Functional Analysis of Two L-Arabinose Transporters from Filamentous Fungi Reveals Promising Characteristics for Improved Pentose Utilization in Saccharomyces cerevisiae
Functional Analysis of Two L-Arabinose Transporters from Filamentous Fungi Reveals Promising Characteristics for Improved Pentose Utilization in Saccharomyces cerevisiae
复制标题
丝状真菌中两种 L-阿拉伯糖转运蛋白的功能分析揭示了改善酿酒酵母戊糖利用的有希望的特征
DOI：
10.1128/aem.00165-15
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发表时间：
2015-06-01
影响因子：
4.4
通讯作者：
Tian, Chaoguang
中科院分区：
文献类型：
--
作者：
Li, Jingen;Xu, Jing;Tian, Chaoguang
木质纤维素酶解物对L-阿拉伯糖的吸收有限是酿酒酵母工程菌发酵木质纤维素酶解物的瓶颈之一。本研究表征了两种新的L-阿拉伯糖转运蛋白，粗糙脉孢菌的LAT-1和嗜热毁丝霉的MtLAT-1。虽然这两种蛋白具有较高的同源性（约83%），但它们显示出不同的底物特异性。糖转运测定使用S.酿酒酵母菌株EBY。VW 4000表明LAT-1接受广泛的底物谱。相比之下，MtLAT-1对L-阿拉伯糖的特异性更高。两种转运蛋白动力学性质的测定显示，LAT-1和MtLAT-1对L-阿拉伯糖的Km值分别为58.12 +/- 4.06 mM和29.39 +/- 3.60 mM，相应的V-max值分别为116.7 +/- 3.0 mmol/h/g干细胞重量（DCW）和10.29 +/- 0.35 mmol/h/g DCW，分别此外，这两种转运蛋白被发现使用质子偶联的同向转运机制，并显示在L-阿拉伯糖摄取过程中D-葡萄糖仅部分抑制。LAT-1和MtLAT-1在S.酿酒酵母菌株BSW 2AP含有L-阿拉伯糖代谢途径。与对照菌株相比，两种重组菌株均表现出更快的L-阿拉伯糖利用率、更大的生物量积累和更高的乙醇产量。总之，由于更高的最大速度和D-葡萄糖的抑制减少，两个特征性转运蛋白的基因是有前途的目标，提高L-阿拉伯糖利用和发酵的S。啤酒。
Limited uptake is one of the bottlenecks for L-arabinose fermentation from lignocellulosic hydrolysates in engineered Saccharomyces cerevisiae. This study characterized two novel L-arabinose transporters, LAT-1 from Neurospora crassa and MtLAT-1 from Myceliophthora thermophila. Although the two proteins share high identity (about 83%), they display different substrate specificities. Sugar transport assays using the S. cerevisiae strain EBY. VW4000 indicated that LAT-1 accepts a broad substrate spectrum. In contrast, MtLAT-1 appeared much more specific for L-arabinose. Determination of the kinetic properties of both transporters revealed that the Km values of LAT-1 and MtLAT-1 for L-arabinose were 58.12 +/- 4.06 mM and 29.39 +/- 3.60 mM, respectively, with corresponding V-max values of 116.7 +/- 3.0 mmol/h/g dry cell weight (DCW) and 10.29 +/- 0.35 mmol/h/g DCW, respectively. In addition, both transporters were found to use a proton-coupled symport mechanism and showed only partial inhibition by D-glucose during L-arabinose uptake. Moreover, LAT-1 and MtLAT-1 were expressed in the S. cerevisiae strain BSW2AP containing an L-arabinose metabolic pathway. Both recombinant strains exhibited much faster L-arabinose utilization, greater biomass accumulation, and higher ethanol production than the control strain. In conclusion, because of higher maximum velocities and reduced inhibition by D-glucose, the genes for the two characterized transporters are promising targets for improved L-arabinose utilization and fermentation in S. cerevisiae.