Co-production of 11[alpha]-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases

• Published in: Biotechnology for biofuels 2017-09, Vol.10 (1)
• Main Authors: Hull, Claire M, Warrilow, Andrew G. S, Rolley, Nicola J, Price, Claire L, Donnison, Iain S, Kelly, Diane E, Kelly, Steven L
• Format: Article
• Language: English
• Subjects: Chemical properties; Complications and side effects; Dosage and administration; Ethanol; Hydroxylases; Hydroxyprogesterone; Production processes; Yeast (Food product)
• ISSN: 1754-6834; 1754-6834
• DOI: 10.1186/s13068-017-0904-z

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. Genes encoding the 11[alpha]-steroid hydroxylase enzymes from Aspergillus ochraceus (11[alpha]-SH.sup.Aoch) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11[alpha]-SH.sup.Aoch and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 [micro]M progesterone) producing either 11[alpha]-hydroxyprogesterone as the sole metabolite (AH22:p11[alpha]-SH.sup.Aoch) or a 7:1 mixture of 11[alpha]-hydroxyprogesterone and 6[beta]-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11[alpha]-SH.sup.Aoch and AH22:p509C12 were comparable resulting in [greater than or equai to]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 11[alpha]-SH.sup.Aoch gene into the yeast genome (AH22:11[alpha]-SHAoch+K) resulted in a 36% reduction in yield of 11[alpha]-hydroxyprogesterone to 174 [micro]mol/L using 300 [micro]M progesterone. However, increasing progesterone concentration to 955 [micro]M and optimizing growth conditions increased 11[alpha]-hydroxyprogesterone production to 592 [micro]mol/L product formed. The progesterone 11[alpha]-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 v