一株易转化、高生物量地衣芽孢杆菌的筛选与鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2024-0080

摘要/Abstract

摘要：

【目的】地衣芽孢杆菌（Bacillus licheniformis）是发酵工业中表达异源蛋白的重要底盘细胞，筛选易转化和高生物量的B. licheniformis菌株，为有效提升宿主的改造效率和蛋白表达水平提供菌种资源。【方法】经过富集、gyrB扩增与16S rDNA序列分析从土壤中筛选与地衣芽孢杆菌同源性较高的菌株，将所筛菌株的电转效率和生物量与工业常用底盘细胞B. licheniformis 2709进行比较分析。对筛选出的易电转和高生物量菌株进行形态学与生理生化鉴定，并进行抗生素敏感性测定和高温碱性环境生长情况测定。【结果】筛选出10株与地衣芽孢杆菌同源性较高的菌株，其中菌株1-33电转化效率为6 700 CFU/μg DNA，是B. licheniformis 2709的13.7倍。在豆粕玉米粉半固体培养基和SR液体培养基中培养，菌株1-33最大生物量达到5.2×10¹⁰ CFU/mL和6.8 g/L，分别为B. licheniformis 2709的1.71倍和1.3倍。进一步经过形态学、生理生化特性实验将菌株1-33最终鉴定为B. licheniformis，并表现出多种常用抗生素的敏感性和良好的耐碱与耐高温的能力。【结论】筛选出一株易电转和高生物量的B. licheniformis 1-33，为开发成高效表达外源蛋白的宿主菌奠定基础。

关键词: 地衣芽孢杆菌, 易转化, 高生物量, 筛选, 鉴定

Abstract:

【Objective】Bacillus licheniformis is an important chassis cell for expressing heterologous proteins in the fermentation industry. Screening B. licheniformis strains with easy electro-transfection and high biomass provides strain resources for effectively improving the host modification efficiency and protein expression level. 【Method】Screening of strains with high homology to B. licheniformis from soil was carried out through enrichment, gyrB amplification, and 16S rDNA sequence analysis. The electro-transfection efficiency and biomass of the screened strains were compared and analyzed with the commonly used chassis cell B. licheniformis 2709 in the industry. The screened strains with easy electro-transfection and high biomass were identified morphologically and physiologically and biochemically as well as were subjected to antibiotic susceptibility assay and growth in high temperature and alkaline environment. 【Result】Ten strains with high homology to B. licheniformis were screened. Among them, the strain 1-33 presented the highest electro-transfection efficiency with a maximum value of 6 700 transformants per μg DNA, which was 13.7 times higher than that of B. licheniformis 2709. Cultured in semi-solid soymeal-corn flour medium and SR liquid medium, the maximum biomass of strain 1-33 was found to be 5.2×10¹⁰ CFU/mL and 6.8 g/L, respectively, surpassing B. licheniformis 2709 by 1.71 and 1.3-fold. Further morphological and physiological experiments identified strain 1-33 as Bacillus licheniformis, which had sensitivity to various commonly used antibiotics and good alkaline and heat resistance. 【Conclusion】A B. licheniformis strain 1-33 with high electro-transfection efficiency and elevated biomass is screened, thereby foundation for its further development as an efficient host for exogenous protein expression in industrial process is established.

Key words: Bacillus licheniformis, easy electro-transfection, high biomass, screening, identification

杜薇, 李志敏, 邢晏铭, 刘蒲临, 缪礼鸿. 一株易转化、高生物量地衣芽孢杆菌的筛选与鉴定[J]. 生物技术通报, 2024, 40(9): 181-189.

DU Wei, LI Zhi-min, XING Yan-ming, LIU Pu-lin, MIAO Li-hong. Screening and Identification of a Bacillus licheniformis Strain with High Electro-transfection Efficiency and Elevated Biomass[J]. Biotechnology Bulletin, 2024, 40(9): 181-189.

