Cell and Tissue Journal

Abstract 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 chemical
Conclusion: 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).
                                                           

Abstract Aim: Lignocellulosic biomass such as agricultural wastes (corn stover, sugar beet pulp and citrus peel) is a widely abundant and attractive source for the production of biofuels and chemicals.
biofuels are sources of clean and renewable energy that are considered as a potential substitute for non-renewable oil fuels. various methods and processes have been tested by scientists and researchers in this field and the most favorable conditions for producing biofuels from biomass. however, this biomass has not been fully exploited in many parts of the world for biofuel production, especially in developing countries, and there is little relation with crop residues and forest and waste in this area. so much work is still needed to replace fossil fuels with biofuels from biomass. lignocellulosic waste biomass such as cassava peels, sugar beet pulp, and Ulva lactuca are suitable materials for bioethanol synthesis.
D-xylose, is the second most abundant sugar in lignocellulosic hydrolysates. Nowadays, considerable efforts have been made to expand microbial cell factories to use D-xylose for the production of value-added chemicals. D-1,2,4-butanetriol (BT) is an extremely important intermediate chemical, is widely used in many fields, such as pharmaceuticals, paper, polymer materials, and military applications.A molecule 1,2,4-butanteriol (BT) is a polyol with unique chemical properties, which has a stereocenter and can be divided into D-BT (the S-enantiomer) and L-BT(the R-enantiomer). BT is widely used in the military industry, medicine, tobacco, polymer.  A synthetic pathway involving four enzymes—D-xylose dehydrogenase (XDH), D-xylonate dehydratase (XD), 2-keto acid decarboxylase (KDC), and aldehyde reductase (ALR)—has been proposed and implemented to produce BT from D-xylose, highlighting its significant role in bioproduction. And because in most studies, the xylonate dehydratase was used in the case of Caulobacter.crescentus, and given the very high genetic similarity between Caulobacter.crescentus and Caulobacter.vibrioides, the aim of this study is to clone and express xylonate dehydratase gene of C.vibrioides in the E.coli. For this purpose, the research was carried out with the aforementioned methods.
Material and methods:  The xylonate dehydratase gene was retrieved from the NCBI database and amplified using PCR with specific primers after extracting the C. vibrioides genome. The piece of the gene was cloned in the pET28 expression vector and then transferred to the E.coli prepared cells using chemical methods. After the induction of the cells, recombinant protein expression was examined using SDS-PAGE. Results: By using restriction enzymes, Colony PCR and sequencing, the cloning process and the entry of the gene into the pET28 expression vector was confirmed. The presence of the recombinant protein was tested by SDS-PAGE gel with a molecular weight of approximately 68 KDa and the expression rate of the recombinant protein, estimated by Image J software, was 54 percent.
Conclusion: The bioproduction of butanetriol requires the construction of a metabolic pathway consisting of several enzymes. The presence of the bacterium E.coli as the target strain and the use of cheap substrate such as xylose-containing biomass and the existence of the enzyme xylonate dehydratase are essential for the production of high-speed and high-volume D-1,2,4 butanetriol.   

Abstract Aim: In this century, the production of recombinant drugs such as growth hormone has increased. Different problems existed in the expressions of cytoplasmic and Periplasmic types of growth hormone. Therefore, finding a way to optimize expression is very necessary. In this study, we optimized expression of growth hormone in the form of solution state by trx-tag method. This method increase protein expression (Periplasmic problem) and also prevents formation of inclusion body (problem cytoplasmic).
Material and methods: Gene synthesis and gene cassette was done in pET 32a expression vector. Gene cassette contains trx tag for protein solubilization, His tag for purification and enterokinase for separate rHgh from previous tags.
 Results: After cloning the gene in vector, its expression was confirmed by Western blot technique. The results showed that the expression of fusion protein was done well.
Conclusion: the obtained finding proved that protein can be soluble by Trx-tag and increased its expression levels. Moreover, better results can be achieved in the fermentation and downstream processing.

Abstract Aim: The aim of this research was cloning and expression of human Midkine coding gene (mdk) in Escherichia coli that achieved in a laboratory-scale experiment.
Material and Methods: Methods were included cell culture, RNA extraction, cDNA synthesis, cloning techniques, induction of expression by IPTG (isopropyl thiogalactosidase), expression evaluation using polyacrylamide gel and confirmation by Western blot techniques.
Results: Midkine gene was cloned in pET-21a (+) and then transformed into Origami strain of E. coli. This growth factor was expressed in cytoplasmic level by a colony containing pETmdk recombinant after 16 hours incubation at 18°C and 250 rpm mixing. Expression of histidine tagged 13 kD protein confirmed by Western blotting technique.
Conclusion: Because Origami strain is trxB and gor genes mutant strain its cytoplasm is an oxidizing environment. Due to this, it enhances disulfide bond forming, therefore it seems that after expression of midkine, cysteine residues make an intra-molecular disulfide bridge and remains in soluble form. These conditions provide suitable environment for the proper folding of the protein and consequently solubilization of the protein.