Construction Of Saccharomyces Cerevisiae Of Production Of Tyrosol From Xylose

Tyrosol is the derivatives of aromatic amino acid tyrosine,it is widely found in fermented products,such as olive oil,wine and sake.As a natural antioxidant,tyrosol has multiple biological activities and medical value such as anti-oxidation,anti-aging,regulating metabolism,reducing pigment deposition,preventing cell damage and so on.Traditonal methods for obtaining tyrosol are plant extraction and chemical synthesis,but these two methods have the disadvantages of high cost,low yield,and serious environmental pollution.Microbial synthesis is a method through constructing an efficient microbial cell factory by the genetic engineering technology to produce high-yield target production.Microbial synthesis shows excellent potential as a short cycle,high transformation efficiency,and environmentally friendly method.Saccharomyces cerevisiae is generally recognized as a safe and it's a potential cell factory with a robust metabolism,clear genetic background,simple and mature genetic manipulation.In S.cerevisiae,aromatic amino acids such as tyrosine synthesized via the shikimate pathway.The first step of the shikimate pathway is the condensation of phosphoenolpyruvate(PEP)and erythrose 4-phosphate(E4P),which are the important intermediate metabolite of glycolytic pathway(EMP)and pentose phosphate pathway(PPP),respectively.The major limiting factor of the shikimate pathway is the supply of E4P.For instance,there are relatively few studies on the synthesis of tyrosol by microbial fermentation.Xylose is one of the main components of lignocellulose,and it's the second most abundant sugar after glucose in nature.Fermentation of xylose to produce various products is beneficial to the full utilization of lignocellulose raw materials.However,wild-type S.cerevisiae itself cannot metabolize xylose directly.In previous work of the laboratory,through the introduction of exgenous xylose metabolism pathways,enhancement of PPP,and adaptive domestication,a recombinant S.cerevisiae strain with high xylose metabolism efficiency was constructed.According to the metabolic pathway,xylose first enters the PPP that produces E4P,and the Crabtree effect(produces ethanol in aerobic conditions and high external glucose concentrations rather than producing biomass via the tricarboxylic acid cycle)is weaker,which is more conducive to the synthesis of tyrosol.However,no research has been reported on S.cerevisiae using xylose as a carbon source for tyrosol production and fermentation.Based on the above research background,this study is mainly aimed at obtaining xylose-utilizing recombinant S.cerevisiae strain with high yield of tyrosol.The construction of high-yield tyrosol strains and the screening system for high-yield tyrosol strains were carried out resprctively.The specific research content and related prospects are as follows:1.Construction and optimization of xylose-utilizing recombinant S.cerevisiae strains to produce tyrosol.Based on the xylose-utilizing recombinant S.cerevisiae strain constructed in the previous work of the laboratory,the aromatic aldehyde synthase gene(aas)from Petroselinum crispum combined with the alcohol dehydrogenase gene(adh)from Escherichia coli was introduced.The fermentation results revealed that the tyrosol yield of the recombinant strain increased by 219.9%compared with the control strain,indicating that the introduction of this pathway can promote the production of tyrosol.The results of fermentation using glucose and xylose as carbon sources,respectively,showed that the tyrosol yield of recombinant strain was 5.13 mg g-1 when using xylose as the carbon source,which was 95.3%higher than that of glucose which verified our conjecture.After that,we deregulate the feedback inhibition(ARO4K229L?AR07G141S)by point mutation,orientate the intermediate metabolite by fusion protein(TALI-ARO4K229L),overexpress the dehydrogenase gene(ADH6)from S.cerevisiae to replace the adh gene,from all of these strategies can increase the tyrosol production,among them,the strain over-expressed ADH6 gene had the strongest capacity for tyrosol production,reached 172.33 mg L-1.Subsequently,we tested the effect of the introduction of the aldehyde reductase gene(yahK)from E.coli combined with the phenylpyruvate decarboxylase gene(ipdC)from Azospirillum brasilense,and the enhancement of the endogenous phenylpyruvate decarboxylase gene(ARO10)on tyrosol production.The results showed that the strain over-expressed AROIO gene had the strongest capacity for tyrosol production,reached 170.63 mg L-1 These advantageous strategies require further combined expression to obtain high-yield tyrosol-producing recombinant S.cerevisiae strains.2.Preliminary construction of screening system for tyrosol high production strains.In this part of work,transcriptional responses of S.cerevisiae to tyrosol were analyzed by using RNA-seq techniques.The results demonstrated that 34 genes(21 up-regulated and 13 down-regulated)were significantly differentially expressed,among them,two genes(CHA1,PDR5)were selected which the transcriptional level gradually increased with the increase of tyrosol concentration using real-time fluorescent quantitative PCR(qPCR).After that,the fluorescent proteins were expressed under the control of the CHA1 and PDR5 promoter,respectively,to test the fluorescence intensity of strains with different tyrosol concentrations in the environment.The results showed that the fluorescence intensity of strains increased with the increase of tyrosol concentration,indicating that the screening system for tyrosol high production strains was initially constructed.In summary,the xylose-utilizing recombinant S.cerevisiae strains to produce tyrosol were constructed in this study,we tried the effectiveness of multiple strategies.These advantageous strategies require further combined expression and the production performance of the strains need to be further improved.At the same time,we constructed a screening system for tyrosol high production strains.Further optimization of the system is needed in the future work,and with this system,tyrosol high production strains can be screen out easily and quickly according to the change of fluorescence intensity after rational or irrational transformation.