Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.
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.
Department of Chemistry, University of California, Berkeley, California.
Luke N
LN
Latimer
Department of Bioengineering, University of California, 2151 Berkeley Way, Berkeley, California 94720.
John E
JE
Dueber
D020650:Combinatorial Chemistry Techniques; D003198:Computer Simulation; D024861:Genetic Enhancement / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis / Q000379:methods*; D008660:Metabolism; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*
D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D014994:Xylose
combinatorial expression; metabolic engineering; synthetic biology; xylose utilization