Skip to main content
SpringerLink
Log in

Improving the utilization rate of soybean meal for efficient production of bacitracin and heterologous proteins in the aprA-deficient strain of Bacillus licheniformis

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Soybean meal is commonly applied as the raw material in the bio-fermentation industry, and bacitracin is a widely used feed additive in the feed industry. In this study, we investigated the influence of subtilisin enhancement on soybean meal utilization and bacitracin production in Bacillus licheniformis DW2, an industrial strain for bacitracin production. Firstly, blocking sRNA aprA expression benefited bacitracin synthesis, and the bacitracin yield produced by aprA-deficient strain DW2△PaprA reached 931.43 U/mL, 18.92% higher than that of DW2 (783.25 U/mL). The bacitracin yield was reduced by 14.27% in the aprA overexpression strain. Furthermore, our results showed that deficiency of aprA led to a 6.54-fold increase of the aprE transcriptional level and a 1.84-fold increase of subtilisin activity, respectively, which led to the increases of soybean meal utilization rate (28.86%) and precursor amino acid supplies for bacitracin synthesis. Additionally, strengthening the utilization rate of soybean meal also benefited heterologous protein production, and the α-amylase and nattokinase activities were respectively enhanced by 59.81% and 50.53% in aprA-deficient strains. Collectively, this research demonstrated that strengthening subtilisin production could improve the utilization rate of soybean meal and thereby enhance bacitracin and target protein production; also, this strategy would be useful for the improvement of protein/peptide production using soybean meal as the main nitrogen source in the fermentation process.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Abe S, Yasumura A, Tanaka T (2009) Regulation of Bacillus subtilis aprE expression by glnA through inhibition of scoC and sigma(D)-dependent degR expression. J Bacteriol 191(9):3050–3058

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barbieri G, Albertini AM, Ferrari E, Sonenshein AL, Belitsky BR (2016) Interplay of CodY and ScoC in the regulation of major extracellular protease genes of Bacillus subtilis. J Bacteriol 198(6):907–920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cai D, Chen Y, He P, Wang S, Mo F, Li X, Wang Q, Nomura CT, Wen Z, Ma X, Chen S (2018) Enhanced production of poly-gamma-glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis. Biotechnol Bioeng 115(10):2541–2553

    Article  CAS  PubMed  Google Scholar 

  • Cai D, He P, Lu X, Zhu C, Zhu J, Zhan Y, Wang Q, Wen Z, Chen S (2017a) A novel approach to improve poly-γ-glutamic acid production by NADPH Regeneration in Bacillus licheniformis WX-02. Sci Rep 7:43404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cai D, Wang H, He P, Zhu C, Wang Q, Wei X, Nomura CT, Chen S (2017b) A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis. Microb Cell Fact 16(1):70

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cai D, Wei X, Qiu Y, Chen Y, Chen J, Wen Z, Chen S (2016) High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase. J Appl Microbiol 121(3):704–712

    Article  CAS  PubMed  Google Scholar 

  • Chen J, Fu G, Gai Y, Zheng P, Zhang D, Wen J (2015) Combinatorial Sec pathway analysis for improved heterologous protein secretion in Bacillus subtilis: identification of bottlenecks by systematic gene overexpression. Microb Cell Fact 14:92

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen X, Xie F, Zhang X, Li D, Chen S, Li J, Wang Z (2014) Supplementations of ornithine and KNO3 enhanced bacitracin production by Bacillus licheniformis LC-11. Ann Microbiol 64:509–514

    Article  CAS  Google Scholar 

  • Chen Y, Cai D, He P, Mo F, Zhang Q, Ma X, Chen S (2018) Enhanced production of heterologous proteins by Bacillus licheniformis with defective D-alanylation of lipoteichoic acid. World J Microbiol Biotechnol 34(9):135

    Article  CAS  PubMed  Google Scholar 

  • Chen Y, Wang Z, Chu J, Zhuang Y, Zhang S, Yu X (2013) Significant decrease of broth viscosity and glucose consumption in erythromycin fermentation by dynamic regulation of ammonium sulfate and phosphate. Bioresour Technol 134:173–179

    Article  CAS  PubMed  Google Scholar 

  • Cui W, Suo F, Cheng J, Han L, Hao W, Guo J, Zhou Z (2018) Stepwise modifications of genetic parts reinforce the secretory production of nattokinase in Bacillus subtilis. Microb Biotechnol 11(5):930–942

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Durban MA, Silbersack J, Schweder T, Schauer F, Bornscheuer UT (2007) High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis. Appl Microbiol Biotechnol 74(3):634–639

  • Gu Y, Lv X, Liu Y, Li J, Du G, Chen J, Rodrigo LA, Liu L (2018) Synthetic redesign of central carbon and redox metabolism for high yield production of N-acetylglucosamine in Bacillus subtilis. Metab Eng 51:59–69

