Biofuel production with a stress-resistant and growth phase-independent promoter: mechanism revealed by in vitro transcription assays

The carbon and energy needed for bioconversion processes face trade-offs between cell reproduction and chemical synthesis. In most processes, microbial cells containing overexpressed pathway enzymes were accumulated in exponential phase before the productions of value-added chemicals dominate the carbon and energy fluxes in stationary phase. The pathway enzymes need to be continuously supplied to compensate their degradation, but the promoters driving their overexpressions are downregulated under stationary phase or stressed conditions. In this study, the gadA promoter enabled stress-resistant and growth phase-independent isobutanol production up to 10–28 g l⁻¹. To investigate the activation mechanism of gadA promoter and its potential in metabolic engineering, an in vitro transcription system was established. Results showed that gadA promoter could be transcribed efficiently under environments that inhibit the transcription of ribosomal promoters, while under moderate to rapid growth conditions, the large majority of new cellular transcripts are ribosomal. This differential transcription relies on the accumulation of environmental glutamate and/or the loss of supercoiling. These results implied that the gadA promoter could be functional or even dominate the cellular transcription under an exceedingly wide range of physiological conditions. Therefore, the gadA promoter is a novel candidate for driving pathway enzyme overexpressions under not only exponential phase but also stationary phase and stressed conditions, which is important for achieving efficient biofuel production.