- Open Access
Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization
© The Author(s). 2016
- Received: 25 August 2016
- Accepted: 3 October 2016
- Published: 17 October 2016
Alkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168.
The activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of B. subtilis was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in B. subtilis 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in B. subtilis 168 mut-16#.
The results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in B. subtilis. Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using B. subtilis cultures as microbial cell factories.
- B. subtilis
- ARTP mutagenesis
- Alkaline amylase
- Fermentation optimization
Amylases (EC 3.2.1) are important industrial enzymes, one of which is alkaline amylase, which is stable under alkaline conditions. Alkaline amylase has significant potential for applications in the textile, paper, and detergent industries. Alkaline amylase is mainly present in alkalophilic microorganisms (e.g., Bacillus licheniformis and Bacillus sp.) [1–3]. Many alkaline amylases have been heterologously expressed in recombinant hosts to improve their yield and optimize their properties [4, 5]. Murakami et al. heterologously expressed alkaline amylase from B. halodurans MS-2-5 in recombinant Escherichia coli, and under optimized cultivation conditions, the amylase yield increased 104-fold compared with yield from the wild-type strain .
B. subtilis, a gram-positive bacterial strain, is an important industrial microorganism with a clear genetic background, is generally recognized as safe, has a superior secretion level, and is applicable for large-scale industrial products [6, 7]. B. subtilis is generally used to overexpress industrial enzymes (e.g., aminopeptidase, amylase, nattokinase, and protease) [6, 8–10]. Ploss et al. overproduced the industrially relevant amylase AmyM from Geobacillus stearothermophilus in B. subtilis 168 based on the secretion stress response . There have been many strategies used to improve the expression of recombinant proteins in B. subtilis, including mutagenesis, screening highly efficient expression systems, strong promoters, peptides with high secretion level, and fermentation optimization [7, 9, 12–14].
ARTP (atmospheric and room temperature plasma) has been used as a novel mutagenesis technology in mutagenesis breeding of microorganisms (e.g., bacteria, actinomycetes, and fungi) to improve the yield of industrial products [9, 15]. In our previous work, a B. subtilis WB600 mutant with a high yield of recombinant alkaline amylase was screened by ARTP mutagenesis technology and a high-throughput screening technique (HTS) . Fed-batch culture is frequently applied to improve product yield and productivity in industrial microbial processes by preventing catabolite repression and substrate inhibition . Park et al. analyzed the effect of controlling amino acid composition in a fed-batch culture on amylase production in recombinant B. brevis, the maximum yield of which was obtained by controlling high asparagine and isoleucine concentrations and low other amino acids concentrations, increased from 5.14 kU/mL to 12.01 kU/mL .
In our previous work, B. subtilis WB600 without any recombinant plasmids was induced by ARTP mutagenesis, which increased the difficulty of high-throughput screening because of the low efficiency of recombinant plasmid injection into B. subtilis . In this work, B. subtilis 168 with recombinant plasmids was induced by ARTP mutagenesis. This strategy avoided the recombinant plasmid by injecting a B. subtilis mutant library after mutagenesis, which significantly improved the efficiency of mutagenesis and screening of B. subtilis mutants with a high expression level of recombinant proteins. The yield and specific production rate of recombinant alkaline amylase and the growth behavior of the mutant were determined and characterized. Moreover, fermentation optimization strategy was used to improve the production yield of recombinant alkaline amylase in B. subtilis mutant in a 3-L fermenter.
ARTP mutagenesis and high throughput screening (HTS)
A recombinant plasmid containing the alkaline amylase gene in B. subtilis 168 mut-16# was obtained and sequenced, and the results showed that the plasmid had no mutations (data not shown). The genetic stability of B. subtilis 168 mut-16# was also evaluated by continuous subcultivation. The results suggest that this strain exhibited good genetic stability (data not shown). In previous studies, Streptomyces albulus A-29 and Enterobacter cloacae (MU-1) mutants also exhibited high genetic stability after ARTP mutagenesis [19, 20]; this indicates that ARTP mutagenesis is a promising tool for generating genetically stable mutants.
