Abstract
Objective
Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by multiple fungi. A key enzyme in the SW synthesis pathway is a hybrid swnk/nrps. To analyze the role of swnk in the SW biosynthesis pathway of Metarhizium anisopliae.
Results
The concentration of SW and the swnk expression in M. anisopliae fermentation from 1st to 7th day were determined using LC–MS and RT-qPCR, respectively. M. anisopliae had the highest SW content and swnk expression on the 5th day of fermentation; Mutant strain (MT) were obtained by PEG-mediated homologous recombination (HR) which knocked out swnk in the wild-type (WT) strain. Complemented-type (CT) strain were obtained by transforming a modified PUC19 complementation vector containing the geneticin (G418) resistance gene and swnK. SW was not detected in the MT strain and reverted to its original level in the CT strain; A Psilent-1 plasmid with Benomyl (ben)-resistant that was used interfered with swnk of WT strain. The level of SW was markedly diminished in the RNAi strain. RNAi of swnk affects the formation of the cell wall in M. anisopliae.
Conclusion
These results indicate that swnk plays a crucial role in the SW biosynthesis of M. anisopliae.
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Data availability
All data generated or analysed during this study are included in this published article.
Code availability
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Acknowledgements
The authors are grateful to Prof. Zekun Guo, College of Life Sciences, Northwest A&F University for his valuable help and advice during the experiment.
Supporting information
Table S1—Primers are used to measure the expression of the swnk gene.
Table S2—Primers for the swnk gene of M. anisopliae to mutation vector, complementary vector and RNAi vector.
Table S3—Identification primers for the swnk gene MT, CT and RNAi strain.
Figure S1—Comparison of swnk and pks genes.
Figure S2—Construction of knockout vector, complement vector and RNAi vector of the swnk gene in M. anisopliae.
Figure S3—Fragments of the swnk gene mutation vector, complementary vector and RNAi vector of M. anisopliae.
Figure S4—Validation of the swnk gene mutation vector, complementary vector and RNAi vector of M. anisopliae and its specific fragments.
Figure S5—Identification figure for swnk gene MT, CT and RNAi strain.
Funding
This work was supported by the Grants from the National Natural Science Foundation of China (No. 32072929) and the Science and Technology Special Fund Aid to Qinghai province (No. 2020-QY-210) and Advanced Foreign Expert Introduction Program (No. G2022172001).
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Conceptualization: [HL, BZ and CM]; Methodology: [EH, YZ, LS, YZ]; Formal analysis and investigation: [EH, LS, ST, CM]; Writing—original draft preparation: [EH, LS]; Writing—review and editing: [EH, YZ, LS]; Funding acquisition: [HL]; Resources: [BZ, HL]; Supervision: [HL]. All authors contributed to revision, read and approved the submitted version of the manuscript.
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Huang, E., Zhang, Y., Sun, L. et al. swnk plays an important role in the biosynthesis of swainsonine in Metarhizium anisopliae. Biotechnol Lett 45, 509–519 (2023). https://doi.org/10.1007/s10529-023-03356-0
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DOI: https://doi.org/10.1007/s10529-023-03356-0