Expanding the Genetic Code of a Synthetic Yeast

The proposed project aims to develop an efficient yeast-based protein expression platform with an expanded genetic code. The standard genetic code of natural organisms comprises 20 canonical amino acids. This set of amino acids together with post-translational modifications enables the enormous range of protein functions found in nature. However, nature’s alphabet has now been further expanded by engineering cellular translation to incorporate hundreds of structurally diverse non-canonical amino acids (ncAAs) into proteins (1,2). This expanded code opens the door to the development of proteins with new functions, and offers new chemical probes of protein structure and function (3).

Site-specific incorporation of non-canonical amino acids requires an orthogonal translation system consisting of an aminoacyl-tRNA-synthetase (aaRS) / tRNA pair that is orthogonal to the host organism. The aaRS has to specifically load the desired ncAA onto the tRNA. The tRNA’s anticodon typically targets UAG, leading to ncAA incorporation at this codon. Since in natural organisms UAG/TAG is the amber stop codon, introduction of an orthogonal translation system targeting UAG leads to readthrough of these stops (amber suppression). E. coli has been engineered to remove all amber stops to free the codon altogether (4). Saccharomyces cerevisiae has a considerably higher usage of the amber stop codon (24% of stops), resulting in a strong growth defect upon efficient amber suppression. The Sc2.0 project is re-writing the S. cerevisiae genome (Cai is the international coordinator for Sc2.0) according to a number of design rules including the replacement of all TAG stop codons. Freeing this codon will allow its efficient repurposing with an orthogonal translation system.

The PhD student will establish site-specific ncAA incorporation in a fully synthetic yeast strain devoid of UAG stops. S/he will subsequently exploit this synthetic strain to efficiently incorporate of non-canonical amino acids into proteins to develop enzymes with new catalytic functions.

Academic background of candidates:
Applicants are expected to hold, or about to obtain, a minimum upper second class undergraduate degree (or equivalent) in biology, chemistry, genetics, and/or biochemistry. A Masters degree in a relevant subject and/or experience in synthetic biology/ biotechnology subject area is desirable.

Contact for further Information:
Prof. Patrick Yizhi Cai, [Email Address Removed], www.cailab.org
Dr. Anthony Green, [Email Address Removed]