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

开发生物燃料以取代我们对基于石油化学的燃料的依赖是一个具有挑战性的经济问题。基础科学可以通过提高这一过程的效率，从而提高经济竞争力，为解决这一问题做出重大贡献。用更可持续的基于木质纤维素的材料替代用于生产生物燃料（如生物丁醇）的基于淀粉的原料是具有挑战性的，这是由于该材料的复杂性，以有效地分解成可由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工程化为两种生物技术上重要的细菌，即在半纤维素衍生的木糖低聚物上生长的能力。木糖低聚物如木四糖（X4）、木三糖（X3）和木二糖（X2）的形成发生在半纤维素被木聚糖酶酶促分解期间。进一步分解为单糖木糖（X1）需要β-木糖醇酶的作用，然后游离木糖通过主动转运蛋白转运到细菌细胞中。木糖单糖的利用一直是微生物工程中最受关注的焦点，用于有效利用半纤维素，而直接使用木糖低聚物尚未被广泛考虑。在这个项目中，我们希望工程细菌更有效地利用这种材料。通过使用X2-X4的转运蛋白，其以一个单位的能量吸收这些分子中的一个，细菌比那些必须每2-4个单位的能量来吸收以单体形式存在的相同量的糖的细菌更有效。在内部将X2-X4分解为X1没有能量成本。在用于制备生物丁醇的厌氧发酵条件下生长的细菌是能量有限的，因此这将使它们的生长速率明显增加，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。我们还将研究使用更有效的次级转运蛋白与初级ABC转运蛋白，以进一步提高生长产量。在生物工艺水平上，使用木糖低聚物而不是完全水解的木糖允许在半纤维素预处理中使用更温和的化学水解步骤，这也导致随后抑制发酵的化学品的生产速率更低。木糖低聚物簇Xylo 4将在大肠杆菌中产生和测试，然后转移到绿色生物制品公司使用的工业梭菌菌株中，绿色生物制品公司的合作伙伴目前正在中国使用这些菌株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.