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精確調控技術方面獲得的新進展。
基因表達精確調控技術是代謝工程和合成生物學的核心技術之一,,對優(yōu)化目標產品合成途徑的效率,,提高人工合成細胞的生產能力起著至關重要的作用。傳統(tǒng)的提高目標產品合成能力的策略是采用質粒過表達的方法對基因表達進行調控,,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劑來進行誘導,;2)質粒的維持對宿主細胞會造成很大的代謝負荷;3)很多質粒的遺傳穩(wěn)定性不好,;4)很多化合物的生產需要在細胞中構建出一條復雜的合成途徑,,因此需要引入包含多個基因的長DNA片段,而大部分質粒都比較難攜帶長DNA片段,;5)為了使微生物合成目標產品的代謝流達到最大,,必須精確控制合成途徑中各個基因的表達強度,使它們達到協同表達的狀態(tài),。
目前廣泛使用的誘導型啟動子其強度都是固定的,,不能滿足精確調控基因表達強度的需求。通過使用具有不同表達強度的人工調控元件,,直接在染色體上對基因表達進行精確調控,,可以很好地解決上述質粒過表達的問題。
中國科學院天津工業(yè)生物技術研究所張學禮研究員領導的微生物代謝工程研究組通過Red重組技術,,在大腸桿菌的染色體上分別構建了啟動子,、信使RNA穩(wěn)定區(qū)和核糖體結合位點的標準化調控元件庫,并精確定量了文庫中每個元件的強度,。研究人員最終獲得一個包含400個調控元件的標準化數據庫,,元件的強度差異達到4個數量級。在此基礎上,,他們開發(fā)了一步法基因表達精確調控技術,,可以使用一對通用引物,快速高效地將待調控的基因表達替換為各種強度,。該研究已經申請了一項中國專利。
張學禮研究組使用該技術,,優(yōu)化了非PTS葡萄糖轉運途徑,。通過對galP和glk的基因表達的組合精確調控,篩選出一個最佳的調控強度組合,,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,。(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.