Protein-based biorefining driven by nitrogen-responsive transcriptional machinery

Background: Protein-based bioconversion has been demonstrated as a sustainable approach to produce higher alcohols and ammonia fertilizers. However, owing to the switchover from transcription mediated by the bacterial RNA polymerase σ⁷⁰ to that mediated by alternative σ factors, the biofuel production driven by σ⁷⁰-dependent promoters declines rapidly once cells enter the stationary phase or encounter stresses. To enhance biofuel production, in this study the growth phase-independent and nitrogen-responsive transcriptional machinery mediated by the σ⁵⁴ is exploited to drive robust protein-to-fuel conversion. Results: We demonstrated that disrupting the Escherichia coli ammonia assimilation pathways driven by glutamate dehydrogenase and glutamine synthetase could sustain the activity of σ⁵⁴-mediated transcription under ammonia-accumulating conditions. In addition, two σ⁵⁴-dependent promoters, argTp and glnAp2, were identified as suitable candidates for driving pathway expression. Using these promoters, biofuel production from proteins was shown to persist to the stationary phase, with the net production in the stationary phase being 1.7-fold higher than that derived from the optimal reported σ⁷⁰-dependent promoter P _LlacO₁. Biofuel production reaching levels 1.3-to 3.4-fold higher than those of the σ⁷⁰-dependent promoters was also achieved by argTp and glnAp2 under stressed conditions. Moreover, the σ⁵⁴-dependent promoters realized more rapid and stable production than that of σ⁷⁰-dependent promoters during fed-batch fermentation, producing up to 4.78 g L ^-1 of total biofuels. Conclusions: These results suggested that the nitrogen-responsive transcriptional machinery offers the potential to decouple production from growth, highlighting this system as a novel candidate to realize growth phase-independent and stress-resistant biofuel production.