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Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases

Claire Hull, Andrew Warrilow, Nicola Rolley, Claire Price Orcid Logo, Iain S. Donnison, Diane Kelly, Steven Kelly

Biotechnology for Biofuels, Volume: 10, Issue: 1

Swansea University Authors: Claire Hull, Andrew Warrilow, Nicola Rolley, Claire Price Orcid Logo, Diane Kelly, Steven Kelly

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DOI (Published version): 10.1186/s13068-017-0904-z

Abstract

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 firs...

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Published in: Biotechnology for Biofuels
ISSN: 1754-6834
Published: Springer Science and Business Media LLC 2017
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URI: https://cronfa.swan.ac.uk/Record/cronfa64804
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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 11&#x3B1;-steroid hydroxylase enzymes from Aspergillus ochraceus (11&#x3B1;-SHAoch) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11&#x3B1;-SHAoch and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 &#xB5;M progesterone) producing either 11&#x3B1;-hydroxyprogesterone as the sole metabolite (AH22:p11&#x3B1;-SHAoch) or a 7:1 mixture of 11&#x3B1;-hydroxyprogesterone and 6&#x3B2;-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11&#x3B1;-SHAoch and AH22:p509C12 were comparable resulting in &#x2265;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&#x3B1;-SHAoch gene into the yeast genome (AH22:11&#x3B1;-SHAoch+K) resulted in a 36% reduction in yield of 11&#x3B1;-hydroxyprogesterone to 174 &#xB5;mol/L using 300 &#xB5;M progesterone. However, increasing progesterone concentration to 955 &#xB5;M and optimizing growth conditions increased 11&#x3B1;-hydroxyprogesterone production to 592 &#xB5;mol/L product formed. Conclusions: The progesterone 11&#x3B1;-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. 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spelling 2023-11-14T17:20:54.1621561 v2 64804 2023-10-24 Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases 6c495cb489b67bc9bb3f746975c4ff19 Claire Hull Claire Hull true false f066e233e8d0136c9f547b86fa43747f Andrew Warrilow Andrew Warrilow true false bf3e225536b328599743bf455cea51e0 Nicola Rolley Nicola Rolley true false 9a4e4dfa37f4318c6fa67933d4fc9a17 0000-0002-6045-4835 Claire Price Claire Price true false 5ccf81e5d5beedf32ef8d7c3d7ac6c8c Diane Kelly Diane Kelly true false b17cebaf09b4d737b9378a3581e3de93 Steven Kelly Steven Kelly true false 2023-10-24 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-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. 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. Journal Article Biotechnology for Biofuels 10 1 Springer Science and Business Media LLC 1754-6834 Bioconversion, Bioethanol, Biorefinery, 11α-Hydroxyprogesterone, Ryegrass, Yeast 31 12 2017 2017-12-31 10.1186/s13068-017-0904-z http://dx.doi.org/10.1186/s13068-017-0904-z COLLEGE NANME COLLEGE CODE Swansea University Funding for this study was provided by the BEACON Convergence project supported by Welsh Government and the European Regional Development Fund (ERDF) of the European Union. 2023-11-14T17:20:54.1621561 2023-10-24T09:57:36.7994664 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Claire Hull 1 Andrew Warrilow 2 Nicola Rolley 3 Claire Price 0000-0002-6045-4835 4 Iain S. Donnison 5 Diane Kelly 6 Steven Kelly 7 64804__29020__0681001b0e0c449f8d167534e5839a5b.pdf 64804.VOR.pdf 2023-11-14T17:18:08.1039910 Output 1544353 application/pdf Version of Record true © The Author(s) 2017. Distributed under the terms of a Creative Commons Attribution 4.0 International License (CC BY 4.0). true eng https://creativecommons.org/licenses/by/4.0/
title Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases
spellingShingle Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases
Claire Hull
Andrew Warrilow
Nicola Rolley
Claire Price
Diane Kelly
Steven Kelly
title_short Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases
title_full Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases
title_fullStr Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases
title_full_unstemmed Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases
title_sort Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases
author_id_str_mv 6c495cb489b67bc9bb3f746975c4ff19
f066e233e8d0136c9f547b86fa43747f
bf3e225536b328599743bf455cea51e0
9a4e4dfa37f4318c6fa67933d4fc9a17
5ccf81e5d5beedf32ef8d7c3d7ac6c8c
b17cebaf09b4d737b9378a3581e3de93
author_id_fullname_str_mv 6c495cb489b67bc9bb3f746975c4ff19_***_Claire Hull
f066e233e8d0136c9f547b86fa43747f_***_Andrew Warrilow
bf3e225536b328599743bf455cea51e0_***_Nicola Rolley
9a4e4dfa37f4318c6fa67933d4fc9a17_***_Claire Price
5ccf81e5d5beedf32ef8d7c3d7ac6c8c_***_Diane Kelly
b17cebaf09b4d737b9378a3581e3de93_***_Steven Kelly
author Claire Hull
Andrew Warrilow
Nicola Rolley
Claire Price
Diane Kelly
Steven Kelly
author2 Claire Hull
Andrew Warrilow
Nicola Rolley
Claire Price
Iain S. Donnison
Diane Kelly
Steven Kelly
format Journal article
container_title Biotechnology for Biofuels
container_volume 10
container_issue 1
publishDate 2017
institution Swansea University
issn 1754-6834
doi_str_mv 10.1186/s13068-017-0904-z
publisher Springer Science and Business Media LLC
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
url http://dx.doi.org/10.1186/s13068-017-0904-z
document_store_str 1
active_str 0
description 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-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. 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.
published_date 2017-12-31T14:08:15Z
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