Novel industrial bioprocesses for production of key valuable steroid precursors from phytosterols

Steroids are specific structure terpenoid lipids that occur widely in living systems. Over 250 sterols and related compounds
have been reported in plants, insects, vertebrates and lower eukaryotes such as yeasts. Steroid hormones control cell
proliferation and tissue differentiation and modulate gene expression. Hence, steroid based pharmaceuticals are important
for human and animal health in; (a) disease prevention (antitumor, anti-estrogenic); (b) disease therapy (cancer, obesity)
and (c) life-style choices (menopause, human fertility). As a result, about 300 approved steroid drugs exist to date and the
numbers are growing. Indeed the market for steroid drugs lies second only to that for antibiotics; the global market for
steroids is in excess of US $10 billion and more than 1,000,000 tons annually. In recent years the conversion of
agricultural, food or cellulose industrial wastes containing phytosterols into high value steroid compounds has become a
highly attractive goal. However studies to date indicate that improvements are needed in phytosterol culture broth solubility,
microbial strains, and product recovery. Project MySterI will focus on improvement of the known bioconverting bacterium,
Mycobacterium sp. NRRL B-3085, to replace the existing microbial/chemical multistep processes with single-step
production processes for androst-4-ene-3,17-dione (AD) (intermediary precursor), 11alpha hydroxyandrost-4-ene-3,17-
dione (11-alpha-OH-AD), 3beta-hydroxyandrost-5-ene-17-one (DHEA) or testosterone from phytosterols. The use of singlestep
bioconversion processes will reduce costs and loss of yield associated with the multistep processes, and will be
environmentally cleaner. The MySterI pipeline includes the development of genetic engineering tools, strain improvement
supported by 'omics information, scale down and up process and simplified product recovery, all of which comprise a
complete improvement of the production process.

The aim of this project is to develop strains of the fast-growing Mycobacterium sp. NRRL B-3085 that will convert
phytosterols from cheap agricultural plant waste into high value steroids in a single step, thus reducing costs and loss of
yield due to processing of intermediates. Mycobacterium sp. NRRL B-3085 metabolises steroids through the action of
cholesterol oxidases to modify the A-ring of the core and then cleaves the side chain, by multiple enzymes, to produce
androst-4-ene-3,17-dione (AD), a precursor for valuable C19-steroids. Our first goal is to modify this strain to optimize this
conversion by tailoring the innate catabolic enzymes. This will require an understanding of the phytosterol catabolic
pathway of Mycobacterium sp; NRRL B-3085 to facilitate its rational design as a platform for phytosterol bioconversion.
Consequently genomic technologies will be used to characterize the pathways in steroid metabolism. The second goal is to
introduce the genes expressing 11alpha-hydroxylase (and its associated oxidoreductase) required to convert AD to 11-
alpha-OH-AD, DHEA or testosterone. The fungus Aspergillus ochraceus strain Ph34 (isolated by Donova, partner 2)
converts AD to 11-alpha-OH-AD with high efficiency and this strain will be used to source the genes (by cDNA sequencing)
for the 11alpha-hydroxylase and oxidoreductase. Tools for genetic manipulation of Mycobacterium sp; NRRL B-3085 will
be developed. Thirdly bioconversion of steroid faces particular problems due to the hydrophobic nature of both substrates
and products. Therefore the project will address fermentation broth design, reactor design and product purification.

This project aims to develop a novel, environmentally clean, industrial process for the bioconversion of phytosterols in
cheap agricultural plant waste into high value steroids for use in medicine. There are therefore opportunities for scientists,
industry and the general public to benefit from this research.
1. Communication to beneficiaries
As for academic beneficiaries, scientists from the biotechnology industries and environmental agencies will read about our
work from our peer reviewed publications, web sites and from conference presentations. Two of our project partners are
industrial biotechnology companies (Gadea Biopharma S.L. and Pharmins Ltd) and they will be keen to publicise
developments in the project after protection of intellectual property. The University of York Research Administration Unit
will work with us and our partners to negotiate fair intellectual property arrangements and to set up a formal collaboration
agreement.
Communication of the work to the public will be through the Communications Office at the University of York whose press
releases are frequently then taken up in local and national newspapers, TV channels and web sites. The University has
Open Days for visitors, mostly for those interested in studying at York.
2. What are the benefits?
The major benefit to industry will be innovative use of microbial manipulation for the development of new organisms for
bioconversion in an industrial process. The genomics and 'omics data will provide valuable information on the nature of a
microbial saprophyte. The research on the use and applicability of cheap feedstocks for microbial conversion is of great
benefit to many in the industrial biotechnology field. The project in particular addresses important problems on the
hydrophobic nature of both the feedstock and the product. One of the major benefits of the process under development in
this project over previous methods for steroid conversion will be environment cleanliness as the downstream processing
after fermentation will involve fewer steps and less chemical contamination. The benefits of project MySterI will be
economic as well as quality of life and wellbeing, which will affect both industry and the general public. Communication of
the work to the public also imparts benefits in terms of education, understanding and accountability.