产Surfactin贝莱斯芽胞杆菌C5A-1的鉴定和所产Surfactin对植物的促生效果

doi:10.13560/j.cnki.biotech.bull.1985.2025-0364

摘要/Abstract

摘要：

目的筛选surfactin高产菌株，从基因组水平了解菌株的遗传背景和surfactin的生物合成过程，探索surfactin的植物促生功能。方法利用选择性分离技术从植物根际土中筛选surfactin产生菌；分别采用高效液相色谱和MALDI-TOF质谱对surfactin进行定量和结构类型分析；利用Illumina 结合 PacBio三代测序平台进行全基因组测序，利用生物信息学进行分类鉴定、功能基因注释、次级代谢基因簇和surfactin的生物合成过程分析；利用盆栽实验检测surfactin对植物幼苗的促生功能。结果从采集自山西大同杨树根际土壤中筛选到排油能力和抑菌能力强的菌株C5A-1，所产surfactin结构类型为C14-和C15-surfactin A，产量为1 208.16 mg/L。C5A-1与Bacillus velezensis FZB42的基因组平均核苷酸一致性（ANI）和数字DNA-DNA杂交值分别为98.35%和85.4%。C5A-1的基因组大小为3 929 585 bp，GC含量为46.5%，共编码3 747个基因，包含12个次级代谢产物基因簇。surfactin生物合成基因簇包含了核心基因srfAA、srfAB和srfAC。C5A-1所产surfactin能够显著提高毛白杨、玉米和大豆的株高、茎粗、地上和地下部的干重。结论 C5A-1为贝莱斯芽胞杆菌的一株新菌株，所产C14-和C15-surfactin A具有显著的植物促生功能，为代谢工程法改善surfactin产量提供了优良底盘细胞工厂，为surfactin在农林业的实际应用提供了理论基础。

关键词: 贝莱斯芽胞杆菌, surfactin, MALDI-TOF MS, 全基因组测序, 次级代谢产物基因簇

Abstract:

Objective This research is aimed to screen the high surfactin-producing strains, understand the genetic background and surfactin biosynthesis of the strains from the whole genome, and explore the functions of surfactin produced by the strains in plant growth-promoting. Method Surfactin-producing strains were isolated from plant rhizosphere soil by selective separation technique. The quantification and structural characterization of surfactin were performed using high-performance liquid chromatography and MALDI-TOF MS, respectively. The whole-genome was sequenced using Illumina + PacBio third-generation sequencing platform; and species identification, functional gene annotation, secondary metabolic gene clusters and surfactin biosynthesis were analyzed by bioinformatics method. Pot experiments were performed to detect the plant growth-promoting effect by surfactin. Result Strain C5A-1 with strong oil-displacing and antifungal activity was screened from poplar rhizosphere soil collected from Datong, Shanxi province. Surfactin homologues from C5A-1 were identified as C14- and C15-surfactin A, with the yield of 1 208.16 mg/L. The genomic average nucleotide identity (ANI) and digital DNA-DNA hybridization values between C5A-1 and B. velezensis FZB42 were 98.35% and 85.4%, respectively. C5A-1 genome size was 3 929 585 bp with 46.5% guanine-cytosine content, encoding 3 747 genes and harboring 12 secondary metabolite biosynthetic gene clusters. The surfactin biosynthetic gene cluster includes the core genes srfAA, srfAB, and srfAC. Surfactin produced by C5A-1 significantly enhanced the plant height, stem diameter, and aboveground and underground dry biomass of Populus tomentosa, Zea mays and Glycine max. Conclusion C5A-1 with high-producing surfactin was a novel strain belonging to B. velezensis, and the C14- and C15-surfactin A isoforms produced by C5A-1 demonstrated significant plant growth-promoting activity, which provides an optimized chassis cell factory for improving the surfactin yield by metabolic engineering strategies, and established a theoretical foundation for the practical application of surfactin in agricultural and forestry systems.

