Computer-aided engineering of adipyl-CoA synthetase for enhancing adipic acid synthesis
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ORIGINAL RESEARCH PAPER
Computer-aided engineering of adipyl-CoA synthetase for enhancing adipic acid synthesis Ju Yang . Yafeng Wei . Guohui Li . Shenghu Zhou . Yu Deng
Received: 1 June 2020 / Accepted: 27 July 2020 Ó Springer Nature B.V. 2020
Abstract Objective To enhance adipic acid production, a computer-aided approach was employed to engineer the adipyl-CoA synthetase from Thermobifida fusca by combining sequence analysis, protein structure modeling, in silico site-directed mutagenesis, and molecular dynamics simulation. Results Two single mutants of T. fusca adipyl-CoA synthetase, E210bN and E210bQ, achieved a specific enzyme activity of 1.95 and 1.84 U/mg, respectively, which compared favorably with the 1.48 U/mg for the wild-type. The laboratory-level fermentation experiments showed that E210bN and E210bQ achieved a maximum adipic acid titer of 0.32 and 0.3 g/L. In contrast, the wild-type enzyme yielded a titer of 0.15 g/L under the same conditions. Molecular
dynamics (MD) simulations revealed that the mutants (E210bN and E210bQ) could accelerate the dephosphorylation process in catalysis and enhance enzyme activity. Conclusions The combined computational-experimental approach provides an effective strategy for enhancing enzymatic characteristics, and the mutants may find a useful application for producing adipic acid. Keywords Adipic acid Adipyl-CoA synthetase Biosynthesis Enzyme activity Molecular dynamics simulation
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-02978-y) contains supplementary material, which is available to authorized users. J. Yang Y. Wei G. Li S. Zhou Y. Deng (&) National Engineering Laboratory for Cereal Fermentation Technology (NELCF), School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, People’s Republic of China e-mail: [email protected] J. Yang Y. Wei G. Li S. Zhou Y. Deng Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, People’s Republic of China
Adipic acid is an important chemical that is widely used in medicine, food, and manufacturing (Babu et al. 2015). To date, the large-scale production of adipic acid still relies on chemical synthesis which results in a large number of contaminants and green gas. Therefore, it is desirable to develop environmentally friendly biological processes for producing adipic acid (Kircher 2006; Niu et al. 2002; She et al. 2011). Recently, we constructed a new five-step metabolic pathway to synthesize adipic acid from D-glucose (Fig. 1), which achieved a titer of 0.16 g/L and 0.2 g/ L in Thermobifida fusca and E. coli, respectively
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Fig. 1 The pathway for synthesizing adipic acid. The critical enzyme adipyl-CoA synthetase focused in the study is highlighted in bold
(Deng and Mao 2015; Zhao et al. 2018), but the relatively low titer still limits its applic
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