    Article  CAS  PubMed  Google Scholar 

  • Hambraeus G, Persson M, Rutberg B (2000) The aprE leader is a determinant of extreme mRNA stability in Bacillus subtilis. Microbiology 146(Pt 12):3051–3059

    Article  CAS  PubMed  Google Scholar 

  • He P, Zhang Z, Cai D, Chen Y, Wang H, Wei X, Li S, Chen S (2017) High-level production of alpha-amylase by manipulating the expression of alanine racamase in Bacillus licheniformis. Biotechnol Lett 39(9):1389–1394

    Article  CAS  PubMed  Google Scholar 

  • Hertel R, Meyerjurgens S, Voigt B, Liesegang H, Volland S (2017) Small RNA mediated repression of subtilisin production in Bacillus licheniformis. Sci Rep 7(1):5699

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Islam SMM, Loman AA, Ju LK (2018) High monomeric sugar yields from enzymatic hydrolysis of soybean meal and effects of mild heat pretreatments with chelators. Bioresour Technol 256:438–445

    Article  CAS  PubMed  Google Scholar 

  • Jakob F, Martinez R, Mandawe J, Hellmuth H, Siegert P, Maurer KH, Schwaneberg U (2013) Surface charge engineering of a Bacillus gibsonii subtilisin protease. Appl Microbiol Biotechnol 97(15):6793–6802

    Article  CAS  PubMed  Google Scholar 

  • Li M, Chen Z, Zhang X, Song Y, Wen Y, Li J (2010) Enhancement of avermectin and ivermectin production by overexpression of the maltose ATP-binding cassette transporter in Streptomyces avermitilis. Bioresour Technol 101(23):9228–9235

    Article  CAS  PubMed  Google Scholar 

  • Li Y, Jin K, Zhang L, Ding Z, Gu Z, Shi G (2018a) Development of an inducible secretory expression system in Bacillus licheniformis based on an engineered xylose operon. J Agric Food Chem 66(36):9456–9464

    Article  CAS  PubMed  Google Scholar 

  • Li Y, Wu F, Cai D, Zhan Y, Li J, Chen X, Chen H, Chen S, Ma X (2018b) Enhanced production of bacitracin by knocking out of amino acid permease gene yhdG in Bacillus licheniformis DW2. Sheng Wu Gong Cheng Xue Bao 34(6):916–927

    PubMed  Google Scholar 

  • Lin J, Pantalone VR, Li G, Chen F (2011) Molecular cloning and biochemical characterization of an endo-beta-mannanase gene from soybean for soybean meal improvement. J Agric Food Chem 59(9):4622–4628

    Article  CAS  PubMed  Google Scholar 

  • Liu Z, Yu W, Nomura CT, Li J, Chen S, Yang Y, Wang Q (2018) Increased flux through the TCA cycle enhances bacitracin production by Bacillus licheniformis DW2. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-018-9133-z

  • Loman AA, Islam SMM, Li Q, Ju LK (2017) Enzyme recycle and fed-batch addition for high-productivity soybean flour processing to produce enriched soy protein and concentrated hydrolysate of fermentable sugars. Bioresour Technol 241:252–261

    Article  CAS  PubMed  Google Scholar 

  • Rao MB, Tanksale AM, Ghatge MS, Deshpande VV (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62(3):597–635

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shi C, Zhang Y, Lu Z, Wang Y (2017) Solid-state fermentation of corn-soybean meal mixed feed with Bacillus subtilis and Enterococcus faecium for degrading antinutritional factors and enhancing nutritional value. J Anim Sci Biotechnol 8:50

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shu CC, Wang D, Guo J, Song JM, Chen SW, Chen LL, Gao JX (2018) Analyzing AbrB-Knockout effects through genome and transcriptome sequencing of Bacillus licheniformis DW2. Front Microbiol 9:307

    Article  PubMed  PubMed Central  Google Scholar 

  • Strauch MA, Spiegelman GB, Perego M, Johnson WC, Burbulys D, Hoch JA (1989) The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein. EMBO J 8(5):1615–1621

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang D, Thakker C, Liu P, Bennett GN, San KY (2015) Efficient production of free fatty acids from soybean meal carbohydrates. Biotechnol Bioeng 112(11):2324–2333

    Article  CAS  PubMed  Google Scholar 

  • Wang D, Wang Q, Qiu Y, Nomura CT, Li J, Chen S (2017a) Untangling the transcription regulatory network of the bacitracin synthase operon in Bacillus licheniformis DW2. Res Microbiol 168(6):515–523

