Abstract
Previously, AoamyA, the alpha amylase-encoding gene from Aspergillus oryzae, was heterologously expressed in Monascus ruber CICC41233 to promote starch hydrolysis and increase the production of Monascus pigments. The target of this study is to screen the effective alpha-amylases from M. ruber for starch fast degradation and investigated for Monascus pigments production. The 13 types of predicted alpha-amylases in the M. ruber NRRL1597 genome were divided into four classes based on EC number and into five groups based on the glycoside hydrolase sub-family. The predicted alpha-amylases MrAMY1 (protein ID 440333) and MrAMY2 (protein ID 324551) showed the closest match with AOamyA by phylogenetic analysis. The genes encoding alpha-amylase, Mramy1and Mramy2, were cloned from M. ruber CICC41233. However, the gene sequence of Mramy1 from M. ruber CICC41233 differed from that of M.ruber NRRL1597 in the length of the intron sequence. Furthermore, the Mramy1-overexpressed strain M.ruber 440333-6A completely degraded the starch of rice grain in 2 d; in contrast, starch (40.32 mg/mL) remained when rice grain was incubated with the Mramy2-overexpressed strain, M. ruber 324551-D even after 2 d, while 45.43 mg/mL and 10.48 mg/mL starch remained after 2 d and 6 d, respectively, in wild type M. ruber CICC41233. Compared to that of M. ruber CICC41233, the total Monascus pigments and ethanol-soluble pigments in M.ruber 440333-6A increased by 71.69% and 119.33% after 6d, respectively; however, it decreased by 21.40%and 26.58% after 6d, respectively, in M. ruber 324551-D. This study demonstrated that alpha-amylase MrAMY1 was superior to MrAMY2, as it effectively degraded the starch of rice grain and enhanced Monascus pigments production.
Electronic supplementary material
The online version of this article (10.1007/s13205-019-2026-8) contains supplementary material, which is available to authorized users.
Keywords: Alpha-amylase, Monascus ruber, Monascus pigments, Starch degradation
Introduction
Natural pigments such as Monascus pigments (MPs), carotenoids, melanins, flavins, quinones, monascins, violacein, phycocyanin, and indigo are usually derived from microorganisms (Dufosséet al. 2005; Vendruscolo et al. 2016). The MPs manufactured by Monascus species have been used worldwide as natural food colorants for over 1000 years, especially in China (Patakova 2013; Chen et al. 2015), as they possess several advantages, such as ease of production from cheap substrates, high bioactive metabolite content, and good solubility in water and ethanol (Mostafa and Abbady 2014; Chen et al. 2015, 2017). Therefore, developing methods of increasing the production of MPs is important. Although improvement of MPs production can be affected by various factors, such as strains, fermentation substrate, nitrogen source, pH, dissolved oxygen, temperature, and light intensity (Feng et al. 2012; Vendruscolo et al. 2016), genetic engineering can best improve MPs production (Shao et al. 2014; Chen et al. 2015).
Rice grains, which contain abundant starch, are often used to produce MPs (Chen et al. 2015). We have previously reported that the alpha-amylase-encoding gene AoamyA of Aspergillus oryzae can be heterologously expressed in Monascus ruber CICC41233 (Long et al. 2018a). Alpha-amylase activity, starch degradation, and MPs production increased significantly in the transformant (Long et al. 2018a). However, the complete genome sequence of Monascus purpureus YY-1 showed that it harbors 13 putative alpha-amylase-encoding genes (Yang et al. 2015). The M. ruber NRRL1597 genome database (https://genome.jgi.doe.gov/Monru1/Monru1.home.html) also predicted it to contain13 types of alpha-amylases (protein ID P440333, P324551, P379161, P411620, P435885, P63242, P454978, P460054, P464710, P469192, P469571, P501041, and P472279) from the KOG functional classification for carbohydrate transport and metabolism. In this study, the aim was to screen the effective alpha-amylase from the above predicted proteins for starch degradation and evaluate the effect on MPs production. We introduced a valid method involving phylogenetic tree analysis and molecular experiments to screen an effective alpha-amylase. The alpha-amylase genes (Mramy1 and Mramy2) from Monascus ruber CICC41233 were cloned and overexpressed to evaluate the relationship between starch hydrolysis and MPs production. To the best of our knowledge, this is the first study on alpha-amylases from Monascus species for the production of MPs.
