Heterologous signal peptides-directing secretion of Streptomyces mobaraensis transglutaminase by Bacillus subtilis

Mu, Dongdong; Lu, Jiaojiao; Qiao, Mingqiang; Kuipers, Oscar P.; Zhu, Jing; Li, Xingjiang; Yang, Peizhou; Zhao, Yanyan; Luo, Shuhong; Wu, Xuefeng; Jiang, Shaotong; Zheng, Zhi

doi:10.1007/s00253-018-9000-y

Cited by 26 publications

(29 citation statements)

References 44 publications

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“…Genes coding for Sec- or Tat-secreted proteins contain a 5′ sequence encoding the N-terminal signal peptide [ 44 ]. Remarkably, a given signal peptide can direct the secretion of different recombinant proteins [ 45 , 46 ].…”

Section: Resultsmentioning

confidence: 99%

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis

et al. 2020

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Background Bacillus subtilis is widely used for the industrial production of recombinant proteins, mainly due to its high secretion capacity, but higher production yields can be achieved only if bottlenecks are removed. To this end, a crucial process is translation initiation which takes place at the ribosome binding site enclosing the Shine Dalgarno sequence, the start codon of the target gene and a short spacer sequence in between. Here, we have studied the effects of varying spacer sequence lengths in vivo on the production yield of different intra- and extracellular proteins. Results The shuttle vector pBSMul1 containing the strong constitutive promoter P HpaII and the optimal Shine Dalgarno sequence TAAGGAGG was used as a template to construct a series of vectors with spacer lengths varying from 4 to 12 adenosines. For the intracellular proteins GFPmut3 and β-glucuronidase, an increase of spacer lengths from 4 to 7–9 nucleotides resulted in a gradual increase of product yields up to 27-fold reaching a plateau for even longer spacers. The production of secreted proteins was tested with cutinase Cut and swollenin EXLX1 which were N-terminally fused to one of the Sec-dependent signal peptides SPPel, SPEpr or SPBsn. Again, longer spacer sequences resulted in up to tenfold increased yields of extracellular proteins. Fusions with signal peptides SPPel or SPBsn revealed the highest production yields with spacers of 7–10nt length. Remarkably, fusions with SPEpr resulted in a twofold lower production yield with 6 or 7nt spacers reaching a maximum with 10–12nt spacers. This pattern was observed for both secreted proteins fused to SPEpr indicating a dominant role also of the nucleotide sequence encoding the respective signal peptide for translation initiation. This conclusion was corroborated by RT qPCR revealing only slightly different amounts of transcript. Also, the effect of a putative alternative translation initiation site could be ruled out. Conclusion Our results confirm the importance of the 5′ end sequence of a target gene for translation initiation. Optimizing production yields thus may require screenings for optimal spacer sequence lengths. In case of secreted proteins, the 5′ sequence encoding the signal peptide for Sec-depended secretion should also be considered.

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Section: Resultsmentioning

confidence: 99%

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis

et al. 2020

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“…It is critical to the optimization of SP for the heterologous protein. S. mobaraensis TG was expressed in B. subtilis using SP wapA and SP amyQ [13]. Our study demonstrated that for S. ladakanum TG, the SP sacB and SP abnA were more suitable than SP wapA (Fig.…”

Section: Involvement Of the N-terminal Region Of Tg In Increasing Enzmentioning

confidence: 76%

“…In our previous study, active S. mobaraense TG was secreted by B. subtilis through the introduction of VMM into zymogen [14]. S. mobaraense TG was also secreted by B. subtilis through the Tat pathway [13]. These studies indicated that B. subtilis was a promising host for TG production.…”

Section: Sift Deskmentioning

confidence: 87%

“…Some wild-type B. subtilis strains can produce TG, but the yield is too low to be industrial used [12]. It has been reported that S. mobaraensis TG expressed in B. subtilis showed similar enzymatic activities to that produced from E. coli and catalyzed the cross-linking reactions [13]. In a previous report, we demonstrated that active-form S. mobaraensis TG was efficiently secreted by B. subtilis when a modified Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein was introduced into TG zymogen [14].…”

Section: Introductionmentioning

confidence: 99%

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Extracellular production of Streptomyces ladakanum transglutaminase in a food-grade strain, Bacillus subtilis

