BIOTEK2021-Bioteknologi for verdiskaping

The Gram-negative bacterium Escherichia coli is the most widely used organism for the production of recombinant proteins with applications in industry, pharmacy, medicine and molecular biology reagents. Protein synthesis is the most energy and resource exhaustive process for the cells and therefore needs to be optimized to achieve maximal productivities of functional proteins. Natural environment of E. coli is much harsher compared to the near optimal growth conditions used in industrial production processes. For their natural lifestyle cells produce many native proteins that could be considered unnecessary for the cells in industrial production conditions. In LeanProt project, our aim has been to map and remove the most resource exhaustive unnecessary proteins from the host cells to free up resources for maximized recombinant protein production. This was accomplished by a systems biology drive approach combining omics analyses, modeling, host cell physiology characterization, and process and genetic engineering technologies. The Norwegian (NTNU) role in LeanProt was to design and construct a novel three reporter system enabling careful and preside evaluation of mutant E. coli host strains. We chose to use a broad-host range replicon extending the application range of this tool to include any Gram-negative bacteria. As one important knowledge basis for this part, a review on the field of relevant genetic tools was written and published in Microbial Biotechnology (Gawin and Brautaset et al 2017). The properties and application potential of the constructed reporter system was demonstrated in three different bacterial, species and published in Microbial Cell Factories (Gawin and Brautaset et al 2018). The constructed reporter system was also used by our LeanProt partners to test more mutant E. coli strains constructed in LeanProt (Manuscripts in preparation). In addition to taking leading role in generation of E. coli Lean mutant strains (see above), we also focused on targeted mutagenesis of E. coli chromosomal genes encoding proteins with predicted roles in folding, disulphide bridge formation and degradation of secreted recombinant proteins. Totally nine mutant strains were constructed with altered regulation of such genes, and tested for improved secretion of two pharmaceutically proteins. Four mutant strains displayed significantly improved production of functional proteins were achieved and this work is under publishing now (Gawin and Brautaset; manuscript in preparation). NTNU partner also took role as guest-editor for a special issue in the journal Microorganisms on "Recombinant protein production in Microorganisms", directly related to and relevant for LeanProt (Brautaset and Valla, Microorganisms 2019).

LeanProt prosjektet har representert et nytt nettverk av sterke europeiske partnere vi (NTNU) ikke har hatt samarbeid med før innenfor system biologi. Særlig har samarbeidet mot partnere i Latvia og Tyskland fungert veldig bra med god utveksling av biologiske materialer, metoder og co-publiseringer. Disse nettverkene vil vi beholde for fremtidige nye søknader. Rekombinant protein produksjon er et stort og viktig felt og resultatene oppnådd i prosjektet har betydelighet for bedre design av selve vertstammenn, samt bedre systemer for å evaluere dem. Vi har opparbeidet ny forståelse innenfor verts-egenskaper som er avgjørende for folding og produksjon av sekterete proteiner i E. coli. Dette er kunnskap og teknologi av industriell interesse. NTNU har lang erfaring innenfor rekombinant protein produksjon og deltagelse i leanProt har tatt dette videre og i nye spennende retninger.

LEANPROT aims to develop an iterative systems biology-based strain engineering platform based on a novel approach of proteome optimization in iterative cycles of modeling and biological experiments which carries a substantial potential for addressing the aforementioned challenges in bioprocess development. That potential arises from the fact that cells express proteins unnecessary for growth under well-controlled optimal conditions, typically realized in biotechnological processes (e.g. flagellar, heat or acid stress proteins). This leads to non-efficient use of protein synthesis capacity (translation machinery) and energy for bioprocesses. Thus removing the expression burden of unnecessary proteins i.e. creation of lean-proteome strains, could enable to specifically manipulate the allocation of ribosomes for higher synthesis of proteins leading to increased target molecule production. One could theoretically greatly increase the key metrics of bioprocess performance (titer, yield, productivity) by deleting as few as ca 10 unnecessary genes with the highest translational burden in E. coli (in total 7% of proteome) and substituting the freed 7% of the total proteome with target molecule-related proteins. In LEANPROT we will demonstrate the potential of this concept as proof-of-principle by creating superior E. coli strains of recombinant protein production through reducing the expression of unnecessary proteins. LEANPROT will yield E. coli strains with superior performance of recombinant protein production - theoretically at least 50% higher productivity and titer - as a result of proteome optimization. Since targeted optimization of the layer of protein synthesis capacity should be applicable to a broad range of organisms, from bacteria to yeasts to higher eukaryotes, LEANPROT has potentially a big impact in the advancement of cell design overall.