Smit A, Moses S G, Pretorius I S, Cordero Otero R R
Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Matieland, South Africa.
J Appl Microbiol. 2008 Apr;104(4):1103-11. doi: 10.1111/j.1365-2672.2007.03671.x. Epub 2007 Dec 20.
The main objective of this study was to identify amino acid residues in the AGT1-encoded alpha-glucoside transporter (Agt1p) that are critical for efficient transport of maltotriose in the yeast Saccharomyces cerevisiae.
The sequences of two AGT1-encoded alpha-glucoside transporters with different efficiencies of maltotriose transport in two Saccharomyces strains (WH310 and WH314) were compared. The sequence variations and discrepancies between these two proteins (Agt1p(WH310) and Agt1p(WH314)) were investigated for potential effects on the functionality and maltotriose transport efficiency of these two AGT1-encoded alpha-glucoside transporters. A 23-amino-acid C-terminal truncation proved not to be critical for maltotriose affinity. The identification of three amino acid differences, which potentially could have been instrumental in the transportation of maltotriose, were further investigated. Single mutations were created to restore the point mutations I505T, V549A and T557S one by one. The single site mutant V549A showed a decrease in maltotriose transport ability, and the I505T and T557S mutants showed complete reduction in maltotriose transport.
The amino acids Thr(505) and Ser(557), which are respectively located in the transmembrane (TM) segment TM(11) and on the intracellular segment after TM(12) of the AGT1-encoded alpha-glucoside transporters, are critical for efficient transport of maltotriose in S. cerevisiae.
Improved fermentation of starch and its dextrin products, such as maltotriose and maltose, would benefit the brewing and whisky industries. This study could facilitate the development of engineered maltotriose transporters adapted to starch-efficient fermentation systems, and offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer and whisky industries.
本研究的主要目的是确定酿酒酵母中AGT1编码的α-葡萄糖苷转运蛋白（Agt1p）中对麦芽三糖高效转运至关重要的氨基酸残基。
比较了两种酿酒酵母菌株（WH310和WH314）中具有不同麦芽三糖转运效率的两种AGT1编码的α-葡萄糖苷转运蛋白的序列。研究了这两种蛋白质（Agt1p(WH310)和Agt1p(WH314)）之间的序列变异和差异对这两种AGT1编码的α-葡萄糖苷转运蛋白的功能和麦芽三糖转运效率的潜在影响。结果表明，23个氨基酸的C末端截短对麦芽三糖亲和力并非至关重要。进一步研究了三个可能对麦芽三糖转运起作用的氨基酸差异。逐个创建单突变以恢复点突变I505T、V549A和T557S。单位点突变体V549A的麦芽三糖转运能力下降，而I505T和T557S突变体的麦芽三糖转运完全降低。
分别位于AGT1编码的α-葡萄糖苷转运蛋白跨膜（TM）区段TM(11)和TM(12)之后的细胞内区段的苏氨酸（505）和丝氨酸（557）氨基酸对酿酒酵母中麦芽三糖的高效转运至关重要。
改善淀粉及其糊精产物（如麦芽三糖和麦芽糖）的发酵将有利于酿造和威士忌行业。本研究有助于开发适用于淀粉高效发酵系统的工程化麦芽三糖转运蛋白，并为开发具有改善的麦芽糖和麦芽三糖摄取能力的酵母菌株提供了前景，进而可以提高啤酒和威士忌行业的整体发酵效率。
