Article pubs.acs.org/JAFC
Transcriptional Characteristics Associated with Lichenysin Biosynthesis in Bacillus licheniformis from Chinese Maotai-Flavor Liquor Making Qun Wu, Rong Zhang, Suqin Peng, and Yan Xu* State Key Laboratory of Food Science and Technology, The Key Laboratory of Industrial Biotechnology, Ministry of Education, Synergetic Innovation Center of Food Safety and Nutrition, and School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China 214122 S Supporting Information *
ABSTRACT: This work investigated the biosynthetic mechanism of lichenysin, the newly identified nonvolatile matrix component in Chinese liquors. Transcriptomes were analyzed in three producers, Bacillus licheniformis CGMCC 3961, 3962, and 3963, which were isolated from Maotai-flavor liquor-making process and produced 386.3, 553.5, and 795.2 μg/L lichenysin in a simulative liquor fermentation process. Lichenysin synthetase genes lchAA−AD in these three producers were expressed much more highly than those of the nonproducer B. licheniformis ATCC 14580 (>18.4-fold). In addition, ABC transporters were the most significant responsive metabolic pathway, and the expression levels of peptide transporter genes dppABCDE all increased more than 19.2-fold. When B. licheniformis CGMCC 3963 was cultured in synthetic medium, the expression of dppABCDE and lichenysin both increased with the addition of casein hydrolysate (containing various peptides). This indicated that peptide would act as a substrate for lichenysin synthesis. This work sheds new light on the mechanism for lichenysin biosynthesis. KEYWORDS: Bacillus licheniformis, Chinese liquor, dipeptide transport, lichenysin, transcriptome
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INTRODUCTION Lipopeptides are an important class of biosurfactants. They consist of a fatty acid chain and a peptide chain with several amino acids, and they exhibit various interesting biological properties, including surface properties, emulsifying, foaming, antiviral, antitumoral, antimicrobial, and antimycoplasmal activities.1 These properties make lipopeptides of increasing interest in the food, pharmaceutical, cosmetic, and petroleum industries.2,3 Recently, these compounds have been identified and applied in several fermented foods. For example, lichenysin has been newly identified in varieties of Chinese liquor. It would act as a nonvolatile matrix component and efficiently suppress the volatility of off-flavor phenolic compounds.4,5 In addition, the producer, Bacillus amyloliquefaciens ssp. plantarum, exhibiting broad-spectrum antifungal and antibacterial properties, has been applied in food fermentation for biological control of pathogenic bacteria.6 Among the lipopeptide family, surfactin has been reputed as the most powerful and bestcharacterized biosurfactant. However, lichenysin, having the same properties as surfactin, outperforms surfactin in terms of certain efficacy.7 Therefore, it is important to reveal the biosynthetic mechanism of lichenysin in the food-making process. Several strains of Bacillus have been described as lichenysin producers, including Bacillus mojavensis JF-2,8 Bacillus licheniformis 86,9 B. licheniformis BAS50,10,11 B. licheniformis IM1307,12 and B. licheniformis ATCC10716.13 Medium composition plays an important role in the production of lichenysin, including carbon source, nitrogen source, and metal ions.3,14 However, most of the work has focused on fermentation optimization, and less work has revealed the © 2015 American Chemical Society
biosynthetic mechanism of lichenysin. The synthetase genes and their expression regulators comprise the only knowledge of the lichenysin biosynthetic process.15 Lichenysin is synthesized by a multienzyme peptide synthetase complex, containing three peptide synthetase genes lchAA, lchAB, and lchAC and the thioesterase/acyltransferase gene lchAD, which stimulates the initiation of this process.15,16 It has also been reported that transcription of the biosynthetic machinery of lichenysin is activated by the phosphorylated form of ComA,17 which was activated by the gene of the kinase ComP.18 In addition, the regulatory pathway was further investigated for surfactin, the homologue of lichenysin.1 ComX plays a role in the initial regulation of surfactin in Bacillus subtilis. It becomes active after being modified by ComQ and then controls srfA (surfactin synthetase operon) expression when it interacts with ComP and ComA. ComP donates a phosphate to the response regulator ComA, and then the resulting ComA-P tetramer activates transcription of srfA.1,18 Although the synthetase genes have been described, the substance supply and the related genes have not been revealed. The lack of understanding of the lichenysin synthetic mechanism has inhibited development of lichenysin production. Therefore, it is important to reveal the biochemistry and genetics of lichenysin synthesis, which is significant for fermented food production industries. In this work, three lichenysin producers were isolated from Chinese Maotai-flavor liquor-making process. Maotai-flavor Received: Revised: Accepted: Published: 888
July 31, 2014 December 23, 2014 January 6, 2015 January 6, 2015 DOI: 10.1021/jf5036806 J. Agric. Food Chem. 2015, 63, 888−893
Article
Journal of Agricultural and Food Chemistry
Figure 1. Time profile of lichenysin produced by different strains of B. licheniformis cultured in sorghum extract. (A) Biomass; (B) lichenysin concentration. valine, and L-glutamine (the component unit of lichenysin). The synthetic medium was prepared according to a previous report24 with minor modifications. It included 20 g/L glucose, 24 g/L (NH4)2HPO4, 14.2 g/L Na2HPO4·12H2O, 3.0 g/L KH2PO4, 0.5 g/L NaCl, 0.24 g/L MgSO4·7H2O, and 0.014 g/L CaCl2, with the addition of 10 mL of metal ions mixture. Metal ions mixture contained 1.6 g/L FeSO4· 7H2O, 0.1 g/L MnCl2·H2O, 0.17 g/L ZnCl2, 0.043 g/L CuCl2·H2O, 0.06 g/L CoCl2·H2O, and 0.06 g/L Na2MoO4·2H2O. Strains were inoculated in 50 mL of the synthetic medium and incubated at 37 °C with an agitation speed of 150 rpm. The strain was incubated for 32 h to determine lichenysin yield and incubated for 24 h to determine the gene expression levels by quantitative real-time polymerase chain reaction (qRT-PCR). RNA Extraction and cDNA Preparation. Total RNA was extracted from B. licheniformis CGMCC 3961, 3962, and 3963 and B. licheniformis ATCC 14580 cells cultured in sorghum extract and synthetic medium by use of RNeasy micro kits (Qiagen). DNase I was used to remove DNA contamination. The obtained RNA was determined by a nanobiosizing assay (Agilent) and then amplified and labeled by use of a one-color DNA labeling kit (NimbleGen) to obtain the cDNA sample. Gene Expression Arrays. The obtained cDNA samples from strains cultured in sorghum extract were used for gene expression array processing. The expression array chip used contained 385 000 probes representing 4196 genes (NimbleGen). Target cDNA was hybridized to microarrays, and the processed slides after hybridization were scanned as reported.22 The raw data of fluorescent intensity were extracted, normalized, background-corrected, and statistically analyzed as reported.22 Genes with an absolute change >2.0-fold were identified as significantly differentially expressed genes. Quantitative Real-Time Polymerase Chain Reaction. To verify the microarray results, two differentially expressed genes (dppA and dppC) were further analyzed by qRT-PCR. To investigate the effect of substrate on lichenysin production, strains were inoculated in synthetic medium with different substrates for 24 h. The obtained cDNA samples were also used for qRT-PCR. Relative transcript abundance was detected by SYBRGreen (Invitrogen) on the ABI 7900HT thermal cycler by use of gene-specific primers. The primer pairs were as follows: dppA (BLi01392), 5′-GGCTTATTTCGGGAGATGT-3′ (forward) and 5′-TTTCTTTGACGGCTGCTG-3′ (reverse); dppB (BLi01393), 5′-CACGACATTGACCTTTGTGA-3′ (forward) and 5′-ACGAAACGGGAAATCCTC-3′ (reverse); dppC (BLi01394), 5′ATCGTCCAGATGACCTTG-3′ (forward) and 5′CAGTCCGTCTCCAACAAC-3′ (reverse); dppD (BLi01395), 5′TTGAGGCAGTATCCGCAC-3′ (forward) and 5′-TCACGGCGATTCTGTCTG-3′ (reverse); dppE (BLi01396), 5′-CGAAAGAAACGGCTGGAG-3′ (forward) and 5′-CATTTGGCATCATCACGG-3′ (reverse); 16S rDNA (reference gene), 5′GTAACCTGCCTGTAAGACTG-3′ (forward) and 5′TGTCTCAGTCCCAGTGTG-3′ (reverse); ypqE (BLi02359), 5′TGATGGGTGACGGAATG-3′ (forward) and 5′-
