Effect of ECF-Sig16 on Avermectin Production in Streptomyces avermitilis

doi:10.13304/j.nykjdb.2022.0105

Abstract

Abstract:

Streptomyces avermitilis produces avermectin， which is a kind of anthelmintic antibiotics with broad-spectrum， low toxicity activities and high potency and widely used in agricultural and medical fields. Extracytoplasmic function Sigma （ECF-Sigma） regulators play key roles for adaptation to changes of living environment and activating stress responses efficiently. To investigate the function of ECF-Sig16 regulator in avermectin biosynthesis， shaking flask fermentation of sig16 gene deletion， complementary， and overexpression strains were test. The results indicated that Sig16 inhibited avermectin biosynthesis， but exerted no function on cell growth. Phenotype observation experiment showed that Sig16 didn’t affect Streptomyces avermitilis morphological differentiation in some conditions. Electrophoretic mobility shift assay （EMSA） showed that Sig16 binded to the promoter of branched-chain amino acid ABC transporter operon SAV1190~SAV1194， which indicated Sig16 could inhibit avermectin production by affecting avermectin precursor supply.

Key words: avermectin, Streptomyces avermitilis, ECF-sigma regulators, Sig16

摘要：

阿维菌素是阿维链霉菌产生的一种广谱、高效、低毒的杀虫抗生素，在医药、农业和畜牧业领域有着广泛的应用。ECF-Sigma因子在细菌适应生存环境变化和高效响应激活压力中起重要作用。为研究阿维链霉菌中ECF-Sig16因子对阿维菌素合成的影响，通过对sig16基因缺失、回补和过表达，利用摇瓶发酵试验初步证实Sig16能够抑制阿维菌素的合成，但不影响菌株的生长。表型观察试验显示Sig16不影响阿维链霉菌的形态分化。通过凝胶阻滞试验（EMSA）确定Sig16直接结合在支链氨基酸ABC转运体操纵子SAV1190~SAV1194的启动子区，暗示Sig16可能通过影响阿维菌素生物合成的前体物质，抑制阿维菌素的合成。

关键词: 阿维菌素, 阿维链霉菌, ECF-Sigma调控蛋白, Sig16

CLC Number:

S182

Shuai LUO, Yaqin WEN, Hua ZHU, Rong ZHANG, Xiaowen WANG, Lili LIU, Jianya ZHU. Effect of ECF-Sig16 on Avermectin Production in Streptomyces avermitilis[J]. Journal of Agricultural Science and Technology, 2023, 25(7): 113-121.

罗帅, 问亚琴, 朱华, 张蓉, 王晓雯, 刘丽丽, 朱建亚. 阿维链霉菌中ECF-Sig16对阿维菌素合成的影响[J]. 中国农业科技导报, 2023, 25(7): 113-121.

Figures/Tables 7

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类型Type	名称Name	特征Character	来源Source or reference
质粒Plasmid	pKC1139	大肠杆菌-链霉菌穿梭质粒，多拷贝，温敏型E.coli-Streptomyces shuttle vector， multi-copy and temperature-sensitive	［32］
	pSET152	大肠杆菌-链霉菌穿梭质粒，整合型E.coli-Streptomyces shuttle vector， integrative	［32］
	pET-28a（+）	蛋白大量表达载体 Vector for heterologous protein overexpression	Novegen
	pJL117	携带红霉素抗性基因启动子的pIJ2925衍生质粒 pIJ2925 derivative carrying promoter with erythromycin resistance	［33］
菌株Strain	阿维链霉菌 S. avermitilis	野生型阿维链霉菌Wild-type （WT） S. avermitilis	实验室保存 Laboratary stock
大肠杆菌E. coli	JM109	基因克隆宿主菌 General cloning host	Novegen
	ET12567	甲基缺陷型菌株 MetHylation-defective strain	［34］
	BL21（DE3）	蛋白异源表达宿主菌 Host for protein heterologous overexpression	Novegen

类型Type	名称Name	特征Character	来源Source or reference
质粒Plasmid	pKC1139	大肠杆菌-链霉菌穿梭质粒，多拷贝，温敏型E.coli-Streptomyces shuttle vector， multi-copy and temperature-sensitive	［32］
	pSET152	大肠杆菌-链霉菌穿梭质粒，整合型E.coli-Streptomyces shuttle vector， integrative	［32］
	pET-28a（+）	蛋白大量表达载体 Vector for heterologous protein overexpression	Novegen
	pJL117	携带红霉素抗性基因启动子的pIJ2925衍生质粒 pIJ2925 derivative carrying promoter with erythromycin resistance	［33］
菌株Strain	阿维链霉菌 S. avermitilis	野生型阿维链霉菌Wild-type （WT） S. avermitilis	实验室保存 Laboratary stock
大肠杆菌E. coli	JM109	基因克隆宿主菌 General cloning host	Novegen
	ET12567	甲基缺陷型菌株 MetHylation-defective strain	［34］
	BL21（DE3）	蛋白异源表达宿主菌 Host for protein heterologous overexpression	Novegen

