启动子篇:芽孢杆菌启动子

代谢工程可以通过改变遗传调控元器件的表达强度达到优化代谢途径的目的,进而实现目的产物的高效合成。遗传调控元器件主要包括启动子、核糖体结合位点、终止子以及其他一些调控基因表达的元件。

启动子的定义:

启动子是指一段能使基因进行转录的DNA 序列,包括核心启动子区 (−35 区、−10 区) 和调控区域。在转录起始时,启动子与RNA 聚合酶、调控基因转录的转录因子产生相互作用, 进而控制基因转录的起始时间和程度[1]

启动子结构

启动子的类型:

按照其诱导的方式可以分为组成型和诱导型启动子。
组成型启动子(Constitutive promoter)具有强启动、不需添加诱导剂、可直接产生目的蛋白以及成本低等优势,常见的如P43、PHpaII、Plaps及PamyQ;根据表达时期的不同,又可分为指数阶段、对数中期和早期平稳阶段、滞后对数和平稳阶段以及平稳阶段4 种类型。
诱导型启动子(Inducible promoter)可在特定的时期调控异源蛋白的表达量,具有可控、快速、大量等特点,如Pgrac、Pspac、Pxyl等[2]


组成型启动子

1.1 组成型启动子P43

原始文献:
Wang PZ, Doi RH. Overlapping promoters transcribed by bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases. J Biol Chem. 1984 Jul 10;259(13):8619-25. PMID: 6330116.
应用文献:

  1. Zhou C, Ye B, Cheng S, Zhao L, Liu Y, Jiang J, Yan X. Promoter engineering enables overproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis. Microb Cell Fact. 2019 Jun 14;18(1):111. doi: 10.1186/s12934-019-1159-0. PMID: 31200722; PMCID: PMC6570832.
  2. Chen J, Gai Y, Fu G, Zhou W, Zhang D, Wen J. Enhanced extracellular production of α-amylase in Bacillus subtilis by optimization of regulatory elements and over-expression of PrsA lipoprotein. Biotechnol Lett. 2015 Apr;37(4):899-906. doi: 10.1007/s10529-014-1755-3. Epub 2014 Dec 17. PMID: 25515799.

启动子特征:可在对数生长期和稳定期持续表达基因(logarithmic growth phase and stationary phase)
序列信息:K02174.1,B.subtilis P43 fragment containing two overlapping promoters
可选择载体:pP43NMK、pUBC19

组成型启动子P43

pP43NMK

pUBC19

1.2 组成型启动子PHpaII

原始文献:
Zyprian E, Matzura H. Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid pUB110 and development of a gram-positive expression vector system. DNA. 1986 Jun;5(3):219-25. doi: 10.1089/dna.1986.5.219. PMID: 3013549.
启动子特征:
可选择载体:pMA5
序列信息:

ggtggagattttttgagtgatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgacagcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgattta


1.3 组成型启动子Pylb

原始文献:
Yu X, Xu J, Liu X, Chu X, Wang P, Tian J, Wu N, Fan Y. Identification of a highly efficient stationary phase promoter in Bacillus subtilis. Sci Rep. 2015 Dec 17;5:18405. doi: 10.1038/srep18405. PMID: 26673679; PMCID: PMC4682092.
应用文献:

  1. Zhou C, Ye B, Cheng S, Zhao L, Liu Y, Jiang J, Yan X. Promoter engineering enables overproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis. Microb Cell Fact. 2019 Jun 14;18(1):111. doi: 10.1186/s12934-019-1159-0. PMID: 31200722; PMCID: PMC6570832.

启动子特征:平稳阶段,效果优于P43
可选择载体:根据pUBC19改造
序列信息:

Pylb(F4 strain)
ACTTCTCAAAGATCCCATGTGCTTAAAATTAAAGTTTAAATATTGGATTTTTTAAATAAAGCGTTTACAATATATGTAGAAACAACAAAGGGGGAGATTTGTTTGACAAAGGTAGAACGT

升级进化版本,升级编号NBP3510
原始文献:
Promoter engineering enablesoverproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis

1.4 组成型启动子PsrfA

原始文献:
Guan C, Cui W, Cheng J, Zhou L, Guo J, Hu X, Xiao G, Zhou Z. Construction and development of an auto-regulatory gene expression system in Bacillus subtilis. Microb Cell Fact. 2015 Sep 21;14:150. doi: 10.1186/s12934-015-0341-2. PMID: 26392346; PMCID: PMC4578258.
应用文献:

  1. Zhou C, Ye B, Cheng S, Zhao L, Liu Y, Jiang J, Yan X. Promoter engineering enables overproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis. Microb Cell Fact. 2019 Jun 14;18(1):111. doi: 10.1186/s12934-019-1159-0. PMID: 31200722; PMCID: PMC6570832.
    特征:stationary phase promoter

诱导型启动子

  • image.png

2.1 诱导型启动子Pgrac

文献出处:Phan TT, Nguyen HD, Schumann W. Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements. J Biotechnol. 2012 Jan;157(1):167-72. doi: 10.1016/j.jbiotec.2011.10.006. Epub 2011 Nov 10. PMID: 22100269.

