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 PLlacO1. 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.


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Primers for promoter selection To clone pLM1, primers pLM1-F and pLM1-R were used to amplify PLlacO1: alsS-ilvC-ilvD from pSA69, primers avta-F and avta-R were used to amplify avtA from the E. coli genomic DNA, the PCR products were assembled by Gibson Assembly.
To clone pLM2, primers rrnBp-F and rrnBp-R were used to amplify rrnBp1 from the E. coli genomic DNA, primers pLM-F and pLM-R were used to amplify alsS-ilvC-ilvD-avtA from pLM1, the PCR products were assembled by Gibson Assembly.
To clone pLM3, primers rrnBp-F and rrnBp-R were used to amplify rrnBp1 from the E. coli genomic DNA, primers pLM-F and pLM-R were used to amplify leuDH-kivD-yqhD; lacI from pYX97, the PCR products were assembled by Gibson Assembly.
To clone pLM6, primers pLM6-F and pLM6-R were used to amplify glnAp2 from the E. coli genomic DNA, primers pLM-F and pLM-R were used to amplify alsS-ilvC-ilvD-avtA from pLM1, the PCR products were assembled by Gibson Assembly.
To clone pLM7, primers pLM6-F and pLM6-R were used to amplify glnAp2 from the E. coli genomic DNA, primers pLM-F and pLM-R were used to amplify leuDH-kivD-yqhD; lacI from pYX97, the PCR products were assembled by Gibson Assembly.
To clone pLM8, primers pLM8-F and pLM8-R were used to amplify argTp from the E.
coli genomic DNA, primers pLM-F and pLM-R were used to amplify alsS-ilvC-ilvD-avtA from pLM1, the PCR products were assembled by Gibson Assembly.
To clone pLM9, primers pLM8-F and pLM8-R were used to amplify argTp from the E.