Materials and methods
Strains
M.ruber CICC41233 was purchased from the China Center of Industrial Culture Collection and grown on malt-peptone-starch (MPS) agar (10 g/L malt extract, 10 g/L peptone, 40 g/L soluble starch, and 2 g/L agar) medium (Long et al. 2018b). The strains Escherichia coli DH5a and Agrobacterium tumefaciens EHA105 were used for DNA manipulation.
Phylogenetic tree analyses
The phylogenetic tree was constructed based on the sequences of alpha-amylase AOamyA from A.oryzae (Long et al. 2018a) and the predicted 13 alpha-amylases from M. ruber NRRL1597 (Supplementary Sequence S1) using ClustalX and Mega 4 software.
Vector construction and creation of positive transformants
Mramy1and Mramy2 (encoding alpha-amylases) were amplified via polymerase chain reaction (PCR) from the cDNA and total DNA of M. ruber CICC41233 as the template. The primers 440333-Hind III-F and 440333-SacI-R were used to amplify Mramy1, and 324551-Hind III-F and 324551-SacI-R were used to amplify Mramy2 (Supplementary Table S1). Then, Mramy1 and Mramy2 were sequenced by BGI, analyzed using BLAST on NCBI database, and aligned with the sequence from M. ruber NRRL1597. The plasmid pNeo0380 (Long et al. 2018b) and the gene fragments Mramy1 and Mramy2 were digested with HindIII and SacI, respectively. Then, the digested gene fragments Mramy1 and Mramy2 were ligated to the digested plasmid pNeo0380 to construct the binary expression vector pNeo0380-440333 and pNeo0380-324551, respectively. The vectors pNeo0380-440333 and pNeo0380-324551 were transferred into M. ruber CICC41233 via A. tumefaciens EHA105.The positive strains were identified using PCR.
Analysis of Monascus pigments, enzyme activity, and starch concentration
Five million freshly harvested spores were inoculated into 50 mL medium for Monascus pigments fermentation (Long et al. 2018a). The medium component per liter was 90 g rice powder (78.5 g carbohydrate per 100 g rice, 7.8 g protein per 100 g rice, 0.6 g lipid per 100 g rice), 2 g NaNO3, 1 g KH2PO4, 2 g MgSO4·7H2O, and 0.2% acetate. The fermentation samples of 2 d and 6 d were analyzed. The water-soluble pigments, ethanol-soluble pigments, and biomass were determined using a published method (Long et al. 2018a, b).
The α-amylase (AMS) assay kit (C016-1-1, Nanjing Jiangcheng Bioengineering Institute, China) was used to assay alpha-amylase activity, and the starch concentration was determined using iodine solution (2.6 g L−1 I2, 5.0 g L−1 KI) as described previously (Long et al. 2018a).
Gene expression analysis
Quantitative reverse transcription-PCR was performed as described previously (Long et al. 2018a, b). The primer sequences of the control gene Actin and alpha-amylase genes Mramy1 and Mramy2, and the key genes pks and mppr1 of the MPs gene cluster are shown in Supplementary Table S1.
Results and discussion
Predicted information regarding alpha-amylases from the M.ruber NRRL1597 genome
M. ruber NRRL1597 was predicted to harbor 13 types of alpha-amylases from the KOG functional classification for carbohydrate transport and metabolism (https://genome.jgi.doe.gov/cgi-bin/kogBrowser?class=G&models=1&type=KOG&db=Monru1) (Table 1). Apart from the protein ID 435885, which belonged to KOG3625, the others belonged to KOG0471.They were divided into four classes, namely, EC:3.2.1.1(P440333, P324551, P472279), EC:3.2.1.20 (P379161, P411620, P63242, P454978, P460054, P501041), EC:2.4.1.183 (P464710, P469192, P469571), and EC:3.2.1.33 (P435885) based on the EC number. In addition, they were all part of the glycoside hydrolase family 13, but belonged to different sub-families, such as subf1(P440333, P324551), subf5(P472279), subf40(P379161, P411620, P63242, P454978, P460054, P501041), subf22(P464710, P469192, P469571), and subf25(P435885).
