21ENGBIO- DEVELOPMENT OF BIOENGINEERED MICROBIAL CELLS FOR CONVERSION OF WASTE HYDROCARBONS TO HIGH VALUE COMPOUNDS

石化行业提取原油、天然气或煤炭，生产汽油、柴油和其他碳氢化合物燃料。然而，在这些过程中，通常会产生大量含有长链直链烷烃的废气。使用传统的废水处理设施很难处理这些污水，因此对下行水道构成了威胁。这些长链烷烃(无论是在废物中，甚至在产品切割中)的价值都相对较低。然而，如果烷烃上添加了氧部分(例如，为了生产醇、羧酸或醛)，那么产品就会从低价值变成可以作为各种高价值产品的基础。长链醇和羧酸是生产特种表面活性剂、香水和化妆品的重要化学品。因此，烷烃活化(通过添加氧气部分)是为低价值废流增加价值的一条有吸引力的途径。然而，由于烷烃的稳定性，活化可能是一种困难的化学转化。工业过程完全绕过了活化，而是选择通过齐聚来生产这些化合物-这会产生一系列醇，然后需要大量且昂贵的分离过程。另一方面，烃类降解菌表现出显著的产生特定长度的醇的能力，这取决于用于新陈代谢的烷烃流。然后，这些醇被转化为特定长度的醛和羧酸，这两种化合物也是高价值的化合物，然后作为能量和碳的来源进行代谢。但如果这种新陈代谢的改变允许在进一步降解之前收集含氧醇、醛和羧酸，那么一条新的生产路线将是可能的。在这个项目中，我们将开发工具来精确地对波尔库姆链藻SK2进行基因改造，以产生产生这些化合物的菌株。Alcanivorax物种在碳氢化合物和塑料的环境退化中发挥着主要作用。尽管它们很重要，但还没有发表过对Alcanivorax物种进行精确基因改造的公开方法。我们将开发一种系统，使用两步无标记敲除方法重复删除博尔库马氏菌SK2中的目标基因，该方法已被我们的团队在一系列细菌中成功使用。我们将针对编码与酒精、醛和羧酸降解有关的蛋白质的基因，这将导致在烷烃存在下生长时积累这些产物的突变株。为了产生从细胞外分泌这些化合物的菌株，从而使产品的收获更具商业可行性，我们将使用相同的方法将基因插入并表达到博尔库姆链藻SK2染色体中，编码转运蛋白的物种来自其他已知出口长链醇和羧酸的物种。因此，这个系统将被用来测试染色体的插入和缺失，如果成功，可以用来产生单核苷酸染色体变化。由于波氏链球藻SK2的快速生长，预计在转化后的一周内可以迅速产生突变株。这将使Alcanivorax borkumensis SK2成为研究碳氢化合物和塑料降解的优秀新模式生物，这将引起海洋微生物学家对这些化合物在环境中，特别是海洋中的生物修复的极大兴趣。最后，我们将在更大规模的反应器中培养任何能够产生和分泌大量所需化合物的菌株，以证明该方法有潜在的商业生产可行性。

The petrochemical industry takes crude oil, natural gas, or coal, and produces petrol, diesel and other hydrocarbon fuel cuts. However, significant waste streams, containing long chain linear alkanes, are commonly produced in these processes. These are difficult to treat using traditional wastewater treatment facilities, and so pose a threat to down-steam waterways. These long chain alkanes (both in the wastes, and even the product cuts) are comparatively low value. However, if an alkane has an oxygen moiety added to it (to produce, for example, an alcohol, a carboxylic acid or an aldehyde), then the product goes from a low value to one which can be the basis for various high value products. Long chain alcohols and carboxylic acids are important chemicals in the production of specialised surfactants, fragrances, and cosmetics. Alkane activation (by the addition of an oxygen moiety) is therefore an attractive route to adding value to a low value waste stream. However, due to the stability of alkanes, activation can be a difficult chemical conversion. Industrial processes bypass activation entirely, opting to rather produce these compounds via oligimerization - this produces a suite of alcohols that then require significant and expensive separation processes. On the other hand, hydrocarbon degrading organisms show a remarkable ability to produce alcohols of specific length, which is dependent on the alkane stream used for metabolism. These alcohols are then converted to aldehydes and carboxylic acids of specific length, both also high value compounds, before being metabolised as a source of energy and carbon. But if modification of this metabolism allowed for collection of the oxygenated alcohols, aldehydes and carboxylic acids before further degradation, then a new route to production would be possible.In this project we will develop tools for precise genetic modification of Alcanivorax borkumensis SK2, a fast-growing species capable of degrading alkanes ranging from 8-30 carbons, in order to generate strains producing these compounds. Alcanivorax species play a major role in environmental degradation of hydrocarbons and plastics. Despite their importance, no published method for precise genetic modification of an Alcanivorax species has been published. We will develop a system for repeated deletion of target genes in Alcanivorax borkumensis SK2 using a two-step unmarked knockout method that has been successfully utilised by our group in a range of bacteria. We will target genes encoding the proteins involved in alcohol, aldehyde and carboxylic acid degradation, which will result in mutants that accumulate these products when grown in the presence of alkanes. In order to generate strains that secrete these compounds out of the cell, which makes harvesting of the products more commercially viable, we will use the same method to insert and express genes into the Alcanivorax borkumensis SK2 chromosome encoding transporters derived from other species known to export long-chain alcohols and carboxylic acids. This system will therefore be used to test both chromosomal insertion and deletion and if successful, could be utilised to generate single nucleotide chromosomal alterations. Due to the fast growth of Alcanivorax borkumensis SK2, it is expected that mutants could be generated rapidly, within a week following transformation. This would establish Alcanivorax borkumensis SK2 as an excellent new model organism for investigating hydrocarbon and plastic degradation, which would be of great interest to marine microbiologists interested in bioremediation of these compounds in the environment, especially the oceans. Finally, we will culture any strains demonstrated to produce and secrete large amounts of the desired compounds in larger scale reactors to demonstrate that the process is potentially viable for commercial production.