Application Of DNA Assembler In Biosynthesis Of Cis, Cis - Adipic Acid And Siderophere

The first part:The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Here, we present the production of muconic acid in Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Klebsiella pnemoniae, protocatechuic acid decarboxylase from K. pnemoniae, and catechol 1,2-dioxygenase from Acinetobacter baylyi imported into yeast. Because protocatechuic acid decarboxylase is the rate-limiting step in the pathway, an integration cassette, containing a selectable maker(G418) and protocatechuic acid decarboxylase gene(AroY),was designed and integrated into chromosomal delta-sequences by yeast transformation. Through selection on increasing concentrations of G418 after repeated transformation, clones with increasing copies of the inserted genes could be isolated. Finally, strains with 9 copies of the integration construct per genome were obtained. The corresponding muconic acid production increased up to 272mg/L, which is about 76% increase compared to the parental strain.The second part:Ferrimycins are antibiotics containing siderophore and function with special mechanism. The siderophore can target to the specific iron transport system in bacteria and contribute to the active transport of antibiotics with high efficiency, which can enhance the efficacy of the antibiotic significantly. Albomycin is a typical example of ferrimycin family which contains siderophore and produced by Streptomyces. Based on the proposed gene cluster of albomycin, we constructed the biosynthetic pathway for siderophore via DNA assembler and tried to heterologously express in S. lividans TK64 to produce siderophore.The third part:Optically pure amino acids have been widely used as intermediates in the synthesis of antibiotics, antifungal agents, pesticides, and sweeteners. Of particular importance, D-p-hydroxyphenylglycine(D-HPG) can be used for the production of antibiotics like amoxicillin and cefadroxil and is one of the most needed pharmaceutical intermediate in China. It can be produced from D, L-hydroxyphenly hydantoin (D, L-HPH) in a two-step reaction mediated by D-hydantoinase and N-carbamoyl-D-amino acid amidohydrolase. To make this production more industrially appealing, we engineer a fusion protein of the two enzymes with a chitin binding domain and heterogenously expressed in Escherichia coli. Fortunately, we get a soluble fusion protein which can catalyze production of D-HPG in one step. Latter, we try to immobilize the fusion protein to chitin, which allows enzyme reutilization. Due to the instability of N-carbamoyl-D-amino acid amidohydrolase, error-prone PCR is used to create a protein mutant library, and by using the High-throughput screening method to get an enzyme exhibits high tolerance to heat and hydrogen peroxide.