Barbi Florian, Vallon Laurent, Guerrero-Galán Carmen, Zimmermann Sabine D, Melayah Delphine, Abrouk Danis, Doré Jeanne, Lemaire Marc, Fraissinet-Tachet Laurence, Luis Patricia, Marmeisse Roland
Univ Lyon, Université Claude Bernard Lyon 1, CNRS, INRAE, VetAgro Sup, UMR Ecologie Microbienne, F-69622, Villeurbanne, France.
Department of Soil and Environment, Swedish University of Agricultural Sciences, Box 7014, SE-750 07, Uppsala, Sweden.
Appl Microbiol Biotechnol. 2021 Jan;105(2):647-660. doi: 10.1007/s00253-020-11076-y. Epub 2021 Jan 4.
Sugar transporters are essential components of carbon metabolism and have been extensively studied to control sugar uptake by yeasts and filamentous fungi used in fermentation processes. Based on published information on characterized fungal sugar porters, we show that this protein family encompasses phylogenetically distinct clades. While several clades encompass transporters that seemingly specialized on specific "sugar-related" molecules (e.g., myo-inositol, charged sugar analogs), others include mostly either mono- or di/oligosaccharide low-specificity transporters. To address the issue of substrate specificity of sugar transporters, that protein primary sequences do not fully reveal, we screened "multi-species" soil eukaryotic cDNA libraries for mannose transporters, a sugar that had never been used to select transporters. We obtained 19 environmental transporters, mostly from Basidiomycota and Ascomycota. Among them, one belonged to the unusual "Fucose H Symporter" family, which is only known in Fungi for a rhamnose transporter in Aspergillus niger. Functional analysis of the 19 transporters by expression in yeast and for two of them in Xenopus laevis oocytes for electrophysiological measurements indicated that most of them showed a preference for D-mannose over other tested D-C6 (glucose, fructose, galactose) or D-C5 (xylose) sugars. For the several glucose and fructose-negative transporters, growth of the corresponding recombinant yeast strains was prevented on mannose in the presence of one of these sugars that may act by competition for the binding site. Our results highlight the potential of environmental genomics to figure out the functional diversity of key fungal protein families and that can be explored in a context of biotechnology. KEY POINTS: • Most fungal sugar transporters accept several sugars as substrates. • Transporters, belonging to 2 protein families, were isolated from soil cDNA libraries. • Environmental transporters featured novel substrate specificities.
糖转运蛋白是碳代谢的重要组成部分，人们对其进行了广泛研究，以控制发酵过程中使用的酵母和丝状真菌对糖的摄取。基于已发表的有关已鉴定真菌糖转运蛋白的信息，我们发现该蛋白家族包含系统发育上不同的进化枝。虽然几个进化枝包含似乎专门转运特定“糖相关”分子的转运蛋白（例如，肌醇、带电荷的糖类似物），但其他进化枝主要包含单糖或二糖/寡糖低特异性转运蛋白。为了解决糖转运蛋白底物特异性这一蛋白质一级序列无法完全揭示的问题，我们在“多物种”土壤真核生物cDNA文库中筛选甘露糖转运蛋白，甘露糖是一种从未用于筛选转运蛋白的糖。我们获得了19种环境转运蛋白，大部分来自担子菌门和子囊菌门。其中一种属于不寻常的“岩藻糖H同向转运蛋白”家族，该家族在真菌中仅因黑曲霉中的鼠李糖转运蛋白而为人所知。通过在酵母中表达对这19种转运蛋白进行功能分析，并对其中两种在非洲爪蟾卵母细胞中进行电生理测量，结果表明它们中的大多数对D-甘露糖的偏好高于其他测试的D-C6（葡萄糖、果糖、半乳糖）或D-C5（木糖）糖。对于几种对葡萄糖和果糖呈阴性反应的转运蛋白，在存在可能通过竞争结合位点起作用的这些糖之一的情况下，相应重组酵母菌株在甘露糖上的生长受到抑制。我们的结果突出了环境基因组学在揭示关键真菌蛋白家族功能多样性方面的潜力，并且这种潜力可以在生物技术背景下进行探索。要点：•大多数真菌糖转运蛋白接受多种糖作为底物。•从土壤cDNA文库中分离出属于2个蛋白家族的转运蛋白。•环境转运蛋白具有新的底物特异性。
