Metabolic engineering of Saccharomyces cerevisiae for (-)-α-bisabolol production

다국어 초록 (Multilingual Abstract)

Isoprenoids are value-added biomaterials with various healthy functions useful for food and healthcare industries. In yeast, isoprenoids and their derivatives are synthesized through the mevalonate pathway. In this study, several strategies were applied to redirect carbon fluxes to the mevalonate pathway. First, in order
to enhance acetyl-CoA supply, aldehyde dehydrogenase (ALD6p) and acetyl-CoA synthetase (ACS1p) were co-expressed. Overexpression of acetoacetyl-CoA thiolase (ERG10p) converting acetyl-CoA into acetoacetyl-CoA was undertaken to enhance the carbon flux from acetyl-CoA to mevalonate. The HMG1 gene encoding a crucial regulatory enzyme of 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase was exchanged by CRISPR/Cas9 system into a truncated HMG1 gene, of which protein is not deactivated considerably by feedback inhibition under mevalonate accumulation. The MrBBS gene encoding (-)-α-bisabolol synthase, one of the isorenoids, was expressed. Product inhibition test was performed to determine whether the final product of (-)-α-bisabolol affected the growth of S. cerevisiae D452-2 host cells. As a result of spot assay, it was found that S. cerevisiae D452-2 in 2% YPD medium containing 1 g/L (-)-α-bisabolol showed similar colony
forming unit to the same strain grown without (-)-α-bisabolol. In flask batch culture using 50 g/L glucose at 100 rpm and 30 ℃, the DtEMA strains produced 296.6 mg/L (-)-α-bisabolol. A 1.28 g/L (-)-α-bisabolol was produced in fedbatch culture.

목차 (Table of Contents)

• 1.INTRODUCTION 1
• 2.MATERIALS AND METHODS 4
• 2.1.Strains and plasmids 4
• 2.2.Genetic manipulation 6
• 2.2.1.Using CRISPR/Cas9 system for gene editing 12
• 2.3.Product inhibition test 16
• 2.4.Fermentation condition 16
• 2.4.1.Batch culture 16
• 2.4.2.Fed-batch culture 17
• 2.5.(-)-α-Bisabolol qualification and quantification 19
• 3.RESULTS 21
• 3.1.Overexpression of genes involved in (-)-α-bisabolol production 21
• 3.2.Product inhibition test 22
• 3.3.Effect of codon optimized MrBBS genes in S. cerevisiae 24
• 3.4.Fermentation using strains inserted with (-)-α –bisabolol relates genes 26
• 3.5.Fed-batch culture 29
• 4.DISCUSSIONS 31
• 5.REFERENCES 32