Project Details
Description
The aim of this project is to develop strains of a fast-growing Mycobacterium 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 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 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 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 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.
Layman's description
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 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 single-step 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.
Key findings
We are successfully growing Mycobacterium with sterol as a Carbon source and we can analyse the breakdown of sterols using analytical methods. This strain of Mycobacterium can be transformed with plasmid DNA and the creation of gene knockouts and over expression strains is underway.
| Acronym | MySterI |
|---|---|
| Status | Finished |
| Effective start/end date | 10/06/13 → 9/06/16 |
Funding
- BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL): £312,246.00