Effect of agitation speed on alkaline amylase production in B. subtilis 168 mut-16#
The DCW of B. subtilis 168 mut-16# was highest when grown at 550 rpm: 5.7 g/L (Fig. 3c). Meanwhile, the highest specific growth rate of B. subtilis 168 mut-16# was the highest at 550 rpm, indicating that these conditions favored quick growth of this strain. High agitation speed (650 rpm) favored quick growth of B. subtilis 168 mut-16# at initial phases, but the culture quickly reached stationary phase. These results suggested that high agitation speed promoted faster growth of B. subtilis 168 mut-16# at initial phases, but the higher shear force may also negatively affect growth at later phases.
Effect of different soluble starch/soy peptone concentrations on alkaline amylase production by B. subtilis 168 mut-16#
Effect of different feeding compositions on alkaline amylase production in B. subtilis 168 mut-16#
Feeding with appropriate nutrients favors the optimal expression of transcription promoters and effective secretion of heterologous proteins by B. subtilis . Media comprising the required nutrients for supporting strain growth and preventing the inhibition of growth were generally fed to the bacteria to improve protein yield . Batch feeding of carbon sources with catabolite-repressing could significantly improve the expression level of proteins (e.g., amylase) with carbon catabolite repression . Based on the above optimum conditions, a fed-batch strategy was used to improve the yield of alkaline amylase in B. subtilis 168 mut-16# in this study. The different feeding compositions included glucose, hydrolyzed starch, and a concentrated mixture of hydrolyzed starch and soy peptone. Following application of these carbon sources, the pH of the fermentation medium increased, and fed-batch cultures controlled by pH change have been used for producing recombinant proteins and chemical products [28, 30, 31]. Based on changes in pH, the effect of different feeding compositions on the production of recombinant alkaline amylase in B. subtilis 168 mut-16# was investigated.
Effect of different feeding methods and times on alkaline amylase production in B. subtilis 168 mut-16#
B. subtilis 168 mut-16#, the mutant with the highest yield of alkaline amylase, was obtained by novel ARTP mutagenesis-screening method. The cell growth and recombinant alkaline amylase production capacity in B. subtilis 168 mut-16# were significantly enhanced by ARTP mutagenesis in this study. An agitation speed of 550 rpm favored alkaline amylase production by B. subtilis 168 mut-16# and resulted in fast growth. A high concentration of soluble starch and soy peptone was preferred for cell growth and recombinant enzyme production by B. subtilis 168, while excessively higher concentrations promoted faster cell growth but inhibited recombinant enzyme production. Feeding hydrolyzed starch promoted the growth and recombinant alkaline amylase production by B. subtilis 168 mut-16#. Glucose, a quickly utilized carbon source, inhibited recombinant alkaline amylase expression in this B. subtilis strain. Feeding with a nitrogen source (soy peptone) promoted the growth of B. subtilis 168 mut-16#, but inhibited the yield of alkaline amylase. Single pulse feeding-based fed-batch promoted the expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. Feeding too early or too late could result in outgrowth or hypotrophy and inhibit the production of alkaline amylase in B. subtilis 168 mut-16#. In the future, we will investigate the effect of integration of more rounds of ARTP mutagenesis with systems-level fermentation optimization including as many high-producing cells on high-level gene expression in B. subtilis.
Microorganisms and media
The wild-type strain B. subtilis 168 and the shuttle plasmid pMA0911-amy (containing an alkaline amylase gene) were maintained in our culture collection center. Luria-Bertani (LB) medium was used for the B. subtilis starter culture. The trypan blue-starch agar plate included 10.0 g/L soluble starch, 0.2 g/L trypan blue, and 100.0 μg/mL kanamycin. The initial fermentation medium included 10.0 g/L soluble starch, 5.0 g/L NaCl, 30.0 g/L soy peptone, 20.0 g/L soybean meal, and 100.0 μg/mL kanamycin.
The working volume of flask culture was 25 mL/250 mL. The B. subtilis starter culture was grown at 37 °C and 200 rpm for 10 h. The starter culture inoculum was 4.0 % when transferred into the fermentation medium in the 3-L fermenter. B. subtilis was cultured at 37 °C to produce recombinant alkaline amylase.
