۲-hpp production by the yeast saccharomyces cerevisiae

۲-hydroxypropiophenone (۲-hpp) is an important chemical in organic chemistry and pharmacology. Production of ۲-hpp was explained by Ward and Wilkocs in ۱۹۹۲. They found out that Pseudomonas putida and Acinetobacter calcoaceticus can grow on mandelate. They also discovered that the last enzyme of mandelate pathway is benzoylformate decarboxylase which produces benzaldehyde from benzoyllformate by decarboxylation reaction (Wilcocks and Ward ۱۹۹۲), on the other hand in presence of acetaldehyde this enzyme can do a carboligation reaction and attach benzoylformre and acetaldehyde but the rate of decarboxylation reaction is ۴۰-fold more than carboligation reaction (Wilcocks and Ward ۱۹۹۲, Wilcocks, Ward et al. ۱۹۹۲, Prosen, Ward et al. ۱۹۹۳). CRISPR-Cas۹ system is the best way to engineer genome and metabolome of the yeast. In this system a donor DNA can be easily integrated in the yeast’s genome (Stovicek, Borodina et al. ۲۰۱۵). One of the advantages of CRISPR-Cas۹ system is that it can be used for diploid or polyploid industrial strains without any dominant marker. in ۲۰۱۶ Brodina et al. introduced a vector set for integration biobrieks into the genome of S. cerevisiae without any marker with mostly ۱۰۰% efficiency (Jessop‐Fabre, Jakočiūnas et al. ۲۰۱۶). These integrative vectors have bidirectional promoter site and biobrieaks joined together by USER cloning. USER cloning is a Ligation Independent Cloning (LIC), therefore no ligase is used in this reaction. Steaky ends of vector and biobreaks are homolog and join together by hydrogen bonds of inorganic bases (Bitinaite, Rubino et al. ۲۰۰۷). The yeast Saccharomyces cerevisiae is an old friend of humans. It is one of the GRAS (Generally Regard as Safe) organisms and men have been using it to produce wine, beer and bread for thousands of Years (Feldmann ۲۰۱۱, Borodina and Nielsen ۲۰۱۴). Furthermore, today the yeast is one of the most known cell factories of all time. This cell factory makes proteins, chemicals, drugs etc. for us every day. By metabolic engineering of this cell factory we can increase production of lots of metabolites (Borodina and Nielsen ۲۰۱۴). Here a modified strain of S. cerevisiae which has BFD gene in its genome is introduced. A TDH۳ promoter is constructed upstream the gene for better transcription. A wild type gene sequence of BFD from P. putida ATCC ۲۴۴۰ and the promoter was extracted from S. cerevisiae CEN.PK ۱۱۳-۷D. All of the methods and protocols of this paper are based on EasyClone-MarkerFree method. Generally, ۳ vectors were transformed to the yeast, an integrative linearized vector (pCfB۲۹۰۴) with the gene and the promoter, a helper vector (pCfB۳۰۴۵) with gRNA and Nourseothricin (NTC) resistance, and an expression vector (pCfB۱۲۲۳) possessing Cas۹ gene and G۴۱۸ resistance.

کلیدواژه ها:

Pseudomonas Putida ، Saccharomyces cerevisiae ، ۲-hydroxypropiophenine ، Benzoylformate decarboxylase ، CRISPR-Cas۹.