Effects of S-adenosylmethionine on production of secondary metabolites in Streptomycesdiastatochromogenes 1628

Effects of S -adenosylmethionine on production of secondary metabolites in Streptomyces diastatochromogenes 1628 S -腺苷甲硫氨酸对淀粉酶产色链霉菌1628次级代谢产物产量的影响 1 first-author 1 1 1 corresp Zheng MA, mazheng520@163.com Zheng MA, mazheng520@163.com mazheng520@163.com https://orcid.org/0000-0002-1446-0708 2 1 Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou310018, China Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou310018, China Institute for Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, University of Freiburg, 79104Freiburg, Germany Institute for Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, University of Freiburg, 79104Freiburg, Germany 目的 2 考察 S -腺苷甲硫氨酸（SAM）对淀粉酶产色链霉菌（ Streptomyces diastatochromogenes ）1628次级代谢产物产量的影响。 创新点 2 SAM是甲基的重要供体，可为次级代谢产物提供前体，同时也可以作为信号增强次级代谢产物合成基因的表达。因此，本研究首次探究了SAM与 S. diastatochromogenes 1628次级代谢产物合成的相关性。 方法 2 以 S. diastatochromogenes 1628为对象，通过体外添加SAM以及在 S. diastatochromogenes 1628体内过表达、敲除和回补SAM编码基因 metK sd ，考察 S. diastatochromogenes 1628次级代谢产物产量的变化。 结论 2 由于渗透性差，外源添加SAM对 S. diastatochromogenes 1628合成次级代谢产物（丰加霉素和三种四烯大环内酯类抗生素：四霉素A、四霉素P和四烯菌素B）没有影响；敲除、回补和过表达基因 metK sd 证实增加胞内SAM可提高 S. diastatochromogenes 1628三种四烯大环内酯类抗生素的产量，且可促进四烯大环内酯类化合物合成关键基因的表达，而胞内SAM浓度的变化对丰加霉素的产量和及其关键基因的表达均无明显影响。 淀粉酶产色链霉菌1628 S -腺苷甲硫氨酸 MetK 丰加霉素 四烯大环内酯类 Streptomyces are famous for their ability to synthesize a large number of bioactive compounds as secondary metabolites containing antibiotics, enzyme inhibitors, and other small molecules with potential physiological activity ( Niu et al., 2016 Niu et al., 2016 ; Song et al., 2019 Song et al., 2019 ; Yin et al., 2019 Yin et al., 2019 ). Secondary metabolites are produced by a multi-step reaction of a primary metabolite as a precursor ( Liu et al., 2013 Liu et al., 2013 ; Li et al., 2021 Li et al., 2021 ). Therefore, it is of great research significance to increase the overall synthesis level of antibiotics by increasing the amount of synthesis of precursors. S -adenosylmethionine (SAM), which is synthesized by a synthase (MetK) from L-methionine and adenosine triphosphate, is the major donor of methyl groups ( Zhao XQ et al., 2010 Zhao XQ et al., 2010 ) and plays an important role in primary and secondary metabolisms ( Liu et al., 2020 Liu et al., 2020 ). In the last few years, some research groups have shown that exogenous addition of SAM and/or overexpression of the metK gene are effective strategies to increase secondary metabolite production in Streptomyces strains ( Okamoto et al., 2003 Okamoto et al., 2003 ; Wang et al., 2012 Wang et al., 2012 ; Gu et al., 2016 Gu et al., 2016 ); examples of such strains are actinorhodin ( Kim et al., 2003 Kim et al., 2003 ), pristinamycin ( Huh et al., 2004 Huh et al., 2004 ), avermectin ( Tian et al., 2017 Tian et al., 2017 ), nosiheptide ( Zhang et al., 2008 Zhang et al., 2008 ), pikromycin ( Maharjan et al., 2008 Maharjan et al., 2008 ), and doxorubicin ( Oh et al., 2010 Oh et al., 2010 ). However, although the SAM in Streptomyces species has been studied extensively, no studies have yet been conducted on the antagonistic strain Streptomyces diastatochromogenes 1628. S . diastatochromogenes 1628 was isolated and deposited in the China General Microbiological Culture Collection Center (CGMCC, No. 2060) and exhibits good bioactivity against a variety of phytopathogenic fungi ( Ma et al., 2014a Ma et al., 2014a ). In our previous study, toyocamycin (TM) was identified in the fermentation broth of S . diastatochromogenes 1628 ( Ma et al., 2014a Ma et al., 2014a ). TM, a member of the nucleoside antibiotics family, is