Genome shuffling for improving the activity of alkaline pectinase in Bacillus subtilis FS105 and its molecular mechanism

  • Ping YuEmail author
  • Xinxin Wang
  • Qian Ren
  • Xingxing Huang
  • Tingting Yan
Original Paper


Genome shuffling for improving the activity of alkaline pectinase in Bacillus subtilis FS105 and its molecular mechanism were investigated. The fused strain B. subtilis FS105 with the highest activity of alkaline pectinase was obtained after two rounds of genome shuffling. The activity of alkaline pectinase in B. subtilis FS105 was 499 U/ml, which was improved by 1.6 times compared to that in original strain. To elucidate its molecular mechanism, rpsL gene sequences from original and fused strains were cloned and aligned, and the space structure of their coding proteins were also analyzed and compared. The alignment of the rpsL gene sequences indicated that three bases G, G and C were respectively replaced by A, A and G in the positions 52, 408 and 409 after genome shuffling. This resulted in the substitution of two amino acid residues in ribosomal protein S12: D18N and P137A, and therefore improving the biosynthesis of alkaline pectinase. This study lays a foundation for improving the activity of alkaline pectinase by genome shuffling and understanding its molecular mechanism.


Bacillus subtilis FS105 Alkaline pectinase Genome shuffling rpsL gene Ribosomal protein S12 



This study was supported by the Food Science and Engineering - the Most Important Discipline of Zhejiang Province, China (No. 2017SIAR214).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Research involving human participants and/or animals