Table 1.
The 13 predicted alpha-amylases of M.ruber NRRL1597 from the KOG functional classification
| Protein ID | EC number | Best hit | CAZy database | Length of gene (bp) | Number of exons |
|---|---|---|---|---|---|
| 440333 | EC:3.2.1.1 |
gi|212,541,000|, alpha-amylase, putative, Penicillium marneffei ATCC 18,224 Identity: 65% |
GH13_1a | 1706 | 2 |
| 324551 | EC:3.2.1.1 |
gi|350,636,595|, alpha-amylase,Aspergillus niger ATCC 1015 Identity: 57% |
GH13_1 | 2145 | 8 |
| 472279 | EC:3.2.1.1 |
gi|317,140,381|, alpha-amylase,Aspergillus oryzae RIB40 Identity: 68% |
GH13_5 | 2125 | 8 |
| 379161 | EC:3.2.1.20 |
aor:AO090026000034, alpha-amylase, Aspergillus oryzae Identity: 72% |
GH13_40 | 1860 | 3 |
| 411620 | EC:3.2.1.20 |
afm:Afu3g07380,oligo-1,6-glucosidase, Aspergillus fumigatus Af293 Identity:79% |
GH13_40 | 1834 | 2 |
| 63242 | EC:3.2.1.20 |
gi|242,806,039|, maltase, Talaromyces stipitatus ATCC 10,500 Identity: 65% |
GH13_40 | 1907 | 1 |
| 454978 | EC:3.2.1.20 |
afm:Afu2g11620, oligo-1,6-glucosidase,Aspergillus fumigatus Af293 Identity: 79% |
GH13_40 | 2389 | 10 |
| 460054 | EC:3.2.1.20 |
gi|345,564,184|, hypothetical protein AOL_s00097g3, Arthrobotrys oligospora ATCC 24,927 Identity: 48% |
GH13_40 | 1934 | 3 |
| 501041 | EC:3.2.1.20 |
gi|119,481,197|, alpha-glucosidase/alpha-amylase, putative, Neosartorya fischeri NRRL 181 Identity: 76% |
GH13_40 | 2295 | 9 |
| 464710 | EC:2.4.1.183 |
gi|242,818,376, alpha-1,3-glucan synthase, putative, Talaromyces stipitatus ATCC 10,500 Identity: 57% |
GH13_22 | 7859 | 11 |
| 469192 | EC:2.4.1.183 |
afm:Afu2g11270, alpha-1,3-glucan synthase, Aspergillus fumigatus Af293 Identity: 71% |
GH13_22 | 7595 | 6 |
| 469571 | EC:2.4.1.183 |
aor:AO090010000106, glycogen synthase, Aspergillus oryzae Identity: 80% |
GH13_22 | 7428 | 4 |
| 435885 | EC:3.2.1.33 |
gi|119,497,357|, amylo-alpha-1,6-glucosidase, putative, Neosartoryafischeri NRRL 181 Identity:73% |
GH13_25 | 4761 | 3 |
aThe GH13_1 represented Glycoside Hydrolase Family 13 / Sub-family 1
Selection of alpha-amylase from M. ruber NRRL1597 based on the phylogenetic tree and cloning the target alpha-amylase gene from M. ruber CICC41233
Previous reports showed that heterologous expression of AoamyA in M. ruber CICC41233 accelerated starch hydrolysis and enhanced Monascus pigments production (Long et al. 2018a). This indicated that AOamyA from A.oryzae was an effective alpha-amylase associated with MPs production. We predicted that M. ruber NRRL1597 (Supplementary Sequence S1) contains 13 alpha amylases, which is consistent with that observed for M. purpureus (Yang et al. 2015). Phylogenetic analysis showed that the protein P440333 (named as MrAMY1) was closest to AOamyA (79% confidence level) (Fig. 1a). P440333 and AOamyA were closest to the protein P324551 (named as MrAMY2) (Fig. 1a). The results of phylogenetic tree analysis were consistent with the classification of predicted amylase based on glycoside hydrolase sub-family (Table 1).
Fig. 1.
