Biosynthesis of Natural Acylsucroses from Sucrose and Short Branched-Chain Fatty Acids via Artificially Engineered <i>Escherichia coli</i>

Natural acylsucrose, often found in the glandular trichomes of <i>Solanaceae</i> plants, has potential applications in many industries, including food, cosmetics, and pharmaceuticals. In this study, we engineered an <i>Escherichia coli</i> strain to complete the biosynthesis of acylsucroses through whole-cell transformation. Using acylsucrose acyltransferases and CoA ligases, acylsucroses, including monoacylsucrose S1:5 ("S" represents an acylsucrose backbone, the number before the colon indicates the number of acyl chains, and the number after the colon indicates the sum of carbons in all acyl chains), diacylsucrose S2:10, triacylsucrose S3:14, and triacylsucrose S3:15 were synthesized from the substrate sucrose and short branched-chain fatty acids by the engineered <i>E. coli</i> EcoSE07, of which S3:15 was the primary product. Several strategies were applied to improve acylsucrose production, including codon optimization, constitutive promoter replacement, and serial resting cell assays. The use of fed-batch fermentation with an engineered <i>E. coli</i> strain of EcoSE22 containing a constitutive promoter further improved the production of acylsucroses. Serial resting cell assays with an optical density of 50 at 600 nm significantly increased the production of acylsucroses S3:15 and S2:10. These findings will facilitate the synthesis of natural acylsucroses through whole-cell transformations and provide the potential for future industrial applications.