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


  1. Angayarkanni J, Palaniswamy M, Murugesan S, Swaminathan K (2002) Improvement of tea leaves fermentation with Aspergillus spp. pectinase. J Biosci Bioeng 94:299–303PubMedGoogle Scholar
  2. Chalopagorn P, Charoenpanich J, Choowongkomon K (2014) Genome shuffling enhances lipase production of thermophilic Geobacillus sp. Appl Biochem Biotechnol 174:1444–1454PubMedGoogle Scholar
  3. Chen X, Wei P, Fan L, Yang D, Zhu X, Shen W, Xu Z, Cen P (2009) Generation of high-yield rapamycin-producing strains through protoplasts-related techniques. Appl Microbiol Biotechnol 83:507–512PubMedGoogle Scholar
  4. Cheng C, Almario MP, Kao KC (2015) Genome shuffling to generate recombinant yeasts for tolerance to inhibitors present in lignocellulosic hydrolysates. Biotechnol Lett 37:2193–2200PubMedGoogle Scholar
  5. Dixit VS, Kumar AR, Pant A, Khan MI (2004) Low molecular mass pectate lyase from Fusarium moniliforme: similar modes of chemical and thermal denaturation. Biochem Biophys Res Commun 315:477–484PubMedGoogle Scholar
  6. Dong Y, Chen JC, Jiang W, Yang GH (2009) A study on quality improvement of APMP by pectinase. Paper Chem 4:4–9Google Scholar
  7. Du W, Huang D, Xia M, Wen J, Huang M (2014) Improved FK506 production by the precursors and product-tolerant mutant of Streptomyces tsukubaensis based on genome shuffling and dynamic fed-batch strategies. J Ind Microbiol Biotechnol 41:1131–1143PubMedGoogle Scholar
  8. Esquivel JCC, Hours RA, Voget CE, Mignone CF (1999) Aspergillus kawachii produces an acidic pectin releasing enzyme activity. J Biosci Bioeng 88:48–52Google Scholar
  9. Fernández-González M, Ubeda J, Vasudevan T, Otero RC, Briones A (2004) Evaluation of polygalacturonase activity in Saccharomyces cerevisiae wine strains. FEMS Microbiol Lett 237:261–266PubMedGoogle Scholar
  10. Giacobbe S, Pepe O, Ventorino V, Birolo L, Vinciguerra R, Faraco V (2014) Identification and characterisation of a pectinolytic enzyme from Paenibacillus xylanolyticus. BioResources 9:4873–4887Google Scholar
  11. Gong J, Zheng H, Wu Z, Chen T, Zhao X (2009) Genome shuffling: progress and applications for phenotype improvement. Biotechnol Adv 27:996–1005PubMedGoogle Scholar
  12. Grassino AN, Brnčić M, Vikić-Topić D, Roca S, Dent M, Brnčić SR (2016) Ultrasound assisted extraction and characterization of pectin from tomato waste. Food Chem 198:93–100PubMedGoogle Scholar
  13. Gu HY, Qi HY, Zhang HX (2002) Fermentation conditions of pectinase by Mucor mucedo. Chin J Proc Eng 2:254–256Google Scholar
  14. Han GG, Song AA, Kim EB, Yoon SH, Bok JD, Cho CS, Kil DY, Kang SK, Choi YJ (2017) Improved antimicrobial activity of Pediococcus acidilactici against Salmonella gallinarum by UV mutagenesis and genome shuffling. Appl Microbiol Biotechnol 101:5353–5363PubMedGoogle Scholar
  15. Hoondal G, Tiwari R, Tewari R, Dahiya N, Beg Q (2002) Microbial alkaline pectinases and their industrial applications: a review. Appl Microbiol Biotechnol 59:409–418PubMedGoogle Scholar
  16. Horikoshi K (1972) Production of alkaline enzymes by alkalophilic microorganisms: Part III. alkaline pectinase of Bacillus No. P-4-N. Agric Biol Chem 36:285–293Google Scholar
  17. Hosaka T, Tamehiro N, Chumpolkulwong N, Hori-Takemoto C, Shirouzu M, Yokoyama S, Ochi K (2004) The novel mutation K87E in ribosomal protein S12 enhances protein synthesis activity during the late growth phase in Escherichia coli. Mol Genet Genom 271:317–324Google Scholar
  18. Iconomou D, Arapoglou D, Israilides C (2010) Improvement of phenolic antioxidants and quality characteristics of virgin olive oil with the addition of enzymes and nitrogen during olive paste processing. Grasasyaceites 61:303–311Google Scholar
  19. Ingallinera B, Barbagallo RN, Spagna G, Palmeri R, Todaro A (2005) Effects of thermal treatments on pectinesterase activity determined in blood oranges juices. Enzyme Microb Technol 36:258–263Google Scholar
  20. Jin Z, Xu B, Lin S, Jin Q, Cen P (2009) Enhanced production of spinosad in Saccharopolyspora spinosa by genome shuffling. Appl Biochem Biotechnol 159:655–661PubMedGoogle Scholar
  21. Kang JX, Chen XJ, Chen WR, Li MS, Fang Y, Li DS, Ren YZ, Liu DQ (2011) Enhanced production of pullulan in Aureobasidium pullulans by a new process of genome shuffling. Process Biochem 46:792–795Google Scholar
  22. Kapoor M, Beg QK, Bhushan B, Singh K, Dadhich K, Hoondal G (2001) Application of an alkaline and thermostable polygalacturonase from Bacillus sp. MG-cp-2 in degumming of ramie (Boehmeria nivea) and sunn hemp (Crotalaria juncea) bast fibres. Process Biochem 36:803–807Google Scholar
