Abstract
Objective
O-acetylhomoserine (OAH) is an important platform chemical to produce high-valuable chemicals. To improve the production of O-acetylhomoserine from glycerol, the glycerol-oxidative pathway was investigated and the optimization of fermentation with crude glycerol was carried out.
Results
The glycerol-uptake system and glycerol-oxidative pathway were modified and O-acetyltransferase from Corynebacterium glutamicum was introduced into the engineered strain to produce O-acetylhomoserine. It was found that overexpression of glycerol 3-phosphate dehydrogenase improved the OAH production to 6.79 and 4.21 g/L from pure and crude glycerol, respectively. And the higher OAH production depending on higher level of transcription of glpD. Two-step statistical approach was employed to optimize the fermentation conditions. The significant effects of glycerol, ammonium chloride and yeast extract were screened applying Plackett–Burman design and were optimized further by employing the Response Surface Methodology. Under optimized conditions, the OAH production was up to 9.42 and 7.01 g/L when pure and crude glycerol were used in shake flask cultivations, respectively.
Conclusions
The enzymatic step catalyzing the oxidation of glycerol through GlpD was the key step for OAH production, which served the foundation for realization of a consistent OAH production from crude glycerol in the future.
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Acknowledgements
This research was supported by the National Natural Science Foundation of China (Nos. 31971342 and 31700095).
Supporting information
Supplementary Table 1—Bacterial strains and plasmids used in this study.
Supplementary Table 2—Primers used in this study.
Supplementary Table 3—2-Level fractional factorial design for OAH production.
Supplementary Table 4—Box–Behnken factorial design for OAH production.
Supplementary Table 5—Analysis of variance (ANOVA) for the experimental results of the BBD.
Supplementary Fig. 1—The mass spectrometry results of standard and biosynthetic OAH (a) Standard OAH (b) Fermentative OAH.
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Liu, P., Liu, JS., Zhang, B. et al. Increasement of O-acetylhomoserine production in Escherichia coli by modification of glycerol-oxidative pathway coupled with optimization of fermentation. Biotechnol Lett 43, 105–117 (2021). https://doi.org/10.1007/s10529-020-03031-8
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DOI: https://doi.org/10.1007/s10529-020-03031-8