生物技术通报 ›› 2022, Vol. 38 ›› Issue (2): 205-217.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0447
邱益彬1,3(), 马艳琴2, 沙媛媛2, 朱逸凡2, 苏二正1, 雷鹏2, 李莎2, 徐虹2
收稿日期:
2021-04-07
出版日期:
2022-02-26
发布日期:
2022-03-09
作者简介:
邱益彬,男,博士,讲师,研究方向:微生物合成生物学与代谢工程;E-mail: 基金资助:
QIU Yi-bin1,3(), MA Yan-qin2, SHA Yuan-yuan2, ZHU Yi-fan2, SU Er-zheng1, LEI Peng2, LI Sha2, XU Hong2
Received:
2021-04-07
Published:
2022-02-26
Online:
2022-03-09
摘要:
解淀粉芽孢杆菌是FDA认定的安全级(generally recognized as safe,GRAS)菌株,在工业酶制剂、高分子聚合物、大宗化学品、绿色生物农药等方面的生产具有突出的优势。近年来,随着解淀粉芽孢杆菌的分子遗传操作技术越来越成熟,对利用该菌开发成微生物发酵平台化菌株用于合成生物学制造领域提出了更迫切的需求。文中围绕解淀粉芽孢杆菌的遗传操作工具、代谢改造应用和未来发展前景等方面进行了详细综述,为进一步推动解淀粉芽孢杆菌合成生物学技术的创新与发展提供借鉴和 参考。
邱益彬, 马艳琴, 沙媛媛, 朱逸凡, 苏二正, 雷鹏, 李莎, 徐虹. 解淀粉芽孢杆菌分子遗传操作及其应用研究进展[J]. 生物技术通报, 2022, 38(2): 205-217.
QIU Yi-bin, MA Yan-qin, SHA Yuan-yuan, ZHU Yi-fan, SU Er-zheng, LEI Peng, LI Sha, XU Hong. Research Progress in Molecular Genetic Manipulation Technology of Bacillus amyloliquefaciens and Its Application[J]. Biotechnology Bulletin, 2022, 38(2): 205-217.
Plasmid | Promoter | Terminator | Replicon | Reference |
---|---|---|---|---|
pUBXC | PxylA | t0 | repB | [ |
pDR | PHpaII | Tfd | repF | [ |
pMA5 | PHpaII | Tfd | repB | |
pNX01 | P43 | Tamy | Ori-rep(p2Sip) | [ |
pWH1520 | PxylA | pBC16 ori | [ | |
pKSV7 | P43 | Temperature-sensitive replication from pE194ts | ||
pNW33N | ctaB、qcr、qox、resD promoter | repB | [ | |
pLY-3 | Promoter from amyE of B. subtilis 168 | [ | ||
pLakr | PQ | [ | ||
pHT01 | Pgrac | Theta replicon | [ |
表1 B. amyloliquefaciens中所使用的合成生物学基因元件
Table 1 Synthetic biological genetic elements used in B. amyloliquefaciens
Plasmid | Promoter | Terminator | Replicon | Reference |
---|---|---|---|---|
pUBXC | PxylA | t0 | repB | [ |
pDR | PHpaII | Tfd | repF | [ |
pMA5 | PHpaII | Tfd | repB | |
pNX01 | P43 | Tamy | Ori-rep(p2Sip) | [ |
pWH1520 | PxylA | pBC16 ori | [ | |
pKSV7 | P43 | Temperature-sensitive replication from pE194ts | ||
pNW33N | ctaB、qcr、qox、resD promoter | repB | [ | |
pLY-3 | Promoter from amyE of B. subtilis 168 | [ | ||
pLakr | PQ | [ | ||
pHT01 | Pgrac | Theta replicon | [ |
图2 CRISPR-Cas9n基因编辑技术在B. amyloliquefaciens中的操作流程示意图 A:CRISPR-Cas9n系统的工作原理;B:CRISPR-Cas9n与传统同源重组技术操作流程的对比
Fig. 2 A schematic diagram of operation while CRISPR-Cas9n gene editing technology in B. amyloliquefaciens A:The gene editing mechanism of CRISPR-Cas9n. B:Comparison of CRISPR-Cas9n and conventional homologous recombination techniques
图3 CRISPR-dCas9n基因沉默技术在B. amyloliquefaciens中的构建 A:CRISPR-dCas9系统的工作原理;B:CRISPR-dCas9双质粒系统组成;C:靶向egfp表达框的sgRNA设计;不同sgRNA介导的egfp基因的转录水平;不同sgRNA调控下荧光共聚焦显微镜观察
Fig. 3 Construction of CRISPR-Cas9n gene silencing technology in B. amyloliquefaciens A:The catalytic mechanism of CRISPR-dCas9. B:Schematic representation of the CRISPR-dCas9 double plasmid system. C:Design of sgRNA targeting to egfp expression region. Transcription level of different sgRNA-mediated egfp genes,observed by fluorescence confocal microscopy under different sgRNA regulation
Strain | Transformation method | Concrete operation | Transformation plasmid | Transformation efficiency /(CFU·μg-1) |
---|---|---|---|---|
