Co-production of ethanol and squalene using a Saccharomyces cerevisiae ERG1 (squalene epoxidase) mutant and agro-industrial feedstock

Background: Genetically customised Saccharomyces cerevisiae that can produce ethanol and additional bio-based chemicals from sustainable agro-industrial feedstocks (for example, residual plant biomass) are of major interest to the biofuel industry. We investigated the microbial biorefinery concept of ethanol and squalene co-production using S. cerevisiae (strain YUG37-ERG1) wherein ERG1 (squalene epoxidase) transcription is under the control of a doxycycline-repressible tet0(7)-CYC1 promoter. The production of ethanol and squalene by YUG37-ERG1 grown using agriculturally sourced grass juice supplemented with doxycycline was assessed. Results: Use of the tet0(7)-CYC1 promoter permitted regulation of ERG1 expression and squalene accumulation in YUG37-ERG1, allowing us to circumvent the lethal growth phenotype seen when ERG1 is disrupted completely. In experiments using grass juice feedstock supplemented with 0 to 50 mu g doxycycline mL(-1), YUG37-ERG1 fermented ethanol (22.5 [+/- 0.5] mg mL(-1)) and accumulated the highest squalene content (7.89 +/- 0.25 mg g(-1) dry biomass) and yield (18.0 +/- 4.18 mg squalene L-1) with supplements of 5.0 and 0.025 mu g doxycycline mL(-1), respectively. Grass juice was found to be rich in water-soluble carbohydrates (61.1 [+/- 3.6] mg sugars mL(-1)) and provided excellent feedstock for growth and fermentation studies using YUG37-ERG1. Conclusion: Residual plant biomass components from crop production and rotation systems represent possible substrates for microbial fermentation of biofuels and bio-based compounds. This study is the first to utilise S. cerevisiae for the co-production of ethanol and squalene from grass juice. Our findings underscore the value of the biorefinery approach and demonstrate the potential to integrate microbial bioprocess engineering with existing agriculture.