Designing a whole-cell biosensor applicable for S-adenosyl-l-methionine-dependent methyltransferases.

Autor: Zhen Z; Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China; University of Chinese Academy of Sciences, 100049, Beijing, China., Xiang; Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China., Li S; Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China; University of Chinese Academy of Sciences, 100049, Beijing, China., Li H; Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China; University of Chinese Academy of Sciences, 100049, Beijing, China., Lei Y; Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China; University of Chinese Academy of Sciences, 100049, Beijing, China., Chen W; Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China., Jin JM; Beijing Key Laboratory of Plant Resources Research and Development, Beijing Technology and Business University, 100048, Beijing, China. Electronic address: jinjianming@btbu.edu.cn., Liang C; Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China. Electronic address: laraineliang@163.com., Tang SY; Department of Microbial Physiological & Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China. Electronic address: tangsy@im.ac.cn.
Jazyk: angličtina
Zdroj: Biosensors & bioelectronics [Biosens Bioelectron] 2025 Jan 15; Vol. 268, pp. 116904. Date of Electronic Publication: 2024 Nov 01.
DOI: 10.1016/j.bios.2024.116904
Abstrakt: This study was undertaken to develop a high-throughput screening strategy using a whole-cell biosensor to enhance methyl-group transfer, a rate-limiting step influenced by intracellular methyl donor availability and methyltransferase efficiency. An l-homocysteine biosensor was designed based on regulatory protein MetR from Escherichia coli, which rapidly reported intracellular l-homocysteine accumulation resulted from S-adenosyl-l-homocysteine (SAH) formation after methyl-group transfer. Using S-adenosyl-l-methionine (SAM) as a methyl donor, this biosensor was applied to caffeic acid 3-O-methyltransferase derived from Arabidopsis thaliana (AtComT). After several rounds of directed evolution, the modified enzyme achieved a 13.8-fold improvement when converting caffeic acid to ferulic acid. The best mutant exhibited a 5.4-fold improvement in catalytic efficiency. Characterization of beneficial mutants showed that improved O-methyltransferase dimerization greatly contributed to enzyme activity. This finding was verified when we switched and compared the N-termini involved in dimerization across different sources. Finally, with tyrosine as a substrate, the evolved AtComT mutant greatly improved ferulic acid biosynthesis, yielding 3448 mg L -1 with a conversion rate of 88.8%. These results have important implications for high-efficiency O-methyltransferase design, which will greatly benefit the biosynthesis of a wide range of natural products. In addition, the l-homocysteine biosensor has the potential for widespread applications in evaluating the efficiency of SAM-based methyl transfer.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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