Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning. Issue 6 (20th February 2017)

• Record Type: Journal Article
• Authors: Latimer, Luke N.; Dueber, John E.
• Is Part Of: Biotechnology and bioengineering. Volume 114:Issue 6(2017)
• Page Start: 1301
• Page End: 1309
• Publication Date: 2017-02-20
• Subjects: metabolic engineering -- xylose utilization -- combinatorial expression -- synthetic biology; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26262
• Languages: English
• ISSNs: 0006-3592

Abstract

ABSTRACT: A common challenge in metabolic engineering is rapidly identifying rate‐controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae . For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight‐enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ∼70% increase in biomass yield and ∼240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301–1309. © 2017 Wiley Periodicals, Inc. Abstract : Identifying and engineering rate‐controlling enzymes in longer, heterologous metabolic pathways remains an outstanding challenge in metabolic engineering. To address this challenge, we propose an iterative workflow that uses combinatorial expression libraries to map the expression space and demonstrate its utility in improving an eight‐enzyme xylose utilization pathway in S accharomyces cerevisiae .