Background Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts. Results We constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal. Conclusion This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism.

中文翻译：

---

基于结构的定向进化通过影响体内酶的性能来改善酿酒酵母在木糖上的生长。

背景通过酿酒酵母从半纤维素原料高效生产生物乙醇需要利用木糖。尽管酿酒酵母不代谢木糖，但表达木糖异构酶的工程菌株可以通过将木糖转化为木酮糖来代谢木糖。为此，使用来自 Piromyces (PirXI) 的 II 型木糖异构酶，但体内活性相当低，并且木糖代谢需要非常高水平的酶。在这项研究中，我们探索使用蛋白质工程和体内选择来提高 PirXI 的性能。最近解决的晶体结构被用于集中诱变工作。结果 我们通过替换底物和金属结合位点周围的第二个壳残基构建了 Piromyces 木糖异构酶的集中突变文库。图书馆转移到 S. 酿酒酵母和在好氧和厌氧条件下增强木糖支持生长的选择，获得了两个新的木糖异构酶突变体，对其进行纯化并进行生化和结构分析。除了对金属可用性的响应存在微小差异外，在各种体外测定条件下，新突变体和前面描述的突变体都没有表现出催化性能的显着变化。然而，体内性能明显改善。由于酶负载有钙，酶在体内的功能似乎次优，这导致木糖转化动力学较差。结果表明，在体外用单一类型的添加金属测定的木糖异构化动力学参数中，较好的体内酶性能很少反映。结论本研究表明，体内选择可以鉴定木糖异构酶突变体，在标准条件下测量的催化性能只有很小的变化。在酵母中表达的木糖异构酶的金属负载不是最理想的，并且强烈影响动力学特性。金属吸收、分布和与木糖异构酶的结合与木糖的快速转化高度相关，可能是优化酵母木糖代谢的重要目标。