considered to be a promising fungicide in the plant protection field ( Battaglia et al., 2011 Battaglia et al., 2011 ). Therefore, some efforts have been made to improve the TM production of S . diastatochromogenes 1628 ( Ma et al., 2014b, Ma et al., 2014b, , 2016 2016 , 2020 2020 ; Xu et al., 2019 Xu et al., 2019 ). In addition, recent studies have demonstrated that the fermentation products of S . diastatochromogenes 1628 contain three other tetraene macrolides (tetramycin A, tetramycin P, and tetrin B) ( Fan et al., 2020 Fan et al., 2020 ). Tetramycin, a 26-member tetraene antibiotic, has been applied to treat some plant-pathogenic fungi diseases in China ( Zhao XH et al., 2010 Zhao XH et al., 2010 ) and to control the growth of the brown planthopper, which is one of the most destructive insect pests in rice ecosystems ( Shentu et al., 2016 Shentu et al., 2016 ). Therefore, we developed an interest in improving the tetraene macrolide production of S . diastatochromogenes 1628. Improvement in production of these three tetraene macrolides has been accomplished by introducing cumulative drug-resistance mutation ( Fan et al., 2020 Fan et al., 2020 ). However, how SAM affects the biosynthesis of TM and tetraene macrolides in S . diastatochromogenes 1628 remains unknown. The objective of this study was to determine the effects of SAM on production of secondary metabolites in S . diastatochromogenes 1628. First, we investigated the effect of SAM on TM and on the production of three tetraene macrolides (tetramycin A, tetramycin P, and tetrin B) by exogenous supplementation of SAM. Subsequently, we further investigated the relationship between SAM and secondary metabolite biosynthesis in S . diastatochromogenes 1628 by experiments involving metK sd overexpression, disruption, and complementation. In addition, we determined the effects of SAM on transcriptional levels of key genes involved in secondary metabolite biosynthesis. We initially investigated the effect of external addition of SAM on biosynthesis of secondary metabolites in S . diastatochromogenes 1628. The strain was cultured in the fermentation medium with varying concentrations of SAM (2, 10, 25, and 50 µmol/L), and the levels of tetraene macrolides (tetramycin A, tetramycin P, and tetrin B) and TM were measured. As shown in Fig. 1 Fig. 1 , production of tetraene macrolides was not increased with any experimental concentration of SAM ( Fig. 1 Fig. 1 a). Similarly, under the final concentrations of SAM from 2 to 50 µmol/L in the fermentation medium, the TM production of S . diastatochromogenes 1628 showed no significant change ( Fig. 1 Fig. 1 a). In addition, we found that extracellular SAM concentrations generally remained constant during the whole fermentation process ( Fig. 1 Fig. 1 b). The intracellular level of SAM did not vary depending on the amount of added SAM ( Fig. 1 Fig. 1 c). We cloned the metK sd gene which encodes SAM synthetase of S . diastatochromogenes 1628 by polymerase chain reaction (PCR). The nucleotide sequence was submitted to the GenBank database under accession No. KX689260. To increase intracellular concentration of SAM in S . diastatochromogenes 1628, the obtained 1209-bp metK sd was placed under the control of promoter ermE * in plasmid pIB139 to create pIB139- metK sd (Fig. S1). Then, the plasmid pIB139- metK sd was introduced into S . diastatochromogenes 1628 by intergeneric conjugation to generate recombinant strain 1628-MetK. Both the control strain (1628) and the recombinant strain (1628-MetK) were cultured in fermentation medium as described in the supplementary materials. As shown in Fig. 2 Fig. 2 a, we observed no significant differences