    Article  CAS  PubMed  Google Scholar 

  • Wang Q, Zheng H, Wan X, Huang H, Li J, Nomura CT, Wang C, Chen S (2017b) Optimization of inexpensive agricultural by-products as raw materials for bacitracin production in Bacillus licheniformis DW2. Appl Biochem Biotechnol 183(4):1146–1157

    Article  CAS  PubMed  Google Scholar 

  • Wang Z, Cui Y, Liu P, Zhao Y, Wang L, Liu Y, Xie J (2017c) Small peptides isolated from enzymatic hydrolyzate of fermented soybean meal promote endothelium-independent vasorelaxation and ACE inhibition. J Agric Food Chem 65(50):10844–10850

    Article  CAS  PubMed  Google Scholar 

  • Watchararuji K, Goto M, Sasaki M, Shotipruk A (2008) Value-added subcritical water hydrolysate from rice bran and soybean meal. Bioresour Technol 99(14):6207–6213

    Article  CAS  PubMed  Google Scholar 

  • Wei X, Zhou Y, Chen J, Cai D, Wang D, Qi G, Chen S (2015) Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization. J Ind Microbiol Biotechnol 42(2):287–295

    Article  CAS  PubMed  Google Scholar 

  • Wiegand S, Dietrich S, Hertel R, Bongaerts J, Evers S, Volland S, Daniel R, Liesegang H (2013) RNA-Seq of Bacillus licheniformis: active regulatory RNA features expressed within a productive fermentation. BMC Genomics 14:667

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wohlleben W, Mast Y, Muth G, Rottgen M, Stegmann E, Weber T (2012) Synthetic biology of secondary metabolite biosynthesis in actinomycetes: engineering precursor supply as a way to optimize antibiotic production. FEBS Lett 586(15):2171–2176

    Article  CAS  PubMed  Google Scholar 

  • Yang J, Wu XB, Chen HL, Sun-Waterhouse D, Zhong HB, Cui C (2019) A value-added approach to improve the nutritional quality of soybean meal byproduct: enhancing its antioxidant activity through fermentation by Bacillus amyloliquefaciens SWJS22. Food Chem 272:396–403

    Article  CAS  PubMed  Google Scholar 

  • Zhan Y, Sheng B, Wang H, Shi J, Cai D, Yi L, Yang S, Wen Z, Ma X, Chen S (2018) Rewiring glycerol metabolism for enhanced production of poly-gamma-glutamic acid in Bacillus licheniformis. Biotechnol Biofuels 11:306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhou S, Ding R, Chen J, Du G, Li H, Zhou J (2017) Obtaining a panel of cascade promoter-5'-UTR complexes in Escherichia coli. ACS Synth Biol 6(6):1065–1075

    Article  CAS  PubMed  Google Scholar 

  • Zhu J, Cai D, Xu H, Liu Z, Zhang B, Wu F, Li J, Chen S (2018a) Enhancement of precursor amino acid supplies for improving bacitracin production by activation of branched chain amino acid transporter BrnQ and deletion of its regulator gene lrp in Bacillus licheniformis. Synth Syst Biotechnol 3(4):236–243

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhu S, Cai D, Liu Z, Zhang B, Li J, Chen S, Ma X (2018b) Enhancement of bacitracin production by NADPH generation via overexpressing glucose-6-phosphate dehydrogenase Zwf in Bacillus licheniformis. Applied Biochem Biotechnol. https://doi.org/10.1007/s12010-018-2894-0

Download references

Funding

This study was funded by the National Program on Key Basic Research Project (973 Program, No. 2015CB150505), the Technical Innovation Special Fund of Hubei Province (No. 2018ACA149), and the China Postdoctoral Science Foundation (2018 M642802).

Author information

Authors and Affiliations

  1. State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, 368 Youyi Avenue, Wuchang District, Wuhan, 430062, Hubei, People’s Republic of China

    Dongbo Cai, Bowen Zhang, Yi Rao, Lingfeng Li, Jiang Zhu, Xin Ma & Shouwen Chen

  2. Lifecome Biochemistry Co. Ltd, Nanping, 353400, People’s Republic of China

    Junhui Li

Authors
  1. Dongbo Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bowen Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yi Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lingfeng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiang Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Junhui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xin Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shouwen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D Cai and S Chen designed and supervised the study. D Cai, B Zhang, Y Rao, L Li, and J Zhu performed the experiments. D Cai, B Zhang, J Li, X Ma, and S Chen analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Shouwen Chen.

Ethics declarations

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 578 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cai, D., Zhang, B., Rao, Y. et al. Improving the utilization rate of soybean meal for efficient production of bacitracin and heterologous proteins in the aprA-deficient strain of Bacillus licheniformis. Appl Microbiol Biotechnol 103, 4789–4799 (2019). https://doi.org/10.1007/s00253-019-09804-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-019-09804-0

Keywords

Search

Navigation