Phylogenetic analysis of the amylase from M.ruber NRRL1597 (a) and comparative analysis of target alpha-amylase gene sequence from M.ruber NRRL1597 and M.ruber CICC41233(b). (a The amylase AOamyA was from A.oryzae. The 13 amylases were from M.ruber NRRL1597. The arrow indicates the target protein in this study. b The length of the intron of the Mramy1 gene of M. ruber CICC41233 differed from that of M.ruber NRRL1597)
To further investigate the effect of the protein on starch hydrolysis and MPs production, full length Mramy1 (encoding protein P440333) and Mramy2 (encoding protein P324551) were first amplified from M. ruber CICC41233 using PCR with DNA and cDNA as templates, respectively, followed by sequencing. Mramy1 from M. ruber CICC41233 possessed an intron from 994 bp to1050 bp (Fig. 1b, Supplementary Figure S1), while the intron of M. ruber NRRL1597 Mramy1 was from 994 bp to1037 bp (Fig. 1b, Supplementary Figure S1). The sequence of Mramy2 from M. ruber CICC41233 was similar to the sequence from M. ruber NRRL1597. The deduced 548 amino acid sequence of MrAMY1 from M. ruber CICC41233 exhibited the closest match to the alpha-amylase (69% identity) of Rasamsonia emersonii CBS 393.64 (GenBank Accession No. XP013323067.1), and also matched with the sequence of taka-amylase A (69% identity) of A.oryzae (GenBank accession no. AAA32708.1). The deduced 571 amino acid sequence of MrAMY2 from M. ruber CICC41233 exhibited the closest match to the alpha-amylase (64% identity) of Rasamsonia emersonii CBS 393.64 (GenBank accession no. XP013325801.1), and also matched with the sequence of taka-amylase A (48% identity) of A.oryzae (GenBank Accession No. AAA32708.1).
Alpha-amylase MrAMY1 was superior to MrAMY2 with respect to starch degradation and Monascus pigments production
Mramy1 and Mramy2 were over-expressed in wild type strain M. ruber CICC41233, respectively. The transformants named M. ruber 440333-6A (Mramy1 overexpression) and M. ruber324551-D (Mramy2 overexpression) were verified using PCR and MPs fermentation analysis (data not shown).
M. ruber CICC41233, M. ruber 440333-6A, and M. ruber 324551-D were simultaneously cultivated on fermentation medium for MPs production. The 2 d and 6 d samples were further analyzed. The alpha-amylase activity was detected using the alpha-amylase assay kit and the absorbance values are shown in Table 2. The alpha-amylase activities of M. ruber 440333-6A were 63.98 and 64.01 U/dL after 2 d and 6 d, respectively (Supplementary Figure S2). However, the alpha-amylase activities of M. ruber CICC41233 and M. ruber 324551-D could not be accurately measure owing to the presence of large starch residues (Supplementary Figure S3). After 2 d and 6 d, 45.43 mg/mL and 10.48 mg/mL starch was left in M. ruber CICC41233, respectively (Fig. 2a). In contrast, 0.06 mg/mL starch remained after 2 d and no starch was detected after 6 d in M. ruber 440333-6A (Fig. 2a). However,40.32 mg/mL and 6.73 mg/mL starch remained in M. ruber 324551-D after 2 d and 6 d, respectively (Fig. 2a), indicating that the alpha-amylase MrAMY1 was superior to MrAMY2 in starch degradation.
Table 2.
The absorbance values of α-amylase at 660 nm
| Samples | 48 h | 144 h |
|---|---|---|
| Control | 0.53 | 0.498 |
| M. ruber CICC41233 | 5.33 ± 0.34 | 1.29 ± 0.11 |
| M. ruber 440333-6A | 0.09 ± 0.01 | 0.03 ± 0.00 |
| M. ruber 324551-D | 4.73 ± 0.01 | 0.829 ± 0.22 |
Fig. 2.