in cell growth between the control strain and recombinant strain. As expected, the SAM synthetase activity of recombinant strain 1628-MetK was found to be markedly increased compared to that of the wild-type strain. Compared with the control strain, recombinant strain 1628-MetK successively exhibited a 140.0%, 63.3%, 57.3%, and 96.2% increase in intracellular SAM during the fermentation periods of 24, 48, 72, and 96 h, respectively ( Table 1 Table 1 ). Consistent with the increase of intracellular SAM, recombinant strain 1628-MetK showed higher production levels of tetramycin A (201.3 mg/L vs. 135.8 mg/L), tetramycin P (155.5 mg/L vs. 108.6 mg/L), and tetrin B (57.2 mg/L vs. 40.3 mg/L) ( Fig. 2 Fig. 2 b). In contrast, overexpression of metK sd had no obvious stimulating effect on TM production (138.4 mg/L vs. 131.7 mg/L) ( Fig. 2 Fig. 2 b). Since the SAM synthetase level slightly affected the growth of the transformants, the specific production per mg dry cell weight (DCW) was also compared between recombinant strain 1628-MetK and wild-type strain 1628. The experimental data were as follows: tetramycin A (1628-MetK vs. 1628: 63.3 mg/g vs. 46.1 mg/g), tetramycin P (1628-MetK vs. 1628: 48.9 mg/g vs. 36.8 mg/g), tetrin B (1628-MetK vs. 1628: 18.0 mg/g vs. 13.7 mg/g), and TM (1628-MetK vs. 1628: 44.5 mg/g vs. 44.6 mg/g), suggesting that the effect on production and specific production per mg DCW was very similar. a One unit of activity is defined as the amount of enzyme that produced a change in optical density at 340 nm ( Okamoto et al., 2003 Okamoto et al., 2003 ). b Strains were cultured in fermentation medium. Samples were taken at the indicated time points, and SAM synthetase activity and intracellular levels were determined as described in Materials and methods ( Okamoto et al., 2003 Okamoto et al., 2003 ). SAM: S -adenosylmethionine. 2 Time (h) 4 SAM synthetase activity a (mU/mg protein) 3 Intracellular level of SAM b (pmol/mg wet weight) 2 1628 1628-ΔmetK sd 1628-MetK 1628 1628-ΔmetK sd 1628-MetK 2 24 1.00±0.06 0.20±0.01 3.1±0.3 10.1±1.4 3.6±0.5 24.4±2.2 2 48 2.30±0.15 0.40±0.03 6.7±0.4 69.2±6.2 12.3±1.6 113.3±8.3 2 72 1.50±0.08 0.20±0.02 5.3±0.4 76.4±6.8 8.3±1.1 120.2±7.6 2 96 1.30±0.07 0.10±0.02 4.3±0.2 55.1±5.4 7.5±0.7 108.1±6.2 To further determine whether SAM was involved in tetraene macrolide biosynthesis in S . diastatochromogenes 1628, knock-out plasmid pKC1132-‍Δ metK sd was constructed as described in the supplementary materials ( Fig. 3 Fig. 3 a). We disrupted the metK sd gene in the S . diastatochromogenes 1628 chromosome via double-crossover homologous recombination ( Fig. 3 Fig. 3 a) to generate mutant S . diastatochromogenes 1628-‍ΔmetK sd . As shown in Fig. 3 Fig. 3 b, a 1209-bp metK sd gene could be amplified from control strain 1628, but not from 1628-‍ΔmetK sd . In addition, amplicons of 5.4 kb and 4.2 kb represented products of the control strain and the mutant strain, respectively, suggesting that a double-crossover homologous recombinant event had occurred and that the metK sd gene was disrupted in the mutant 1628-‍ΔmetK sd . Subsequently, we determined the effect of disrupting the metK sd gene on secondary metabolite production of S . diastatochromogenes 1628. The results showed that intracellular concentrations of both SAM ( Table 1 Table 1 ) and tetraene macrolides ( Fig. 2 Fig. 2 b) in mutant 1628-‍ΔmetK sd were decreased sharply compared to those in control strain 1628. Complementation of 1628-‍ΔmetK sd with pSET152:: metK sd recovered SAM biosynthesis and tetraene macrolide production. Moreover, the results showed that disruption or complementation of metK sd in S . diastatochromogenes 1628 had no effect on TM production ( Fig. 2 Fig. 2 b) or on cell growth ( Fig. 2 Fig. 2 a). 