Optimization Of Fermentation Process For Bioconversion Of Eugenol To Vanillin And Transcriptome Analysis

Vanillin has the reputation of "the king of spices" and is one of the most productive spices in the world.It is widely used in food,medicine,cosmetics,agriculture and other industries.At present,there are three main preparation methods for vanillin:the first is the natural extraction method.Although the vanillin produced by this method is a natural product,the supply of raw vanilla pods is limited,the processing cycle is long,and the product is expensive,which is far from meeting the market demand.The second is the chemical synthesis method,in which unnecessary racemic mixtures are produced,it also lacks substrate selectivity,and it is restricted by the environmentally harmful process.The third is the biotransformation method,which includes plant cell culture method,enzyme transformation method and microbial transformation method.The vanillin produced by the microbial transformation method is superior in price and quality.The chemically synthesized vanillin meets people's pursuit of naturalness and health.This study studied the production of vanillin by microbial transformation,aiming to reveal the internal mechanism of the increase in the production of vanillin.In this study,strains that can tolerate eugenol and biotransform it into vanillin were obtained by separating and screening from the soil.The strain with the highest yield of vanillin was used as the dominant strain;morphological,physiological and biochemical tests and 16S rDNA sequence identification were performed on it,and construction of a phylogenetic tree;by optimizing the fermentation medium formula and fermentation conditions of the dominant strain to determine the best production conditions;at the same time,the transcriptome analysis of the strains before and after optimization will provide a certain theoretical basis for the targeted increase of product yield in the future,and lay a good foundation for the realization of industrialized production.The main findings are as follows:(1)43 strains that can tolerate eugenol obtained through strain screening,10 strains can bioconvert eugenol into vanillin;among them,the strain with the highest vanillin yield was used as the target strain.This strain can bioconvert 600 mg/L eugenol into 22.75±2.5 mg/L vanillin,and the molar conversion rate was 4.09±0.45%,and it was named S000450-3.The strain was identified as Bacillus cereus.(2)By optimizing the fermentation medium formula and fermentation conditions,the experiment finally obtained that the bacteria in the fermentation medium formula was 0.2 g/L complex inorganic salt(FeSO4·7H2O,K2HPO4,MgSO4·7H2O,CaCl2),15 g/L peptone;the initial pH 8.0,temperature 34?,inoculation volume 5 mL,a rotation speed of 180 r/min,and the volume of the culture medium 25 mL,600 mg/L of eugenol can be bioconverted into 114.42 ±1.44 mg/L vanillin,and the molar conversion rate was 20.58±0.21%,which was about five times higher than before optimization.(3)Through transcriptome sequencing of Bacillus cereus S000450-3 before optimization(D1)and after optimization(D2),GO showed that the differential genes were mainly enriched in "structural constituent of ribosome"(molecular functions),"sporulation resulting in formation of a cellular spore"(the biological process involved),and "ribosomes"(cellular component).Among them,the enrichment of "sporulation resulting in formation of a cellular spore" was the most significant.KEGG enrichment analysis showed that most of the differential genes were concentrated in the”metabolic pathway"(54.34%),and the enrichment of "oxidative phosphorylation" was the most significant.COG analysis showed that a total of 1213 differential genes were aligned to different functional classification annotations:the up-regulated differential genes between D1 and D2 were mainly enriched in the "pyrroline-5-carboxylate reductase"(amino acid transport and metabolism),"permeases of the drug/metabolite transporter(drug/metabolite transporters,DMT)superfamily"(general function prediction only),"uncharacterized conserved protein"(unknown function).These results provided a certain reference basis for further improving the production of vanillin.