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Applied Microbiology and Biotechnology

, Volume 103, Issue 23–24, pp 9593–9606 | Cite as

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

  • Zhituo Yu
  • Huihui Lv
  • Yuanjie Wu
  • Tengyun Wei
  • Songbai Yang
  • Dianwen JuEmail author
  • Shaoxin ChenEmail author
Applied genetics and molecular biotechnology
  • 206 Downloads

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.

Keywords

FK520 Streptomyces hygroscopicus Mutagenesis Metabolic engineering 

Notes

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 information

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

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10192_MOESM1_ESM.pdf (565 kb)
ESM 1 (PDF 565 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biological MedicinesFudan University School of PharmacyShanghaiChina
  2. 2.Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical IndustryShanghaiChina

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