B. amyloliquefaciens TA208[ | Electroproration | Adding DL-threonine or glycine to weaken the cell wall in hypertonic medium | pUB110 | (1.13 ± 0.34)×107 |
Adding DL-threonine or glycine to weaken the cell wall in hypertonic medium;heat shock treatment | pHCMC02 | (8.94 ± 0.77)×105 | ||
B. amyloliquefaciens NB[ | Electroproration | Demethylation | pDR | 4.94 ± 0.42)×104 |
Demethylation | pMA5 | (6.15 ± 0.19)×103 | ||
B. amyloliquefaciens LL3[ | Electroproration | Demethylation | pWH1520 | 7.6×102 |
B. amyloliquefaciens[ | Chemical transformation | Overexpressing ComK regulator to induce the formation of competent | pUBXC | [(129 ± 20.6)- (1.7 ± 0.1)]×105 |
PCR fragment-mediated knockout | [(3.2 ± 0.76)- (3.5 ± 0.42)]×104 | |||
B. amyloliquefaciens[ | Transformation of protoplasts | Mediating by polyethylene glycol | pUB110 | (2-4)×105 |
表2 不同方法转化B. amyloliquefaciens转化效率的研究
Table 2 Study on the transformation efficiency of B. amyloliquefaciens by different methods
Strain | Transformation method | Concrete operation | Transformation plasmid | Transformation efficiency /(CFU·μg-1) |
---|---|---|---|---|
B. amyloliquefaciens TA208[ | Electroproration | Adding DL-threonine or glycine to weaken the cell wall in hypertonic medium | pUB110 | (1.13 ± 0.34)×107 |
Adding DL-threonine or glycine to weaken the cell wall in hypertonic medium;heat shock treatment | pHCMC02 | (8.94 ± 0.77)×105 | ||
B. amyloliquefaciens NB[ | Electroproration | Demethylation | pDR | 4.94 ± 0.42)×104 |
Demethylation | pMA5 | (6.15 ± 0.19)×103 | ||
B. amyloliquefaciens LL3[ | Electroproration | Demethylation | pWH1520 | 7.6×102 |
B. amyloliquefaciens[ | Chemical transformation | Overexpressing ComK regulator to induce the formation of competent | pUBXC | [(129 ± 20.6)- (1.7 ± 0.1)]×105 |
PCR fragment-mediated knockout | [(3.2 ± 0.76)- (3.5 ± 0.42)]×104 | |||
B. amyloliquefaciens[ | Transformation of protoplasts | Mediating by polyethylene glycol | pUB110 | (2-4)×105 |
Products | Strategies | Yield | Reference | |
---|---|---|---|---|
γ-PGA | Knock out genes involved in by-products,degrading enzymes and optimize the endogenous glutamate synthesis | 20.3 g/L | [ | |
Modification of substrate inulin utilization,sugar metabolism and by-product pathways | 32.14 g/L | [ | ||
Using the CRISPRi system’s multiple sgRNA combination strategy to control the expression of degrading enzymes,that achieve multiple molecular weights of γ-PGA | Different molecular weights of γ-PGA High(>800 kD)、middle(400-600 kD)、low(50-100 kD);25-27 g/L | [ | ||
To control the stereochemical configuration | Low-molecular-weight(<10 kD)γ-PGA;28.35 g/L | [ | ||
EPS | Optimization of fermentation conditions | 4.46 g/L | [ | |
Levan | Removing six protease-related genes,the biofilm matrix protein TasA and γ-PGA synthase genes | 31.1 g/L | [ | |
Optimization of levansucrase expression based on promoters and signal peptides | 102 g/L | [ | ||
Enzymes production | α-amylase | Optimization of the promoters and host | 2714 U/mL | [ |
Pullulanase | Cloning and expression optimization | 2.8 ASPU/mL | [ | |
Keratinase | Cloning and expression optimization | 1 361.54 U/mL | [ | |
