Dataset Information

Tuning transcription factor DegU for developing extracellular protease overproducer in Bacillus pumilus.

ABSTRACT:

Background

Global transcription machinery engineering (gTME) is an effective approach employed in strain engineering to rewire gene expression and reshape cellular metabolic fluxes at the transcriptional level.

Results

In this study, we utilized gTME to engineer the positive transcription factor, DegU, in the regulation network of major alkaline protease, AprE, in Bacillus pumilus. To validate its functionality when incorporated into the chromosome, we performed several experiments. First, three negative transcription factors, SinR, Hpr, and AbrB, were deleted to promote AprE synthesis. Second, several hyper-active DegU mutants, designated as DegU(hy), were selected using the fluorescence colorimetric method with the host of the Bacillus subtilis ΔdegSU mutant. Third, we integrated a screened degU(L113F) sequence into the chromosome of the Δhpr mutant of B. pumilus SCU11 to replace the original degU gene using a CRISPR/Cas9 system. Finally, based on transcriptomic and molecular dynamic analysis, we interpreted the possible mechanism of high-yielding and found that the strain produced alkaline proteases 2.7 times higher than that of the control strain (B. pumilus SCU11) in LB medium.

Conclusion

Our findings serve as a proof-of-concept that tuning the global regulator is feasible and crucial for improving the production performance of B. pumilus. Additionally, our study established a paradigm for gene function research in strains that are difficult to handle.

SUBMITTER: Xie CY

PROVIDER: S-EPMC10464342 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Publications

Tuning transcription factor DegU for developing extracellular protease overproducer in Bacillus pumilus.

Xie Chao-Ying CY Li Wen-Jin WJ Feng Hong H

Microbial cell factories 20230827 1

<h4>Background</h4>Global transcription machinery engineering (gTME) is an effective approach employed in strain engineering to rewire gene expression and reshape cellular metabolic fluxes at the transcriptional level.<h4>Results</h4>In this study, we utilized gTME to engineer the positive transcription factor, DegU, in the regulation network of major alkaline protease, AprE, in Bacillus pumilus. To validate its functionality when incorporated into the chromosome, we performed several experiment ...[more]

PMID: 37635205

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Project description:BackgroundSince volatile and rising cost factors such as energy, raw materials and market competitiveness have a significant impact on the economic efficiency of biotechnological bulk productions, industrial processes need to be steadily improved and optimized. Thereby the current production hosts can undergo various limitations. To overcome those limitations and in addition increase the diversity of available production hosts for future applications, we suggest a Production Strain Blueprinting (PSB) strategy to develop new production systems in a reduced time lapse in contrast to a development from scratch.To demonstrate this approach, Bacillus pumilus has been developed as an alternative expression platform for the production of alkaline enzymes in reference to the established industrial production host Bacillus licheniformis.ResultsTo develop the selected B. pumilus as an alternative production host the suggested PSB strategy was applied proceeding in the following steps (dedicated product titers are scaled to the protease titer of Henkel's industrial production strain B. licheniformis at lab scale): Introduction of a protease production plasmid, adaptation of a protease production process (44%), process optimization (92%) and expression optimization (114%). To further evaluate the production capability of the developed B. pumilus platform, the target protease was substituted by an α-amylase. The expression performance was tested under the previously optimized protease process conditions and under subsequently adapted process conditions resulting in a maximum product titer of 65% in reference to B. licheniformis protease titer.ConclusionsIn this contribution the applied PSB strategy performed very well for the development of B. pumilus as an alternative production strain. Thereby the engineered B. pumilus expression platform even exceeded the protease titer of the industrial production host B. licheniformis by 14%. This result exhibits a remarkable potential of B. pumilus to be the basis for a next generation production host, since the strain has still a large potential for further genetic engineering. The final amylase titer of 65% in reference to B. licheniformis protease titer suggests that the developed B. pumilus expression platform is also suitable for an efficient production of non-proteolytic enzymes reaching a final titer of several grams per liter without complex process modifications.

Project description:BACKGROUND:Mesophilic alkaline serine proteases from various bacteria have been commercially applied in a range of industries owing to their high catalytic efficiency and wide substrate specificity. However, these proteases have an optimal catalytic temperature of approximately 50 °C, and their activity decreases significantly at low temperature. Therefore, to enhance their cold activity, it is necessary to improve the catalytic performance of these proteases at low temperature. The alkaline serine protease (DHAP) from Bacillus pumilus BA06 is a typical mesophilic enzyme, which has demonstrated great potential in various industrial applications. Here we attempted to improve the cold activity of DHAP via directed evolution. RESULTS:Seven variants (P9S, A1G/K27Q, A38V, A116T, T162I, S182R, and T243S) of DHAP from B. pumilus were obtained via directed evolution. The results showed that all of the variants had increased proteolytic activity at 15 °C towards both the casein and synthetic peptide substrates. With the exception of variant T243S, the thermostability of these variants did not decrease in comparison with the wild-type enzyme. Kinetic analysis indicated that the increase in catalytic efficiency was largely attributed to the increase in turnover number (kcat). Furthermore, the combined variants generated by site-directed mutagenesis showed a further increase in specific caseinolytic activity and the kcat value for hydrolysis of the synthetic peptide. The combined variants of P9S/K27Q and P9S/T162I exhibited an approximate 5-fold increase in caseinolytic activity at 15 °C and almost no loss of thermostability. Finally, the possible mechanism responsible for the change in catalytic properties for these variants was interpreted based on structural modeling. CONCLUSIONS:Directed evolution and site-directed mutagenesis were combined to engineer variants of the DHAP from B. pumilus. All of the variants exhibited an increase in hydrolytic efficiency at low temperature towards both of the substrates, casein and synthetic peptide, without any loss of thermostability compared with the wild-type. These data suggest that engineering low-temperature activity for a bacterial protease is not always associated with the loss of thermostability. Furthermore, our findings demonstrate that enhanced cold activity and thermostability could be integrated into a single variant.

Dataset Information

Tuning transcription factor DegU for developing extracellular protease overproducer in Bacillus pumilus.

Background

Results

Conclusion

Publications

Tuning transcription factor DegU for developing extracellular protease overproducer in Bacillus pumilus.

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