  23. Kashyap D, Vohra P, Chopra S, Tewari R (2001) Applications of pectinases in the commercial sector: a review. Bioresour Technol 77:215–227PubMedGoogle Scholar
  24. Kashyap DR, Soni SK, Tewari R (2003) Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation. Bioresour Technol 88:251–254Google Scholar
  25. Klug-Santner BG, Schnitzhofer W, Vršanská M, Weber J, Agrawal PB, Nierstrasz VA, Guebitz GM (2006) Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2. J Biotechnol 121:390–401PubMedGoogle Scholar
  26. Kovtunovych G, Lytvynenko T, Negrutska V, Lar O, Brisse S, Kozyrovska N (2003) Identification of Klebsiella oxytoca using a specific PCR assay targeting the polygalacturonase pehX gene. Res Microbiol 154:587–592PubMedGoogle Scholar
  27. Kuhad RC, Kapoor M, Rustagi R (2004) Enhanced production of an alkaline pectinase from Streptomyces sp. RCK-SC by whole-cell immobilization and solid-state cultivation. World J Microbiol Biotechnol 20:257–263Google Scholar
  28. Kurosawa K, Hosaka T, Tamehiro N, Inaoka T, Ochi K (2006) Improvement of α-amylase production by modulation of ribosomal component protein S12 in Bacillus subtilis 168. Appl Environ Microbiol 72:71–77PubMedPubMedCentralGoogle Scholar
  29. Lee BU, Choi MS, Kim DM, Oh KH (2017) Genome shuffling of Stenotrophomonas maltophilia OK-5 for improving the degradation of explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). Curr Microbiol 74:268–276PubMedGoogle Scholar
  30. Li Z, Bai Z, Zhang B, Xie H, Hu Q, Hao C, Xue W, Zhang H (2005) Newly isolated Bacillus gibsonii S-2 capable of using sugar beet pulp for alkaline pectinase production. World J Microbiol Biotechnol 21:1483–1486Google Scholar
  31. Li S, Li F, Chen XS, Wang L, Xu J, Tang L, Mao ZG (2012) Genome shuffling enhanced ε-poly-l-lysine production by improving glucose tolerance of Streptomyces graminearus. Appl Biochem Biotechnol 166:414–423PubMedGoogle Scholar
  32. Liu Z, Zhao X, Bai F (2013) Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering. Appl Microbiol Biotechnol 97:4361–4368PubMedGoogle Scholar
  33. Luo L, Yang Y, Wei H, Rang J, Tang Q, Hu S, Sun Y, Yu Z, Ding X, Xia L (2016) Effect of ribosome engineering on butenyl-sspinosyns synthesis of Saccharopolyspora pogona. Chin J Biotechnol 32:259–263Google Scholar
  34. Midoro-Horiuti T, Mathura V, Schein CH, Braun W, Yu S, Watanabe M, Lee JC, Brooks EG, Goldblum RM (2003) Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1 map to the pectate lyase catalytic site. Mol Immunol 40:555–562PubMedGoogle Scholar
  35. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428Google Scholar
  36. Mohnen D (2008) Pectin structure and biosynthesis. Curr Opin Plant Biol 11:266–277PubMedGoogle Scholar
  37. Nair J, Rouse DA, Bai GH, Morris SL (1993) The rpsL gene and streptomycin resistance in single and multiple drug-resistant strains of Mycobacterium tuberculosis. Mol Microbiol 10:521–527PubMedGoogle Scholar
  38. Ochi K, Okamoto S, Tozawa Y, Inaoka T, Hosaka T, Xu J, Kurosawa K (2004) Ribosome engineering and secondary metabolite production. Adv Appl Microbiol 56:155–184PubMedGoogle Scholar
  39. Okamoto-Hosoya Y, Hosaka T, Ochi K (2003) An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3 (2). Microbiology 149:3299–3309PubMedGoogle Scholar
  40. Olano C, Lombo F, Mendez C, Salas JA (2008) Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering. Metab Eng 10:281–292PubMedGoogle Scholar
  41. Oliveira TÍS, Rosa MF, Cavalcante FL, Pereira PHF, Moates GK, Wellner N, Mazzetto SE, Waldron KW, Azeredo HM (2016) Optimization of pectin extraction from banana peels with citric acid by using response surface methodology. Food Chem 198:113–118PubMedGoogle Scholar
  42. Ortega N, De Diego S, Perez-Mateos M, Busto M (2004) Kinetic properties and thermal behaviour of polygalacturonase used in fruit juice clarification. Food Chem 88:209–217Google Scholar
  43. Otte B, Grunwaldt E, Mahmoud O, Jennewein S (2009) Genome shuffling in Clostridium diolis DSM 15410 for improved 1,3-propanediol production. Appl Environ Microbiol 75:7610–7616PubMedPubMedCentralGoogle Scholar
  44. Patnaik R, Louie S, Gavrilovic V, Perry K, Stemmer WP, Ryan CM, del Cardayré S (2002) Genome shuffling of Lactobacillus for improved acid tolerance. Nat Biotechnol 20:707–713PubMedGoogle Scholar
  45. Rashmi R, Murthy K, Sneha G, Shabana S, Syama A, Radhika V (2008) Partial purification and biochemical characterization of extracellular pectinase from Aspergillus niger isolated from groundnut seeds. J Appl Biosci 9:378–384Google Scholar
