Rational Design of the N-Terminal Coding Sequence for Regulating Enzyme Expression in <i>Bacillus subtilis</i>

Xu, Kuidong; Tong, Yi; Li, Yi; Tao, Jin; Li, Jianghua; Zhou, Jingwen; Liu, Song

doi:10.1021/acssynbio.0c00309

Cited by 20 publications

(22 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This result was consistent with autoinduction characteristics. 15 However, the RFI remained around 44,853 after 30 h of fermentation (Figure 1B), which was only 29% of the RFI achieved using the strong promoter P lytR in B. subtilis 29 (Figures 1B and 2C). Therefore, the efficiency of P srfA required to be improved further.…”

Section: ■ Results and Discussionmentioning

confidence: 97%

Efficient, Flexible Autoinduction Expression Systems with Broad Initiation in Bacillus subtilis

Tong

et al. 2021

ACS Synth. Biol.

Self Cite

View full text Add to dashboard Cite

Low expression levels and inflexible induction initiation have been the main obstacles to produce proteins using bacterial quorum sensing (QS). The typical QS system in Bacillus subtilis, ComQXPA, activates the promoter PsrfA using ComX and ComA as an auto-inducer and a promoter activator, respectively. Here, we developed a series of flexible autoinduction expression systems in B. subtilis WB600 based on ComQXPA using a super-folder green fluorescent protein as the reporter. The −35 region of PsrfA was replaced with corresponding conserved sequences of σA-dependent promoters, yielding P1 with 85% enhanced strength. We then applied a semi-rational design within the spacer between the −35 and −15 regions of P1 to generate the QS promoter PS1E, which generated 8.22-fold more expression than PsrfA. Based on PS1E, we finally obtained three types of autoinduction expression systems with initiation ranging from 1.5–9.5 h by optimizing the combination of the promoters for ComX and ComA. The yield of Bacillus deramificans pullulanase generated using autoinduction expression systems in B. subtilis reached 80.2 U/mL, which was 36% more than that of the most powerful constitutive promoter P566. Flexible autoinduction expression systems with diverse dynamic features have considerable potential for improving protein expression and metabolite production in B. subtilis.

show abstract

Section: ■ Results and Discussionmentioning

confidence: 97%

Efficient, Flexible Autoinduction Expression Systems with Broad Initiation in Bacillus subtilis

Tong

et al. 2021

ACS Synth. Biol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…This may be because the signal peptides not only guided transmembrane transport but also affected protein translation [ 25 ]. For example, the interaction time with ectopic molecules can affect processing after protein synthesis, including folding, binding of chaperone molecules, and integration of transmembrane regions [ 26 , 27 ]. Furthermore, the efficiency of protein secretion depends on the host strain, signal sequence, and target protein [ 13 ].…”

Section: Resultsmentioning

confidence: 99%

Enhancing extracellular production of lipoxygenase in Escherichia coli by signal peptides and autolysis system

et al. 2022

Self Cite

View full text Add to dashboard Cite

Background Lipoxygenase (LOX) is a non-heme iron containing dioxygenase that is widely used to improve food quality and produce active drug intermediates and biodiesel. Escherichia coli is one of the most widely used host microorganisms for recombinant protein expression; however, its weak extracellular secretion ability precludes its effective production of recombinant proteins into the extracellular environment. To facilitate subsequent characterization and application of LOX, improving its secretion efficiency from E. coli is a major challenge that needs to be solved. Results Several strategies were adopted to improve the extracellular secretion of LOX based on the signal peptides and cell wall permeability of E. coli. Here, we studied the effect of signal peptides on LOX secretion, which increased the secretory capacity for LOX marginally. Although surfactants could increase the permeability of the cell membrane to promote LOX secretion, the extracellular LOX yield could not meet the requirements of industrialization production. Subsequently, an autolysis system was constructed in E. coli based on the bacteriophage lysis gene ΦX174-E to enhance the production of extracellular proteins. Thus, the extracellular production of LOX was achieved and the content of inclusion bodies in the cell was reduced by optimizing cell lysis conditions. The extracellular LOX yield reached 368 ± 1.4 U mL−1 in a 5-L bioreactor under optimized lysis conditions that is, an induction time and temperature, and arabinose concentration of 5 h, 25 °C, and 0.6 mM, respectively. Conclusions In this study, the different signal peptides and cell autolysis system were developed and characterized for extracellular LOX production in E. coli. Finally, the cell autolysis system presented a slight advantage on extracellular LOX yield, which also provides reference for other protein extracellular production.

