Fundamental Insight and Biotechnological Optimisation to Enable Commercial Production of Renewable Propane

丙烷是一种用作可燃燃料的短链烷烃，在全球拥有良好的市场。近年来，新的合成途径被描述为丙烷的生物生产。虽然这些技术目前还不具备商业可行性，但一种真正的可再生丙烷资源将减轻我们目前对不可持续的化石燃料的依赖。与BDS燃料公司和曼彻斯特大学（Nigel Scrutton集团）一起，我们已经确定了开发可再生丙烷生产的机会，作为现有天然气系统的补充。然而，为了实现可再生丙烷的商业化生产，该工艺需要首先得到改进。烷烃生物合成的途径集中在醛转化为烷烃的最后一步。该反应可以由蓝藻醛脱甲酰加氧酶（cADO）催化，利用铁和氧从醛底物中去除甲酸分子，产生碳氢化合物产物。然而，cADO的催化活性很低，因此这一步限制了碳通量到丙烷。虽然通过合理的工程设计提高cADO结合袋对短链底物的特异性的努力已经提高了碳氢化合物产品的滴度，但丁醛的有效利用还需要进一步优化。商业规模的可再生丙烷生物生产需要解决三个方面的问题；首先，提高我们对天然体系的理解，增加cADO的功能，从而直接或通过发展更有利的环境来增加烷烃的产量。其次，提高对烷烃前体的通量，最后，扩大可再生丙烷生物合成的规模和实施。该项目将研究天然环境下的烷烃生物合成系统，以了解并潜在地利用新的应用见解。我们估计，从糖到丙烷的碳通量至少需要增加10倍才能实现商业可行性。利用相同底物的竞争途径可以被删除。然而，这可能对细胞的整体代谢平衡产生负面影响。代谢网络建模可以深入了解途径改变的影响。当结合对使用CRISPR干扰的变体库下调的系统调查时，建模可以用来帮助实现最佳平衡。蛋白质和废物分析可用于验证变体的功能，生成的数据可用于反馈并用于测试模型的有效性。工程菌株将与曼彻斯特大学和BDS燃料公司合作，在实验室和中试规模上进行评估。

Propane, a short chain alkane used as a combustible fuel, has a well-established global market. Recently, novel synthetic pathways have been described for the biological production of propane. Though these are currently not yet commercially viable, a genuinely renewable source of propane would alleviate some of our current dependence on unsustainable fossil fuels.Together with BDS Fuels and University of Manchester (Nigel Scrutton group) we have identified an opportunity to develop renewable propane production as a complement to existing natural gas systems. However, in order to implement renewable propane production commercially the process needs to first be improved.Pathways for alkane biosynthesis converge on the terminal step where aldehydes are converted to alkanes. This reaction can be catalysed by cyanobacterial Aldehyde Deformylating Oxygenase (cADO) using iron and oxygen to remove a formate molecule from an aldehyde substrate producing hydrocarbon product. However, the catalytic activity of cADO is very low and therefore this step is limiting carbon flux to propane. Whilst efforts to improve the specificity of the cADO binding pocket towards short chain substrates through rational engineering have already improved titres of hydrocarbon products, efficient utilisation of butyraldehyde would require further optimisation.Biological production of renewable propane on a commercial scale requires three aspects to be addressed; first, improving our understanding of the native system and increasing the function of cADO and therefore also increasing the production of alkanes, either directly or by developing a more favourable environment. Secondly, enhancing flux to alkane precursors and finally, the scale up and implementation of renewable propane biosynthesis.This project will study the alkane biosynthetic system in its native environment in order to understand and potentially also utilise new insight for application. We estimate that at least a 10-fold increase in carbon flux from sugar to propane is required for commercial viability. Competing pathways utilising the same substrates can be deleted. However, this can have negative implications for the overall metabolic balance of the cell. Metabolic network modelling could provide insight into the impact of pathway alterations. When combined with systematic investigations into down regulation of a library of variants using CRISPR interference, modelling could be used to help move towards an optimal balance. Protein and waste product analysis can be used to verify the functionality of variants and data generated can be fed back and used to test the efficacy of models.Engineered strains will be evaluated at lab and pilot scale in collaboration with the University of Manchester and BDS Fuels.