Wasserstrom Lisa, Portugal-Nunes Diogo, Almqvist Henrik, Sandström Anders G, Lidén Gunnar, Gorwa-Grauslund Marie F
Division of Applied Microbiology, Department of Chemistry, Lund University, PO Box 124, 221 00, Lund, Sweden.
Harboes Bryggeri A/S, Spegerborgvej 34, 4230, Skælskør, Denmark.
AMB Express. 2018 Mar 5;8(1):33. doi: 10.1186/s13568-018-0564-9.
Engineering of the yeast Saccharomyces cerevisiae towards efficient D-xylose assimilation has been a major focus over the last decades since D-xylose is the second most abundant sugar in nature, and its conversion into products could significantly improve process economy in biomass-based processes. Up to now, two different metabolic routes have been introduced via genetic engineering, consisting of either the isomerization or the oxido-reduction of D-xylose to D-xylulose that is further connected to the pentose phosphate pathway and glycolysis. In the present study, cytosolic D-xylose oxidation was investigated instead, through the introduction of the Weimberg pathway from Caulobacter crescentus in S. cerevisiae. This pathway consists of five reaction steps that connect D-xylose to the TCA cycle intermediate α-ketoglutarate. The corresponding genes could be expressed in S. cerevisiae, but no growth was observed on D-xylose indicating that not all the enzymes were functionally active. The accumulation of the Weimberg intermediate D-xylonate suggested that the dehydration step(s) might be limiting, blocking further conversion into α-ketoglutarate. Although four alternative dehydratases both of bacterial and archaeon origins were evaluated, D-xylonate accumulation still occurred. A better understanding of the mechanisms associated with the activity of dehydratases, both at a bacterial and yeast level, appears essential to obtain a fully functional Weimberg pathway in S. cerevisiae.
在过去几十年里，对酿酒酵母进行工程改造以实现高效的D-木糖同化一直是一个主要研究重点，因为D-木糖是自然界中第二丰富的糖类，将其转化为产品可以显著提高基于生物质的工艺的经济性。到目前为止，通过基因工程引入了两条不同的代谢途径，包括将D-木糖异构化或氧化还原为D-木酮糖，后者进一步连接到磷酸戊糖途径和糖酵解。在本研究中，通过在酿酒酵母中引入来自新月柄杆菌的魏伯格途径，转而研究胞质D-木糖氧化。该途径由五个反应步骤组成，将D-木糖连接到三羧酸循环中间体α-酮戊二酸。相应的基因可以在酿酒酵母中表达，但在D-木糖上未观察到生长，这表明并非所有酶都具有功能活性。魏伯格中间体D-木糖酸的积累表明脱水步骤可能是限制因素，阻碍了进一步转化为α-酮戊二酸。尽管评估了四种来自细菌和古菌的替代脱水酶，但D-木糖酸积累仍然发生。更好地理解与脱水酶活性相关的机制，无论是在细菌还是酵母水平上，对于在酿酒酵母中获得完全功能的魏伯格途径似乎至关重要。
