Abstract
BackgroundProtein-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.ResultsWe 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.ConclusionsThese 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.
Highlights
Protein-based bioconversion has been demonstrated as a sustainable approach to produce higher alcohols and ammonia fertilizers
The engineered biosynthetic pathways in bacteria for the production of value-added chemicals are mostly governed by σ70-dependent promoters [7], the transcription of which is determined by the number of the RNA polymerase (RNAP) carrying the σ70 subunit (Eσ70)
Influence of ammonia assimilation on sustaining σ54‐mediated transcription In general, to maintain active σ54-mediated transcription, the E. coli cells must be maintained under nitrogen starvation conditions
Summary
Protein-based bioconversion has been demonstrated as a sustainable approach to produce higher alcohols and ammonia fertilizers. 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 promot‐ ers declines rapidly once cells enter the stationary phase or encounter stresses. The relative advantage of σ70 over other σ factors in recruiting the core enzyme is highly compromised once the cells enter the stationary phase or encounter stresses This derives in part from the sharp increase in the number of alternative σ factors (e.g., σ38 and σ24) in response to both intra- and extracellular disturbances. Regulatory molecules such as Hofmeister salts, regulator of σD, and guanosine pentaphosphate or tetraphosphate [9, 10] simultaneously accumulate, whereas chromosomal DNA supercoiling decreases [11] Together, these physiological shifts suppress the association between the core RNAP and σ70, facilitating core RNAP interaction with alternative σ factors at the expense of Eσ70 [9]. To overcome the innate drawbacks of σ70-mediated transcription, we posited that metabolic engineering could transform the intrinsic transcriptional regulation process into a driving force for the robust biorefining of waste protein
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