图/表 11

表1 本实验用引物序列

Table 1 Primer sequences used in this experiment

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
gyrB-F^[19]	GCCGGCTTCATGGGTTCCG
gyrB-R^[19]	GCGTCGGTGCTTCTGTTG
27f^[20]	AGAGTTTGATCCTGGCTCAG
1492r^[20]	GGTTACCTTGTTACGACTT
GFP-F	CTGCGGCCGGTGCACATATGATGGTGAGCAAGGGCGAG
GFP-R	CTGCAGGTCGACAAGCTTCTACTTGTACAGCTCGTCCATG
pBES-F	AAGCTTGTCGACCTGCAGTC
pBES-R	CATATGCACCGGCCGC

图1 所筛选菌株的亲缘关系分析

Fig. 1 Phylogenetic classification of screened strains

图2 荧光显微镜观察菌体绿色荧光 A：明场观察野生菌株1-33；B：明场观察转入pBES-P43-egfp的菌株1-33；C：荧光激发观察野生菌株1-33；D：荧光激发观察转入pBES-P43-egfp的菌株1-33

Fig. 2 Fluorescence microscope observation of green fluorescence of bacteriophage A: Bright field observation of wild strains 1-33. B: Bright field observation of strains 1-33 transfected with pBES-P43-egfp. C: Fluorescence-excited observation of wild strains 1-33. D: Fluorescence-excited observation of strains 1-33 transferred to pBES-P43-egfp

图3 不同电压对B. licheniformis 2709转化效率的影响不同小写字母表示差异显著（P < 0.05），下同

Fig. 3 Effects of different voltages on the electro-transfection efficiency of B. licheniformis 2709 Different lowercase letters indicate significant differences（P < 0.05）, the same below

表2 不同菌株的转化效率

Table 2 Electro-transfection efficiency of different strains

菌株编号 Strain No.	转化效率Electro-transfection efficiency/ （CFU·μg^-1 DNA）	菌株编号 Strain No.	转化效率Electro-transfection efficiency/（CFU·μg^-1 DNA）
1-4	1 420±20	1-25	1 570±81
1-5	3 650±61	1-27	855±56
1-8	0±0	1-33	6 700±89
1-16	270±22	1-37	260±35
1-17	118±9	2709	490±29
1-19	416±46

图4 发酵培养基中不同菌株活菌数的测定

Fig. 4 Determination of the viable count of different strains in fermentation medium

表3 菌株1-33与B. licheniformis 2709干湿重对比

Table 3 Comparison of wet and dry weights of strain 1-33 and B. licheniformis 2709

菌株 Strain	湿重 Wet weight/（g·L^-1）	干重 Dry weight/（g·L^-1）
1-33	42.8±1.05	6.8±0.74
B. licheniformis 2709	33.1±0.42	5.2±0.11

图5 菌株1-33形态学特征 A：1-33的菌落形态；B：1-33的革兰氏染色菌体特征

Fig. 5 Morphological characteristics of strains 1-33 A: Colony morphology of 1-33. B: Gram-stained bacterial characteristics of 1-33（10×100）

表4 菌株1-33生理生化特征

Table 4 Physiological and biochemical characterization of strain 1-33

实验项目 Experimental project	结果 Result	实验项目 Experimental project	结果 Result
利用木糖	+	厌氧生长	+
利用阿拉伯糖	+	V-P实验	+
甘露醇	+	pH=5.7生长	+
明胶液化	+	柠檬酸盐	+
7% NaCl生长	+	丙酸盐	-
淀粉水解	+	硝酸盐还原	+

表5 四种抗生素对B. licheniformis 1-33的抑菌浓度

Table 5 Inhibitory concentrations of four antibiotics against B. licheniformis 1-33

抗生素种类 Antibiotic type	抑菌浓度 Inhibitory concentration/（μg·mL^-1）
氨苄青霉素Benzylpenicillin	10
四环素Tetracycline	25
卡那霉素Kanamycin	5
红霉素Erythromycin	2

图6 高温（A）和碱性条件（B）下B. licheniformis 1-33与B. licheniformis 2709的生长情况比较

Fig. 6 Comparison of the growth of B. licheniformis 1-33 and B. licheniformis 2709 under high temperature（A）and alkaline conditions（B）