lytic polysaccharide monooxygenase | Establishing a high-throughput screening system for expression and secretion | 12.17 U/g | [ | |
Nucleotides | Guanosine | Relieving the purine operon of the purine biosynthetic pathway and optimizing the energy of the respiratory chain | 19 g/L | [ |
Inosine | Protoplast fusion | 6 g/L | [ | |
Antimicrobial lipopeptides | Surfactin | Using nano iron particles to improve the permeability of cell membranes | 7.15 g/L | [ |
Iturin A | Overexpression of the iturin A biosynthesis genes | 37.35 mg/L | [ | |
Fengycin | Genome shuffling | 450.51 mg/L | [ | |
Bacillomycin D | Knocking out of the gene rapC | (360.8±30.7)mg/L | [ | |
Bulk chemicals | Acetoin | Compound mutagenesis | 85.2 g/L | [ |
2, 3-butanediol | Overexpression of glyceraldehyde-3-phosphate dehydrogenase and 2, 3-butanediol dehydrogenase | 132.9 g/L | [ |
表3 基于B. amyloliquefaciens合成的生物基产品汇总
Table 3 Summary of the synthesis of biobased products based on B. amyloliquefaciens species
Products | Strategies | Yield | Reference | |
---|---|---|---|---|
γ-PGA | Knock out genes involved in by-products,degrading enzymes and optimize the endogenous glutamate synthesis | 20.3 g/L | [ | |
Modification of substrate inulin utilization,sugar metabolism and by-product pathways | 32.14 g/L | [ | ||
Using the CRISPRi system’s multiple sgRNA combination strategy to control the expression of degrading enzymes,that achieve multiple molecular weights of γ-PGA | Different molecular weights of γ-PGA High(>800 kD)、middle(400-600 kD)、low(50-100 kD);25-27 g/L | [ | ||
To control the stereochemical configuration | Low-molecular-weight(<10 kD)γ-PGA;28.35 g/L | [ | ||
EPS | Optimization of fermentation conditions | 4.46 g/L | [ | |
Levan | Removing six protease-related genes,the biofilm matrix protein TasA and γ-PGA synthase genes | 31.1 g/L | [ | |
Optimization of levansucrase expression based on promoters and signal peptides | 102 g/L | [ | ||
Enzymes production | α-amylase | Optimization of the promoters and host | 2714 U/mL | [ |
Pullulanase | Cloning and expression optimization | 2.8 ASPU/mL | [ | |
Keratinase | Cloning and expression optimization | 1 361.54 U/mL | [ | |
lytic polysaccharide monooxygenase | Establishing a high-throughput screening system for expression and secretion | 12.17 U/g | [ | |
Nucleotides | Guanosine | Relieving the purine operon of the purine biosynthetic pathway and optimizing the energy of the respiratory chain | 19 g/L | [ |
Inosine | Protoplast fusion | 6 g/L | [ | |
Antimicrobial lipopeptides | Surfactin | Using nano iron particles to improve the permeability of cell membranes | 7.15 g/L | [ |
Iturin A | Overexpression of the iturin A biosynthesis genes | 37.35 mg/L | [ | |
Fengycin | Genome shuffling | 450.51 mg/L | [ | |
Bacillomycin D | Knocking out of the gene rapC | (360.8±30.7)mg/L | [ | |
Bulk chemicals | Acetoin | Compound mutagenesis | 85.2 g/L | [ |
2, 3-butanediol | Overexpression of glyceraldehyde-3-phosphate dehydrogenase and 2, 3-butanediol dehydrogenase | 132.9 g/L | [ |
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