Bilateral NSF/BIO-BBSRC: Synthetic gene circuits to measure and mitigate translational stress during heterologous protein expression

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In this project, an interdisciplinary team of biologists and physicists will establish novel technologies to improve the ability of a cell to make recombinant proteins (rPs) at higher efficiency and with greater accuracy. A majority of the biotechnology industry is dedicated to the production of rPs for use as pharmaceuticals, vaccines and food additives. These proteins are made in living cells using the normal process of protein synthesis, whereas the industrial production of these same proteins requires unusually high levels of the protein - such that the desired protein accounts for the majority of all protein mass in the cell. At such a high production level, proteins are made with errors, when incorrect amino acids are inserted into the growing protein chain. This project will investigate how these errors accumulate, and will create genetic circuits that can sense any significant increase in the frequency of protein synthesis errors and can deploy mechanisms to restore normal highly accurate protein synthesis. The research team consists of the lab at University of Maryland Baltimore County, working with a model bacterium E.coli, and the lab at the University of Aberdeen, UK, working with baker`s yeast. The two teams will also collaborate with a major biotechnology company in the United Kingdom to insure that the products of the project will be directly applicable to use in industrial scale protein production.Amino acids in proteins are encoded by three nucleotide codons in mRNA; translation involves three nucleotide base pairs between the mRNA codon and a tRNA anticodon. Errors occur when a decoding tRNA forms only two base pairs with the codon. For each correct (cognate) tRNA there are about fourteen incorrect (near-cognate) tRNAs that could induce such an error. The decoding process is normally highly accurate, with near-cognate tRNAs introducing incorrect amino acids at frequencies from about one in 10,000 to as low as one in 1,000,000. Mistranslation can also result from frameshifting in which a codon overlapping the correct codon is selected for decoding. The frequency of these mistranslation errors increase sharply during translation of codons served by low abundance tRNA, or during ribosomal pausing caused by depletion of charged tRNAs. The high-level expression of biotechnological proteins can creates exactly these conditions and thus increasing translational error frequencies. Indeed, many reports describe biotechnological protein expression generating a range of undesirable mistranslation events, compromising product yield and quality, and thus the safety and efficacy of biologics. In this project we will pursue a better understanding of the system-wide causes of translational error through the design and application of novel reporters of mistranslation, capable of producing either reporter enzyme activities, or regulatory transcription factors. We combine these experimental approaches with global mathematical modelling of translation and tRNA competition to predict when system stress will stimulate mistranslation. We then use synthetic biology gene circuits to transcriptionally couple the output from these new mistranslation sensors to control rP expression, to reduce mistranslation and increase rP product quality. Our industrial partner will test these synthetic gene circuits to maximize the opportunities for realizing the impact of this research on biotechnology. This research will reveal for the first time the role of rPs in triggering translation system stress, and identify novel ways in which stress can be ameliorated.