Sustainable Bioremediation: Transforming Saccharomyces Cerevisiae Using CRISPR/Cas9 for Oil Spill Cleanup

Oil spills continue to cause significant environmental damage because existing remediation approaches are often slow, inefficient, or environmentally disruptive. The engineering design goal investigated whether the fast-growing, commonly available inexpensive baker’s yeast, Saccharomyces cerevisiae, can be genetically engineered to degrade hydrocarbons to advance sustainable bioremediation aligned with UN Sustainability Development Goal 9.

CRISPR gRNAs were designed in-silico using ChopChop and Benchling platforms to target two genes in S.cerevisiae. The alkane hydroxylase (alkM) gene from Acinetobacter baumannii was codon-optimized for yeast, commercially synthesized and cloned into a yeast expression plasmid with independent promoters and terminators for CRISPR/Cas9-mediated Knock-in. The ADE2 gene in S.cerevisiae was designed for knock-out via CRISPR/Cas9 homology-directed repair, producing red colonies that enabled visual and quantitative transformation efficiency assessment. Hydrocarbon degradation was evaluated gravimetrically over a 20-day incubation in sterile diesel-amended soil inoculated with the yeast having Cas9-only control plasmid and transformed with Cas9+gRNA plasmid. All in-vitro experiments were conducted under BSL1 conditions using commercially prepared competent yeast cells and plasmids.

Transformation efficiency exceeded 75% based on red versus white colony counts. Expressions of AlkM alone did not yield efficient hydrocarbon degradation. Co-expression of AlkG (rubredoxin) and AlkT (rubredoxin reductase) genes with AlkM into a single multigene plasmid (re-synthesized) enabled measurable diesel degradation (~49% with 95% confidence interval of +/- 3%).

Limited degradation efficiency reflects the absence of optimized downstream β-oxidation pathways for long-chain hydrocarbons in yeast. Future work could integrate peroxisomal β-oxidation pathway engineering. Results demonstrate the feasibility of engineering fast-growing yeast for scalable, innovative hydrocarbon bioremediation