دانشگاه اراک سلول و بافت 2228-7035 17 1 2026 03 21 Cloning and surface display of xylose dehydrogenase from Caulobacter vibrioides Via E.coli transmembrane protein YiaT کلون و بیان سطحی آنزیم زایلوزدهیدروژناز از کلوباکتر ویبریوئیدس متصل به پروتئین عرض غشایی Yia Tدر باکتری اشریشیا کلی 1 16 732547 10.61882/jct.2025.2069888.2105 FA هانیه کرمی پور دانش آموخته کارشناسی ارشد، مجتمع آمایش و پدافند غیرعامل، دانشگاه مالک اشتر، تهران، ایران محمد جواد دهقان عصمت آبادی دانشگاه صنعتی مالک اشتر- مجتمع دانشگاهی پدافند غیرعامل-پژوهشکده علوم و فناوری زیستی 0000-0003-4961-4583 علی اصغر دلدار استادیار، مجتمع آمایش و پدافند غیرعامل، دانشگاه مالک اشتر، تهران، ایران 0000-0003-1894-8089 فاطمه بزرگمهر دانش آموخته کارشناسی ارشد، مجتمع آمایش و پدافند غیرعامل، دانشگاه مالک اشتر، تهران، ایران 0000-0002-7709-1604 Journal Article 2025 09 06 Introduction: Concern over fossil energy costs and environmental deterioration, along with energy security, has created a strong motivation for research and development of routes to provide sustainable, renewable fuels. In recent years, the use of biomass to produce highly valued chemicals has attracted widespread attention. lignocellulosic biomass, as a promising renewable resource for biofuel production, has distinct advantages in terms of economic and environmental aspects. The conversion of renewable raw materials to hydrocarbon fuels is an attractive alternative to fossil fuels from an economic and environmental point of view. The production process of lignocellulosic biomass mainly consists of biomass accumulation, biomass decomposition, simple sugars, and conversion of sugars to biofuel. One of the crucial steps in the economic success of lignocellulosic biofuels depends on the inhibition of competitive metabolism in microorganisms to achieve high productivity. To date, a growing focus is on the use of S. cerevisiae and E. coli as cell lines. These two cellular factories have the benefits that are well known. <br><br>Aim: The organic compound D-1,2,4-Butanetriol is a valuable chemical with wide-ranging applications in various fields. The chemical synthesis routes for BT have many drawbacks. By genetically modifying microorganisms, the metabolic pathway for producing many substances, including BT, can be engineered. The organic compound D-1,2,4-butanediol (BT) is an important intermediate chemical widely used in fields such as pharmaceuticals, paper, polymer materials, and military applications. When D-xylose sugar is provided to the bacterium, it is first converted to an intermediate compound called xylonolactone. This compound itself slowly converts into xylonate through a non-enzymatic reaction. To produce xylonate, the engineered bacteria have received xylose and initially, by a dehydrogenase reaction by the xylose dehydrogenase enzyme, that converts it into an intermediate substance: xylonolactone. The xylonolactone is converted slowly and in a nonenzymatic reaction to xylonate. Xylonate is a five-carbon organic acid. Over the past few years, xylonate has been increasingly being considered as an important chemical due to its potential as an important chemical component. Xylonate has many applications that can be used in the food, chemical, and pharmaceutical industries. Specifically, xylonate can act as a precursor for the synthesis of D-1,2,4-Butanetriol and a decrease in concrete water. E.coli due to fast growth in cheap culture medium, having two enzymes for the bto synthesis and product production in lower 24 hours of fermentation was chosen as the target strain of genetic engineering and metabolism. This study aimed to clone and express xylose dehydrogenase from Caulobacter vibrioides in E.coli. Materials and Methods: At first, to access the bacterial gene sequence, the genome of the target bacterium was extracted. Then, to create a strain expressing the enzymes xylose dehydrogenase and xylonolactonase, the genes for these proteins were amplified from Caulobacter vibrioides CB1 and transferred to E. coli. For this purpose, the target genes were amplified using specifically designed primers via the Polymerase Chain Reaction (PCR) method and initially cloned into a pTZ57cloning vector and then subcloned into pET 26b expression vector. At the final step, the expression of the enzyme was assessed by SDS-PAGE, and the other confirmation was the reduction of NAD+ to NADH, which was used as an activity indicator of the enzyme, as investigated by a change in NADH absorbance at 340 nm. Results: Confirmatory tests were performed to ensure the presence of the gene in the vectors (using restriction enzymes and colony PCR for gene amplification). The expression and activity of the enzyme were analysed. The recombinant protein's presence was confirmed by SDS-PAGE for the xylose dehydrogenase gene, with a molecular weight of 52.2 kDa. The estimated recombinant protein expression levels were approximately 25%. Conclusion: The objective of this research was solely to establish the metabolic pathway for xylonate production in E. coli by surface expression of enzymes in this pathway (xylose dehydrogenase). The results obtained in this study confirm that half of the pathway is active at the cell surface, but further experiments are required to determine the precise production levels and complete the pathway. This study aimed to create a metabolic pathway for producing xylonate in E.coli. هدف: ماده D-1,2,4 بوتان تریول یک ماده شیمیایی ارزشمند با کاربردهای گسترده در زمینه های مختلف است. هدف از انجام این مطالعه کلون‌سازی و بیان سطحی ژن آنزیم زایلوزدهیدروژناز از کلوباکتر ویبریوئیدس در ای. کلی میباشد. مواد و روش‌ها: برای دسترسی به باکتری ابتدا ژنوم باکتری مورد نظر استخراج شد. به منظور ساخت سویه‌ی بیان کننده آنزیم زایلوز دهیدروژناز ژن این پروتئین از باکتری کلوباکترویبریوئیدسCB1 تکثیر و به باکتری ای. کلی انتقال یافت. سپس ژن مورد نظربا استفاده از پرایمرهای طراحی شده به روش PCR تکثیر و ابتدا در وکتور کلونینگ pTZ57و سپس در وکتور بیانی pET 26 کلون شد. سپس بیان این آنزیم به وسیله‌ی SDS-PAGE و عملکرد آن با تغییر در جذب طول موج 340 نانومتر توسط NADH تولید شده مورد بررسی قرار گرفت. نتایج: با انجام آزمایش های تاییدی و اطمینان از وجود ژن در وکتورها(با استفاده از آنزیم‌های محدودالاثر و تکثیر ژن به کمک واکنش زنجیره‌ای پلی‌مراز (Colony PCR) و فرآیند کلونینگ)، بیان و عملکرد این آنزیم مورد بررسی قرار گرفت. وجود پروتئین نوترکیب توسط SDS-PAGE برای ژن زایلوز دهیدروژناز با وزن مولکولی 52.2 کیلودالتون بررسی و میزان بیان پروتئین نوترکیب با نرم افزار ImageJ ، تقریبا 25 درصد تخمین زده شد. نتیجه گیری:هدف از این تحقیق تنها ایجاد مسیر متابولیسمی تولید زایلونات در سویه ای. کلی به وسیله ی سطحی کردن اولین آنزیم این مسیر(زایلوزدهیدروژناز) بود که نتایج بدست آمده در این تحقیق موید فعال بودن این آنزیم در سطح سلول می‌باشد و برای تعیین میزان دقیق تولید و تکمیل کردن مسیرنیازمند بررسی های دیگرمی‌باشد. https://jct.araku.ac.ir/article_732547_18d8bb9199ecb9c42ed50826c6b83882.pdf