Construction of high polyoxin-producing strains by ultraviolet mutagenesis and duplication of a biosynthetic gene cluster

doi:10.12211/2096-8280.2020-032

Abstract

Abstract:

Modern agriculture urgently demands for green biological pesticides. Polyoxins are a class of nucleoside antibiotics with a broad spectrum of biological activities. Polyoxins show remarkable potency towards diverse crop diseases due to their competitive inhibition of the chitin synthetase's activity during the building of fungal cell wall and insect crust. This study aimed to construct a high polyoxin B-producing strain, which is one of the most bioactive ingredients in polyoxin derivatives. First, a high polyoxin B-producing mutant strain Pol-12, showing a 1.2-fold higher yield of polyoxin B than the wild-type strain, was obtained from the random mutants generated by ultraviolet mutagenesis of the starting strain Streptomyces ansochromogenes that was isolated from soil and stored by this laboratory. Second, the polyoxin biosynthetic gene cluster pol was directly cloned into p15A vector by ExoCET direct cloning method; and the original promoter and the kasOp* strong promoter were respectively added upstream of the first gene of pol. Third, the resulting shuttle vectors were used to transform the Pol-12 strain by interspecies conjugation and the gene cluster pol was integrated into the chromosome by integrase phiC31, leading to the pol-duplicated strains S. ansochromogenes Pol-12::P _ori -pol (M1) and S. ansochromogenes Pol-12::P _kasOp* -pol (M2). Compared with Pol-12, the yield of polyoxin B was increased by 22 and 33 times in M1 and M2, respectively. These results indicate that UV mutagenesis together with genetic engineering breeding can be applied in construction of high polyoxin-producing strains. Increase of the copy number of biosynthetic gene cluster and strong promoter insertion are effective for titer-improvement of polyoxin B.

Key words: polyoxins, Streptomyces, UV mutagenesis, ExoCET direct cloning, genetic engineering breeding

摘要：

多氧霉素是一种抑制几丁质生物合成的广谱抗真菌类抗生素，对多种真菌引起的农作物病害具有显著的防治效果，且对人和动植物无害，是一种绿色安全的生物农药，目前仍然是全球应用最广泛的抗真菌农药之一。多氧霉素的主要作用机制在于竞争性抑制真菌细胞壁合成中几丁质合成酶的活性，因此对农作物真菌病害具有显著的防治效果。现代农业的发展对于绿色生物农药的需求日益增长，本研究的目的是构建多氧霉素关键活性成分——多氧霉素B的高产菌株。从一株自土壤环境中分离得到的金色链霉菌（Streptomyces ansochromogenes）出发，首先通过紫外诱变初步筛选多氧霉素B的高产突变菌株；然后利用ExoCET直接克隆技术对多氧霉素基因簇pol进行克隆，并在基因簇第1个基因上游分别添加原始启动子和kasOp*强启动子，通过整合酶phiC31将基因簇整合到突变株染色体上构建pol倍增菌株，HPLC-MS检测比较多氧霉素B的产量。通过紫外诱变育种和筛选获得了链霉菌突变株Pol-12菌株，其产量较野生型菌株提高了1.2倍。为进一步提高多氧霉素产量，利用ExoCET直接克隆技术将pol克隆至p15A载体，并通过接合转移转化Pol-12菌株获得pol倍增菌株S. ansochromogenes Pol-12：：P _ori -pol（M1）和S. ansochromogenes Pol-12：：P _kasOp* -pol（M2）。与受体菌Pol-12相比，菌株M1和M2多氧霉素B的产量分别提高了22倍和33倍。因此得出结论：紫外随机诱变育种联合基因工程定向育种可有效应用于多氧霉素高产菌株的构建，增加基因簇的拷贝数以及强启动子插入有效提高了多氧霉素B的产量。

关键词: 多氧霉素, 链霉菌, 紫外诱变, ExoCET直接克隆, 基因工程育种

CLC Number:

Q 939.9

LIU Ruxin, DU Lei, XU Xiaoqing, DING Jinpeng, ZHANG Wei, LI Shengying. Construction of high polyoxin-producing strains by ultraviolet mutagenesis and duplication of a biosynthetic gene cluster[J]. Synthetic Biology Journal, 2020, 1(5): 609-620.

