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Background: Bioethanol production from sustainable sources of biomass that limit effect on food production are needed and in a biorefinery approach co-products are desirable, obtained from both the plant material and from the microbial biomass. Fungal biotransformation of steroids was among the first industrial biotransformations allowing corticosteroid production. In this work, the potential of yeast to produce intermediates needed in corticosteroid production is demonstrated at laboratory scale following bioethanol production from perennial ryegrass juice., Results: Genes encoding the 11α-steroid hydroxylase enzymes from Aspergillus ochraceus (11α-SH Aoch ) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11α-SH Aoch and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 µM progesterone) producing either 11α-hydroxyprogesterone as the sole metabolite (AH22:p11α-SH Aoch ) or a 7:1 mixture of 11α-hydroxyprogesterone and 6β-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11α-SH Aoch and AH22:p509C12 were comparable resulting in ≥75% conversion of glucose to alcohol. Co-production of bioethanol together with efficient production of the 11-OH intermediate for corticosteroid manufacture was then demonstrated using perennial ryegrass juice. Integration of the 11α-SH Aoch gene into the yeast genome (AH22:11α-SHAoch+K) resulted in a 36% reduction in yield of 11α-hydroxyprogesterone to 174 µmol/L using 300 µM progesterone. However, increasing progesterone concentration to 955 µM and optimizing growth conditions increased 11α-hydroxyprogesterone production to 592 µmol/L product formed., Conclusions: The progesterone 11α-steroid hydroxylases from A. ochraceus and R. oryzae , both monooxygenase enzymes of the cytochrome P450 superfamily, have been functionally expressed in S. cerevisiae . It appears that these activities in fungi are not associated with a conserved family of cytochromes P450. The activity of the A. ochraceous enzyme was important as the specificity of the biotransformation yielded just the 11-OH product needed for corticosteroid production. The data presented demonstrate how recombinant yeast could find application in rural biorefinery processes where co-production of value-added products (11α-hydroxyprogesterone and ethanol) from novel feedstocks is an emergent and attractive possibility.

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 μg 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 μg 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.