Study Of Increasing Production Of Mevalonic Acid In Saccharomyces Cerevisiae Using Artificial Non-oxidized Glycolysis Pathway

Saccharomyces cerevisiae(S.cerevisiae),which has complete genomic sequences to make genetic manipulations simple and practicable,is the model eukaryote to synthesize products,but it is hard to build a cell factory with genetic stability.Mevalonic acid(MVA)is an essential intermediate metabolite in the S.cerevisiae and a crucial function to become the precursory compounds of some additional high value substances.In this study,S.cerevisiae was designed into a cell factory to produce MVA with the core concept of biological refining.A thorough and meticulous research has been established focusing on how to build an efficient biosynthetic pathway of MVA within S.cerevisiae.Contemporary studies using biological refining on MVA biosynthesis have mostly constructed heterogenous pathways into Escherichia coli(E.coli)and rarely studied on S.cerevisiae.There are three aspects to accumulate the production of MVA,which contain reducing carbon loss within the original pathway,weakening competitive branch pathway with fewer by-product and less consumption of MVA by autophage as well as keeping the balance of electron within the metabolic reaction.First,the pta,encoding glycerol-3-phosphate acyltransferase,was obtained from different strains comprised of E.coli and Bacillus subtilis(B.subtilis).The fpk and fbp respectively encoded phosphoketolase and fructose-1,6-diphosphatase,which were both cloned from E.coli.Three cloned genes,which were constructed on pESC vectors with the yeast 2 ? origin,were introduced into cells by electroporation to induce NOG pathway with over-accumulation of acetyl coenzyme A(acetyl-CoA).Secondly,zwf and gnd,both of them encoding phosphogluconic dehydrogenase,were cloned from S.cerevisiae genome to enhance pentose phosphate(PPP)pathway.As a common product called xylulose-5-phosphate(X5P)occured between NOG and PPP pathway,enhancing the PPP pathway can make the metabolic flux transfer toNOG pathway to increase the yield of acetyl-CoA.In addition,with the single enhancement of PPP pathway,the concentration of NADPH in the fermentation solution increased from 0.085 mM to 0.17 mM,which was twice as much as that of original strain BY4741.Thirdly,over expression of tHMG1,encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR),caused prominent increase of rate-limiting step efficiency in the MVA pathway,which can catalyze more cumulative acetyl-CoA above to generate MVA.During the establishment of NOG pathway,the parameters of MVA production and cell physiological states were setted up as a criterion to select the better one of two strains different from the origin of pta.The strain BYPNM-EC whose pta was originally from E.coli,had a higher production of MVA up to 123mg/l.Therefore,strain BYPNM-EC was used to accomplish further construction.In the end,weaken the competitive pathway.Knockout of pyk1 in strain BYPNM-EC make the yield of ethanol decrease from 1.86 g/l to 0.66 g/l,compared with strain BY4741,and the yield of MVA increase to 138 mg/l.Moreover,further knockout ERG8 weaken part downstream metabolic flux to conform a new strain called BYM02.Its MVA yield is 180 mg/l which is 4.3 times as much as that of strain BY4741.MVA is the key precursor of isoprene and terpenoids.Biological refining make it come true to synthesize MVA in industrialization,which can lower the cost of isoprene and terpenoids synthesis based on MVA.S.cerevisiae that is generally recognized as safe(GRAS),can effectively avoid introducing endotoxin through the process of producing MVA.This method simplifies the production process and reduces the safety risk of manufacturing oral health care products and medical drugs.