Integrated Automation for Continuous High-Throughput Synthetic Chromosome Assembly and Transformation to Identify Improved Yeast Strains for Industrial Production of Biofuels and Bio-based Chemicals

Abstract

An exponential increase in our understanding of genomes, proteomes, and metabolomes provides greater impetus to address critical biotechnological issues such as sustainable production of biofuels and bio-based chemicals and, in particular, the development of improved microbial biocatalysts for use in industrial biorefineries. Because these studies involve 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, and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving fungal strains for industrial production of biofuels and bio-based chemicals. We describe a system of four robotic platforms required for continuous operation of this process: (1) synthesis and screening of mutagenized gene ORFs by systematically replacing codons using an amino acid scanning mutagenesis algorithm, which evaluates all codons for functionality, in a multiplexed format to produce a library of optimized 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 required for profitable and environmentally friendly operations at a biorefinery 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 produces 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 improved host strains for use in production of biofuels via biorefinery operations.