刘如欣, 杜磊, 徐晓庆, 丁金鹏, 张伟, 李盛英. 基于紫外诱变与生物合成基因簇倍增的多氧霉素高产菌株构建[J]. 合成生物学, 2020, 1(5): 609-620.

Figures/Tables 10

References 32

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	通讯作者：作者简介:李盛英（1978—），男，博士，教授。研究方向为合成生物学，微生物学及生物化学。

Type	Characteristics	Source
Strains
S. ansochromogenes	Wild type strain	This laboratory
S. ansochromogenes　Pol-12	High polyoxin B-producing strain prepared by ultraviolet mutagenesis	This study
S. ansochromogenes　Pol-12::P _ori-pol	High polyoxin B-producing strain upon integration of pol into the S. ansochromogenes Pol-12 chromosome	This study
S. ansochromogenes　Pol-12::P _kasOp-*pol	High polyoxin B-producing strain upon integration of pol into the S. ansochromogenes Pol-12 chromosome, and the strong promoter kasOp* is inserted upstream of the first gene of pol	This study
E. coli GB2005	E. coli DH10B, fhuA::IS2, ΔrecET, ΔybcC; endogenous recET and DLP12 phage ybcC genes are deleted, and fhuA is mutated to make it resistant to T1 phage	Gifted from Professor Zhang Youming at Shandong University
E. coli GB05-dir	E. coli GB2005, P_BAD-ETγA; recE, recT, redγ and recA regulated by the arabinose-induced promoter are integrated at the ybcC site on chromosome
E. coli GB08-red	E. coli GB2005, P_BAD-αβγA, ΔlacZ; redα, redβ, redγ, and recA regulated by the arabinose-induced promoter are integrated at the ybcC site on chromosome, and lacZ is knocked out
E. coli ET12567/　pUZ8002	Donor strain for conjugation, methylated defective strain, pUZ8002 plasmid contains the tra gene, which encodes the transfer protein Tra, and contains chloramphenicol and kanamycin resistance genes	This laboratory
Plasmids
pSC101-BAD-ETgA	The plasmid contains ETgA operon regulated by the arabinose-induced promoter and tetracycline resistance gene; the pSC101 origin is temperature-sensitive (replicates at 30 ℃ and stops at 37 ℃)	Gifted from Professor Zhang Youming at Shandong University
p15A-cm-tetR-tetO- 　hyg-ccdB	The plasmid contains ccdB, chloramphenicol, hygromycin and tetracycline resistance genes with a p15A origin
pR6K-oriT-phiC31	The plasmid contains oriT, integrase phiC31 and apramycin resistance genes with a pR6k origin

Type	Characteristics	Source
Strains
S. ansochromogenes	Wild type strain	This laboratory
S. ansochromogenes　Pol-12	High polyoxin B-producing strain prepared by ultraviolet mutagenesis	This study
S. ansochromogenes　Pol-12::P _ori-pol	High polyoxin B-producing strain upon integration of pol into the S. ansochromogenes Pol-12 chromosome	This study
S. ansochromogenes　Pol-12::P _kasOp-*pol	High polyoxin B-producing strain upon integration of pol into the S. ansochromogenes Pol-12 chromosome, and the strong promoter kasOp* is inserted upstream of the first gene of pol	This study
E. coli GB2005	E. coli DH10B, fhuA::IS2, ΔrecET, ΔybcC; endogenous recET and DLP12 phage ybcC genes are deleted, and fhuA is mutated to make it resistant to T1 phage	Gifted from Professor Zhang Youming at Shandong University
E. coli GB05-dir	E. coli GB2005, P_BAD-ETγA; recE, recT, redγ and recA regulated by the arabinose-induced promoter are integrated at the ybcC site on chromosome
E. coli GB08-red	E. coli GB2005, P_BAD-αβγA, ΔlacZ; redα, redβ, redγ, and recA regulated by the arabinose-induced promoter are integrated at the ybcC site on chromosome, and lacZ is knocked out
E. coli ET12567/　pUZ8002	Donor strain for conjugation, methylated defective strain, pUZ8002 plasmid contains the tra gene, which encodes the transfer protein Tra, and contains chloramphenicol and kanamycin resistance genes	This laboratory
Plasmids
pSC101-BAD-ETgA	The plasmid contains ETgA operon regulated by the arabinose-induced promoter and tetracycline resistance gene; the pSC101 origin is temperature-sensitive (replicates at 30 ℃ and stops at 37 ℃)	Gifted from Professor Zhang Youming at Shandong University
p15A-cm-tetR-tetO- 　hyg-ccdB	The plasmid contains ccdB, chloramphenicol, hygromycin and tetracycline resistance genes with a p15A origin
pR6K-oriT-phiC31	The plasmid contains oriT, integrase phiC31 and apramycin resistance genes with a pR6k origin

