Construction And Regulation Of β-amyrin Biosynthetic Pathway In Saccharomyces Cerevisiae

β-amyrin, a kind of pentacyclic triterpenoids and skeleton of oleanane type triterpenoids such as glycyrrhetinic acid and soyasaponin, has anti-inflammatory, anti-bacteria and fungi, anti-virus infection and cancer prevention. In recent years, it has been found that it also has the good hypoglycemic and hypolipidemic effects. However, similar to a lot of terpenoids, the very low content of β-amyrin in plants and low yield of chemical synthesis have greatly limited its large-scale production and wide application. In order to improve the production of β-amyrin, metabolic engineering and synthetic biology are employed to synergistically express milti genes from various species for construction of β-amyrin biosynthetic pathway in Saccharomyces cerevisiae. Through the optimization of pathway and fermentation process, β-amyrin is expected to efficiently be produced. The results are as follows.The gene of β-amyrin synthase from Glycyrrhiza glabra was firstly chosen and codonoptimized. After expressed using different plasmids in yeast, GC/MS analysis showed that β-amyrin was successfully produced by S. cerevisiae, and the titer was 0.75±0.06 mg/L with a yield of 0.07±0.02 mg/g DCW. The study indicated that it is feasibe to produce β-amyrin by constructing β-amyrin biosynthetic pathway in S. cerevisiae, and providing a foundation for furthure improving the production of β-amyrin through pathway modification.Squalene is a branch point between triterpenoid pathway and sterol biosynthesis, which give us a chance to optimize triterpenoid pathway by balancing the pool of squalene in yeast. Accordingly to this hypothesis, we designed a "push-pull" approach to optimize the β-amyrin production. Through the expression of IPP isomerase from Escherichia coli, FPP synthase and squalene synthase from S. cerevisiae, squalene supply was pushed, and the transformation of squalene to downstream 2, 3-oxidesqualene was pulled by expresseing a squalene monooxygenase from Candida albicans. This metabolic engineering manipulation resulted in the β-amyrin production increased by 49 times, and reach to 36.50±1.99 mg/L. In addition, Based on the analysis of global regulation mechanism of transcription factor UPC2, UPC2 binding site sequence(SRE sequence) was consciously refactored to the upteam of ADH1 p, ALA1 p, GPM1 p, TYS1 p and FBA1 p promoters controlling β-amyrin biosynthetic pathway, resulting in their strength increased by 42.2%~67.5% and the titer of β-amyrin increased to 48.50±2.55 mg/L, which increased by 32.9% compared with the titer produced by "push-pull" metabolic engineering manipulation.To down-regulate the competition of endogenous sterol and acetyl coenzyme A metabolism in yeast on β-amyrin production, CRISPR/dCas9 system was designed and constructed in S. cerevisiae. Transcriptional repression of CRISPR/dCas9 to LacZ showed that gRNA structure is an important factor. A method of one-step assemblying multiple gRNA was develepoed for down-regulation of multiple genes. Through this method, assembly of 7 gRNAs for lanosterol synthase gene ERG7, alcohol dehydrogenase ADH1/4/5/6, citrate synthase CIT2 and malate synthase gene MLS1 were achieved with an assembly efficiency of 40%. The average repression ratio on the expression of 7 genes reach to 75.5%, leading to the production of β-amyrin increased by 42.2%, which showed the application potential of CRISPR/dCas9 in pathway engineering of yeast.The culture conditions of engineered S. cerevisiae were optimized to produce β-amyrin, and the optimal conditions were obtained as pH 5.0, inoculation of OD 0.30 and initial glucose concentration 35 g/L. Fed-batch fermentation using ethanol as substrate resulted in the production of β-amyrin increased by 90.2%. The gradient supplement of methyl-β-cyclodextrin not only exported β-amyrin but also promoted the production of β-amyrin. In the ethanol fed-batch fermentation with the gradient supplement of methyl-β-cyclodextrin, β-amyrin titer was up to156.7±8.62 mg/L with a yield of 18.44±1.91 mg/g DCW, which was increased by 209 and 121 times compared with the engineered strain SpKb, respectively.