Generation and Characterization of Two Acid-resistant Macrocin -methyltransferase Variants with a Higher Enzyme Activity at 30 °C from

Overview

Journal Comput Struct Biotechnol J

Specialty Biotechnology

Date 2024 Sep 11

PMID 39257526

Authors

Chaoyue Yan

Yujun Tao

Jingyan Fan

Jun Dai

Shuo Li

Qi Huang

Rui Zhou

Affiliations

Soon will be listed here.

Abstract

Tylosin is an important macrolide antibiotic produced by . In the biosynthesis of tylosin, macrocin -methyltransferase TylF catalyzes the conversion of the side-product tylosin C (macrocin) to the primary component tylosin A (C/A conversion). This conversion is the rate-limiting step in the biosynthesis of tylosin, and affects the quality of the end product. To find a high activity and environment-adapted TylF enzyme, a TylF variant pool has been constructed protein evolution approach in our previous study (Fan et al., 2023 [41]). In this study, the TylF variants with higher C/A conversion rates were expressed in and purified. The variants TylF, TylF and TylF were shown to have a higher C/A conversion rate at 30 °C than that of TylF at 38 °C. Moreover, they had a greater acid resistance and showed more adaptable to the pH change during fermentation. Further protein structural and substrate-binding affinity analyses revealed that the T36S, V54A, Q138H, Y139F, and F232Y mutations enlarged the volume of the substrate-binding pocket, thereby increasing the affinity of enzyme variants for their substrates of SAM and macrocin, and decreasing the inhibition of SAH. Three of the TylF variants were overexpressed in the industrial tylosin-producing strain, and the recombinant strains showed the highest C/A conversion at 30 °C without heating up to 38 °C during the last 24 h of fermentation. This is of great energy-saving significance for tylosin industrial production.

References

Tong L, Zheng J, Wang X, Wang X, Huang H, Yang H . Improvement of thermostability and catalytic efficiency of glucoamylase from Talaromyces leycettanus JCM12802 via site-directed mutagenesis to enhance industrial saccharification applications. Biotechnol Biofuels. 2021; 14(1):202. PMC: 8520190. DOI: 10.1186/s13068-021-02052-3. View

Zubieta C, Ross J, Koscheski P, Yang Y, Pichersky E, Noel J . Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell. 2003; 15(8):1704-16. PMC: 167163. DOI: 10.1105/tpc.014548. View

Martin J, McMillan F . SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold. Curr Opin Struct Biol. 2002; 12(6):783-93. DOI: 10.1016/s0959-440x(02)00391-3. View

Seno E, Baltz R . Properties of S-adenosyl-L-methionine:macrocin O-methyltransferase in extracts of Streptomyces fradiae strains which produce normal or elevated levels of tylosin and in mutants blocked in specific O-methylations. Antimicrob Agents Chemother. 1981; 20(3):370-7. PMC: 181703. DOI: 10.1128/AAC.20.3.370. View

Stopa N, Krebs J, Shechter D . The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond. Cell Mol Life Sci. 2015; 72(11):2041-59. PMC: 4430368. DOI: 10.1007/s00018-015-1847-9. View

Loke M, Ingerslev F, Halling-Sorensen B, Tjornelund J . Stability of Tylosin A in manure containing test systems determined by high performance liquid chromatography. Chemosphere. 2000; 40(7):759-65. DOI: 10.1016/s0045-6535(99)00450-6. View

Arrowsmith C, Bountra C, Fish P, Lee K, Schapira M . Epigenetic protein families: a new frontier for drug discovery. Nat Rev Drug Discov. 2012; 11(5):384-400. DOI: 10.1038/nrd3674. View

Kristensen J, Felby C, Jorgensen H . Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol Biofuels. 2009; 2(1):11. PMC: 2699335. DOI: 10.1186/1754-6834-2-11. View

Abdelraheem E, Thair B, Varela R, Jockmann E, Popadic D, Hailes H . Methyltransferases: Functions and Applications. Chembiochem. 2022; 23(18):e202200212. PMC: 9539859. DOI: 10.1002/cbic.202200212. View

10.

Baltz R, Seno E, Stonesifer J, WILD G . Biosynthesis of the macrolide antibiotic tylosin. A preferred pathway from tylactone to tylosin. J Antibiot (Tokyo). 1983; 36(2):131-41. DOI: 10.7164/antibiotics.36.131. View

11.

Hamidian K, Amini M, Samadi N . Consistency evaluation between matrix components ratio and microbiological potency of tylosin major components. Daru. 2018; 26(2):155-164. PMC: 6279661. DOI: 10.1007/s40199-018-0220-6. View

12.

Siddiqui K . Some like it hot, some like it cold: Temperature dependent biotechnological applications and improvements in extremophilic enzymes. Biotechnol Adv. 2015; 33(8):1912-22. DOI: 10.1016/j.biotechadv.2015.11.001. View

13.

Bennett M, Shepherd S, Cronin V, Micklefield J . Recent advances in methyltransferase biocatalysis. Curr Opin Chem Biol. 2017; 37:97-106. DOI: 10.1016/j.cbpa.2017.01.020. View

14.

DE LA HABA G, CANTONI G . The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine. J Biol Chem. 1959; 234(3):603-8. View

15.

Yu H, Ma S, Li Y, Dalby P . Hot spots-making directed evolution easier. Biotechnol Adv. 2022; 56:107926. DOI: 10.1016/j.biotechadv.2022.107926. View

16.

Cheng F, Zhu L, Schwaneberg U . Directed evolution 2.0: improving and deciphering enzyme properties. Chem Commun (Camb). 2015; 51(48):9760-72. DOI: 10.1039/c5cc01594d. View

17.

Choi J, Han S, Kim H . Industrial applications of enzyme biocatalysis: Current status and future aspects. Biotechnol Adv. 2015; 33(7):1443-54. DOI: 10.1016/j.biotechadv.2015.02.014. View

18.

Yao Z, Fan J, Dai J, Yu C, Zeng H, Li Q . A High-Throughput Method Based on Microculture Technology for Screening of High-Yield Strains of Tylosin-Producing . J Microbiol Biotechnol. 2023; 33(6):831-839. PMC: 10331940. DOI: 10.4014/jmb.2210.10023. View

19.

Shields K, Tooley J, Petkowski J, Wilkey D, Garbett N, Merchant M . Select human cancer mutants of NRMT1 alter its catalytic activity and decrease N-terminal trimethylation. Protein Sci. 2017; 26(8):1639-1652. PMC: 5521588. DOI: 10.1002/pro.3202. View

20.

He C . Grand challenge commentary: RNA epigenetics?. Nat Chem Biol. 2010; 6(12):863-5. DOI: 10.1038/nchembio.482. View