show abstract

“…As an important element of the gene 5′-end mRNA secondary structure, 5′-proximal coding sequences strongly affects gene translation efficiency [ 33 ]. Thus, it was an effective approach to optimize the translation efficiency by changing the 5′-proximal coding sequences [ 13 ]. To increase the free energy of AmyZ1 mRNA secondary structure, the sequences of the first 11 codons of SP amyQ were changed according to the prediction results of UTR Designer (see “ Materials and methods ”).…”

Section: Resultsmentioning

confidence: 99%

“…However, there are still factors that limit the high-level expression of the target proteins in B . subtilis , such as secretion of multiple proteases, lack of strong promoter and efficient signal peptide (SP), and inefficient translation [ 11 – 13 ]. Many strategies have been developed to improve the recombinant expression level of target proteins in B. subtilis , such as host strain optimization [ 14 ], promoter and signal peptide optimization [ 15 ], and translation initiation efficiency optimization [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

“…Many studies have shown that the secondary structures of mRNA present at the translation initiation region, including 5′-untranslated region and 5′-proximal coding sequence, greatly impact on the gene translation initiation efficiency [ 28 ]. Previous studies have reported that the recombinant expression level of the target proteins in B. subtilis can be improved by optimizing the translation initiation efficiency [ 13 , 29 ]. For example, when a mutation occurred at the 5′-untranslated region of mRNA, the extracellular diacetylchitobiose deacetylase activity of the corresponding strain was 82-fold higher than that of the original strain [ 29 ].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Enhanced extracellular raw starch-degrading α-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization

Yao

Pan

et al. 2022

Microb Cell Fact

View full text Add to dashboard Cite

Background A raw starch-degrading α-amylase from Pontibacillus sp. ZY (AmyZ1), previously screened by our laboratory, showed a promising application potential for starch-processing industries. However, the AmyZ1 secretory production still under investigation, which seriously restricts its application in the starch-processing industry. On the other hand, Bacillus subtilis is widely used to achieve the extracellular expression of target proteins. Results AmyZ1 secretory production was achieved in B. subtilis and was enhanced by promoter engineering and translation initiation efficiency optimization. First, based on the different phase-dependent promoters, the dual-promoter PspoVG–PspoVG142 was constructed by combining dual-promoter engineering and promoter modification. The corresponding strain BZd34 showed an extracellular AmyZ1 activity of 1437.6 U/mL during shake flask cultivation, which was 3.11-fold higher than that of the original strain BZ1 (PgroE). Then, based on translation initiation efficiency optimization, the best strain BZd343 containing optimized 5'-proximal coding sequence (opt3) produced the highest extracellular α-amylase activity of 1691.1 U/mL, which was 3.65-fold higher than that of the strain BZ1. Finally, cultivation of BZd343 in 3-L fermenter exhibited an extracellular AmyZ1 activity of 14,012 U/mL at 48 h, with productivity of 291.9 U/mL·h. Conclusions This is the first report of recombinant expression of AmyZ1 in B. subtilis and the expression level of AmyZ1 represents the highest raw starch-degrading α-amylase level in B. subtilis to date. The high-level expression of AmyZ1 in this work provides a foundation for its industrial production. The strategies used in this study also provide a strategic reference for improving the secretory expression of other enzymes in B. subtilis.

show abstract

Rational Design of the N-Terminal Coding Sequence for Regulating Enzyme Expression in Bacillus subtilis

Cited by 20 publications

References 39 publications

Efficient, Flexible Autoinduction Expression Systems with Broad Initiation in Bacillus subtilis

Efficient, Flexible Autoinduction Expression Systems with Broad Initiation in Bacillus subtilis

Enhancing extracellular production of lipoxygenase in Escherichia coli by signal peptides and autolysis system

Enhanced extracellular raw starch-degrading α-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization

Contact Info

Product

Resources

About