Key words: Bacillus velezensis, surfactin, MALDI-TOF MS, whole-genome sequencing, secondary metabolite biosynthetic gene clusters

吕镇, 甘恬, 霍思羽, 赵晨笛, 赵梦瑶, 李亚涛, 马玉超, 耿玉清. 产Surfactin贝莱斯芽胞杆菌C5A-1的鉴定和所产Surfactin对植物的促生效果[J]. 生物技术通报, 2025, 41(9): 265-276.

LYU Zhen, GAN Tian, HUO Si-yu, ZHAO Chen-di, ZHAO Meng-yao, LI Ya-tao, MA Yu-chao, GENG Yu-qing. Identification of Surfactin-producing Bacillus Velezensis C5A-1 and Evaluation of the Plant Growth-promoting Effects of Its Surfactin[J]. Biotechnology Bulletin, 2025, 41(9): 265-276.

图/表 10

表1 采样地情况

Table 1 Sampling sites

样品名称 Sample name	采样地 Sampling location	植物种类 Plant species	经纬度 Longitude and latitude
A5A	湖南省岳阳市	番茄	112°90′E 28°68′N
A5C	云南省丽江市	苹果	100°25′E 26°86′N
C5A	山西省大同市	杨树	113°20′E 40°3′N
A6A	北京市昌平区	黄豆	116°25′E 40°7′N
A6B	湖北省仙桃市	白萝卜	112°20′E 30°18′N
A6D	湖南省常德市	蒜苗	108°93’E 34°23’N

图1 Surfactin产生菌的筛选A：分离菌株的排油能力；B：分离菌株对立枯丝核菌的抑菌活性

Fig. 1 Screening of surfactin-producing strainsA: Oil-displacing e capacity of the isolated strains. B: Inhibitory activity of the isolated strains against R. solani

图2 C5A-1所产surfactin的结构鉴定

Fig. 2 Structural identification of surfactin from C5A-1

图3 基于16S rRNA基因构建的系统发育树

Fig. 3 Phylogenetic tree based on 16S rRNA genes

图4 C5A-1的基因组圈图

Fig. 4 Genome circle map of C5A-1

图5 C5A-1基因组的COG功能注释

Fig. 5 COG function annotation of the C5A-1 genome

图 6 C5A-1基因组的KEGG功能注释

Fig. 6 KEGG function annotation of C5A-1 genome

表2 C5A-1全基因组次级代谢基因簇分析

Table 2 Genome-wide secondary metabolite gene clusters from C5A-1

Cluster	Type	Start	End	Similar cluster	Similarity （%）
1	NRPS	323409	387387	Surfactin	82
2	PKS-like	923849	926504	Butirosin A/B	7
3	Terpene	1049972	1067381	-	-
4	Lanthipeptide-class-ii	1188370	1217259	-	-
5	TransAT-PKS	1383378	1471714	Macrolactin H	100
6	TransAT-PKS	1691242	1791808	Bacillaene	100
7	NRPS	1865549	1999860	Fengycin	100
8	Terpene	2028497	2025381	-	-
9	T3PKS	2113698	2154799	-	-
10	TransAT-PKS	2282174	2375967	Difficidin	100
11	NRPS	300670	3052462	Bacillibactin	100
12	Other	3588771	3630190	Bacilysin	100

图7 C5A-1的surfactin生物合成基因簇

Fig. 7 Surfactin biosynthetic gene cluster of C5A-1

图8 C5A-1所产的不同浓度surfactin对植物的促生功能A：毛白杨 84K：B：大豆；C：玉米。不同小写字母代表处理间差异显著（P<0.05）

Fig. 8 Plant-growth promoting by different concentrations of surfactin from C5A-1A: Populus tomentosa 84K. B: Glycine max. C: Zea mays. Different lowercase letters indicate significant differences between treatments (P<0.05)

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