ERA-NET: Synthetic Biology for Industrial Production of Steroids

Steroid pharmaceuticals are, after antibiotics, the largest class of drugs marketed for medical applications. They are important for life quality, healthy development, and ageing. Currently more than 300 steroidal compounds are clinically approved representing a 10 billion USD market. Examples are hydrocortisone, testosterone, and prednisolone. Some bacteria in the phylum Actinobacteria can turn plant-based sterols (phytosterols) into key steroid intermediates that subsequently can be modified to the final steroid products. The production of key steroid intermediates from phytosterols has been industrially implemented, but there is still considerable room for improvement and many challenges remain. The overall goal of the Syntheroids project is to establish an eco-friendly and economical steroid production process. Important aims are: (1) to create novel steroid products in a single-step bioprocess, (2) to improve bioprocess efficiency, (3) to create new strains with higher tolerance for the steroid end products, and (4) to improve product recovery by creating strains that form less and fewer by-products. The project is a cooperation between industry and research institutions with partners from Norway, Spain, Germany and Russia. Relevant strains were sent to SINTEF by our collaboration partners. AT SINTEF, we have established cultivation protocols at different scales (from multiwell plates over shake flasks to fermenters) using improved chemically defined media. These kinds of media without any complex compounds are used to be able to better understand a bioprocess. In the Syntheroids project, cultivations in laboratory scale fermenters using defined media were performed to increase understanding of the bacterial phytosterol conversion process by in-depth characterization of the strains and analysis of the bioprocess (using 'omics technology). SINTEF performed fermentations and collected samples. Metabolites were analysed in-house using mass spectrometry (MS)-based methods. Samples for proteomics and transcriptomics analyses were sent to partners for analysis to find targets for direct strain improvement with the overall goal to create mutants that produce fewer by-products. Another task was to identify genes in a selected bacterium that can be modified to achieve higher tolerance towards the steroid products. First, a mutant library of a selected strain was prepared using a chemical mutagen. In parallel, selection protocols to identify mutants with increased tolerance were developed. These included an enrichment of mutants on media containing a key steroid product followed by fluorescence staining and sorting of surviving bacteria using fluorescence-activated cell sorting (FACS) as well as using agar-based media containing increasing concentrations of the steroid product. The whole mutant library was screened and promising mutants from this screen were characterized in phytosterol- and/or steroid- containing media. Growth of the mutants was followed both in multiwell plates and microbioreactors. Several mutants showed improved growth in presence of the steroid product. To monitor phytosterol conversion, samples were taken in regular time intervals and analysed. For this, robotic extraction of the sterols and steroids followed by use of a rapid (RapidFire, ca. 8 sec. per analysis) MS-based analysis for key compounds was applied. Finally, genomes of 5 mutants were sequenced using SINTEF's Illumina MiSeq instrument and the underlying mutations in the genome were mapped to find target genes for future genetic engineering to create strains with higher steroid tolerance. Some selected genes from this approach were also identified previously in proteomics-based approaches performed by one of our Spanish partners. Additionally, SINTEF contributed with both MS and NMR analyses to analyze samples from project partners to identify unknown steroid products in mutant strains.

- Prosjektet har vært en fortsettelse av et tidligere prosjekt (MySterI ERA-IB-2) og har dermed økt samarbeidet mellom forsknings- og industripartnerne ytterligere. Konsortiet ser etter mulighet for videre samarbeid gjennom nye prosjekt. - Prosjektet kunne demonstrere at fornybare råstoffer kan bli omdannet til verdifulle medisinske produkter. - Prosjektet har skapt økt kunnskap og vitenskapelig kompetanse både mht metodeetablering (f.eks. screening pipeline) og resultater som har blitt publisert og skal publiseres både i tidskrifter og en bok. - Prosjektets resultater i sin helhet bidrar med å gi industripartnerne økt konkurransekraft på markedet.

Steroids are the second largest class of drugs marketed for medical applications, only exceeded by antibiotics. Currently, there are more than 300 clinically approved steroidal compounds, representing a 10 billion USD market. Although the production of key steroid intermediates (mainly C19-steroids) from phytosterols has been industrially implemented, there is still considerable room for improvement and many challenges remain. The generation of effective engineered strains, improved bioprocess efficiency, improved product recovery, and the problem of end-product inhibition are major challenges for the industry. The Syntheroids project aims to develop integrative processes for innovative bioconversion of phytosterols to C22-steroids based on Synthetic Biology of non-pathogenic Actinobacteria (Mycobacterium, Nocardioides). These compounds are key intermediates in the synthesis of several therapeutic steroids applied in gastroenterology and endocrinology. Steroid pharmaceuticals are important for life quality, healthy development and ageing, all major challenges today. Production of key intermediates for the synthesis of therapeutic steroids, in an eco-friendly and economical process, is the main expected result of Syntheroids. A few steroid precursors are today industrially produced from phytosterols, mainly by companies in China, India and the US, although some are located in Europe. All in fierce and keen competition. Innovative ideas that can expand the list of steroids produced from phytosterols in a single-step biotechnological process are wanted, and this is why two European companies (Bionice and Pharmins ltd.) are active partners in the five-partner Consortium Syntheroids. Shorter steroid production pipelines and eco-friendly processes, in compliance with European regulations, will increase the EU-GDP (gross domestic product) as it will increase the companies' competitiveness and reduce the end drug user's medical invoice.