ARTP mutagenesis and high throughput screening (HTS)
The experimental protocols for ARTP and HTS are shown in Fig. 1. Methods and materials used for ARTP mutagenesis were the same as the materials used in our previous study . Trypan blue-starch agar was used to screen for B. subtilis with high amylase expression to improve the screening efficiency. Based on the size of the transparent rings, B. subtilis mutants with high alkaline amylase activity were selected, and were further screened by HTS.
Screening and verification of B. subtilis mutants with high alkaline amylase activity
B. subtilis mutants with high yield of recombinant alkaline amylase were further verified and screened by flask fermentation. After HTS, B. subtilis mutants with a high yield of recombinant alkaline amylase were cultured in 250 mL shaker flasks with 25 mL fermentation medium at 200 rpm and 37 °C. After verification by shaker flask fermentation, the recombinant plasmid in B. subtilis mutants with the highest yield was obtained and sequenced to verify a lack of mutations. B. subtilis mutant genetic stability was examined by subculturing mutants for 20 generations .
Analysis of amylase activity
One unit (U) of amylase was defined as the amount of enzyme required for catalyzing starch to release 1 μmol reducing sugar (glucose) per minute at 50 °C and pH 9.5 . Amylase activity was determined by a modified DNS (3,5-dinitrosalicylic acid) method .
Dry Cell Weight (DCW) was determined to analyze B. subtilis cell concentration. The soybean meal was first sedimented by low-speed centrifugation (600 × g, 30 s). Then, B. subtilis was centrifuged at 12,000 × g for 5 min and washed with NaCl solution (0.9 %, w/v). Cells were dried at 105 °C for 2 h and weighed on an electronic balance.
To study the effect of agitation speed on the production of alkaline amylase in recombinant B. subtilis, agitation speeds during 3-L fermentation were maintained at 450, 550, and 650 rpm. To study the effects of different soluble starch and soy peptone concentrations, soluble starch and soy peptone concentrations in the fermentation medium were 1.0-, 1.5-, 2.0-, 2.5-, and 3.0-fold of the initial concentration. To study the effect of different feeding compositions on alkaline amylase production and growth of B. subtilis, glucose, hydrolyzed starch, and hydrolyzed starch and soy peptone were fed after 10 h of culture. Soluble starch (200 mL 20 % (w/v)) was hydrolyzed by 0.04 g thermostable amylase (20,000 U/g) at 100 °C until the blue color of iodine and potassium iodide solution stabilized. The ratio of hydrolyzed starch to soy peptone used was 2:3. To study the effect of the feeding method on alkaline amylase production and growth of B. subtilis, fed-batch methods included single pulse feeding or constant feed flow rate-based feeding. The constant feed flow rate was 1.0 mL/min. Different feeding times of 8, 10, and 12 h were studied.
Experiments were independently performed 3 times, and the data shown are the mean of these replicates. Errors are shown as the standard deviations (SD).
The authors would like to acknowledge Lab of Biosystem and Bioprocessing Engineering.
This project was financially supported by 863 Program (2014AA021304), Natural Science Foundation of Jiangsu Province (BK20140152), National Natural Science Foundation of China (21406089), the Open Project Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (KLIB-KF201509), 111 Project (111-2-06). The funding supported all of the research including the design of the study and collection, analysis, and interpretation of data and writing of the manuscript.
Availability of data and materials
Since these data has not still been published, and we will prepare a new paper by refining new experiment results based on these data. Authors do not wish to share their data now.
