semi-crystalline elastomers with a low melting point and high elongation at break, allowing for a wide range of applications in domestic, agricultural, industrial, and mainly medical fields. Utilizing low-cost cellulose hydrolyzed sugar as a carbon source and metabolic engineering to enhance synthesis in <i>Pseudomonas putida</i> is a promising strategy for commercializing mcl-PHAs, but little has been attempted to improve the utilization of glucose for synthesizing mcl-PHAs. In this study, a multi-pathway modification was performed to improve the utilization of substrate glucose and the synthesis capacity of PHAs. To enhance glucose metabolism to flow to acetyl-CoA, which is an important precursor of mcl-PHA, multiple genes in glucose metabolism were inactive (branch pathway and negative regulatory) and overexpressed (positive regulatory) in this study. The two genes, <i>gcd</i> (encoding glucose dehydrogenase) and <i>gltA</i> (encoding citrate synthase), involved in glucose peripheral pathways and TCA cycles were separately and jointly knocked out in <i>Pseudomonas putida</i> QSRZ6 (<i>ΔphaZΔhsdR</i>), and the mcl-PHA synthesis was improved in the mutants; particularly, the mcl-PHA titer of QSRZ603 (<i>ΔgcdΔgltA</i>) was increased by 33.7%. Based on the glucose branch pathway truncation, mcl-PHA synthesis was further improved with <i>hexR</i>-inactivation (encoding a negative regulator in glucose metabolism). Compared with QSRZ603 and QSRZ6, the mcl-PHA titer of QSRZ607 (<i>ΔgcdΔgltAΔhexR</i>) was increased by 62.8% and 117.5%, respectively. The mutant QSRZ609 was constructed by replacing the endogenous promoter of <i>gltB</i> encoding a transcriptional activator of the two-component regulatory system GltR/GltS with the ribosome subunit promoter P<sub>33</sub>. The final mcl-PHA content and titers of QSRZ609 reached 57.3 wt% and 2.5 g/L, an increase of and 20.9% and 27.3% over that of the parent strain QSRZ605 and an increase of 110.4% and 159.9% higher as compared to QSRZ6, respectively. The fermentation was optimized with a feeding medium in shaker flacks; then, the mcl-PHA contents and titer of QSRZ609 were 59.1 wt% and 6.8 g/L, respectively. The results suggest that the regulation from glucose to acetyl-CoA by polygenic modification is an effective strategy for enhancing mcl-PHA synthesis, and the mutants obtained in this study can be used as chassis to further increase mcl-PHA production. |
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| ISSN: | 1467-3037 1467-3045 |