1,2; SAM has been found to act as an intracellular signal molecule for regulating the secondary metabolites in Streptomyces species. It was reported that the ttmRI gene, belonging to the large adenosine triphosphate (ATP)-binding regulator of LuxR (LAL) family, is involved in the biosynthetic pathway of tetraene macrolides in Streptomyces hygrospinosus ( Cui et al., 2016 Cui et al., 2016 ). To test whether overexpression of metK sd could stimulate the transcriptional level of ttmRI (GenBank accession No. MW880243), we performed quantitative real-time PCR (qRT-PCR) analysis. The result is shown in Fig. 4 Fig. 4 . As compared with the control strain, the transcriptional level of ttmRI was greatly decreased in the mutant 1628-‍ΔmetK sd . In contrast, in recombinant strain S . diastatochromogenes 1628-MetK, the transcriptional level of ttmRI was obviously increased. This result demonstrated that overexpression of metK sd enhanced the transcriptional level of ttmRI and further improved tetramycin A and tetramycin P production. Moreover, the results also indicated that the disruption or overexpression of metK sd had no obvious effect on the transcriptional levels of toy genes (GenBank accession No. KY022432) ( Fig. 4 Fig. 4 ). 1;2; SAM, the major methyl donor in diverse biological processes, was found to be involved in the secondary metabolism in many Streptomyces species ( Park et al., 2005 Park et al., 2005 ). The major secondary metabolites of the antagonistic strain S . diastatochromogenes 1628 contain TM and three tetraene macrolides (tetramycin A, tetramycin P, and tetrin B). Overexpression of metK sd significantly promoted production of the three tetraene macrolides, whereas production of the three tetraene macrolides in mutant 1628-ΔmetK sd drastically decreased due to disruption of the metK sd gene. Production of the three tetraene macrolides in the complemented strain 1628-ΔmetK sd /pSET152:: metK sd was restored to a level comparable to that in the wild-type strain S . diastatochromogenes 1628. Therefore, increased SAM appears to promote overproduction of these three tetraene macrolides in S . diastatochromogenes 1628. In addition, it is worth mentioning that the slight special activity of SAM synthetase and concentration of intracellular SAM could still be detected in mutant 1628-ΔmetK sd . This suggests that strain cell may have an SAM synthetase isoenzyme that is involved in SAM biosynthesis. In addition, neither overexpression nor disruption of the metK sd gene has any obvious influence on cell growth of S . diastatochromogenes 1628. This observation is not consistent with report that SAM usually acts as a factor affecting morphological differentiation in Streptomyces ( Park et al., 2005 Park et al., 2005 ). On the other hand, TM biosynthesis was not significantly affected by the level of intracellular SAM. This conclusion can be supported by our experimental fermentation data on TM, whether in the ΔmetK sd mutant strain or in the recombinant strain harboring overexpression of metK sd . It has been reported that SAM donates a methyl group to metabolites to affect the production of various types of secondary metabolites ( Gu et al., 2016 Gu et al., 2016 ). So far, some studies have reported that overexpression of metK could enhance the production of polyketides ( Wang et al., 2007 Wang et al., 2007 ) or macrolides ( Wang et al., 2012 Wang et al., 2012 ), whereas the successful enhancement of nucleoside antibiotic production through exogenous addition of SAM or overexpression of metK has yet to be reported. TM is a pyrrolopyrimidine