liquor is a symbolic drink of China, just as whisky is for Scotland and brandy for France. It is produced by a unique spontaneous and solid-state fermentation process with sorghum as the main material.19 This process is rich in nutrition and accumulates a complex microbial community, including bacteria, yeast, and fungi. In this process, fungi produce amylase and glucoamylase to hydrolyze starch in cereal to oligosaccharide and also produce protease and peptidase to hydrolyze protein to oligopeptides and amino acids. These small molecular compounds are then consumed and transformed to ethanol and flavor compounds by yeast and bacteria.20 B. licheniformis is a dominant species in this process. Its biosynthetic activity of lichenysin might be important for its survival and predominance in this community, because the produced lichenysin might show inhibition of other strains. Since there are rich nutritional and complex microbial communities in the liquor-making process, it is important to reveal the mechanism of lichenysin biosynthesis by producers in the Maotai-flavor liquor-making process. Therefore, the comparative transcriptome of these producers and a nonproducer, B. licheniformisstrain ATCC 14580, was carried out in a simulated liquor-making process. It is important to reveal the mechanism of the lichenysin biosynthetic process, including the substance supply and transformation by these strains in this complex process. This work would supplement the knowledge of lichensysin biosynthesis and promote the lichenysin production industry.
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MATERIALS AND METHODS
Strains. Lichenysin producers were isolated from Chinese Maotaiflavor liquor-making process, on the basis of hemolysis zone on blood agar.21 Then the obtained strains were cultured in sorghum extract, and their lichenysin productivity was determined by ultra-performance liquid chromatography (UPLC). Strains B. licheniformis CGMCC 3961, 3962,22 and 396323 showed the highest lichenysin productivity. B. licheniformis ATCC 14580, obtaining from Shanghai Institute of Industrial Microbiology, did not originate from Maotai-liquor fermentation process. Culture Conditions. In order to simulate the fermentation process of liquor making, the fermentation medium was prepared with sorghum. Sorghum extract was prepared as previously reported.19 Strains were inoculated in 50 mL of sorghum extract medium and incubated at 37 °C for 40 h with an agitation speed of 150 rpm. To investigate the effect of different substrates on lichenysin production, strains were inoculated in synthetic medium with different substrates: 20 g/L glucose, amino acids, casein hydrolysate, respectively. The amino acids contained 5 g/L each L-leucine, L-aspartate, L-isoleucine, L889
DOI: 10.1021/jf5036806 J. Agric. Food Chem. 2015, 63, 888−893
Article
Journal of Agricultural and Food Chemistry Table 1. Different Metabolic Pathways Derived from Different Responsive Genes entry
term
differential gene count
upregulated gene count
P-value
bld02010 bld00562 bld00680 bld00281 bld00362 bld02060 bld00280 bld00380 bld00626 bld00051 bld02040
ABC transporters inositol phosphate metabolism methane metabolism geraniol degradation benzoate degradation phosphotransferase system (PTS) valine, leucine, and isoleucine degradation tryptophan metabolism naphthalene degradation fructose and mannose metabolism flagellar assembly
81 11 18 8 10 22 15 10 7 15 17
29 9 8 8 8 16 10 9 5 9 10
5.28 × 10−8 0.000128 0.012394 0.016741 0.017837 0.019500 0.023497 0.032104 0.034043 0.035269 0.042324
and Staphylococcus have been discovered in Chinese liquor.27 The antibacterial activity of lichenysin would not only be beneficial for the dominance of B. licheniformis in Maotai-flavor liquor making but could also prevent liquor fermentation from contamination by those harmful microbes, achieving a successful product. Transcriptome Analysis between Three Producers and Nonproducer. To gain further insight into the metabolic mechanism of overproduction of lichenysin by B. licheniformis CGMCC 3961, 3962 and 3963, the mean global differences of gene expression levels of these three producers were compared with those of nonproducer ATCC 14580. Of the 4196 genes on the microarray, a total of 1574 genes (37.5%), with a change of at least 2.0-fold, were identified as differentially expressed genes: 803 genes were upregulated and 771 genes were downregulated. Metabolic pathways obtained from the KEGG databases were characterized from the 1574 genes. As shown in Table 1 and Table S1 (Supporting Information), 11 metabolic pathways had P-value
