代謝工程可以通過改變遺傳調(diào)控元器件的表達(dá)強(qiáng)度達(dá)到優(yōu)化代謝途徑的目的，進(jìn)而實(shí)現(xiàn)目的產(chǎn)物的高效合成。遺傳調(diào)控元器件主要包括啟動(dòng)子、核糖體結(jié)合位點(diǎn)、終止子以及其他一些調(diào)控基因表達(dá)的元件。

啟動(dòng)子的定義：

啟動(dòng)子是指一段能使基因進(jìn)行轉(zhuǎn)錄的DNA 序列，包括核心啟動(dòng)子區(qū) (?35 區(qū)、?10 區(qū)) 和調(diào)控區(qū)域。在轉(zhuǎn)錄起始時(shí)，啟動(dòng)子與RNA 聚合酶、調(diào)控基因轉(zhuǎn)錄的轉(zhuǎn)錄因子產(chǎn)生相互作用， 進(jìn)而控制基因轉(zhuǎn)錄的起始時(shí)間和程度^[1]。

啟動(dòng)子結(jié)構(gòu)

啟動(dòng)子的類型：

按照其誘導(dǎo)的方式可以分為組成型和誘導(dǎo)型啟動(dòng)子。
組成型啟動(dòng)子(Constitutive promoter)具有強(qiáng)啟動(dòng)、不需添加誘導(dǎo)劑、可直接產(chǎn)生目的蛋白以及成本低等優(yōu)勢(shì)，常見的如P43、PHpaII、Plaps及PamyQ；根據(jù)表達(dá)時(shí)期的不同，又可分為指數(shù)階段、對(duì)數(shù)中期和早期平穩(wěn)階段、滯后對(duì)數(shù)和平穩(wěn)階段以及平穩(wěn)階段4 種類型。
誘導(dǎo)型啟動(dòng)子（Inducible promoter）可在特定的時(shí)期調(diào)控異源蛋白的表達(dá)量，具有可控、快速、大量等特點(diǎn)，如Pgrac、Pspac、Pxyl等^[2]。

組成型啟動(dòng)子

1.1 組成型啟動(dòng)子P43

原始文獻(xiàn)：
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.
應(yīng)用文獻(xiàn)：

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.

啟動(dòng)子特征：可在對(duì)數(shù)生長(zhǎng)期和穩(wěn)定期持續(xù)表達(dá)基因（logarithmic growth phase and stationary phase）
序列信息：K02174.1，B.subtilis P43 fragment containing two overlapping promoters
可選擇載體：pP43NMK、pUBC19

組成型啟動(dòng)子P43

pP43NMK

pUBC19

1.2 組成型啟動(dòng)子PHpaII

原始文獻(xiàn)：
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.
啟動(dòng)子特征：
可選擇載體：pMA5
序列信息：

ggtggagattttttgagtgatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgacagcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgattta

1.3 組成型啟動(dòng)子Pylb

原始文獻(xiàn)：
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.
應(yīng)用文獻(xiàn)：

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.

啟動(dòng)子特征：平穩(wěn)階段，效果優(yōu)于P43
可選擇載體：根據(jù)pUBC19改造
序列信息：

Pylb(F4 strain)
ACTTCTCAAAGATCCCATGTGCTTAAAATTAAAGTTTAAATATTGGATTTTTTAAATAAAGCGTTTACAATATATGTAGAAACAACAAAGGGGGAGATTTGTTTGACAAAGGTAGAACGT

升級(jí)進(jìn)化版本，升級(jí)編號(hào)NBP3510
原始文獻(xiàn)：
Promoter engineering enablesoverproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis

1.4 組成型啟動(dòng)子PsrfA

原始文獻(xiàn)：
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.
應(yīng)用文獻(xiàn)：

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

誘導(dǎo)型啟動(dòng)子

• image.png

2.1 誘導(dǎo)型啟動(dòng)子Pgrac

文獻(xiàn)出處：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.

啟動(dòng)子特征：通過融合groESL操縱子的強(qiáng)啟動(dòng)子和lac操縱子，產(chǎn)生Pgrac啟動(dòng)子，并構(gòu)建了新的表達(dá)系統(tǒng)載體。
可選擇載體：文中還構(gòu)建了新的表達(dá)載體，pHT106、pHT104、pHT205、pHT100……這些載體就是整合了優(yōu)化后的Pgrac啟動(dòng)子元件。
序列信息：

AAAATTTTTTATCTTATCACTTGAAATTGGAAGGGAGATTCTTTATTATAAGAATTGTGG

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

2.2 誘導(dǎo)型啟動(dòng)子Pgrac100

文獻(xiàn)出處：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.

啟動(dòng)子特征：在枯草芽孢桿菌確定合適的誘導(dǎo)型啟動(dòng)子控制重組蛋白表達(dá)，同時(shí)在沒有誘導(dǎo)物的情況下在大腸桿菌中保持相對(duì)低的表達(dá)水平，是構(gòu)建枯草芽孢桿菌表達(dá)載體的重要要求。通過啟動(dòng)子文庫(kù)篩選（Pgrac-promoter library），作者篩選到滿足上述兩點(diǎn)要求的啟動(dòng)子 Pgrac100。
可選擇載體：建了可供使用的表達(dá)載體。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 誘導(dǎo)型啟動(dòng)子PxylA

文獻(xiàn)出處：
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.

應(yīng)用文獻(xiàn)：

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
   
   

   啟動(dòng)子特征：木糖誘導(dǎo)
   可用載體：pAX01

image.png

pX載體中PxylA序列

優(yōu)化啟動(dòng)子
文獻(xiàn)：Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon
木糖啟動(dòng)子對(duì)大部分代謝產(chǎn)物不敏感,受葡萄糖的抑制。

image.png

2.4 誘導(dǎo)型啟動(dòng)子Pspac

原始文獻(xiàn)：
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.
應(yīng)用文獻(xiàn)：
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 誘導(dǎo)型啟動(dòng)子Para

原始文獻(xiàn)：
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.
應(yīng)用文獻(xiàn)：
Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter

image.png

其他誘導(dǎo)型啟動(dòng)子

1. 蔗糖啟動(dòng)子PsacB：?jiǎn)?dòng)子的轉(zhuǎn)錄不受蔗糖的嚴(yán)格調(diào)控，在沒有誘導(dǎo)劑時(shí)也能進(jìn)行轉(zhuǎn)錄，但較有蔗糖誘導(dǎo)時(shí)強(qiáng)度低100倍。
2. 麥芽糖誘導(dǎo)型啟動(dòng)子Pglv：glv啟動(dòng)子以麥芽糖為誘導(dǎo)物，價(jià)格便宜，工業(yè)成本低，安全無毒，使用價(jià)值比較大。但是發(fā)酵中產(chǎn)生的葡萄糖會(huì)對(duì)啟動(dòng)子有強(qiáng)烈的反饋抑制,這是工業(yè)應(yīng)用上面急需解決的一個(gè)問題。

啟動(dòng)子的預(yù)測(cè)

啟動(dòng)子的預(yù)測(cè)軟件有Cpgplot、CpGPrediction、EMBOSS、NNPP、Promoter2.0、Promoter Scan等。

參考文獻(xiàn)：
常見的真核和原核表達(dá)系統(tǒng)的啟動(dòng)子（promoters）

1. 枯草芽孢桿菌代謝工程改造的策略與工具 ?

2. 雙啟動(dòng)子促進(jìn)亮氨酸脫氫酶在Bacillus subtilis中表達(dá)及發(fā)酵研究 ?