Exploring Studies On Microbial Synthesis Of Natural Fragrance Monomer Irone

Irones are major aroma components of the Iris family,widely used in high-end perfumes,tobacco products,foods and toiletries.In order to reduce the cost of production of irones and achieve sustainable supply,this paper would explored three strategies for microbial biosynthesis of irones.The first method:inspired by the traditional aging extraction method,the aged rhizome was fermented using the microorganism isolated from the rhizome of the iris to obtain the irones.A fungus obtained from the dried rhizome by 2 years aged,called Aspergillus fumigatus ZD-J1,which could ferment fresh iris rhizome powder to obtain irones.The HPLC/MS analysis and H NMR hydrogen spectrum identification of the fermentation product confirmed that the configurations of the irones in the products were ?-irone and a-irone.Finally,we optimized the fermentation conditions(nitrogen source,temperature and pH).Under the optimal conditions,the concentration of the product was 7.25 mg/L,and the space-time production efficiency was 12 mg/(kg*d),which was produced by the traditional method.The efficiency has increased by 45 times.The second method:In theory,pseudoionone can be biosynthesized irones by methylation and cyclization,and the production of pseudoionone in E.coli by metabolic engineering has become our new strategy for synthesizing irones.First,the crtEIB gene cluster of the lycopene biosynthesis pathway was inserted into the genome of the Escherichia coli strain Echw2f lyc",and then the vector containing the carotenoid cleavage dioxygenase 1 gene(cmCCD1)was transformed into this strain.This construction made E.coli producing pseudoionone.The fermentation production of pseudoionone can be improved by further two-phase fermentation.Finally,the pBAD promoter replaces the Trc promoter,which tightly regulates the expression of cmCCD1 thereby increasing the production of pseudoionone from 30 ?g/L to 110?g/L.In order to further increase the yield of pseudoionone,promoter-engineering was carried out.The effects of five different intensity promoters on the key genes of NADPH supply and the key genes expression of MEP pathway were investigated by using cat-sacB non-marking recombination.Studies have shown that the promoters of the key genes of TCA and PPP(sdhABCD,sucAB and talB)can be combined to optimize the yield of pseudoionone by 280%.Further combining the promoters of the MEP pathway rate-limiting enzyme genes dxs and idi can further increase the pseudoionone by 176%and the yield to 3.42 mg/g.Finally,random mutations were performed because of the mismatch repair system were destroyed before,which increased the production of pseudoionone to 5.76 mg/g.The yield was further improved by optimizing the fermentation conditions of the shake flask:carbon source,nitrogen source,induction time,inducer concentration,temperature and Fe2+concentration,and the production reached to 24 mg/L and 8.55 mg/g.The final yield was 185 times higher than the original strain.The third method:marnerol is considered to be a naturally precursor of irones synthesized in plant cells.This paper envisages a new strategy for the biosynthesis of marnerol in yeast metabolic engineering.The arabidopsisi-derived and codon-optimized MRN1 gene was integrated into the model S.cerevisiae BY4741 genome with the G418 resistance tag,and a S.cerevisiae strain capable of synthesizing marnerol was obtained.Further experiments showed that the high expression of MRN1 and tHMG gene by high-copy plasmid can greatly increase the content of squalene,but it will reduce the concentration of marnerol in cell.Finally,our efforts were paid to inverstigate whether pseudoionone or mamerol,will be transformed into irone using our isolate fung.In summary,first,a new isolated microorganism were used to realize the high-efficiency synthesis of irones by biotransformation of iris rhizome powder.Second,metabolic engineering research was carried out in Escherichia coli and Saccharomyces cerevisiae to construct the microorganisms capable of efficiently synthesizing pseudoionone and marnerol.Third,further fermentation optimization was performed to improve the production of pseudoionone and marnerol.Although the biotransformation of the irone was not achieved in the end,the establishment of these two potential irone precursors biosynthesis processes had important ground-breaking significance for further improvement of the eficient production process of irone.