Synthetic Saccharomyces cerevisiae tolerate and degrade highly pollutant complex hydrocarbon mixture

Fungal laccase (Lac) has become a very useful biocatalyst in different industries, bio-refineries and, most importantly, bioremediation. Many reports have also linked hydrocarbon tolerance and degradation by various microorganisms with Lac secretion. In this study, Trametes trogii Lac (Ttlcc1) was engineered into Saccharomyces cerevisiae strain CEN.PK2-1 C under the constitutive GPD promoter (pGPD) for multi-fold synthesis with efficient hydrocarbon tolerance and degradation. Protein expression in heterologous hosts is strictly strain-specific, it can also be influenced by the synthetic design and culture conditions. We compared synthetic designs with different shuttle vectors for the yeast strains and investigated the best culture conditions by varying the pH, temperature, carbon, nitrogen sources, and CuSO₄ amount. Two S. cerevisiae strains were built in this study: byMM935 and byMM938. They carry the transcription unit pGPD-Ttlcc1-CYC1t either inside the pRSII406 integrative plasmid (byMM935) or the pRSII426 multicopy plasmid (byMM938). The performance of these two synthetic strains were studied by comparing them to the wild-type strain (byMM584). Both byMM935 and byMM938 showed significant response to different carbon sources (glucose, galactose, lactose, maltose, and sucrose), nitrogen sources (NH₄Cl, NH₄NO₃, KNO₃, malt extract, peptone, and yeast extract), and solid state fermentation of different plant biomasses (bagasse, banana peels, corn cob, mandarin peels, and peanut shells). They performed best in optimized growth conditions with specific carbon and nitrogen sources, and a preferred pH in the range 3.5-4.5, temperature between 30 and 40 ⁰C, and 1 mM CuSO_4. In optimized yeast-growth medium, strain byMM935 showed the highest laccase activities of 1.621 ± 0.063 U/mL at 64 h, whereas byMM938 gave its highest activity (1.417 ± 0.055 U/mL) at 48 h. In this work, we established, by using Bushnell Hass synthetic medium, that the new Ttlcc1-yeast strains tolerated extreme pH and complex hydrocarbon mixture (CHM) toxicity. They degraded 60-90% of the key components in CHM within 48 h, including poly-cyclic aromatic hydrocarbons, alkyl indenes, alkyl tetralines, alkyl benzenes, alkyl biphenyls, and BTEX (Benzene, Toluene, Ethylbenzene, and Xylenes). This is the first report on the hydrocarbon degradation potential of a Ttlcc1-yeast. Compared to the native organism, such synthetic strains are better suited for meeting growing demands and have potentials for application in large-scale in situ bioremediation of hydrocarbon-polluted sites.