引物 Primer	引物序列 Primer sequence （5’-3’）	用途 Usage
LS63	CCCAAGCTTATGCTGGAGATCGACGACGT， HindⅢ	缺失sig16基因 Deletion of sig16
LS64	ATGGCCGTCGGTGTCGAGAGTGAACCGCTACACGCTCA
LS65	TGAGCGTGTAGCGGTTCACTCTCGACACCGACGGCCAT
LS66	GGAATTCGAACCACGACGAACAGCAGG， EcoRⅠ
LS67	CCCAAGGTGCAGGAGGTGTA	检测sig16基因是否缺失成功 Confirmation of sig16 deletion
LS68	GCATCACGTCGTTGAGCCC
LS69	TCCAGGACGCCTTCACC
LS70	GAAGATGAGGTAGACGACGG
LS81	CCCAAGCTTCGAACAGCACGTGGGCTT， HindⅢ	扩增sig16基因开放阅读框用于构建回补和过表达菌株 Amplification of sig16 open reading frame
LS82	GAAGATCTGCGGGATCTGCCTCATCAA， BglⅡ
LS89	GGAATTCCATATGGCCGTCGGTGTCG， NdeⅠ	扩增sig16基因开放阅读框用于大量表达His₆-Sig16蛋白 Amplification of sig16 open reading frame for overexpression of His₆-Sig16
LS90	CGGGATCCTCGAACAGCACGTGGGC， BamHⅠ
GJ105	GGTATTCCATTCGGTGTTGC	探针aveRp
GJ106	TGTTATGAATTTGCCCTGGTG	Probe aveRp
LS52	ATGGTCGGGAACCTCCGCAA	探针aveA1p
LS53	CTGTGTCCTCACCGCTAGGC	Probe aveA1p
GJ91	CCAAGGGCTACAAGTTCTCC	探针hrdB
GJ92	TTGATGACCTCGACCATGTG	Probe hrdB
LS207	CGCCTCGATACTGCTCGC	探针 SAV1196p
LS208	TGTGTCCTCCTCGGTGATCT	Probe SAV1196p
LS209	ACCGTGAACCAGTCGGGC	探针 SAV1188p
LS210	GGCATCCTGCTCCTCATCC	Probe SAV1188p
LS211	TCCACAGTTCCCGCCCTT	探针 SAV1190p
LS212	CGCTGGGACCGCTGGACA	Probe SAV1190p

引物 Primer	引物序列 Primer sequence （5’-3’）	用途 Usage
LS63	CCCAAGCTTATGCTGGAGATCGACGACGT， HindⅢ	缺失sig16基因 Deletion of sig16
LS64	ATGGCCGTCGGTGTCGAGAGTGAACCGCTACACGCTCA
LS65	TGAGCGTGTAGCGGTTCACTCTCGACACCGACGGCCAT
LS66	GGAATTCGAACCACGACGAACAGCAGG， EcoRⅠ
LS67	CCCAAGGTGCAGGAGGTGTA	检测sig16基因是否缺失成功 Confirmation of sig16 deletion
LS68	GCATCACGTCGTTGAGCCC
LS69	TCCAGGACGCCTTCACC
LS70	GAAGATGAGGTAGACGACGG
LS81	CCCAAGCTTCGAACAGCACGTGGGCTT， HindⅢ	扩增sig16基因开放阅读框用于构建回补和过表达菌株 Amplification of sig16 open reading frame
LS82	GAAGATCTGCGGGATCTGCCTCATCAA， BglⅡ
LS89	GGAATTCCATATGGCCGTCGGTGTCG， NdeⅠ	扩增sig16基因开放阅读框用于大量表达His₆-Sig16蛋白 Amplification of sig16 open reading frame for overexpression of His₆-Sig16
LS90	CGGGATCCTCGAACAGCACGTGGGC， BamHⅠ
GJ105	GGTATTCCATTCGGTGTTGC	探针aveRp
GJ106	TGTTATGAATTTGCCCTGGTG	Probe aveRp
LS52	ATGGTCGGGAACCTCCGCAA	探针aveA1p
LS53	CTGTGTCCTCACCGCTAGGC	Probe aveA1p
GJ91	CCAAGGGCTACAAGTTCTCC	探针hrdB
GJ92	TTGATGACCTCGACCATGTG	Probe hrdB
LS207	CGCCTCGATACTGCTCGC	探针 SAV1196p
LS208	TGTGTCCTCCTCGGTGATCT	Probe SAV1196p
LS209	ACCGTGAACCAGTCGGGC	探针 SAV1188p
LS210	GGCATCCTGCTCCTCATCC	Probe SAV1188p
LS211	TCCACAGTTCCCGCCCTT	探针 SAV1190p
LS212	CGCTGGGACCGCTGGACA	Probe SAV1190p

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