بیان ژن آنزیم بنزیل فورمات دکربوکسیلاز در باکتری e.coli

هدف: 1،2،4-بوتان تریول (bt)، یک ماده شیمیایی غیرطبیعی با کاربردهای دارویی و نظامی، از ویژگی‫های این ماده است. تولید زیستی bt هدف این تحقیق است، زیرا روش شیمیایی به‫دلیل محدودیت‌های کاتالیزوری و بازده پایین، اقتصادی نیست. تولید آنزیم بنزیل فورمات دکربوکسیلازبا هدف تکمیل مسیر آنزیمی سنتز زیستی bt می‌باشد که از زایلوز طی چهار واکنش آنزیمی انجام می‌شود و e. coli با دارا بودن دو آنزیم از این مسیر، با افزودن دو ژن دیگر، قادر به تکمیل آن خواهد بود. مواد و روش‫ها: به‫منظور ساخت سویه e. coli بیان‌کننده آنزیم بنزوئیل فرمات دکربوکسیلاز، ژن mdlc از p. putida تکثیر و در وکتورهای pbad و pet28 کلون شد. پس از تایید کلونینگ با آزمایش‌های تاییدی، بیان پروتئین با ژل sds-page و عملکرد آنزیم مورد بررسی قرار گرفت. نتایج: پس از انتقال سازواره بیانی به سویه مناسب، حضور باند پروتئینی 56 کیلودالتونی در ژل sds-page، بیان ژن mdlc را تایید کرد. برای ارزیابی عملکرد آنزیم، وکتور نوترکیب pbad.mdlc به سویه top10 ترنسفرم و تولید bt در محیط کشت با روش hplc آنالیز شد. نتیجه‌گیری: بنزوئیل فرمات دکربوکسیلاز (bfd)، آنزیمی کلیدی در مسیر تولید بوتان تریول (bt) درe. coli  است. در این مطالعه، ژن mdlc کد کننده bfd از p. putida تکثیر و در وکتورهای pbad و pet28 کلون شد. سیستم بیانی pet28 به‫دلیل سهولت استفاده انتخاب شد و وکتور pbad نیز به‫دلیل قابلیت القا مورد استفاده قرار گرفت. بیان پروتئین 56 کیلودالتونی با sds-page تایید و عملکرد آنزیم در تولید bt با hplc بررسی شد.

expression of the benzoylformate decarboxylase enzyme gene in escherichia coli

introduction: the construction of synthetic pathways within the framework of metabolic engineering is considered a modern approach in biotechnology, enabling the production of valuable compounds from natural biological resources. this strategy focuses on utilizing abundant biomaterials—particularly carbohydrates—for the industrial production of chemical compounds by modifying metabolic pathways in microorganisms. these processes can convert biomass derived from biological sources into fuels, chemicals, and polymers, thereby opening new opportunities for the sustainable production of chemical substances from renewable resources.aim: this study specifically focuses on the enzymatic production of benzoylformate decarboxylase (bfd) with the overarching goal of completing the enzymatic pathway for the biosynthesis of bt. this intricate pathway initiates with xylose as the primary carbon source and proceeds through a cascade of four distinct enzymatic reactions. notably, escherichia coli (e. coli), possessing two endogenous enzymes integral to this pathway, holds the potential for complete bt biosynthesis upon the introduction of the remaining two requisite genes. this research thus seeks to engineer e. coli as a robust biocatalyst for sustainable bt production. the strategic implementation of a fully functional enzymatic pathway within a well-characterized microbial host, such as  e. coli, promises a more environmentally benign and potentially more efficient route to bt synthesis compared to traditional chemical methods. furthermore, the ability to manipulate and optimize the expression of these key enzymatic components within e. coli offers opportunities to enhance the overall yield and productivity of the bioproduction process. the successful establishment of such a system could pave the way for large-scale, cost-effective, and sustainable production of this valuable chemical intermediate.materials and methods: to construct an e. coli strain capable of expressing the benzoylformate decarboxylase enzyme, the mdlc gene originating from pseudomonas putida was amplified and subsequently cloned into both pbad and pet28 expression vectors. following the confirmation of successful cloning through rigorous confirmatory assays, protein expression was evaluated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page), and the enzymatic activity was assessed.results: benzoylformate decarboxylase (bfd) is a pivotal enzyme within the engineered metabolic pathway for producing 1,2,4-butanetriol (bt) in   e.coli. in this study, the mdlc gene, encoding bfd from pseudomonas putida, was successfully amplified and cloned into the versatile pbad and the robust pet28 expression vectors. the pet28  system was preferred due to its ease of use and established track record in protein production, while the pbad vector was strategically employed for its inducible expression capabilities, allowing for controlled protein synthesis. the expression of the  56 kda target protein was confirmed through sds-page analysis, and the enzymatic function in the production of bt was subsequently verified using the sensitive and accurate hplc method. this work lays a crucial foundation for the further optimization and development of a fully functional and efficient microbial cell factory for the sustainable production of this valuable chemicalconclusion: the successful transfer of the expression construct into an appropriate e. coli host strain was confirmed by the presence of a distinct protein band at approximately 56 kda on the sds-page gel, unequivocally verifying the expression of the mdlc gene. to evaluate the functional capacity of the expressed enzyme, the recombinant vector pbad.mdlc was transformed into the e. coli top10 strain. the subsequent production of bt in the culture medium was meticulously analyzed using high-performance liquid chromatography (hplc).