Metabolically engineered recombinant Saccharomyces cerevisiae for the production of 2-Deoxy- scyllo-inosose (2-DOI)

Ahmed J Al-Fahad; Mohamed B Al-Fageeh; Najeh M Kharbatia; Salim Sioud; Radhakrishnan Mahadevan

doi:10.1016/j.mec.2020.e00134

Metabolically engineered recombinant Saccharomyces cerevisiae for the production of 2-Deoxy- scyllo-inosose (2-DOI)

Metab Eng Commun. 2020 May 31:11:e00134. doi: 10.1016/j.mec.2020.e00134. eCollection 2020 Dec.

Authors

Ahmed J Al-Fahad¹, Mohamed B Al-Fageeh¹, Najeh M Kharbatia², Salim Sioud², Radhakrishnan Mahadevan³

Affiliations

¹ National Center of Biotechnology, King Abdulaziz City for Science and Technology (KACST), Riyadh, 11442, Saudi Arabia.
² Analytical Chemistry Core Laboratory, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Saudi Arabia.
³ Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada.

Abstract

Saccharomyces 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.

Keywords: (−)-Vibo-quercitol; 2-Deoxy-scyllo-inosose; 2-Deoxy-scyllo-inosose synthase; Scyllo-quercitol.