13TSB_CRD - Flexible Engineered Solutions for Xylose Metabolism Using Synthetic Biology (FLEX)

发展生物燃料以取代我们对石化燃料的依赖是一个具有挑战性的经济问题。基础科学可以使这一过程更有效率，从而更具经济竞争力，从而对这一问题做出重大贡献。用更可持续的木质纤维素基材料取代以淀粉为基础的原料以生产生物丁醇等生物燃料具有挑战性，因为这种材料很复杂，要有效地分解成可供生物燃料生产细菌使用的形式。在这个项目中，我们希望使用新的合成生物学方法将一种全新的生物学特性改造成两种具有重要生物技术意义的细菌，即在半纤维素衍生的低聚木糖上生长的能力。在木聚糖酶对半纤维素酶的分解过程中，会形成低聚木糖，如木四糖(X4)、木三糖(X3)和木二糖(X2)。进一步分解成单糖木糖(X1)需要β-木糖苷酶的作用，然后游离的木糖通过活跃的转运蛋白运输到细菌细胞中。木糖单糖的利用一直是微生物工程中最受关注的有效利用半纤维素的方法，而木糖低聚物的直接使用还没有被广泛考虑。在这个项目中，我们希望通过改造细菌来更有效地利用这种材料。通过使用X2-X4的转运体，将这些分子中的一个转化为一个单位的能量，细菌比那些必须每2-4个单位的能量才能吸收相同数量的单体形式的糖的细菌效率要高得多。在内部将X2-X4分解为X1不需要任何能源成本。在厌氧发酵条件下生长的细菌用于生产生物丁醇的能量是有限的，因此这将使它们的生长速度明显增加，因为低聚木聚糖是在半纤维素上生长时可用的主要碳源，因此这些反应具有高通量。我们还将研究使用比主要ABC转运体更有效的辅助转运体来进一步提高生长产量。在生物工艺水平上，使用低聚木糖而不是完全水解的木糖，允许在半纤维素前处理中使用较温和的化学水解步骤，这也导致较低的化学品产量，从而抑制发酵。低聚木质素簇Xylo4将在大肠杆菌中创建和测试，然后转移到绿色生物公司使用的工业梭状芽孢杆菌菌株，绿色生物公司的合作伙伴目前正在中国使用这些菌株来从木质纤维素中生产丁醇。

The development of biofuels to replace our reliance on petrochemical-based fuels is a challenging economic problem. Basic science can make significant contributions to this problem by making the process more efficient and hence more economically competitive. The replacement of starch-based feedstock for the production of biofuels like biobutanol with more sustainable lignocellulose-based material is challenging due to the complexity of this material to efficient breakdown into forms that can used by the biofuel producing bacteria. In this project we wish to use new synthetic biology methods to engineer a whole new biological property into two biotechnologically important bacteria, namely the ability to grow on hemicellulose-derived xylo-oligomers. The formation of xylo-oligomers such as xylotetraose (X4), xylotriose (x3) and xylobiose(X2) occurs during the enzymatic breakdown of the hemicelluloses by xylanases. Further breakdown to the monosaccharide xylose (X1) requires the action of beta-xylosidases and then the free xylose is transported into bacterial cell via active transporters. The utilisation of the xylose monosaccharide has been the focus on most attention in the engineering of microbes for efficient hemicelluloses utilisation and the use of the xylo-oligomers directly has not been considered very widely. In this project we wish to engineer bacteria to use this material more efficiently. By using transporters for X2-X4, which take up one of these molecules for one unit of energy, the bacteria are much more efficient than those which have to each 2-4 units of energy to take up the same amount of sugar present in the monomeric form. There is no energetic costs to break down the X2-X4 to X1 on the inside. Bacteria growing under anaerobic fermentatative conditions used to make biobutanol are energy limited and so this will make a demonstrable increase in their growth rates as oligo-xylans are the primary carbon source available when growing on hemi-cellulose and hence these reactions have high flux. We will also investigate using a more efficient secondary transporter versus a primary ABC transporter to further improve growth yields. At a bioprocess level the use of xylo-oligomers rather than the fully hydrolysed xylose allow the use of milder chemical hydrolysis steps in the hemicelluloses pretreatment which also results in lower rates of production of chemicals that subsequently inhibit the fermentation. The xylo-oligomer clusters, Xylo4, will be created and tested in Escherichia coli and then transferred to industrial Clostridium strains used by Green Biologics that are currently being used in China by partners of Green Biologics to make butanol from lignocellulose.