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Research
Suchada Chanprateep NAPATHORN
Chulalongkorn University Faculty of Science
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3598-9344
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: The present study attempted to increase polyhydroxybutyrate (PHB) production by improving the functional expression of the PhaC gene using various types of promoters, and the effects on PhaC activity in terms of PHB productivity, yield coefficient (YP/S) and molecular weights were investigated.
Results: Here, the PHB biosynthesis operon of Cupriavidus necator A-04, isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO and pUC19 (native promoter) and transformed into E. coli JM109. To alter the expression of phaCAB biosynthesis genes, we optimized parameters in flask experiments to obtain high expression of soluble PhaCA-04 protein with high YP/S and PHB productivity. pColdTF-phaCABA-04-expressing E. coli produced 2.5±0.1 g/L (90.6±4.3%) PHB in 24 h, similar to pColdI-phaCABA-04-expressing E. coli. The amounts of phaC protein and PHB produced from pColdTF-phaCABA-04 and pColdI-phaCABA-04 were significantly higher than those from other promoters. Cultivation in a 5-L fermenter led to PHB production of 7.9±0.7 g/L with 90.0±2.3% PHB content in the cell dry mass (CDM), a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L×h) using pColdTF-phaCABA-04. The PHB from pColdTF-phaCABA-04 had Mw 5.79×105 Da, Mn 1.86×105 Da and PDI 3.11 and the film exhibited high transparency, Young’s modulus and tensile strength, possibly due to the trigger factor (TF) chaperones. Interestingly, when pColdI-phaCABA-04-expressing E. coli was used to produce PHB from crude glycerol and compared with pUC19-nativeP-phaCABA-04-expressing E. coli, the amounts of PHB were similar, but Mw 1.1×106 Da, Mn 2.6×105 Da and PDI 4.1 were obtained from pUC19-nativeP-phaCABA-04-expressing E. coli, indicating that slow and low expression could prolong and maintain phaC polymerization activity.
Conclusions: This is the first report to demonstrate that the cspA promoter in a cold-inducible vector can improve PhaCA-04 expression levels, and TF chaperones show obvious effects on enhancing PhaCA-04 solubility. The high level of PhaCA-04 resulted in a high PHB amount, but the chain termination reaction of PhaC polymerization occurred faster than that with the slowed and low expression of phaCA-04 by the native promoter pUC19, which resulted in a low amount of high-molecular-weight PHB produced from crude glycerol.
Keywords
poly(3-hydroxybutyrate), pCold, chaperone, trigger factor, Cupriavidus necator
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This is a preprint. It has not completed peer review.
Research
Suchada Chanprateep NAPATHORN
Chulalongkorn University Faculty of Science
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3598-9344
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
View the published article here.
Version 2
Posted 22 Sep, 2020
No community comments so far
No comments provided
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Biotechnology and Bioengineering
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article here.
Background: The present study attempted to increase polyhydroxybutyrate (PHB) production by improving the functional expression of the PhaC gene using various types of promoters, and the effects on PhaC activity in terms of PHB productivity, yield coefficient (YP/S) and molecular weights were investigated.
Results: Here, the PHB biosynthesis operon of Cupriavidus necator A-04, isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO and pUC19 (native promoter) and transformed into E. coli JM109. To alter the expression of phaCAB biosynthesis genes, we optimized parameters in flask experiments to obtain high expression of soluble PhaCA-04 protein with high YP/S and PHB productivity. pColdTF-phaCABA-04-expressing E. coli produced 2.5±0.1 g/L (90.6±4.3%) PHB in 24 h, similar to pColdI-phaCABA-04-expressing E. coli. The amounts of phaC protein and PHB produced from pColdTF-phaCABA-04 and pColdI-phaCABA-04 were significantly higher than those from other promoters. Cultivation in a 5-L fermenter led to PHB production of 7.9±0.7 g/L with 90.0±2.3% PHB content in the cell dry mass (CDM), a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L×h) using pColdTF-phaCABA-04. The PHB from pColdTF-phaCABA-04 had Mw 5.79×105 Da, Mn 1.86×105 Da and PDI 3.11 and the film exhibited high transparency, Young’s modulus and tensile strength, possibly due to the trigger factor (TF) chaperones. Interestingly, when pColdI-phaCABA-04-expressing E. coli was used to produce PHB from crude glycerol and compared with pUC19-nativeP-phaCABA-04-expressing E. coli, the amounts of PHB were similar, but Mw 1.1×106 Da, Mn 2.6×105 Da and PDI 4.1 were obtained from pUC19-nativeP-phaCABA-04-expressing E. coli, indicating that slow and low expression could prolong and maintain phaC polymerization activity.
Conclusions: This is the first report to demonstrate that the cspA promoter in a cold-inducible vector can improve PhaCA-04 expression levels, and TF chaperones show obvious effects on enhancing PhaCA-04 solubility. The high level of PhaCA-04 resulted in a high PHB amount, but the chain termination reaction of PhaC polymerization occurred faster than that with the slowed and low expression of phaCA-04 by the native promoter pUC19, which resulted in a low amount of high-molecular-weight PHB produced from crude glycerol.
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