近日,，國際雜志Applied Genetics and Molecular Biotechnology在線刊登了天津工業(yè)生物技術(shù)研究所研究人員的最新成果“Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization”，文章中，研究者揭示了在基因表達(dá)精確調(diào)控技術(shù)方面獲得的新進(jìn)展,。
基因表達(dá)精確調(diào)控技術(shù)是代謝工程和合成生物學(xué)的核心技術(shù)之一,，對優(yōu)化目標(biāo)產(chǎn)品合成途徑的效率,，提高人工合成細(xì)胞的生產(chǎn)能力起著至關(guān)重要的作用,。傳統(tǒng)的提高目標(biāo)產(chǎn)品合成能力的策略是采用質(zhì)粒過表達(dá)的方法對基因表達(dá)進(jìn)行調(diào)控，然而這種方法有許多弊端：1）需要添加IPTG和阿拉伯糖等昂貴的誘導(dǎo)劑來進(jìn)行誘導(dǎo),；2）質(zhì)粒的維持對宿主細(xì)胞會造成很大的代謝負(fù)荷,；3）很多質(zhì)粒的遺傳穩(wěn)定性不好；4）很多化合物的生產(chǎn)需要在細(xì)胞中構(gòu)建出一條復(fù)雜的合成途徑,，因此需要引入包含多個基因的長DNA片段,，而大部分質(zhì)粒都比較難攜帶長DNA片段；5）為了使微生物合成目標(biāo)產(chǎn)品的代謝流達(dá)到最大,，必須精確控制合成途徑中各個基因的表達(dá)強(qiáng)度,，使它們達(dá)到協(xié)同表達(dá)的狀態(tài)。
目前廣泛使用的誘導(dǎo)型啟動子其強(qiáng)度都是固定的,，不能滿足精確調(diào)控基因表達(dá)強(qiáng)度的需求,。通過使用具有不同表達(dá)強(qiáng)度的人工調(diào)控元件，直接在染色體上對基因表達(dá)進(jìn)行精確調(diào)控,，可以很好地解決上述質(zhì)粒過表達(dá)的問題,。
中國科學(xué)院天津工業(yè)生物技術(shù)研究所張學(xué)禮研究員領(lǐng)導(dǎo)的微生物代謝工程研究組通過Red重組技術(shù)，在大腸桿菌的染色體上分別構(gòu)建了啟動子,、信使RNA穩(wěn)定區(qū)和核糖體結(jié)合位點(diǎn)的標(biāo)準(zhǔn)化調(diào)控元件庫,，并精確定量了文庫中每個元件的強(qiáng)度。研究人員最終獲得一個包含400個調(diào)控元件的標(biāo)準(zhǔn)化數(shù)據(jù)庫,，元件的強(qiáng)度差異達(dá)到4個數(shù)量級,。在此基礎(chǔ)上，他們開發(fā)了一步法基因表達(dá)精確調(diào)控技術(shù),，可以使用一對通用引物,，快速高效地將待調(diào)控的基因表達(dá)替換為各種強(qiáng)度。該研究已經(jīng)申請了一項中國專利,。
張學(xué)禮研究組使用該技術(shù),，優(yōu)化了非PTS葡萄糖轉(zhuǎn)運(yùn)途徑。通過對galP和glk的基因表達(dá)的組合精確調(diào)控，篩選出一個最佳的調(diào)控強(qiáng)度組合,，使大腸桿菌的生長速率提高了4倍,，葡萄糖消耗速率提高了10倍，并有效地提高了丁二酸合成關(guān)鍵前體磷酸烯醇式丙酮酸（PEP）的供給,，為丁二酸合成途徑的優(yōu)化奠定了基礎(chǔ),。（生物谷Bioon.com）
doi:10.1007/s00253-011-3752-y
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Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization
Jiao Lu, Jinlei Tang, Yi Liu, Xinna Zhu, Tongcun Zhang and Xueli Zhang
Phosphoenolpyruvate (PEP) is an important precursor for anaerobic production of succinate and malate. Although inactivating PEP/carbohydrate phosphotransferase systems (PTS) could increase PEP supply, the resulting strain had a low glucose utilization rate. In order to improve anaerobic glucose utilization rate for efficient production of succinate and malate, combinatorial modulation of galactose permease (galP) and glucokinase (glk) gene expression was carried out in chromosome of an Escherichia coli strain with inactivated PTS. Libraries of artificial regulatory parts, including promoter and messenger RNA stabilizing region (mRS), were firstly constructed in front of β-galactosidase gene (lacZ) in E. coli chromosome through λ-Red recombination. Most regulatory parts selected from mRS library had constitutive strengths under different cultivation conditions. A convenient one-step recombination method was then used to modulate galP and glk gene expression with different regulatory parts. Glucose utilization rates of strains modulated with either galP or glk all increased, and the rates had a positive relation with expression strength of both genes. Combinatorial modulation had a synergistic effect on glucose utilization rate. The highest rate (1.64 g/L h) was tenfold higher than PTS− strain and 39% higher than the wild-type E. coli. These modulated strains could be used for efficient anaerobic production of succinate and malate.
