is a versatile industrial host for chemical production and has been engineered to produce efficiently many valuable compounds. 2-Deoxy--inosose (2-DOI) is an important precursor for the biosynthesis of 2-deoxystreptamine-containing aminoglycosides antibiotics and benzenoid metabolites. Bacterial and cyanobacterial strains have been metabolically engineered to generate 2-DOI; nevertheless, the production of 2-DOI using a yeast host has not been reported. Here, we have metabolically engineered a series of CEN.PK yeast strains to produce 2-DOI using a synthetically yeast codon-optimized gene from . The expression of the 2-Deoxy--inosose synthase (2-DOIS) gene was successfully achieved an expression vector and th... More
is a versatile industrial host for chemical production and has been engineered to produce efficiently many valuable compounds. 2-Deoxy--inosose (2-DOI) is an important precursor for the biosynthesis of 2-deoxystreptamine-containing aminoglycosides antibiotics and benzenoid metabolites. Bacterial and cyanobacterial strains have been metabolically engineered to generate 2-DOI; nevertheless, the production of 2-DOI using a yeast host has not been reported. Here, we have metabolically engineered a series of CEN.PK yeast strains to produce 2-DOI using a synthetically yeast codon-optimized gene from . The expression of the 2-Deoxy--inosose synthase (2-DOIS) gene was successfully achieved an expression vector and through chromosomal integration at a high-expression locus. In addition, the production of 2-DOI was further investigated for the CEN.PK knockout strains of phosphoglucose isomerase , D-glucose-6-phosphate dehydrogenase and a double mutant in a medium consisting of 2% fructose and 0.05% glucose as a carbon source We have found that all the recombinant strains are capable of producing 2-DOI and reducing it into -quercitol and (-)--quercitol. Comparatively, the high production of 2-DOI and its analogs was observed for the recombinant CEN.PK-btrC carrying the multicopy expression vector. GC/MS analysis of culture filtrates of this strain showed 11 times higher response in EIC for the 479 (methyloxime-tetra-TMS derivative of 2-DOI) than the YP-btrC recombinant that has only a single copy of expression cassette integrated into the genomic DNA of the CEN.PK strain. The knockout strains namely Δpgi1-btrC Δpgi1Δzwf1-btrC, that are transformed with the -expression plasmids, have inactive Pgi1 and produced only traces of the compounds. In contrast, Δzwf1-btrC recombinant which has intact yielded relatively higher amount of the carbocyclic compounds. Additionally, H-NMR analysis of samples showed slow consumption of fructose and no accumulation of 2-DOI and the quercitols in the culture broth of the recombinant CEN.PK-btrC suggesting that is capable of assimilating 2-DOI.