  46. Reid I, Ricard M (2000) Pectinase in papermaking: solving retention problems in mechanical pulps bleached with hydrogen peroxide. Enzyme Microb Technol 26:115–123PubMedGoogle Scholar
  47. Schnitzhofer W, Weber HJ, Vršanská M, Biely P, Cavaco-Paulo A, Guebitz G (2007) Purification and mechanistic characterisation of two polygalacturonases from Sclerotium rolfsii. Enzyme Microb Technol 40:1739–1747Google Scholar
  48. Sharma D, Satyanarayana T (2006) A marked enhancement in the production of a highly alkaline and thermostable pectinase by Bacillus pumilus dcsr1 in submerged fermentation by using statistical methods. Bioresour Technol 97:727–733PubMedGoogle Scholar
  49. Shima J, Hesketh A, Okamato S, Kawamoto S, Ochi K (1996) Induction of actinorhodin production by rpsL mutations that confer streptomycin resistance in S. lividans and S. coelicolor A3 (2). J Bacteriol 178:7276–7284PubMedPubMedCentralGoogle Scholar
  50. Shobha M, Kumar AV, Tharanathan R, Koka R, Gaonkar AK (2005) Modification of guar galactomannan with the aid of Aspergillus niger pectinase. Carbohydr Polym 62:267–273Google Scholar
  51. Sreenath HK, Shah AB, Yang VW, Gharia MM, Jeffries TW (1996) Enzymatic polishing of jute/cotton blended fabrics. J Ferment Bioeng 81:18–20Google Scholar
  52. Tamehiro N, Hosaka T, Xu J, Hu H, Otake N, Ochi K (2003) Innovative approach for improvement of an antibiotic-overproducing industrial strain of Streptomyces albus. Appl Environ Microbiol 69:6412–6417PubMedPubMedCentralGoogle Scholar
  53. Tanabe H, Kobayashi Y, Akamatsu I (1988) Pretreatment of pectic wastewater with pectate lyase from an alkalophilic Bacillus sp. Agric Biol Chem 52:1855–1856Google Scholar
  54. Tang M, Bian M, Li Q (2006) Study on solid fermentation production conditions of pectinase from Aspergillus oryzae. J Fuqing Branch Fujian Norm Univ 2:6–9Google Scholar
  55. Vaillant F, Cisse M, Chaverri M, Perez A, Dornier M, Viquez F, Dhuique-Mayer C (2005) Clarification and concentration of melon juice using membrane processes. Innov Food Sci Emerg Technol 6:213–220Google Scholar
  56. Wagner F, Kusserow H, Schäfer W (2000) Cloning and targeted disruption of two polygalacturonase genes in Penicillium olsonii. FEMS Microbiol Lett 186:293–299PubMedGoogle Scholar
  57. Wang PM, Zheng DQ, Liu TZ, Tao XL, Feng MG, Min H, Jiang XH, Wu XC (2012) The combination of glycerol metabolic engineering and drug resistance marker-aided genome shuffling to improve very-high-gravity fermentation performances of industrial Saccharomyces cerevisiae. Bioresour Technol 108:203–210PubMedGoogle Scholar
  58. Xu B, Jin Z, Wang H, Jin Q, Jin X, Cen P (2008) Evolution of Streptomyces pristinaespiralis for resistance and production of pristinamycin by genome shuffling. App Microbiol Biotechnol 80:261–267Google Scholar
  59. Yu L, Pei X, Lei T, Wang Y, Feng Y (2008) Genome shuffling enhanced l-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus. J Biotechnol 134:154–159PubMedGoogle Scholar
  60. Yu G, Hu Y, Hui M, Chen L, Wang L, Liu N, Yin Y, Zhao J (2014) Genome shuffling of Streptomyces roseosporus for improving daptomycin production. Appl Biochem Biotechnol 172:2661–2669PubMedGoogle Scholar
  61. Yu P, Xu CC, Zhu PZ (2018) The protoplast preparation and regeneration conditions of Bacillus subtilis ZGL14 producing alkaline pectinase. Chin J Appl Environ Biol 24:356–361Google Scholar
  62. Zhang YX, Perry K, Vinci VA, Powell K, Stemmer WP, del Cardayré SB (2002) Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415:644–647PubMedGoogle Scholar
  63. Zhang Y, Liu JZ, Huang JS, Mao ZW (2010) Genome shuffling of Propionibacterium shermanii for improving vitamin B12 production and comparative proteome analysis. J Biotechnol 148:139–143PubMedGoogle Scholar
  64. Zhang J, Wang X, Diao J, He H, Zhang Y, Xiang W (2013) Streptomycin resistance-aided genome shuffling to improve doramectin productivity of Streptomyces avermitilis NEAU1069. J Ind Microbiol Biotechnol 40:877–889PubMedGoogle Scholar
  65. Zhao Y, Duan C, Gao L, Yu X, Niu C, Li S (2017) Genome shuffling of Lactobacillus plantarum C88 improves adhesion. Biosci Biotechnol Biochem 81:184–193PubMedGoogle Scholar
  66. Zheng P, Zhang K, Yan Q, Xu Y, Sun Z (2013) Enhanced succinic acid production by Actinobacillus succinogenes after genome shuffling. J Ind Microbiol Biotechnol 40:831–840PubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Ping Yu
    • 1
    Email author
  • Xinxin Wang
    • 1
  • Qian Ren
    • 1
  • Xingxing Huang
    • 1
  • Tingting Yan
    • 1
  1. 1.College of Food Science and BiotechnologyZhejiang Gongshang UniversityHangzhouPeople’s Republic of China

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