nucleoside antibiotic, and its biosynthetic pathway in Streptomyces rimosus was reported by McCarty and Bandarian (2008) McCarty and Bandarian (2008) , who used guanosine triphosphate (GTP) as a precursor. This could explain why SAM also has no significant effect on the biosynthesis of TM. Tetramycin is a 26-member tetraene antibiotic whose biosynthetic pathway in S. hygrospinosus var. beijingensis was reported by Cao et al. (2012) Cao et al. (2012) and Sheng et al. (2020) Sheng et al. (2020) . The extension and maturation of tetraenes backbone were catalyzed by multifunctional type I polyketide synthases (PKSs) with ten malonates, an acetate, and a methylmalonate; and glycosylation and oxidation of PKS backbone were accomplished with a post-PKS tailoring step to generate tetramycin. It seems that methionine is not a precursor of tetramycin. It has been reported that SAM can act as a signal factor to regulate the transcription of genes involved in secondary metabolite biosynthesis and further affect their production ( Huh et al., 2004 Huh et al., 2004 ; Xu et al., 2018 Xu et al., 2018 ). The tetramycin gene cluster has been reported from the genome of S . hygrospinosus var. beijingensis ACCC40068 ( Cao et al., 2012 Cao et al., 2012 ) and Streptomyces ahygroscopicus CGMCC 4.7082 ( Ren et al., 2014 Ren et al., 2014 ; Cui et al., 2015 Cui et al., 2015 ). Cui et al. (2016) Cui et al. (2016) revealed that ttmRI is a positive regulator that activates transcription of the tetramycin biosynthetic gene cluster. Therefore, we employed qRT-PCR to analyze the expression level of ttmRI in the wild-type strain, mutant strain, and recombinant strain. In accord with the production of tetraene macrolides, the recombinant strain 1628-MetK and mutant 1628-ΔmetK sd , respectively, showed remarkably higher and lower expression levels of ttmRI compared with the corresponding value of wild-type strain 1628, indicating that increased SAM promotes transcriptional expression of ttmRI . However, there were no significant differences in the transcriptional level of toy genes involved in TM biosynthesis among the three strains. Therefore, we speculate that SAM acts as a signal molecule to stimulate production of tetramycin, but not of TM. The greatest limitation of this study was that we did not study the regulatory mechanism of TTMRI on tetramycin biosynthesis in depth. In conclusion, our study shows that overexpression of the metK sd gene in S . diastatochromogenes 1628 leads to increased SAM and promotes the production of three tetraene macrolides, but that it has no effect on TM production. 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Arch Microbiol , 192 ( 4 ): 289 - 297 . https://doi.org/10.1007/s00203-010-0548-x https://doi.org/10.1007/s00203-010-0548-x S-adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces Yefeng HU, Juan WANG, and Jie XU conducted experiments. Yefeng HU wrote the original draft. Zheng MA designed the experiments and wrote this article. Andreas BECHTHOLD and Xiaoping YU gave some good suggestions on the revision of the manuscript, polished English and checked the final version. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This study was supported by the Excellent Youth Fund of Zhejiang Province, China (No. LR17C140002). 10.1631/jzus.B2100115 1673-1581（2021）09-0767-07 2021-02-08 2021-04-30 2021-09-15 2022-05-09 2021-07-14 © Zhejiang University Press 2021 Yefeng HU, Juan WANG, Jie XU, Zheng MA, Andreas BECHTHOLD, and Xiaoping YU declare that they have no conflict of interest. This article does not involve any studies with human or animal subjects. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 9 22