Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering
Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering
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DOI：
10.1002/yea.1216
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发表时间：
2005-04-15
期刊：
影响因子：
2.6
通讯作者：
Gorwa-Grauslund, MF
中科院分区：
文献类型：
--
作者：
Karhumaa, K;Hahn-Hägerdal, B;Gorwa-Grauslund, MF
以木糖异构酶（xylose isomerase， XI）表达菌株中木糖的生长和发酵为主要目的，结合多种已知的提高木糖利用的基因修饰，设计了一株酿酒酵母筛选菌株。菌株TMB 3045是由嗜热热菌（Thermus thermophilus）的XI基因在缺失醛糖还原酶GRE3基因的菌株中表达而得到的，该菌株中编码木糖激酶（XK）和非氧化戊糖磷酸途径（PPP）[转醛醇酶（TAL）、转酮醇酶（TKL）、核糖5-磷酸酮异构酶（RKI）和核酮糖5-磷酸外聚酶（RPE）]的基因过表达。以TMB 3045为原料，在木糖培养基上进行重复培养，获得木糖生长发酵菌株TMB 3050。尽管其XI活性较低，但在30℃条件下，TMB 3050具有好氧木糖生长和厌氧乙醇生产的能力，当用多拷贝质粒表达的木糖还原酶（XR）和木糖醇脱氢酶（XDH）基因代替XI时，好氧木糖生长速率达到0.17 l/h，表明筛选体系是有效的。在PPP基因未过表达或PPP基因过表达但有XR和XDH基因染色体整合的菌株中，以前未检测到木糖生长。这表明，为了提高酿酒酵母对木糖的有效利用，必须同时增加木糖向木糖的转化和木糖下游的代谢步骤。版权所有(c) 2005 John Wiley & Sons, Ltd。
A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae. Copyright (c) 2005 John Wiley & Sons, Ltd.