Metabolically engineered recombinant Saccharomyces cerevisiae for the production of 2-Deoxy-scyllo-inosose (2-DOI)
AuthorsAl-Fahad, Ahmed J.
Al-Fageeh, Mohamed B.
Kharbatia, Najeh M.
Online Publication Date2020-05-31
Print Publication Date2020-12
Permanent link to this recordhttp://hdl.handle.net/10754/664209
MetadataShow full item record
AbstractSaccharomyces cerevisiae is a versatile industrial host for chemical production and has been engineered to produce efficiently many valuable compounds. 2-Deoxy-scyllo-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 btrC gene from Bacillus circulans. The expression of the 2-Deoxy-scyllo-inosose synthase (2-DOIS) gene was successfully achieved via 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 (Δpgi1), D-glucose-6-phosphate dehydrogenase (Δzwf1) and a double mutant (Δpgi1, Δzwf1) 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 scyllo-quercitol and (−)-vibo-quercitol. Comparatively, the high production of 2-DOI and its analogs was observed for the recombinant CEN.PK-btrC carrying the multicopy btrC-expression vector. GC/MS analysis of culture filtrates of this strain showed 11 times higher response in EIC for the m/z 479 (methyloxime-tetra-TMS derivative of 2-DOI) than the YP-btrC recombinant that has only a single copy of btrC expression cassette integrated into the genomic DNA of the CEN.PK strain. The knockout strains namely Δpgi1-btrC and Δpgi1Δzwf1-btrC, that are transformed with the btrC-expression plasmids, have inactive Pgi1 and produced only traces of the compounds. In contrast, Δzwf1-btrC recombinant which has intact pgi1 yielded relatively higher amount of the carbocyclic compounds. Additionally, 1H-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 S. cerevisiae is capable of assimilating 2-DOI.
CitationAl-Fahad, A. J., Al-Fageeh, M. B., Kharbatia, N. M., Sioud, S., & Mahadevan, R. (2020). Metabolically engineered recombinant Saccharomyces cerevisiae for the production of 2-Deoxy-scyllo-inosose (2-DOI). Metabolic Engineering Communications, 11, e00134. doi:10.1016/j.mec.2020.e00134
SponsorsWe thank the Infectious Diseases Program, National Center for Biotechnology, King Abdulaziz City for Science and Technology (KACST) for the financial and intellectual support and Mohammed Alarawi (KAUST). Prof. Radhakrishnan Mahadevan would like to acknowledge funding from the Grand Challenges Canada program for this study.
Except where otherwise noted, this item's license is described as This is an open access article under the CC BY-NC-ND license.