Abstract
Objectives
With the help of attB-attP recombination technique, multiple copies of yfjB gene encoding the NAD kinase of Escherichia coli were inserted into the host chromosome to promote NADPH-dependent poly-3-hydroxybutyrate (PHB) production.
Results
The yfjB integration mutant E. coli T2 harbored a similar metabolic profile to that of the wild type control. When PHB biosynthesis operon was introduced, the yfjB integration mutant produced 3 g PHB l−1 from 18.2 g glucose l−1, while the wild type consumed 15.7 g glucose l−1 to afford 2.34 g PHB l−1 in 48 h of shake-flask cultivation. Transcriptional analysis showed that the transcription levels of genes within the PHB biosynthesis operon were increased by six to eightfold with yfj Bover-expression, which may be the primary reason for the improved PHB production.
Conclusion
A practical method is demonstrated to construct genetically-stable strains harboring extra copies of NAD kinase to enhance NADPH-dependent reactions.
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Acknowledgments
This research was supported by Grants from National Natural Science Foundation of China (31100025, 21476014), National Basic Research Program of China (2013CB733600, 2012CB725200), and Beijing Higher Education Young Elite Teacher Project (YETP0523).
Supporting information
Supplementary Table 1—Strains, plasmids, and primers used in this study. All oligonucleotides were synthesized by AuGCT Biotechnology (Beijing, China). Restriction endonuclease digestion sites were underlined.
Supplementary Table 2—qPCR primers used for transcriptional analysis.
Supplementary Table 3—PCR primers used for genome integration verification.
Supplementary Fig. 1—PCR verification of NAD kinase integration. PCR primers used for genome integration verification were listed in Supplementary Table 3. E. coli T2 was inoculated into Luria-Bertani medium and maintained by transferring into fresh medium every 12 h for 7 days. (A), original culture; (B), 7-day culture.
Supplementary Fig. 2—Expression of gfp gene under the control of phaCAB promoter in E. coli JM109 and T2. E. coli JM109 and T2 strains harboring pBHR68-gfp were cultivated in mineral salts medium at 37 °C for 48 h, respectively. Value 100 corresponds to equal gene expression relative to JM109 and values above 100 correspond to fold increase. Data were the means of triplicate experiments.
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Zhang, J., Gao, X., Hong, PH. et al. Enhanced production of poly-3-hydroxybutyrate by Escherichia coli over-expressing multiple copies of NAD kinase integrated in the host genome. Biotechnol Lett 37, 1273–1278 (2015). https://doi.org/10.1007/s10529-015-1797-1
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DOI: https://doi.org/10.1007/s10529-015-1797-1