Conceived and designed the experiments: WS, XZC, LL, ZMZ, HQY. Performed the experiments: YFM. Analyzed the data: YFM, FX, HQY. Contributed reagents/materials/analysis tools: WS, XZC, HQY. Wrote the paper: YFM, FX, HQY. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This paper is in compliance with ethical standards.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Arikan B. Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp isolate A3-15. Bioresour Technol. 2008;99:3071–6.View ArticleGoogle Scholar
- Roy JK, Mukherjee AK. Applications of a high maltose forming, thermo-stable alpha-amylase from an extremely alkalophilic Bacillus licheniformis strain AS08E in food and laundry detergent industries. Biochem Eng J. 2013;77:220–30.View ArticleGoogle Scholar
- Vester JK, Glaring MA, Stougaard P. An exceptionally cold-adapted alpha-amylase from a metagenomic library of a cold and alkaline environment. Appl Microbiol Biotechnol. 2015;99:717–27.View ArticleGoogle Scholar
- Murakami S, Nagasaki K, Nishimoto H, Shigematu R, Umesaki J, Takenaka S, Kaulpiboon J, Prousoontorn M, Limpaseni T, Pongsawasdi P, Aoki K. Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing alpha-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli. Enzym Microb Technol. 2008;43:321–8.View ArticleGoogle Scholar
- Yang HQ, Liu L, Shin H-D, Chen RR, Li JH, Du GC, Chen J. Integrating terminal truncation and oligopeptide fusion for a novel protein engineering strategy to improve specific activity and catalytic efficiency: alkaline alpha-amylase as a case study. Appl Environ Microbiol. 2013;79:6429–38.View ArticleGoogle Scholar
- Guan CR, Cui WJ, Cheng JT, Zhou L, Liu ZM, Zhou ZM. Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis. Microb Cell Fact. 2016;15:66.View ArticleGoogle Scholar
- Liu L, Yang HQ, Shin H-D, Chen RR, Li JH, Du GC, Chen J. How to achieve high-level expression of microbial enzymes Strategies and perspectives. Bioengineered. 2013;4:212–23.View ArticleGoogle Scholar
- Jaouadi NZ, Jaouadi B, Aghajari N, Bejar S. The overexpression of the SAPB of Bacillus pumilus CBS and mutated sapB-L31I/T33S/N99Y alkaline proteases in Bacillus subtilis DB430: New attractive properties for the mutant enzyme. Bioresour Technol. 2012;105:142–51.View ArticleGoogle Scholar
- Ma YF, Yang HQ, Chen XZ, Sun B, Du GC, Zhou ZM, Song JN, Fan Y, Shen W. Significantly improving the yield of recombinant proteins in Bacillus subtilis by a novel powerful mutagenesis tool (ARTP): Alkaline alpha-amylase as a case study. Protein Expr Purif. 2015;114:82–8.View ArticleGoogle Scholar
- Thao Thi N, Thi Dinh Q, Hoang TL. Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800. Microb Cell Fact. 2013;12:79.View ArticleGoogle Scholar
- Ploss TN, Reilman E, Monteferrante CG, Denham EL, Piersma S, Lingner A, Vehmaanpera J, Lorenz P, van Dijl JM. Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions. Microb Cell Fact. 2016;15:57.View ArticleGoogle Scholar
- Degering C, Eggert T, Puls M, Bongaerts J, Evers S, Maurer K-H, Jaeger K-E. Optimization of protease secretion in Bacillus subtilis and Bacillus licheniformis by screening of homologous and heterologous signal peptides. Appl Environ Microbiol. 2010;76:6370–6.View ArticleGoogle Scholar
- Ozturk S, Calik P, Ozdamar TH. Fed-batch biomolecule production by Bacillus subtilis: A state of the art review. Trends Biotechnol. 2016;34:329–45.View ArticleGoogle Scholar
- Trang Thi Phuong P, Hoang Duc N, Schumann W. Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements. J Biotechnol. 2012;157:167–72.View ArticleGoogle Scholar
- Zhang X, Zhang C, Zhou Q-Q, Zhang X-F, Wang L-Y, Chang H-B, Li H-P, Oda Y, Xing X-H. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis. Appl Microbiol Biotechnol. 2015;99:5639–46.View ArticleGoogle Scholar
