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

21ENGBIO-开发用于将废弃碳氢化合物转化为高价值化合物的生物工程微生物细胞

• 批准号: BB/W012731/1
• 负责人: David John Lea-Smith
• 金额: $ 12.68万
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW012731%2F1
• 关键词: 21ENGBIO; DEVELOPMENT; BIOENGINEERED; MICROBIAL; CELLS

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.

石化工业利用原油、天然气或煤炭，生产汽油、柴油和其他碳氢化合物燃料。然而，在这些过程中通常产生含有长链直链烷烃的大量废物流。使用传统的废水处理设施很难处理这些废水，因此对下游水道构成威胁。这些长链烷烃（在废物中，甚至在产品馏分中）的价值相对较低。然而，如果烷烃具有添加到其中的氧部分（以产生例如醇、羧酸或醛），则产品从低值变为可以是各种高值产品的基础的值。长链醇和羧酸是生产特种表面活性剂、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）是一种有吸引力的途径，可以为低价值废物流增加价值。然而，由于烷烃的稳定性，活化可能是困难的化学转化。工业过程完全绕过活化，而是选择通过低聚反应生产这些化合物-这产生了一系列醇，然后需要大量和昂贵的分离过程。另一方面，烃降解生物体显示出产生特定长度的醇的显著能力，这取决于用于代谢的烷烃流。然后这些醇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代谢。但是，如果这种代谢的修改允许在进一步降解之前收集含氧的醇，醛和羧酸，那么一种新的生产途径将是可能的。在这个项目中，我们将开发工具，用于精确的Alcanivorax boreholensis SK 2的基因修饰，一种快速生长的物种，能够降解范围从8-30个碳的烷烃，以产生产生这些化合物的菌株。食烷菌属物种在碳氢化合物和塑料的环境降解中发挥着重要作用。尽管其重要性，但尚未公布用于食烷菌属物种的精确遗传修饰的公开方法。我们将开发一个系统，使用两步未标记的敲除方法重复删除Alcanivorax boreholensis SK 2中的靶基因，该方法已被我们的团队成功地用于一系列细菌。我们将靶向编码参与醇、醛和羧酸降解的蛋白质的基因，这将导致在烷烃存在下生长时积累这些产物的突变体。为了产生将这些化合物分泌到细胞外的菌株，这使得收获产物在商业上更可行，我们将使用相同的方法将基因插入并表达到Alcanivorax boreholensis SK 2染色体中，所述基因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，该系统将用于检测染色体插入和缺失，如果成功，则可用于产生单核苷酸染色体改变。由于食烷菌Borkenensis SK 2的快速生长，预期突变体可以在转化后一周内快速产生。这将使Alcanivorax borhouensis SK 2成为研究碳氢化合物和塑料降解的一种极好的新模式生物，这将引起对环境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，特别是海洋。最后，我们将在更大规模的反应器中培养任何被证明能产生和分泌大量所需化合物的菌株，以证明该方法对商业生产是潜在可行的。