达托霉素高产菌株选育及基因组差异分析

doi:10.16085/j.issn.1000-6613.2024-1483

摘要/Abstract

摘要：

为提高玫瑰孢链霉菌的达托霉素产量，对原始菌株G904进行了甲基磺酸乙酯-氯化锂（EMS-LiCl）复合诱变。成功获得高产菌株G12，其摇瓶发酵产量达到152.47mg/L±0.49mg/L，是出发菌株的1.53倍。经平板传代、菌落形态及BOX-PCR分析显示，G12的高产性能稳定遗传，与G904相比，菌丝形态和基因组DNA在0.1kb处有显著差异，提示基因发生突变。全基因组测序和基因组学分析发现，G12含有大量与基因表达相关的蛋白质及更多的运输蛋白基因，具有更强的抗生素产生和分泌能力。此外，G12基因中检测到4个单核苷酸多态性（SNP），1个多核苷酸多态性（MNP）和1个特有的基因家族成员基因可能与达托霉素高产相关。研究表明，EMS-LiCl复合诱变是选育达托霉素高产菌株的有效方法，为微生物育种及菌株改良提供了技术参考，同时揭示了基因突变与达托霉素高产的关系，为进一步通过基因改造提高达托霉素产量奠定了理论基础。

关键词: 达托霉素, 甲基磺酸乙酯-氯化锂复合诱变, 菌株选育, 基因组学, 高产机制

Abstract:

To improve the daptomycin yield from Streptomyces roseosporus, the original strain G904 was subjected to ethyl methanesulfonate-lithium chloride (EMS-LiCl) combination mutagenesis. High-yielding strain G12 with a 152.47mg/L±0.49mg/L of shake flask fermentation yield was successfully obtained, which was 1.53 times higher than that of the starting strain. Plate passaging, colony morphology, and BOX-PCR analyses showed that the high-yielding performance of G12 was stably inherited, and compared with G904, the mycelial morphology, and genomic DNA were significantly different at 0.1kb, suggesting mutations in the genes. Whole genome sequencing and comparative genomics analyses revealed that G12 contained many proteins related to gene expression and more transport protein genes, and possessed enhanced antibiotic production and secretion. In addition, four single-base mutations (SNPs), one multi-base mutation (MNP), and one exclusive gene family were detected in the G12 gene that may be associated with high daptomycin production. The study demonstrated that EMS-LiCl combination mutagenesis was an effective method for breeding daptomycin high-yielding strains, which provided a technical reference for microbial breeding and strain improvement, and revealed the relationship between gene mutations and high daptomycin yield, which laid a theoretical foundation for further improving daptomycin yield by genetic modification.

Key words: daptomycin, ethyl methanesulfonate-lithium chloride combination mutagenesis, strain selection, genomics, high yield mechanism

中图分类号:

TQ927

郭启超, 刘闪, 席志文, 惠丰立. 达托霉素高产菌株选育及基因组差异分析[J]. 化工进展, 2025, 44(10): 5871-5880.

GUO Qichao, LIU Shan, XI Zhiwen, HUI Fengli. Breeding of high-yielding strains of daptomycin and genomic differences analysis[J]. Chemical Industry and Engineering Progress, 2025, 44(10): 5871-5880.

图/表 16

参考文献 29

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菌株	第一代产量/mg·L^-1	第二代产量/mg·L^-1	第三代产量/mg·L^-1	第四代产量/mg·L^-1	第五代产量/mg·L^-1
G12	151.35±1.21	152.31±0.50	151.95±0.57	152.03±0.32	152.47±0.49
G121	149.46±0.48	149.98±1.53	148.78±1.35	148.01±0.63	147.02±0.85
E4S5-9	148.52±1.40	145.68±2.06	141.99±0.94	139.48±1.92	136.74±1.45
E3L12-20	149.72±1.50	147.59±0.63	145.32±1.61	143.33±0.69	142.69±0.67

菌株	第一代产量/mg·L^-1	第二代产量/mg·L^-1	第三代产量/mg·L^-1	第四代产量/mg·L^-1	第五代产量/mg·L^-1
G12	151.35±1.21	152.31±0.50	151.95±0.57	152.03±0.32	152.47±0.49
G121	149.46±0.48	149.98±1.53	148.78±1.35	148.01±0.63	147.02±0.85
E4S5-9	148.52±1.40	145.68±2.06	141.99±0.94	139.48±1.92	136.74±1.45
E3L12-20	149.72±1.50	147.59±0.63	145.32±1.61	143.33±0.69	142.69±0.67

项目	G12	G904
编码基因数量	6790	6792
基因平均长度/bp	1007.63	1006.72
基因平均密度	0.88	0.89
基因的平均GC含量/%	71.87	71.88
基因占基因组百分比/%	89.11	89.16
基因间区长度/bp	836189	831597
基因间区GC含量/%	68.87	68.83
基因间区占基因组百分比/%	10.89	10.84

项目	G12	G904
编码基因数量	6790	6792
基因平均长度/bp	1007.63	1006.72
基因平均密度	0.88	0.89
基因的平均GC含量/%	71.87	71.88
基因占基因组百分比/%	89.11	89.16
基因间区长度/bp	836189	831597
基因间区GC含量/%	68.87	68.83
基因间区占基因组百分比/%	10.89	10.84

TCDB描述	TCDB家族	TCDB类别
MetQ protein-Bacillus subtilis subsp. subtilis str. 168.MetQ	The ATP-binding Cassette (ABC) Superfamily	Primary Active Transporters
D-methionine-binding lipoprotein MetQ precursor-Escherichia coli.	The ATP-binding Cassette (ABC) Superfamily	Primary Active Transporters
SDR family oxidoreductase [Pseudomonas abietaniphila]	The Pore-forming NADPH-dependent 1-Acyldihydroxyacetone Phosphate Reductase (Ayr1) Family	Channels/Pores