Engineering of an NADPH/NADP+ redox sensor in Saccharomyces cerevisiae

Jie Zhang,1 Thomas P. B. Pihl,1 Michael K. Jensen,1 Jay D. Keasling1,2,3,4,5,*

1 The Novo Nordisk Foundation Center for Sustainability, Technical University of Denmark, Denmark

2 Joint BioEnergy Institute, Emeryville, CA, USA

3 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

4 Department of Chemical and Biomolecular Engineering & 5 Department of Bioengineering University of California, Berkeley, CA, USA

* E-mails: jdkeasling@lbl.gov

Abstract

Last two decades have seen an enormous progress in metabolic engineering as an enabling technology for a biosustainable society. By introducing a heterologous pathway into a microbial host and rewiring the carbon fluxes, it is often achievable that a new compound can be produced with improved titer, rate or yield. However, the improvement may be largely limited by non-optimal flux distribution or imbalanced cofactors, including NADPH, a reducing equivalent for the biosynthesis of many economically important chemicals. Due to the technical challenges, analysis of this cofactor can only be performed at a low throughput. To facilitate high-throughput screening of strains with increased reducing equivalent capacity, we developed an NADPH/NADP+ redox sensor in the yeast Saccharomyces cerevisiae by exploiting the native oxidative stress defense system, namely, the yeast transcription factor Yap1, and the promoter of
its target gene, TRX2. When coupled with yeast enhanced green fluorescent protein (yEGFP), our sensor- reporter can generate an output of up to 10-fold increase induced by diamide, a disulfide generating oxidant, as well as by genetic modifications that hamper NADPH production. In this study, we built a sensor-actuator circuit to demonstrate the capability to select strains with better NADPH regeneration capacity. In principle, this approach can be scaled up for screening of a larger mutant library to identify novel target for metabolic engineering. The sensor can also be applied to drive the expression of
NADPH-generating enzymes to rescue the cofactor deficiency.