Author
 |
Hughes, Stephen |
|
Submitted to: Meeting Abstract
Publication Type: Abstract Only Publication Acceptance Date: 1/22/2015 Publication Date: 1/22/2015 Citation: Hughes, S.R. 2015. Integrated automation for continuous high-throughput synthetic chromosome assembly and transformation to identify improved yeast strains for industrial production of peptide sweetener brazzein. Meeting Abstract. Interpretive Summary: Technical Abstract: Production and recycling of recombinant sweetener peptides in industrial biorefineries involves the evaluation of large numbers of genes and proteins. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly synthesize, clone, and express heterologous gene open reading frames (ORFs) in bacteria, cell-free extracts, or yeast, and to screen large numbers of expressed proteins for functional activity are important technologies for evaluating fungal strains for improved production of new sweetener peptides. We describe a continuous system consisting of four robotic platforms for this process: 1) synthesis and screening of gene ORFs by systematically replacing codons using an amino acid scanning mutagenesis algorithm to evaluate all codons for functionality, in a multiplexed format and to produce a library of mutagenized ORFs; 2) one-step construction of a synthetic yeast artificial chromosome (YAC) containing the optimized ORFs in a polyprotein cassette for expression of multiple genes such as those for enzymes in metabolic pathways or for valuable peptide or protein coproducts behind an optimized promoter with custom expression fusion tags selected for desired expression levels and protein locations inside or outside the industrial strain; 3) selection of a host strain that has been subjected to mutagenesis by irradiation and/or incubation at elevated temperatures anaerobically to produce a strain capable of robust growth in the particular biomass feedstock or waste stream or agricultural product and transformation of this host strain with these collections of synthetic YACs; and 4) high-throughput screening of the transformed strains for desired industrial traits. This system can produce an improved industrial microorganism by manipulation of the host strain, assembly of an optimized synthetic chromosome, and stable transformation of the chromosome into the engineered host strains for use in production of useful new sweeteners. |
