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.
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.
Results and discussion
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.
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