参考文献 35

[1]	Chen JQ, Zhu YM, Fu G, et al. High-level intra- and extra-cellular production of D-psicose 3-epimerase via a modified xylose-inducible expression system in Bacillus subtilis[J]. J Ind Microbiol Biotechnol, 2016, 43(11): 1577-1591.
[2]	张莹, 韩晓静, 蔡逸安, 等. 解淀粉芽孢杆菌内源启动子的筛选及表达碱性果胶酶的应用研究[J]. 微生物学报, 2023, 63(4): 1575-1586.
	Zhang Y, Han XJ, Cai YA, et al. Screening of endogenous promoters of Bacillus amyloliquefaciens and application of them in the expression of alkaline pectinase[J]. Acta Microbiol Sin, 2023, 63(4): 1575-1586.
[3]	Shrestha S, Chio C, Khatiwada JR, et al. Optimization of multiple enzymes production by fermentation using lipid-producing Bacillus sp[J]. Front Microbiol, 2022, 13: 1049692.
[4]	Elemosho R, Suwanto A, Thenawidjaja M. Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase[J]. Electron J Biotechnol, 2021, 53: 71-79.
[5]	Wang Q, Zheng H, Wan X, et al. Optimization of inexpensive agricultural by-products as raw materials for bacitracin production in Bacillus licheniformis DW2[J]. Appl Biochem Biotechnol, 2017, 183(4): 1146-1157. doi: 10.1007/s12010-017-2489-1 pmid: 28593603
[6]	Li YX, Li ZR, Yamanaka K, et al. Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis[J]. Sci Rep, 2015, 5: 9383.
[7]	He FM, Gao BJ, Cheng X, et al. High-level production of poly-γ-glutamic acid by a newly isolated Bacillus sp. YJY-8 and potential use in increasing the production of tomato[J]. Prep Biochem Biotechnol, 2024, 54(5): 637-646.
[8]	Cai D, Rao Y, Zhan Y, et al. Engineering Bacillus for efficient production of heterologous protein: current progress, challenge and prospect[J]. J Appl Microbiol, 2019, 126(6): 1632-1642. doi: 10.1111/jam.14192 pmid: 30609144
[9]	Souza CC, Guimarães JM, Pereira SDS, et al. The multifunctionality of expression systems in Bacillus subtilis: emerging devices for the production of recombinant proteins[J]. Exp Biol Med, 2021, 246(23): 2443-2453.
[10]	Liu HL, Wang S, Song LX, et al. Trehalose production using recombinant trehalose synthase in Bacillus subtilis by integrating fermentation and biocatalysis[J]. J Agric Food Chem, 2019, 67(33): 9314-9324.
[11]	Zhang K, Su LQ, Wu J. Enhanced extracellular pullulanase production in Bacillus subtilis using protease-deficient strains and optimal feeding[J]. Appl Microbiol Biotechnol, 2018, 102(12): 5089-5103. doi: 10.1007/s00253-018-8965-x pmid: 29675805
[12]	Wang Y, Chen ZM, Zhao RL, et al. Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis[J]. Microb Cell Fact, 2014, 13: 129. doi: 10.1186/s12934-014-0129-9 pmid: 25176138
[13]	Wang Q, Yu HM, Wang MM, et al. Enhanced biosynthesis and characterization of surfactin isoforms with engineered Bacillus subtilis through promoter replacement and Vitreoscilla hemoglobin co-expression[J]. Process Biochemistry, 2018, 70:36-44.
[14]	Muras A, Romero M, Mayer C, et al. Biotechnological applications of Bacillus licheniformis[J]. Crit Rev Biotechnol, 2021, 41(4): 609-627.
[15]	Zhou CX, Zhou HY, Zhang HT, et al. Optimization of alkaline protease production by rational deletion of sporulation related genes in Bacillus licheniformis[J]. Microb Cell Fact, 2019, 18(1): 127.
[16]	Shen PL, Niu DD, Liu XL, et al. High-efficiency chromosomal integrative amplification strategy for overexpressing α-amylase in Bacillus licheniformis[J]. J Ind Microbiol Biotechnol, 2022, 49(3): kuac009.
[17]	陈坤. 2709碱性蛋白酶的高产工程菌株构建及应用性能分析[D]. 天津: 天津科技大学, 2018.
	Chen K. Construction of the engineering bacteria with high yield of alkaline protease 2709 and its application performance[D]. Tianjin:Tianjin University of Science & Technology, 2018.
