Development and use of a novel gene expression engineering approach in synthetic biology applications
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Microorganisms have diverse biochemical capabilities that can be exploited to produce metabolites or proteins of interest, such as drugs, vitamins, or biofuels in industrial scale biomanufacturing processes. To enable protein and metabolite production with microorganisms, often, gene expression needs to be manipulated. Gene expression depends on the regulatory sequences located upstream of genes, such as the promoter and the 5′ untranslated region (5′ UTR), which regulate transcription and translation. RNA polymerases bind to promoters to transcribe DNA into RNA and ribosomes bind to the 5′ UTR of mRNAs to translate the coding sequences of genes into proteins. Hence, these 5′ regulatory sequences strongly influence gene expression. Standard gene expression methods make use of organism-specific 5′ regulatory sequences. Consequently, it is difficult to express genes with non-model organisms because the pool of characterized regulatory sequences is limited for these organisms. Moreover, gene expression can be unpredictable because transcription and translation are not fully understood yet. In this Ph.D. project, the goal was to develop a gene expression engineering method that does not require organism-specific knowledge about 5′ regulatory sequences. This means that the method can be used to express any gene of choice with any organism of choice, including non-model organisms. We developed the Gene Expression Engineering (GeneEE) method in which random nucleotides are used as Artificial 5′ Regulatory Sequence (ARES). The ARESs contain 200 random nucleotides that are scarlessly cloned upstream of a gene of interest. This results in a diverse library of constructs that contain unique ARESs driving expression of a gene of interest. To easily identify functional ARESs, the ARESs were used to drive expression of genes encoding resistance to antibiotics or genes encoding fluorescent proteins. This allows identification of clones carrying functional ARESs based on growth/no growth selection or fluorescence emission. The fluorescence reporter system has the advantage that expression strength can be quantified because fluorescence intensity correlates with how much protein the cells produce. We used both types of reporters in our initial proof-of-principle study. In this initial study, we tested the method in a range of model and non-model organisms including Gram-negative bacteria Escherichia coli, Pseudomonas putida and Thermus thermophilus, Gram-positive bacteria Corynebacterium glutamicum, Streptomyces albus and Streptomyces lividans and the eukaryote Saccharomyces cerevisiae (underlined organisms are organism that I worked with, the other organisms were handled by co-authors). The ARESs successfully drove gene expression in all tested hosts. Together with collaborators we performed transcription start site determination experiments in 200 clones (done by coauthors). We found that an ARES can contain multiple transcription start sites. We also showed that the method could be used to fabricate an artificial inducible XylS/Pm system in E. coli (done by co-authors). In a follow-up study, we identified functional ARESs in the nonmodel organism V. natriegens. V. natriegens is currently gathering attention as a potential protein production workhorse due to its short doubling time of 10 minutes. We selected 26 ARESs to be cross-characterized in E. coli and V. natriegens using the fluorescent reporter proteins GFP and mCherry. By cross-characterizing expression strength with two reporters in two hosts, we were able to identify ARESs that led to similar expression in all host-reporter combinations. We also found ARESs that led to host- or gene-dependent expression and identified one ARES that drove expression in a carbon source responsive manner. Thus, we demonstrated that the GeneEE method can be used to drive expression in different host organisms. To develop a reliable method, we went through several rounds of the Design-Build-Test-Learn cycle. We optimized the protocol in several steps to increase the efficiency of the method and to reduce the complexity and time spent on the workflow. We furthermore investigated an issue that we encountered when using the red fluorescent protein mCherry as reporter. We found that a shorter fluorescent mCherry version is encoded in the mCherry gene. This shorter mCherry version is produced besides the longer form. We tested several measures to prevent simultaneous production of the two mCherry versions and demonstrated that either using the shorter form or changing the coding sequence of the long form abolished background fluorescence. Taken together, the GeneEE method that was developed during this Ph.D. project is a useful tool for fabricating regulatory sequences and enabling gene expression of any gene of interest in any microorganism of interest.