Abstract
BackgroundBioethanol 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.ResultsGenes encoding the 11α-steroid hydroxylase enzymes from Aspergillus ochraceus (11α-SHAoch) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11α-SHAoch 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-SHAoch) or a 7:1 mixture of 11α-hydroxyprogesterone and 6β-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11α-SHAoch 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-SHAoch 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.ConclusionsThe 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.
Highlights
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
Recombinant yeast: progesterone bioconversion Metabolite profiles for yeast transformants grown on YM+His with 300 μM progesterone indicate that the optimized gene sequences encode functional proteins that were constitutively expressed and conferred progesterone hydroxylating capabilities on AH22:pCYP509C12 culture supernatant. Control (AH22):p11α-SHAoch and AH22:pCYP509C12 yeast (Table 1; Fig. 2)
During initial growth experiments with YM+His with 300 μM progesterone, almost total bioconversion of progesterone was achieved with AH22:p11α-SHAoch which produced ≥90% 11α-hydroxyprogesterone as the sole metabolite after 48 h of growth (Table 1)
Summary
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. The potential of yeast to produce intermediates needed in corticosteroid production is demonstrated at laboratory scale following bioethanol production from perennial ryegrass juice. Biofuels such as bioethanol are a key focus for sustainability away from fossil fuels and in a biorefinery approach maximum value and product extraction is envisaged. Some of the first industrial biotransformations involved filamentous fungi for production of key intermediates in corticosteroid production that were hard to achieve by chemical synthesis. Structural hydroxylations are often necessary to functionalize steroid-based drugs that exhibit higher efficacy than their non-hydroxy analogs [10], and the ability to perform regio- and stereo-specific modifications using whole-cell microbial biocatalysis is invaluable for largescale steroid manufacturing [11] not least because certain hydroxylations can prove difficult or even impossible to achieve via synthetic chemistry
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