Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET recombineering.
Heterologous expression of biosynthetic pathways is an important way to research and discover microbial natural products. Bacillus subtilis is a suitable host for the heterologous production of natural products from bacilli and related Firmicutes. Existing technologies for heterologous expression of large biosynthetic gene clusters in B. subtilis are complicated. Herein, we present a simple and rapid strategy for direct cloning based heterologous expression of biosynthetic pathways in B. subtilis via Red/ET recombineering, using a 5.2 kb specific direct cloning vector carrying homologous sequences to the amyE gene in B. subtilis and CcdB counterselection marker. Using a two-step procedure, two large biosynthetic pathways for edeine (48.3 kb) and bacillomycin (37.2 kb) from Brevibacillus brevis X23 and B. amyloliquefaciens FZB42, respectively, were directly cloned and subsequently integrated into the chromosome of B. subtilis within one week. The gene cluster for bacillomycin was successfully expressed in the heterologous host, although edeine production was not detectable. Compared with similar technologies, this method offers a simpler and more feasible system for the discovery of natural products from bacilli and related genera.
College of Plant Protection, Hunan Agricultural University, Changsha, 410128, People's Republic of China.|Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.|Hunan Institute of Microbiology, Changsha, 410009, People's Republic of China.
Qingshu
Q
Liu
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Qiyao
Q
Shen
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Xiaoying
X
Bian
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Hanna
H
Chen
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Jun
J
Fu
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Hailong
H
Wang
Hunan Institute of Microbiology, Changsha, 410009, People's Republic of China.
Ping
P
Lei
Hunan Institute of Microbiology, Changsha, 410009, People's Republic of China.
Zhaohui
Z
Guo
College of Plant Protection, Hunan Agricultural University, Changsha, 410128, People's Republic of China.
Wu
W
Chen
College of Plant Protection, Hunan Agricultural University, Changsha, 410128, People's Republic of China.|Hunan Business College, Changsha, 410205, People's Republic of China.
Dingjun
D
Li
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Youming
Y
Zhang
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