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Enhancement of FK520 production in Streptomyces hygroscopicus by combining traditional mutagenesis with metabolic engineering

  • Applied genetics and molecular biotechnology
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Abstract

FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production.

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References

  • Armando JW, Boghigian BA, Pfeifer BA (2012) LC-MS/MS quantification of short-chain acyl-CoA’s in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity. Lett Appl Microbiol 54(2):140–148

    Article  CAS  PubMed  Google Scholar 

  • Bérdy J (2005) Bioactive Microbial Metabolites. J Antibiot 58(1):1–26

    Article  PubMed  Google Scholar 

  • Bierman M, Logan R, Obrien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116(1):43–49

    Article  CAS  PubMed  Google Scholar 

  • Chan YA, Podevels AM, Kevany BM, Thomas MG (2009) Biosynthesis of polyketide synthase extender units. Nat Prod Rep 26(1):90–114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen D, Zhang Q, Zhang Q, Cen P, Xu Z, Liu W (2012) Improvement of FK506 production in Streptomyces tsukubaensis by genetic enhancement of the supply of unusual polyketide extender units via utilization of two distinct site-specific recombination systems. Appl Environ Microbiol 78(15):5093–5103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dunn BJ, Khosla C (2013) Engineering the acyltransferase substrate specificity of assembly line polyketide synthases. J R Soc Interface 10(85):20130297–20130297

    Article  PubMed  PubMed Central  Google Scholar 

  • Eichenfield LF, Lucky AW, Boguniewicz M, Langley RG, Cherill R, Marshall K, Bush C, Graeber M (2002) Safety and efficacy of pimecrolimus (ASM 981) cream 1% in the treatment of mild and moderate atopic dermatitis in children and adolescents. J Am Acad Dermatol 46(4):495–504

    Article  PubMed  Google Scholar 

  • Jung WS, Yoo YJ, Park JW, Park SR, Han AR, Ban YH, Kim EJ, Kim E, Yoon YJ (2011) A combined approach of classical mutagenesis and rational metabolic engineering improves rapamycin biosynthesis and provides insights into methylmalonyl-CoA precursor supply pathway in Streptomyces hygroscopicus ATCC 29253. Appl Microbiol Biotechnol 91(5):1389–1397

    Article  CAS  PubMed  Google Scholar 

  • Jung WS, Kim E, Yoo YJ, Ban YH, Kim EJ, Yoon YJ (2014) Characterization and engineering of the ethylmalonyl-CoA pathway towards the improved heterologous production of polyketides in Streptomyces venezuelae. Appl Microbiol Biotechnol 98(8):3701–3713

    Article  CAS  PubMed  Google Scholar 

  • Kieser T, Bibb MJ, Butter MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics: a laboratory manual. The John Innes Foundation, Norwich

    Google Scholar 

  • Kosec G, Goranovic D, Mrak P, Fujs S, Kuscer E, Horvat J, Kopitar G, Petkovic H (2012) Novel chemobiosynthetic approach for exclusive production of FK506. Metab Eng 14(1):39–46

    Article  CAS  PubMed  Google Scholar 

  • Li L, Wei K, Liu X, Wu Y, Zheng G, Chen S, Jiang W, Lu Y (2019) aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in Actinomycetes. Metab Eng 52:153–167

    Article  CAS  PubMed  Google Scholar 

  • Lin J, Bai L, Deng Z, Zhong J (2011) Enhanced production of ansamitocin P-3 by addition of isobutanol in fermentation of Actinosynnema pretiosum. Bioresour Technol 102(2):1863–1868

    Article  CAS  PubMed  Google Scholar 

  • Liu H, Reynolds KA (1999) Role of crotonyl coenzyme A reductase in determining the ratio of polyketides monensin A and monensin B produced by Streptomyces cinnamonensis. J Bacteriol 181(21):6806–6813

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25(4):402–408

    Article  CAS  Google Scholar 

  • Lu C, Zhang X, Jiang M, Bai L (2016) Enhanced salinomycin production by adjusting the supply of polyketide extender units in Streptomyces albus. Metab Eng 35:129–137

    Article  CAS  PubMed  Google Scholar 

  • Ochi K, Hosaka T (2013) New strategies for drug discovery: activation of silent or weakly expressed microbial gene clusters. Appl Microbiol Biotechnol 97(1):87–98

    Article  CAS  PubMed  Google Scholar 

  • Qi H, Xin X, Li S, Wen J, Chen Y, Jia X (2012) Higher-level production of ascomycin (FK520) by Streptomyces hygroscopicus var. ascomyceticus irradiated by femtosecond laser. Biotechnol Bioprocess Eng 17(4):770–779