[18]	Xue GP, Johnson JS, Dalrymple BP. High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis[J]. J Microbiol Meth, 1999, 34(3): 183-191.
[19]	Huang CH, Chang MT, Huang LN, et al. Development of a novel PCR assay based on the gyrase B gene for species identification of Bacillus licheniformis[J]. Mol Cell Probes, 2012, 26(5): 215-217.
[20]	马凯, 刘光全, 程池. 地衣芽孢杆菌16S rRNA基因的TD-PCR扩增及系统发育分析[J]. 微生物学通报, 2007, 34(4): 709-711.
	Ma K, Liu GQ, Cheng C. The TD-PCR and phylogenetic analysis of Bacillus licheniformis 16S rDNA[J]. Microbiology, 2007, 34(4): 709-711.
[21]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001.
	Dong XZ, Cai MY. Handbook of identification of common bacterial systems[M]. Beijing: Science Press, 2001.
[22]	Brockmeier U, Caspers M, Freudl R, et al. Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria[J]. J Mol Biol, 2006, 362(3): 393-402. pmid: 16930615
[23]	Zhan YY, Xu Y, Zheng PL, et al. Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis[J]. Appl Microbiol Biotechnol, 2020, 104(1): 391-403.
[24]	Zakataeva NP, Nikitina OV, Gronskiy SV, et al. A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains[J]. Appl Microbiol Biotechnol, 2010, 85(4): 1201-1209. doi: 10.1007/s00253-009-2276-1 pmid: 19820923
[25]	He HH, Zhang YP, Shi GY, et al. Recent biotechnological advances and future prospective of Bacillus licheniformis as microbial cell factories[J]. Syst Microbiol Biomanuf, 2023, 3(4): 521-532.
[26]	Ahn S, Jun SM, Ro HJ, et al. Complete genome of Bacillus subtilis subsp. subtilis KCTC 3135^T and variation in cell wall genes of B. subtilis strains[J]. J Microbiol Biotechnol, 2018, 28(10): 1760-1768.
[27]	Kunst F, Ogasawara N, Moszer I, et al. The complete genome sequence of the gram-positive bacterium Bacillus subtilis[J]. Nature, 1997, 390(6657): 249-256.
[28]	Rahimnahal S, Meimandipour A, Fayazi J, et al. Biochemical and molecular characterization of novel keratinolytic protease from Bacillus licheniformis(KRLr₁)[J]. Front Microbiol, 2023, 14: 1132760.
[29]	Priya I, Dhar MK, Bajaj BK, et al. Cellulolytic activity of thermophilic bacilli isolated from tattapani hot spring sediment in North West Himalayas[J]. Indian J Microbiol, 2016, 56(2): 228-231. doi: 10.1007/s12088-016-0578-4 pmid: 27570317
[30]	全爽, 陈涛, 毛宗林, 等. 一株土壤源地衣芽孢杆菌的鉴定及生物学特性研究[J]. 中国畜牧杂志, 2023, 59(1): 217-222.
	Quan S, Chen T, Mao ZL, et al. Identification and biological characterization of a soil-derived Bacillus licheniformis strain[J]. Chin J Anim Sci, 2023, 59(1): 217-222.
[31]	Li YR, Wang HR, Zhang L, et al. Efficient genome editing in Bacillus licheniformis mediated by a conditional CRISPR/Cas9 system[J]. Microorganisms, 2020, 8(5): 754.
[32]	Hoffmann K, Wollherr A, Larsen M, et al. Facilitation of direct conditional knockout of essential genes in Bacillus licheniformis DSM13 by comparative genetic analysis and manipulation of genetic competence[J]. Appl Environ Microbiol, 2010, 76(15): 5046-5057.
[33]	莫静燕, 陈献忠, 王正祥. 地衣芽孢杆菌原生质体的制备、再生及转化研究[J]. 生物技术, 2009, 19(5): 75-77.
	Mo JY, Chen XZ, Wang ZX. Preparation, regeneration and genetic transformation of Bacillus licheniformis protoplasts[J]. Biotechnology, 2009, 19(5): 75-77.
[34]	Zhang Y, Hu JM, Zhang Q, et al. Enhancement of alkaline protease production in recombinant Bacillus licheniformis by response surface methodology[J]. Bioresour Bioprocess, 2023, 10(1): 27.
[35]	肖静, 张虎, 李子源, 等. 抑制菌体自溶的地衣芽孢杆菌工程菌及其构建方法和应用:CN109868253A[P]. 2019-06-11.
	Xiao J, Zhang H, Li ZY, et al. Bacillus licheniformis engineered bacteria that inhibit bacterial autolysis and its construction method and application: CN109868253A[P]. 2019-06-11.