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Recombinant protein production is at the core of biotechnology and numerous molecular tools and bacterial strains have been developed to make the process more efficient. One commonly used generic solution is to supply extra copies of low-abundance tRNAs to compensate for the presence of complementary rare codons in genes-of-interest. Here we show that such extra tRNA, supplied by the commonly used pLysSRARE2 plasmid, can cause two side effects: (1) growth and gene expression can be impaired, and (2) apparent positive effects can be caused by differential expression of the lysozyme gene encoded on the same plasmid and not the tRNAs per se. These phenomena seem to have been largely overlooked despite the huge popularity of the T7/pET-based systems for bacterial protein production.

Den grundlæggende forståelse af biologiske systemer bærer nøglen til en bæredygtig produktion af medicin, kemikalier og materialer, og bioteknologien muliggør dette. Produktion af rekombinante proteiner er helt centralt for den bioteknologiske industri. Talrige molekylære værktøjer og mikroorganismer er blevet udviklet gennem de seneste fire årtier, for at udvikle nye processer og forbedreeksisterende, med henblik på netop rekombinant proteinproduktion. Vejen til at udvikle mikrobielle cellefabrikker går gennem en forståelse af den genetiske kode og vores evne til at manipulere arvemasse: Dette gør os i stand til at skabe bæredygtige og omkostningseffektive systemer. I denne afhandling præsenteres mine kollegers og min fælles indsats for at forbedre præmisserne for udviklingen af cellefabrikker gennem i) øget opmærksomhed på uforudsete effekter i komplekse bakterielle produktionssystemer ii) optimering af kloningsstrategier og iii) udviklingen af en platform til ekspression af en bestemt klasse af plantegener i bakterier. Der redegøres for forskningens relevans for bioteknologi, suppleres med nødvendig baggrundsinformation og et historisk perspektiv. Konkret vises det at øget ekspression af tRNA-gener, der afkoder sjældent forekommende kodons har overraskende lille effekt på membranproteiner i Escherichia coli, og efterfølgende belyses to typer af sideeffekter ved denne fremgangsmåde: I visse tilfælde er vækst og proteinproduktion nedsat som en direkte effekt af de ekstra tRNA-gener. I andre tilfælde ses det at den tilsyneladende positive effekt på proteinproduktion ikke skyldes tRNA-generne, men forskellige niveauer af enzymet T7 lysozym, som er udtrykt fra samme plasmid. Disse fænomener har været overset, på trods af den store popularitet som T7/pET-baserede systemer til proteinproduktion i bakterier har fået. Ydermere addresseres det paradoks, at standardisering af kloning oftest betyder reduceret fleksibilitet.Udviklingen af en kloningsstrategi sigtet mod maksimal standardisering, hvor fleksibiliteten bevares rapporteres. Endelig gennemgås den systematiske optimering af en bakteriel produktionsplatform. Produktionen af seks cytochrom P450 enzymer fra planter optimeres ved brugen af reporter-proteinet GFP (green fluorescent protein). Dette studie antyder, at der ikke er nogen begrænsning for valg af Eschericia coli stammer til produktion af denne type enzymer. Sammenlagt har resultaterne af denne afhandling forbedret præmisserne for udviklingen af cellefabrikker fremover. The sustainable manufacturing of medicines, materials and chemicals is enabled with biotechnology, and the key to the development of new processes, as well as improvement of existing ones, lies in our fundamental understanding of the biological systems we manipulate. Recombinant protein production is at the core of biotechnology and numerous molecular tools and bacterial strains have beendeveloped over the past four decades for this purpose. Understanding of the genetic code and our ability to manipulate genetic material, paves the way for the microbial cell factory development that enables production of protein in a sustainable, costefficient manner. In this thesis I report the joined efforts of my colleagues and myself, to improve the premises for cell factory development by optimizing the cloning strategies, improving the awareness of unforeseen side-effects in complex bacterial expression systems, and building a platform for enhanced expression of certain plant genes in bacteria. The relevance of the conducted research to the field of biotechnology is covered, as well as necessary scientific background and history. Specifically, the surprisingly minor effects of tRNA overexpression on the production of a large number of membrane proteins in Escherichia coli are reported, and also the subsequent work elucidating two types of side effects: in some cases growth and gene expression are directly impaired by the extra tRNA sequences and in other cases the apparent positive effects are instead caused by a differential expression of the lysozyme gene encoded on the same plasmid. These phenomena seem to have been largely overlooked despite the huge popularity of the T7/pet-based systems for bacterial protein production. Additionally, the paradox that standardization comes at the cost of reduced flexibility is addressed. The development of a cloning strategy is covered, aiming at maximized standardization while maintaining high flexibility in choice of cloning technique. Finally, the systematic optimization of production of six plant-derived cytochrome P450 enzymes in E. coli, using a high-throughput workflow based on C-terminal fusions to the green fluorescent protein is also presented. Our work suggests that there are no inherent limitations in using different standard E. coli strains and expression conditions for exploiting cytochrome P450s for biotechnological applications. The results of this thesis have improved the premises for cell factory development in the future.