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


RESULTS
By 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).


CONCLUSIONS
We 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.
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China. wuh2007@gmail.com.
Hang
H
Wu
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China. menstein@126.com.
Meng
M
Chen
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China. 18297938278@163.com.
Yongrong
Y
Mao
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China. 751379833@qq.com.
Weiwei
W
Li
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China. jtliuok@126.com.|Beijing Institute of Cell Biotechnology, Beijing, 100043, China. jtliuok@126.com.
Jingtao
J
Liu
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China. huangxunduan@163.com.
Xunduan
X
Huang
State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China. zhouying@ecust.edu.cn.
Ying
Y
Zhou
State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China. bcye@ecust.edu.cn.
Bang-Ce
BC
Ye
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China. zhanglixin@im.ac.cn.|CAS Key Laboratory of Pathogenic Microbiology & Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. zhanglixin@im.ac.cn.
Lixin
L
Zhang
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China. David.weaver.t@gmail.com.
David T
DT
Weaver
Institute of Health Sciences, School of Life Sciences, Anhui University, Hefei, 230601, China. zhbc@ahu.edu.cn.
Buchang
B
Zhang
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004917:Erythromycin / Q000096:biosynthesis*; D006801:Humans; D060847:Metabolic Engineering; D005810:Multigene Family; D016930:Saccharopolyspora / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D014157:Transcription Factors; D004917:Erythromycin