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

石油化工行业以原油、天然气或煤炭为原料，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含有长链线性烷烃的大量废物流。传统的污水处理设施很难处理这些污水，因此对下游水道构成威胁。这些长链烷烃（无论是在废物中，还是在产品切割中）的价值相对较低。然而，如果烷烃上加了氧（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成为各种高价值产品的基础。长链醇和羧酸是生产专用表面活性剂、香料和化妆品的重要化学品。因此，烷烃活化（通过添加氧部分）是为低价值废物流增加价值的有吸引力的途径。然而，由于烷烃的稳定性，活化可能是一个困难的化学转化。工业过程完全绕过活化，而是选择通过寡聚化来生产这些化合物——这就产生了一套醇，然后需要大量和昂贵的分离过程。另一方面，烃类降解生物显示出产生特定长度的醇的非凡能力，这取决于用于代谢的烷烃流。然后，这些醇被转化为特定长度的醛和羧酸，两者都是高价值的化合物，然后作为能量和碳的来源进行代谢。但是，如果这种代谢的改变允许在进一步降解之前收集含氧醇、醛和羧酸，那么一条新的生产途径将是可能的。在这个项目中，我们将开发工具，以精确的基因改造Alcanivorax borkumensis SK2，一个快速生长的物种，能够降解8-30个碳的烷烃，以产生产生这些化合物的菌株。Alcanivorax物种在烃类和塑料的环境降解中起着重要作用。尽管它们很重要，但目前还没有发表过对Alcanivorax物种进行精确基因改造的方法。我们将开发一个重复删除Alcanivorax borkumensis SK2中目标基因的系统，使用两步无标记敲除方法，该方法已被我们的团队成功地用于一系列细菌。我们将以编码酒精、醛和羧酸降解相关蛋白质的基因为目标，这将导致突变体在烷烃存在的情况下积累这些产物。为了产生能够将这些化合物分泌出细胞的菌株，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Alcanivorax borkumensis SK2染色体中，该染色体编码来自已知输出长链醇和羧酸的其他物种的转运蛋白。因此，该系统将用于测试染色体插入和删除，如果成功，可用于产生单核苷酸染色体改变。由于Alcanivorax borkumensis SK2的快速生长，预计在转化后一周内可以快速产生突变体。这将确立Alcanivorax borkumensis SK2作为研究碳氢化合物和塑料降解的良好新模式生物，这将引起对环境，特别是海洋中这些化合物的生物修复感兴趣的海洋微生物学家的极大兴趣。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，以证明该工艺在商业生产方面具有潜在的可行性。

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.