Paints and coatings are widely used in various fields of modern society, such as automobile, construction, wood, and transportation industries. According to the classification of resin, the coatings are segmented into acrylic, alkyd, epoxy, polyurethane (PU), polyster, and others. Among them, PU contains properties of scratch resistance, good gloss, thermal stability, and excellent adhesion. Due to its properties, the growing demand for PU resin coatings is expected to contribute significantly to the market size increase. 1,5-pentanediol (1,5-PDO) is one typical C5 diol which could be used as a building block for the production of PU, and polyesters and then used for the production of coating. The Global 1,5-Pentanediol Market Size was estimated at USD 48 million in 2022 and is projected to reach USD 62 million by 2029, exhibiting a CAGR of 3.6% during the forecast period.

Considering the important applications and broad market of 1,5-PDO, we decide to take this as the subject of our iGEM project this year. Currently, commercial manufacture of 1,5-PDO entirely relies on petroleum feedstock, mainly by hydrogenation-reduction of glutarate or its diesters. However, the capability of 1,5-PDO production based on the mentioned chemical approaches is constrained by the limited accessible C5 petroleum feedstocks. With the fast advancement of synthetic biology tools, as well as the increasing concerns on the environmental protection, the development of microbial cell factory to directly produce 1,5-PDO from renewable biomass feedstocks via a biological process is highly desired.

In this study, we first designed a metabolic pathway for 1,5-PDO biosynthesis from glucose in Escherichia coli. In the designed pathway, lysine derived from glucose is used as a precursor, which is then converted to 1,5-PDO by 5-hydroxyvaleric acid synthesis and 1,5-PDO synthesis module. The two modules are consisted by L-lysine monooxygenase, 5-aminovaleramide amidohydrolase, 5-aminovalerate aminotransferase, carboxylate reductases, and alcohol dehydrogenase, where four heterologous enzymes are required to express in E. coli. The feasibility of the synthetic pathway was identified by a metabolic network model. Subsequently, to obtain an efficient 1,5-PDO-producing cell factory, we first screened the functional enzymes to make the pathway work in E. coli. Subsequently, based on the metabolic network simulation, the overexpression of genes in synthetic 1,5-PDO pathways and deletion of genes in branched pathways are predicted. Therefore, the optimization of gene expression by changing the copy number of plasmid, deletion of the genes in branched pathways to inhibit acetic acid generation and 1,5-PDO degradation, rational engineering and enzyme assembly by protein scaffold to improve limiting enzymes activity were designed and performed to improve the production of the 1,5-PDO cell factory. Overall, our goal is to developa cell factory for the production of 1,5-PDO, providing insights for the biological production of 1,5-PDO from renewable carbon source.

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