Fu¹,

Ju²,

El-Shora³

et al. 2020

SDRP JFST

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Background: Transglutaminase (TG) is an enzyme of the transferase family with cross-linking properties, which has been widely used in the food industry. Traditionally, TG is isolated from strains of Streptomyces sp. However, the development of a facile and efficient production of commercial TG is always desirable. Purpose: In the current study, we described an efficient route for TG production in a food-grade strain of bacteria, Bacillus subtilis. Method: Two strategies were employed for the extracellular production of S. ladakanum TG in B. subtilis. Sixteen signal peptides were optimized to secret TG into the extracelluar medium. Site-directed mutageneses in pro-peptide were further utilized to improve the enzymatic activity. The enzymatic characteristics of S. ladakanum TG expressed in B. subtilis were analyzed. Results: The N-terminal amino acids played important roles in enzymatic activity. Signal peptides of SacB (SPSacB) and AbnA (SPAbnA) showed good abilities to direct the secretion of TG into the medium. The enzyme was secreted into the medium and exhibited good Ca2+ stability and temperature stability, which were comparable to those produced by commercial strains. The enzymatic activity in the supernatant of culture reached 7.6 U/mg. Conclusion: Our study demonstrated that B. subtilis may be a good candidate for the efficient and stable production of TG and has a much easier purification process. Keywords: Transglutaminase (TG), Streptomyces TG, Bacillus subtilis, Food-grade strain.

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“…The pro-peptide is essential for zymogen folding but must be posttranslationally cleaved to activate the zymogen [14]. Therefore, zymogen consisting of the propeptide and mature part of mTGase was normally expressed rst and the protease treatment was subsequently applied to remove the pro-peptide [15][16][17][18][19][20][21][22][23][24][25]. The protease treatment was either conducted in vitro [15-18, 20, 22, 23, 25] or accomplished through co-expression of the protease in vivo [19,21,24].…”

Section: Introductionmentioning

confidence: 99%

Intein-mediated intracellular production of active microbial transglutaminase in Corynebacterium glutamicum

Zhang

Zhang²,

et al. 2020

Enzyme and Microbial Technology

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Background The microbial transglutaminase (mTGase) from Streptomyces mobaraense has been widely used in the food industry. Recombinant production of mTGase is tricky because the mTGase is synthesized as an inactive zymogen, which needs to be activated by proteolytic processing. Self-cleaving inteins have been applied to activate the zymogen in a simple and highly speci c manner as compared with proteolytic processing. However, self-cleaving inteins suffer from the inherent problem of premature cleavage. Moreover, self-cleaving inteins normally require an additional step of long time incubation to induce the cleavage. These two inherent problems limit self-cleaving inteins for their potential application in the production of mTGase. Results In this study, the premature cleavage of intein Ssp DnaB was observed in Corynebacterium glutamicum when the Ssp DnaB was used to activate mTGase precursor protein. Rather than suppressing it, the premature cleavage was applied to produce active mTGase in C. glutamicum. The SDS-PAGE analysis and the mTGase activity assay indicated that the premature cleavage of intein Ssp DnaB was successfully applied to activate the mTGase intracellularly in C. glutamicum. The subsequent N-terminal amino acid sequencing and site-directed mutagenesis studies demonstrated that the premature cleavage activated the mTGase intracellularly in a highly speci c manner. Finally, in a jar fermentor, the intracellular mTGase activity was up to 49 U/mL, which was the highest intracellular mTGase activity ever reported. Conclusions An e cient and simple approach with great potential for large-scale industrial production of active mTGase was presented in this study. This approach employed premature cleavage of intein Ssp DnaB to activate mTGase in C. glutamicum, resulting in high-level intracellular production of active mTGase. Moreover, this approach did not require any further processing steps such as protease treatment or long time incubation, greatly simplifying the production of active mTGase.

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Heterologous signal peptides-directing secretion of Streptomyces mobaraensis transglutaminase by Bacillus subtilis

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Cited by 26 publications

References 44 publications

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis

Extracellular production of Streptomyces ladakanum transglutaminase in a food-grade strain, Bacillus subtilis

Intein-mediated intracellular production of active microbial transglutaminase in Corynebacterium glutamicum

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