    Article  CAS  Google Scholar 

  • Qi H, Zhao S, Fu H, Wen J, Jia X (2014a) Enhancement of ascomycin production in Streptomyces hygroscopicus var. ascomyceticus by combining resin HP20 addition and metabolic profiling analysis. J Ind Microbiol Biotechnol 41(9):1365–1374

    Article  CAS  PubMed  Google Scholar 

  • Qi H, Zhao S, Wen J, Chen Y, Jia X (2014b) Analysis of ascomycin production enhanced by shikimic acid resistance and addition in Streptomyces hygroscopicus var. ascomyceticus. Biochem Eng J 82:124–133

    Article  CAS  Google Scholar 

  • Qi H, Lv M, Song K, Wen J (2017) Integration of parallel 13C-labeling experiments and in silico pathway analysis for enhanced production of ascomycin. Biotechnol Bioeng 114(5):1036–1044

    Article  CAS  PubMed  Google Scholar 

  • Ryu Y, Butler MJ, Chater KF, Lee KJ (2006) Engineering of primary carbohydrate metabolism for increased production of actinorhodin in Streptomyces coelicolor. Appl Environ Microbiol 72(11):7132–7139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Song K, Wei L, Liu J, Wang J, Qi H, Wen J (2017) Engineering of the LysR family transcriptional regulator FkbR1 and its target gene to improve ascomycin production. Appl Microbiol Biotechnol 101(11):4581–4592

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Zhang ZT, Seo SO, Lynn P, Lu T, Jin Y, Blaschek HM (2016) Gene transcription repression in Clostridium beijerinckii using CRISPR-dCas9. Biotechnol Bioeng 113(12):2739–2743

    Article  CAS  PubMed  Google Scholar 

  • Wang C, Liu J, Liu H, Wang J, Wen J (2017a) A genome-scale dynamic flux balance analysis model of Streptomyces tsukubaensis NRRL18488 to predict the targets for increasing FK506 production. Biochem Eng J 123:45–56

    Article  CAS  Google Scholar 

  • Wang J, Wang C, Song K, Wen J (2017b) Metabolic network model guided engineering ethylmalonyl-CoA pathway to improve ascomycin production in Streptomyces hygroscopicus var. ascomyceticus. Microb Cell Fact 16(1):169

    Article  PubMed  PubMed Central  Google Scholar 

  • Wei K, Wu Y, Li L, Jiang W, Hu J, Lu Y, Chen S (2018) MilR2, a novel TetR family regulator involved in 5-oxomilbemycin A3/A4 biosynthesis in Streptomyces hygroscopicus. Appl Microbiol Biotechnol 102(20):8841–8853

    Article  CAS  PubMed  Google Scholar 

  • Wu K, Chung L, Revill WP, Katz L, Reeves CD (2000) The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene 251(1):81–90

    Article  CAS  PubMed  Google Scholar 

  • Xia M, Huang D, Li S, Wen J, Jia X, Chen Y (2013) Enhanced FK506 production in Streptomyces tsukubaensis by rational feeding strategies based on comparative metabolic profiling analysis. Biotechnol Bioeng 110(10):2717–2730

    Article  CAS  PubMed  Google Scholar 

  • Xie H, Zhao Q, Zhang X, Kang Q, Bai L (2019) Comparative functional genomics of the acarbose producers reveals potential targets for metabolic engineering. Synth Syst Biotechnol 4(1):49–56

    Article  PubMed  PubMed Central  Google Scholar 

  • Yu Z, Shen X, Wu Y, Yang S, Ju D, Chen S (2019) Enhancement of ascomycin production via a combination of atmospheric and room temperature plasma mutagenesis in Streptomyces hygroscopicus and medium optimization. AMB Express 9(1):25

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhao Y, Li L, Zheng G, Jiang W, Deng Z, Wang Z, Lu Y (2018) CRISPR/dCas9-Mediated multiplex gene repression in Streptomyces. Biotechnol J 13(9):1800121

    Article  Google Scholar 

Download references

Acknowledgments

We thank the support from Professor Yinhua Lu (College of Life and Environmental Sciences, Shanghai Normal University), and Professor Weihong Jiang (Institute of Plant Physiology and Ecology, Chinese Academy of Sciences) for kindly providing the plasmid pSET-dCas9, PLCB1-A2BE and PLCBR1-A2BE.

Funding

This study was sponsored by Program of Shanghai Technology Research Leader (19XD1433200).

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Correspondence to Dianwen Ju or Shaoxin Chen.

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Yu, Z., Lv, H., Wu, Y. et al. Enhancement of FK520 production in Streptomyces hygroscopicus by combining traditional mutagenesis with metabolic engineering. Appl Microbiol Biotechnol 103, 9593–9606 (2019). https://doi.org/10.1007/s00253-019-10192-8

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