The Effect Of Cofactors On The Biosynthesis Of 7-dehydrocholesterol In Engineered Saccharomyces Cerevisiae

7-dehydrocholesterol (7-DHC) is a crucial precursor of vitamin D3. In order to produce 7-DHC in the Saccharomyces cerevisiae, its natural ergosterol biosynthesis pathway should be blocked to avoid the competition of the limited carbon sources. As an important cofactor and the core in carbon and energy metabolism in microorganisms, acetyl-CoA takes part in thousands of biochemical reactions including the metabolism of proteins, lipids and respiration. The backbone of 7-DHC comes from mevalonate pathway in Saccharomyces cerevisiae, which initiates from acetyl-CoA. How to enhance the provision of acetyl-CoA has become the key to improve the production of some secondary metabolites that untilize acetyl-CoA as the precursors. Redox state plays important role in the production of heterologous secondary metabolites, which is usually determined by the cofactor system. Cofactors provide redox carriers and energy transfer media in catabolic and anabolic reactions. Besides, cofactors are helpful to increase the function of mitochondria and prolong the lifetime of cells. The cellular redox state relies on the ratio of free NADH/NAD+ to a large extent. The fluctuation in the ratio of free NADH/NAD+ would result in the wild change in cell metabolism.In this study, (1) the deletion of erg5 gene results in the inhibition of ergosterol synthesis. After introducing a human C-24 reducase gene dhcr24 and over-expressing a truncated HMG-CoA reducase gene thmgl, the production of 7-DHC is 2.62 mg/L in the engineered yeast during shake flask culture. (2) The combined strategy of over-expression of alcohol dehydrogenase adh2 and acetaldehyde dehydrogenase ald6 together with the introduction of Salmonella enterica acetyl-CoA synthetase gene acs and mus musculus ATP-citrate lyase acl enhances the provision of acetyl-CoA in cytosol and increase the 7-DHC production to 4.84 mg/L in shake flask culture. (3) The free NADH/NAD+ ratios in 7-dehydrocholesterol producing strains are higher than the control, which reveal redox imbalance. To alleviate the redox imbalance, a water-forming NADH oxidase (NOX) from Streptococcus pneumoniae and an alternative oxidase (AOX1) from Histoplasma capsulatum were employed in our system based on cofactor regeneration strategy. Consequently, the ratio of free NADH/NAD+ is reduced the production of 7-dehydrocholesterol was increased to 8.44 mg/L in shake flask culture. In a 5-L bioreactor, the optimal production of 7-DHC reached 44.49 mg/L, which is the highest production reported so far.This study provides the references for the biosynthesis of 7-DHC and the improvement of the production of 7-DHC, which shows the guilding significance to some extent.