马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

Cloning and characterization of sugar transporter genes of Kluyveromyces marxianus GX-UN120

摘要:

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径，可以高效利用木质纤维素中的木糖，因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA_70145和KLMA_80101基因位点的功能预测，获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性，以此间接评价对应转运蛋白的转运能力。【结果】Km_SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力，但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地，Km_SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力，但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下，重组菌株对各种碳源的利用均受抑制，但Km_SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km_SUT2和Km_SUT3可有效促进木糖等单糖转运，其中Km_SUT3可使重组菌株在低葡萄糖浓度条件下具有同时利用葡萄糖和木糖的能力，但Km_SUT2转运木糖过程完全受葡萄糖的抑制。

Abstract:

[Background] Kluyveromyces marxianus with a complete xylose metabolic pathway can utilize xylose in lignocellulose. The study of its sugar transporter genes may solve the problem of how to efficiently transport xylose.[Objective] To explore the functions of the sugar transporter genes of K. marxianus GX-UN120 obtained according to the function prediction of KLMA_70145 and KLMA_80101 in K. marxianus DMKU3-1042. [Methods] Km_SUT2 and Km_SUT3 were respectively expressed in Saccharomyces cerevisiae EBY.VW4000. We then observed the growth characteristics of different recombinants in the media with different carbon sources to evaluate the transport capacity of corresponding transporters. [Results] The sugar transporter Km_SUT2 improved the ability of host cells to transport xylose, arabinose, sorbose, ribose, lactose and glucose, while the cells did not have the ability to transport mannose, fructose, sucrose or galactose. Similarly, the sugar transporter Km_SUT3 can transport xylose, arabinose, sorbose, galactose, ribose, lactose and glucose, while it cannot transport mannose or fructose. In the medium with glucose, the utilization of carbon sources by the recombinants was inhibited, while the inhibition on the transport of xylose and ribose by Km_SUT3 was weaker than that by Km_SUT2. [Conclusion] The transporters Km_SUT2 and Km_SUT3 in K. marxianus GX-UN120 can promote the transport of monosaccharides such as xylose. More significantly, Km_SUT3 enables the recombinant to utilize both glucose and xylose under the condition of low glucose concentration, while the transport of xylose by Km_SUT2 is entirely inhibited by glucose.