ERA-NET: STREPTOMYCES-BASED CELL FACTORIES FOR THE PRODUCTION OF TACROLOGUES DRUGS

Tacrolimus is a drug widely used in clinical practice as an immunosuppressor to prevent transplant rejection. Tacrolimus is produced industrially by fermentation with a bacterium of the Streptomyces family. The aim of TacroDrugs is to develop tacrolimus derivatives with other biological effects than the original molecule. TacroDrugs have partners in Germany, Spain and Portugal in addition to SINTEF in Norway. Our partners aim is to "re-program" the strain to produce tacrolimus analogues instead of tacrolimus. In addition, we want to increase the yield of tacrolimus and its derivatives by optimizing the biosynthetic pathway. SINTEF's contribution in the project is to 1) identify over-producing strains constructed by our partners 2) identify, produce and purify the tacrolimus derivatives produced by our partners 3) improve the yields of tacrolimus by optimizing the fermentation conditions. The technology developed in the project is also relevant for other fermentation processes where Streptomyces strains are used for drug production. TacroDrugs is now finished after 3.5 years. Our partners have focused on improving the production strain by increasing the precursor supply for tacrolimus production, overexpress regulators that may improve the tacrolimus production, remove biosynthetic gene clusters that are not related to the tacrolimus production, removed the native tacrolimus cluster and identified suitable integration sites for mutated clusters. All the candidates constructed by our partners have been tested in controlled cultivations at SINTEF. One of our partners has also succeeded to construct the first tacrolimus analogue producing strain by CRISPR technology. The compound has successfully been produced, purified and pharmacologically characterized by one of our partners. For optimization of the production process and high throughput evaluation of new strains, SINTEF has established miniaturized fermentations (1.2 ml culture volume) using a microfermentor connected to a robot. Together these two units enables online monitoring of crucial fermentation parameters as well as automatic feeding and sampling. The software for this device has been greatly improved during the last year based on SINTEF's needs and feedbacks in the TacroDrugs project. Through >380 micro-fermentations and 74 1-L/3-L fermentations SINTEF has been able to evaluate the mutants produced by our collaborators and improve the volumetric yield of tacrolimus in the wild type from 4 fold in controlled fermentations. Further improvement in volumetric tacrolimus yield has been obtained with engineered strains developed in the project. With the best straind we have obtained 2X volumetric yield of tacrolimus compared to wildtype strain High throughput mass spectroscopy methods for quantitative analysis of tacrolimus and its derivatives have been developed. The analysis time is around 10 minutes per sample. This enables efficient evaluation of large numbers of mutant strains and conditions. A method for identification of novel tacrolimus derivatives and tacrolimus related by-products has been established. We have also established and used methods for isolation and purification of tacrolimus derivatives from fermentations.

TACRODRUGS har bidratt til å konstruere en universell vert for produksjon av biofarmasøytika. En slik vert kan også benyttes for produksjon av andre biomolekyler enn tacrolimus. Det er videre implementert teknologi, blant annet CrispR-Cas, for å endre på geninformasjon slik at nye biomolekyler med nye egenskaper blir produsert. Det er mange som jobber innen dette feltet, og kunnskapen som er etablert i prosjektet vil derfor være av interesse for mange ulike forskningsmiljøer. I TACRODRUGS har vi studert effekten av en rekke globale regulatorer, og det ble funnet regulatorer som viste positiv effekt på produksjon av tacrolimus. Det er sannsynlig at disse regulatorene også kan øke produksjonen av andre forbindelser produsert av Streptomyceter. Gjennom prosjektet har det blitt generert mye kunnskap om hva som påvirker og ikke påvirker produksjon av tacrolimus. Tacrolimus er en kommersielt viktig forbindelse, og denne kunnskapen vil derfor ha nytteverdi også utenfor prosjektet.

In the last decade significant efforts have been made in the development of a universal hosts for the expression of natural products derived from bacterial specialized metabolism. This approach has originated promising results regarding the identification of novel bioactive compounds. However, the production yields are low unveiling a deficient metabolic flux background and undermining this strategy for downstream industrial applications that rely on high production yields. TACRODRUGS introduces the concept of a specialized expression host that combines the stability features of the universal expression host with the metabolic fitness for producing high added-value products. The main goal of TACRODRUGS is to develop a sustainable and robust microbial industrial platform for the production of new tacrolimus and non-immunosuppressive tacrolimus analogues (tacrologues) through the synergic use of synthetic biology principles and metabolic engineering methodologies, guided by global metabolic network understanding.