Gene Cloning Of Fdc1 From Bacillus Pumilus And Expression In Top-Fermenting Yeast Strain IYS

4-Vinylguaiacol(4-VG)existing in top-fermented beer is a kind of special flavor substance that has the characteristics of "smoke flavor"and "clove flavor",which is usually produced by the thermal decarboxylation or ferulic acid decarboxylase's(Fdclp)decarboxylation of ferulic acid(FA).However,the restriction of fermentation conditions in china is not enough to improve the overall taste of the top-fermented beer,and the activity of ferulic acid decarboxylase from yeast(Sc-Fdclp)is also low.To brew the top-fermented beer with unique flavor,smooth taste and excellent quality,we slected a recombinant yeast strain with the best fermentation performance,for example the IYSIBp?kan.In this study,the ferulic acid decarboxylase gene from Bacillus pumilus(Bp fdc1)and top-fermenting yeast strain IYS(Sc FDC1)were successfully transferred into the genome of IYS yeast strain by ?-integration,and recombinant yeast strains IYSSc?IYSBp?IYSIBp and IYSIBp2 were obtained,respectively.The results of small-scale fermentation experiments were shown that the metabolic time of FA by recombinant yeast strain IYSBp was shorter than IYSsc from 48 hours to 12 hours,and the content of 4-VG was increased by 34%.Therefore,the heterologous expression of ferulic acid decarboxylase from Bacillus pumilus(Bp-Fdc1p)was better than the overexpression of Sc-Fdc1p.The experimental results of intracellular accumulation of FA were shown that the accumulation of FA did not increase with the passage of time,so the transport mode of FA is not simple passive diffusion,and it might also involve carrier-mediated transport.To reduce the transport burden of FA on cells and shorten the decarboxylation time,the secretory expression of Bp-Fdc1p by using the signal peptide secreted by inulinase in Kluyveromyces marxinus was achieved.The results of small-scale fermentation experiments were shown that the consumption rate of FA fermented by IYSIBp was further increased,while the content of 4-VG was increased by nearly 65%.Therefore,the secretory expression of Bp-Fdc1p was better than intracellular expression.To further improve the production ability of 4-VG by the recombinant yeast strain,we improved the secretory expression ability of IYSIBp to generate the recombinant yeast strain of IYSIBp2.The results of small-scale fermentation experiments were shown that the fermentation performance of recombinant yeast strain IYSIBp2 is slightly lower than the IYSIBp,and the production of 4-VG has not been significantly improved.The content of 4-VG in the top-fermented beer brewed by recombinant yeast strain IYSIBp?kan increased by 61%.In conclusions,the appropriate secretion of Bp-Fdc1p into wort could be one of the potential strategies to increase the content of 4-VG in the top-fermented beer,and the innovation of this study is that the secretory expression of Bp-Fdc1p was directly used to decarboxylate FA in wort to increase the content of 4-VG in top-fermented beer.