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Thoroughly Optimised Production Chassis for Advanced Pharmaceutical Ingredients

Periodic Reporting for period 2 - TOPCAPI (Thoroughly Optimised Production Chassis for Advanced Pharmaceutical Ingredients)

Reporting period: 2018-07-01 to 2019-12-31

Chemical products from natural sources, i.e. natural products, are an extremely diverse set of compounds with a seemingly infinite variety of chemical structures and bioactivities. Many of these compounds are very successful pharmaceuticals, and the discovery of novel natural products is obviously of great importance. Infectious diseases are believed to be the second leading cause of death worldwide, with antibiotic-resistant infections affecting 4.1 million patients each year, leading to 147,000 deaths globally.
In TOPCAPI, we will address the problems of:
1) the lack of an industry-strength actinomycete production host for bioactive compounds;
2) the lack of a generic host system for the identification and heterologous production of new antimicrobials/natural products;
3) the need for greener production of bioactive compounds and compounds that are effective to antimicrobial resistant infections.
TOPCAPI will exploit the natural fabrication power of actinomycetes as microbial cell factories to produce three high value compounds: GE2270, a starter compound for the semi-synthesis of NAI-Acne, a new topical anti-acne drug in Phase II clinical trials; tetracycline derivatives, intermediates for semi-synthetic conversion to medically important type II polyketide tetracyclines (TC) some of which have been used for decades and new derivatives at present are in advanced clinical trials, to be used against methicillin-resistant Staphylococcus aureus infections. Our work will focus on two actinomycete species. These host species will be characterised using systems biology approaches, applying integrated data analysis to transcriptomics and metabolomics experiments, combined with predictive mathematical modelling to drive the rapid improvement of these microbial cell factories for industrial drug production using advanced metabolic and biosynthetic engineering approaches. At the same time, we will establish an expanded toolbox for the engineering of actinomycete bacteria as cell factories for other high added-value compounds.
In this period the objectives are:
1. Elucidate the genome sequence of Streptomyces rimosus
2. Collect and analyse transcription and metabolite production data in Planobispora rosea, (GE2270 producer) and S. rimosus (type II polyketide tetracycline producer)
3. Create a metabolic model for P. rosea, (GE2270 producer), S. rimosus (type II polyketide tetracycline producer) and Streptomyces coelicolor (GE2270 heterologous expression host)
4. Improve the metabolic model by incorporating the transcription and metabolite production data.
5. Identify target genes for production improvement in S. rimosus and S. coelicolor
6. Create genome editing and integration methods for S. rimosus and S. coelicolor
• Genome sequencing and analysis of four Streptomyces rimosus strains (WT, HP, M4018, R6), including release of WT and preparation of a joint publication.
• Collected samples for transcriptomics and metabolomics analysis (with 5-10 time points) from fermentation of Planobispora rosea and S. rimosus in industrial conditions.
• Developed an optimal RNA isolation protocol for S. rimosus grown in industrial media conditions.
• Collected and analysed RNAseq data for P. rosea and S. rimosus.
• Validation of RNAseq, dRNAseq results and sRNA analysis completed. Several constructs have been prepared for analysis of overexpression or downregulation in S. rimosus and S. coelicolor.
• Proteomics analysis of sRNA identified proteins differentially regulated.
• Established protocols for optimal metabolomic analysis.
• Validation of Q-TOF and QEx targeted analysis of main compounds in S. rimosus and P. rosea. Untargeted analysis detected other metabolites.
• Collected initial metabolomic data using high resolution LC-MS for P. rosea and S. rimosus.
• Quantitative metabolic flux estimation for P. rosea and for S. rimosus WT and HP strains were determined and used to further constrain metabolic models of the three strains.
• Structures of new compounds have been determined by HRMS, 1D and 2D NMR.
• Created and improved several generations of genome-scale metabolic models of S. coelicolor iAA1259, S. coelicolor M1146-GE2270A, S. lividans TK24-GE2270A, P. rosea, S. rimosus
• Optimisation of genome assembly pipeline tailored to the target organism
• Metabolomics data/flux analysis and integration for P. rosea, S. rimosus WT, S. rimosus HP improved correlation between predicted flux and experimental metabolomics data.
• Transcriptomic-driven analysis identified knockout and/or overexpression gene targets for S. coelicolor and S. rimosus to inform the creation of improved production strains.
• Optimised CRISPR-Cas9 method and integrated cosmid excision in S. coelicolor
• Created two sets of integrative plasmids for S. coelicolor, 11 plasmids in total.
• Constructed 6 integrative vectors for S. rimosus with suitable integrases, promoters and resistance markers.
• Cloned entire oxytetracycline (OTC) gene cluster into YAC/E.coli-Streptomyces shuttle vector and confirmed functionalities.
• Optimised the conditions for GE2270A production (selected the host and developed culture conditions as well as assay conditions).
• Produced a strain that can provide an increased supply of a key molecule for precursor of GE2270A processing.
• Investigated the contribution that exporters might have on resistance.
• (Re)cloned entire OTC clusters and parts of the clusters in suitable vectors and confirmed functionality by heterologous expression in S. rimosus deleted OTC strain and S. lividans, respectively.
• Validation and quantitative analysis of TOPCAPI-1A strain (S. coelicolor).
At present, the anti-infective market is the third-largest pharmaceutical market in the world, with antibiotics sales of over $40 billion annually. Market trends presently show a 4–7% annual growth rate in anti-infective sales in emerging economies, and a global growth of around 0.3%, largely as a result of a growing population of the elderly. TOPCAPI will be targeting this market by producing two competitive compounds using systems and synthesis biology tools for higher yields in a sustainable way.
One important target of TOPCAPI are the tetracyclines (TCs), the fifth most prescribed class of antibiotics. The growing medical problem of drug-resistant bacteria represents an expanding market opportunity. On the other hand, in recent years the cost of antibiotics is rapidly growing. For example, the cost of a 250mg tablet of TC soared by more than 70-fold in just two years. Therefore, competitiveness of the EU-based pharma industry will in coming years greatly depend on the efficacy of fermentation and semi-synthetic processes for the production of these important drugs as addressed in TOPCAPI.
The global market for acne products is around $2B/year. The global market is expected to steadily increase due to lifestyle changes in developing nations. Importantly, none of the currently used antibiotics is particularly effective against acne. In contrast, NAI-Acne, which will be one of the focus drugs of TOPCAPI, is highly selective against Propionibacterium acnes, the etiological agent of acne, and might provide improved efficacy over comparator drugs (e.g. clindamycin, erythromycin).
TOPCAPI Partners 2nd Annual Meeting