启动子特征:通过融合groESL操纵子的强启动子和lac操纵子,产生Pgrac启动子,并构建了新的表达系统载体。
可选择载体:文中还构建了新的表达载体,pHT106、pHT104、pHT205、pHT100……这些载体就是整合了优化后的Pgrac启动子元件。
序列信息:

AAAATTTTTTATCTTATCACTTGAAATTGGAAGGGAGATTCTTTATTATAAGAATTGTGG

Genetic features of the Pgrac promoter fused to the reporter gene bgaB.

2.2 诱导型启动子Pgrac100

文献出处:Phan TT, Tran LT, Schumann W, Nguyen HD. Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli. Microb Cell Fact. 2015 May 21;14:72. doi: 10.1186/s12934-015-0255-z. PMID: 25990516; PMCID: PMC4446860.

启动子特征:在枯草芽孢杆菌确定合适的诱导型启动子控制重组蛋白表达,同时在没有诱导物的情况下在大肠杆菌中保持相对低的表达水平,是构建枯草芽孢杆菌表达载体的重要要求。通过启动子文库筛选(Pgrac-promoter library),作者筛选到满足上述两点要求的启动子 Pgrac100。
可选择载体:建了可供使用的表达载体。其中包括pHT253 (His-tag-MCS), pHT254 (MCS-His-tag) 和 pHT255 (MCS-Strep-tag)
序列信息:

AAAATTTTTTAAAAAATCTCTTGACATTGGAAGGGAGATATGTTATTATAAGAATTGCGG

image.png

2.3 诱导型启动子PxylA

文献出处:
Rygus T, Scheler A, Allmansberger R, Hillen W. Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization. Arch Microbiol. 1991;155(6):535-42. doi: 10.1007/BF00245346. PMID: 1719948.

应用文献:

  1. Development of a new integration site within the Bacillus subtilis chromosome and construction of compatible expression cassettes
  2. Bhavsar AP, Zhao X, Brown ED. Development and characterization of a xylose-dependent system for expression of cloned genes in Bacillus subtilis: conditional complementation of a teichoic acid mutant. Appl Environ Microbiol. 2001 Jan;67(1):403-10. doi: 10.1128/AEM.67.1.403-410.2001. Erratum in: Appl Environ Microbiol 2001 Nov;67(11):5349. PMID: 11133472; PMCID: PMC92592.
  3. Promoter engineering enables overproduction of foreign proteins from a single
    copy expression cassette in Bacillus subtilis

    image.png

    启动子特征:木糖诱导
    可用载体:pAX01

image.png
pX载体中PxylA序列

优化启动子
文献:Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon
木糖启动子对大部分代谢产物不敏感,受葡萄糖的抑制。

image.png

2.4 诱导型启动子Pspac

原始文献:
Yansura DG, Henner DJ. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci U S A. 1984 Jan;81(2):439-43. doi: 10.1073/pnas.81.2.439. PMID: 6420789; PMCID: PMC344692.
应用文献:
Development and characterization of a xylose-dependent system for expression of cloned genes in Bacillus subtilis: conditional complementation of a teichoic acid mutant


2.5 诱导型启动子Para

原始文献:
S-Nogueira I, Nogueira TV, Soares S, de Lencastre H. The Bacillus subtilis L-arabinose (ara) operon: nucleotide sequence, genetic organization and expression. Microbiology (Reading). 1997 Mar;143 ( Pt 3):957-969. doi: 10.1099/00221287-143-3-957. PMID: 9084180.
应用文献:
Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter

image.png

其他诱导型启动子

  1. 蔗糖启动子PsacB:启动子的转录不受蔗糖的严格调控,在没有诱导剂时也能进行转录,但较有蔗糖诱导时强度低100倍。
  2. 麦芽糖诱导型启动子Pglv:glv启动子以麦芽糖为诱导物,价格便宜,工业成本低,安全无毒,使用价值比较大。但是发酵中产生的葡萄糖会对启动子有强烈的反馈抑制,这是工业应用上面急需解决的一个问题。

启动子的预测

启动子的预测软件有Cpgplot、CpGPrediction、EMBOSS、NNPP、Promoter2.0、Promoter Scan等。


参考文献:
常见的真核和原核表达系统的启动子(promoters)


  1. 枯草芽孢杆菌代谢工程改造的策略与工具

  2. 双启动子促进亮氨酸脱氢酶在Bacillus subtilis中表达及发酵研究

版权声明:
作者:玉兰
链接:https://www.techfm.club/p/167062.html
来源:TechFM
文章版权归作者所有,未经允许请勿转载。

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