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

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.

In order to complete each objective (1-4) the following techniques will be employed.
1. Precision genetic engineering of Alcanivorax borkumensis SK2 (SK2) will be conducted by first generating plasmids in which amplicons homologous to the flanking regions on either side of the site of deletion/insertion will be cloned into a conjugation plasmid. Between the flanking regions will be inserted a positive (kan(R)) and negative (sacB) selectable marker. Plasmids will be introduced via conjugation and marked mutants in which a double homologous recombination event have occurred will be selected on agar plates containing kanamycin. A second plasmid containing just the flanking regions will be introduced into this strain and unmarked mutants in which the kan(R)/sacB cassette have been removed via double homologous recombination will be selected on agar plates containing 10% sucrose. Mutants will be verified using primers flanking the modified site.
2. This technique will be used to generate deletion mutants targeting the genes encoding the proteins involved in 1-alcohol to aldehyde conversion (AlkJ1, AlkJ2), aldehyde to carboxylic acid conversion (AlkH1, AlkH2) and carboxylic acid to acyl-CoA conversion (AlkK).
3. The same technique will be used to generate strains for 1-alcohol and carboxylic acid export. Codon-optimised expression cassettes will be inserted into the plasmids containing the AlkJ1 or AlkJ2, and AlkK flanking regions so that the genes are under control of the native promoters/terminators. This plasmid will then be introduced into the marked mutants generated in (1) and mutants selected on agar plates containing 10% sucrose.
4. To determine production of each compound, strains will be cultured in small volume (50 ml) flasks in sodium pyruvate till they reach late exponential phase before alkanes of C8 and C17 are added. Cells will then be cultured for 5 days and the concentration of each compound determined in the cellular mass and supernatant via GC-MS.