- Park YS, Dohjima T, Okabe M. Enhanced alpha-amylase production in recombinant Bacillus brevis by fed-batch culture with amino acid control. Biotechnol Bioeng. 1996;49:36–44.View ArticleGoogle Scholar
- Zhang X, Zhang XF, Li HP, Wang LY, Zhang C, Xing XH, Bao CY. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool. Appl Microbiol Biotechnol. 2014;98:5387–96.View ArticleGoogle Scholar
- Fang MY, Jin LH, Zhang C, Tan YY, Jiang PX, Ge N, Li HP, Xing XH. Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP) and generation of a mutant library with diverse phenotypes. PLoS One. 2013;8:e77046.View ArticleGoogle Scholar
- Lu Y, Wang LY, Ma K, Li G, Zhang C, Zhao HX, Lai QH, Li H-P, Xing X-H. Characteristics of hydrogen production of an Enterobacter aerogenes mutant generated by a new atmospheric and room temperature plasma (ARTP). Biochem Eng J. 2011;55:17–22.View ArticleGoogle Scholar
- Hua XF, Wang J, Wu ZJ, Zhang HX, Li HP, Xing XH, Liu Z. A salt tolerant Enterobacter cloacae mutant for bioaugmentation of petroleum-and salt-contaminated soil. Biochem Eng J. 2010;49:201–6.View ArticleGoogle Scholar
- Veening JW, Igoshin OA, Eijlander RT, Nijland R, Hamoen LW, Kuipers O. Transient heterogeneity in extracellular protease production by Bacillus subtilis. Mol Sys Biol. 2008;4:184.Google Scholar
- Kwon E-Y, Kim KM, Kim MK, Lee IY, Kim BS. Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis. Bioprocess Biosyst Eng. 2011;34:789–93.View ArticleGoogle Scholar
- Wu QL, Chen T, Gan Y, Chen X, Zhao XM. Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs. Appl Microbiol Biotechnol. 2007;76:783–94.View ArticleGoogle Scholar
- Huang HJ, Ridgway D, Gu TY, Moo-Young M. Enhanced amylase production by Bacillus subtilis using a dual exponential feeding strategy. Bioprocess Biosyst Eng. 2004;27:63–9.View ArticleGoogle Scholar
- Yao J, Xu H, Shi NN, Cao X, Feng XH, Li S, Ouyang PK. Analysis of carbon metabolism and improvement of g-polyglutamic acid production from Bacillus subtilis NX-2. Appl Biochem Biotechnol. 2010;160:2332–41.View ArticleGoogle Scholar
- Westers H, Darmon E, Zanen G, Veening JW, Kuipers OP, Bron S, Quax WJ, van Dijl JM. The Bacillus secretion stress response is an indicator for α-amylase levels. Lett Appl Microbiol. 2004;39:65–73.View ArticleGoogle Scholar
- Hyyryläinen HL, Bolhuis A, Darmon E, Muukkonen L, Koski P, Vitikainen M, Sarvas M, Prágai Z, Bron S, van Dijl JM, Kontinen VP. A novel two component regulatory system in Bacillus subtilis for the survival of severe secretion stress. Mol Microbiol. 2001;41:1159–72.View ArticleGoogle Scholar
- Tulin EE, Ueda S, Yamagata H, Udaka S, Yamane T. Effective extracullular production of Bacillus stearothermophilus esterase by pH-stat modal fed-batch culture of recombinant Bacillus brevis. Biotechnol Bioeng. 1992;40:844–50.View ArticleGoogle Scholar
- Pohl S, Harwood CR. Heterologous protein secretion by Bacillus species from the cradle to the grave. Adv Appl Microbiol. 2010;73:1–25.View ArticleGoogle Scholar
- Gao M, Tashiro Y, Wang QH, Sakai K, Sonomoto K. High acetone-butanol-ethanol production in pH-stat co-feeding of acetate and glucose. J Biosci Bioeng. 2016;122:176–82.View ArticleGoogle Scholar
- Li KT, Liu D-H, Chu J, Wang Y-H, Zhuang Y-P, Zhang S-L. An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B(12) by Pseudomonas denitrificans. Bioprocess Biosyst Eng. 2008;31:605–10.View ArticleGoogle Scholar
- Zhang M, Shi ML, Zhou Z, Yang S, Yuan ZY, Ye Q. Production of Alcaligenes faecalis penicillin G acylase in Bacillus subtilis WB600 (pMA5) fed with partially hydrolyzed starch. Enzyme Microb Technol. 2006;39:555–60.View ArticleGoogle Scholar
- Cho Y-H, Song JY, Kim KM, Kim MK, Lee IY, Kim SB, Kim HS, Han NS, Lee BH, Kim BS. Production of nattokinase by batch and fed-batch culture of Bacillus subtilis. New Biotechnol. 2010;27:341–6.View ArticleGoogle Scholar