Historically, fermentation has allowed the production of a myriad of food products that acquired central roles in human society such as bread, beer and wine. Through the centuries, fermentation has grown into a billion dollar industry; gaining traction for the production of pharmaceuticals, renewable plastics and new materials. The yeast Saccharomyces cerevisiae ("baker's yeast"), continues to be used by most industries; however, novel species are necessary to attend the growing and diversifying demand as S. cerevisiae has proven difficult to engineer to expand the carbon sources it can use, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeast species, which frequently contain inherent features addressing many of these issues, remain difficult to manipulate genetically. Here, we engineer one of the most promising candidates - the thermotolerant yeast Kluyveromyces marxianus - to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits - thermotolerance, lipid production, and facile transformation with exogenous DNA - into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals. K. marxianus was also successfully employed as a model organism; providing a new platform for a comprehensive, mechanistic, large-scale study of innate thermotolerance. From a library of mutants to identify genes related to innate thermotolerance, we identified 7 mutants that showed impaired growth at high temperature, indicating a potential role of these genes in the high temperature growth phenotype. This work provides a starting point to elucidate the mechanistic basis underlying the ability to sustain growth at high temperatures, the basis of which is still largely unknown in eukaryotes.

Protein synthesis takes place in four stages: initiation, elongation, termination and recycling. Elongation consists of delivery of a charged tRNA to the ribosome, peptide bond formation and translocation of the mRNA and tRNA, three steps forming a cyclic process, which is repeated for every amino acid added to a growing polypeptide chain. The result of the elongation cycle is the translation of the triplet genetic code contained in an mRNA, into the amino acid sequence of a protein. Since the 1960s it was appreciated that complex conformational changes must occur on the ribosome to accomplish translocation of mRNA and tRNA. Here I report x-ray crystallographic studies which shed light on how mRNA and tRNA are manipulated by the ribosome and mechanisms used by certain antibiotics to inhibit the elongation cycle of protein synthesis.

Specifically research reported here argue that tRNA translocation is a stepwise process that involves discrete structural intermediates of the ribosome. I report structural evidence that the antibiotics clindamycin and chloramphenicol inhibit protein synthesis by interfering with aminoacyl-tRNA positioning in the peptidyl transferase center. Also, I hypothesize based on structural data and phylogenetic analysis, that the identity of the ribosomal RNA residues numbered in E. coli 752, 2055 and 2609 contribute to the specificity of many antibiotics for binding to bacterial, rather than archaeal or eukaryotic ribosomes.