Oligos	Sequence
p15Acm-PO-1	ctccgtcatgtcctacccactctccgccggccccgcatgccgaagccgacAGATCCGAAAACCCCAAGTTACG
p15Acm-PO-2	tcaccacgggcgccggtgaatggctggagggacacgtcgagagcaatgcgAGATCCTTTCTCCTCTTTAGATC
p15A-seq-5	GGAAGCGGAATATATCCCTA
tetR-seq-3	GACCTCATTAAGCAGCTCTA
oripromoter-1	CTCCGTCATGTCCTACCCACTCT
oripromoter-2	CCGGTCCCGCCGACCGCCGCACT
p15Aapra-PO-1	ccccacccccatggccccgacgacgccgcgacggccgcgcccgtcgtgtcAGATCCGAAAACCCCAAGTTACG
p15Aapra-PO-2	aggcgtgctgggcgtggagtgcggcggtcggcgggaccggagatcctttcTCCTCTTTAGATC
p15Aapra-PO-3	CCCCACCCCCATGGCCCCGACGA
p15Aapra-kasOp*-PO-2	gtattctcctggccacgactttACAACACCGCACAGCATGTTGTCAAAGCAGAGACCGTTCGAATGTGAACAAGATCCTTTCTCCTCTTTAGATC
p15Aapra-kasOp*-PO-4	ccgacgcggccggcgtccgcggtggcagcggccgcggggaactccgtcatAACTCCCCCAGTCCTGCACGCTGTCGTATTCTCCTGGCCACGACTTT
attP-up	AAGCTCTAGCGATTCCAGAC
attP-dn	TTCGTTCAGGATGCGTTCGAA
attB-up	CCATCATCTTGTCCTTGGCCG
attB-dn	CTTCCGTCGTTACGGGTAGCA

Oligos	Sequence
p15Acm-PO-1	ctccgtcatgtcctacccactctccgccggccccgcatgccgaagccgacAGATCCGAAAACCCCAAGTTACG
p15Acm-PO-2	tcaccacgggcgccggtgaatggctggagggacacgtcgagagcaatgcgAGATCCTTTCTCCTCTTTAGATC
p15A-seq-5	GGAAGCGGAATATATCCCTA
tetR-seq-3	GACCTCATTAAGCAGCTCTA
oripromoter-1	CTCCGTCATGTCCTACCCACTCT
oripromoter-2	CCGGTCCCGCCGACCGCCGCACT
p15Aapra-PO-1	ccccacccccatggccccgacgacgccgcgacggccgcgcccgtcgtgtcAGATCCGAAAACCCCAAGTTACG
p15Aapra-PO-2	aggcgtgctgggcgtggagtgcggcggtcggcgggaccggagatcctttcTCCTCTTTAGATC
p15Aapra-PO-3	CCCCACCCCCATGGCCCCGACGA
p15Aapra-kasOp*-PO-2	gtattctcctggccacgactttACAACACCGCACAGCATGTTGTCAAAGCAGAGACCGTTCGAATGTGAACAAGATCCTTTCTCCTCTTTAGATC
p15Aapra-kasOp*-PO-4	ccgacgcggccggcgtccgcggtggcagcggccgcggggaactccgtcatAACTCCCCCAGTCCTGCACGCTGTCGTATTCTCCTGGCCACGACTTT
attP-up	AAGCTCTAGCGATTCCAGAC
attP-dn	TTCGTTCAGGATGCGTTCGAA
attB-up	CCATCATCTTGTCCTTGGCCG
attB-dn	CTTCCGTCGTTACGGGTAGCA

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