Dissecting and engineering of the TetR family regulator SACE_7301 for enhanced erythromycin production in Saccharopolyspora erythraea

Microbial Cell Factories

Research

Hang Wu¹  Yongrong Mao¹  Xunduan Huang¹  Meng Chen¹  Weiwei Li¹  Buchang Zhang¹  David T Weaver¹  Jingtao Liu²  Lixin Zhang³  Ying Zhou⁴  Bang-Ce Ye⁴ 

[1] Institute of Health Sciences, School of Life Sciences, Anhui University, 230601, Hefei, China;Institute of Health Sciences, School of Life Sciences, Anhui University, 230601, Hefei, China;Beijing Institute of Cell Biotechnology, 100043, Beijing, China;Institute of Health Sciences, School of Life Sciences, Anhui University, 230601, Hefei, China;CAS Key Laboratory of Pathogenic Microbiology & Immunology, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China;State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, 200237, Shanghai, China;

关键词: Saccharopolyspora erythraea;    Erythromycin;    SACE_7301;    TetR family regulator;    Gene overexpression;    Metabolic engineering;   

DOI  :  10.1186/s12934-014-0158-4

 received in 2014-06-29, accepted in 2014-10-23,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundSaccharopolyspora erythraea was extensively utilized for the industrial-scale production of erythromycin A (Er-A), a macrolide antibiotic commonly used in human medicine. Yet, S. erythraea lacks regulatory genes in the erythromycin biosynthetic gene (ery) cluster, hampering efforts to enhance Er-A production via the engineering of regulatory genes.ResultsBy the chromosome gene inactivation technique based on homologous recombination with linearized DNA fragments, we have inactivated a number of candidate TetR family transcriptional regulators (TFRs) and identified one TFR (SACE_7301) positively controlling erythromycin biosynthesis in S. erythraea A226. qRT-PCR and EMSA analyses demonstrated that SACE_7301 activated the transcription of erythromycin biosynthetic gene eryAI and the resistance gene ermE by interacting with their promoter regions with low affinities, similar to BldD (SACE_2077) previously identified to regulate erythromycin biosynthesis and morphological differentiation. Therefore, we designed a strategy for overexpressing SACE_7301 with 1 to 3 extra copies under the control of PermE* in A226. Following up-regulated transcriptional expression of SACE_7301, eryAI and ermE, the SACE_7301-overexpressed strains all increased Er-A production over A226 proportional to the number of copies. Likewise, when SACE_7301 was overexpressed in an industrial S. erythraea WB strain, Er-A yields of the mutants WB/7301, WB/2×7301 and WB/3×7301 were respectively increased by 17%, 29% and 42% relative to that of WB. In a 5 L fermentor, Er-A accumulation increased to 4,230 mg/L with the highest-yield strain WB/3×7301, an approximately 27% production improvement over WB (3,322 mg/L).ConclusionsWe have identified and characterized a TFR, SACE_7301, in S. erythraea that positively regulated erythromycin biosynthesis, and overexpression of SACE_7301 in wild-type and industrial S. erythraea strains enhanced Er-A yields. This study markedly improves our understanding of the unusual regulatory mechanism of erythromycin biosynthesis, and provides a novel strategy towards Er-A overproduction by engineering transcriptional regulators of S. erythraea.

【 授权许可】

Unknown   
© Wu et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.