Analysis of starch content (a), Monascus pigments production (b), and gene expression fold (c) for M. ruber CICC41233, M. ruber 440333-6A, and M. ruber324551-D. (The asterisks indicate the differences between M. ruber CICC41233, and M. ruber 440333-6A and M. ruber 324551-D in each group. Error bars represent SD from duplicate replicates. The differences among different treatments were analyzed using one-way ANOVA. *p < 0.05, **p < 0.01)
Monascus pigments production did not differ significantly among the three strains after 2d (Fig. 2b). However the total MPs and ethanol soluble pigments for M. ruber CICC41233 were 42.34 U and 31.30 U after 6 d, respectively, whereas they were 72.69 U and 68.65 U after 6 d, respectively, for M. ruber 440333-6A and 33.28 U and 22.98 U for M. ruber 324551-D, respectively, after 6 d. Compared to the wild type strain CICC41233, the total MPs and ethanol soluble pigments in strain 440333-6A increased significantly by71.69% and 119.33%, respectively, after 6d (Fig. 2b), while the total MPs and ethanol soluble pigments in strain M.ruber 324551-D decreased by 21.40% and 26.58%, respectively, after 6d (Fig. 2b). This was consistent with the results of our previous study (Long et al. 2018a). Surprisingly, the proportion of ethanol-soluble pigments in total pigments increased from 73.93% in the parent strain CICC41233 to 94.44% in M. ruber 440333-6A. This showed that ethanol-soluble pigments account for a large proportion of total pigments. This phenomenon was common in Monascus spp. (Kang et al. 2014; Shi et al. 2015; Chen et al. 2017). It also showed that rapid degradation of starch can enhance MPs production and increase the yield of alcohol-soluble pigments. However, due to carbon catabolite repression (CCR), large amounts of glucose were obtained from starch because of increase in amylase activity, which reduced pigments production in Penicillium sp. NIOM-02 (Puttananjaiah and Dhale, 2012). In contrast, the transcription factor creA that negatively regulates CCR was down-regulated in M. purpureus YY-1 for MPs fermentation (Yang et al. 2015).
At the same time, Mramy1 of M. ruber 440333-6A was up-regulated by 4.03- and 1.88-fold compared to that of the wild type strain after 2 d and 6 d, respectively (Fig. 2c). This showed that it mainly contributed to the alpha-amylase activity and starch degradation. Similar result was obtained in our previous report where AoamyA was expressed heterologously in M. ruber (Long et al. 2018a). Although Mramy2 of M. ruber 324551-D was up-regulated by 43.84- and 90.53-fold compared to that of the wild type strain after 2 d and 6 d, respectively (Fig. 2c), M. ruber 324551-D degraded starch only slightly (Table 2, Supplementary Figure S3).
Compared to the wild type strain CICC41233, the expression levels of pks (encoding polyketide synthase) and mppr1 (encoding positive regulatory factor in the MPs biosynthetic gene cluster) in M. ruber 440333-6A increased by 4.95- and 1.42 fold, respectively, after 2 d, and by 16.81- and 1.46 fold, respectively, after 6 d (Fig. 2c). The expression levels of pks and mppr1 in M. ruber 324551-D decreased by 75.03% and 92.54%, respectively, after 2 d; pks expression increased by 38.12-fold and mppr1 expression decreased by 64.46% after 6 d (Fig. 2c). The pks and mppr1 deletions (Supplementary Figure S4) resulted incomplete loss in the ability to produce pigments (Balakrishnan et al. 2013; Xie et al. 2013; Chen et al. 2015).
Therefore, Mramy1 was overexpressed in M. ruber CICC41233, which enhanced amylase activity and accelerated starch hydrolysis. This implied that the supply of metabolic flux contributed to MPs production.
Conclusions
In this study, 13 types of predicted alpha-amylases in the Monascus ruber NRRL1597 genome were divided into four classes based on the EC number and five groups based on the glycoside hydrolase sub-family. Phylogenetic analyses showed that the proteins MrAMY1 (P440333) and MrAMY2 (P324551) among these 13 types of alpha-amylases of M.ruber NRRL1597 were closest to AOamyA. Data showed that the alpha-amylase MrAMY1of M. ruber CICC41233 was superior to MrAMY2 in terms of starch degradation and Monascus pigments production.
Electronic supplementary material
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Acknowledgements
This work was funded by the National Natural Science Foundation of China (grant nos. 31860436, 31171731, 31460447), Natural Science Foundation of Jiangxi Province (grant nos. 20161BAB214177, 20181BAB204001), Science and Technology Research project of Jiangxi Provincial Education Department (grant no. GJJ170676), and Youth Top-notch Talent project of Jiangxi Science and Technology Normal University (grant no. 2018QNBJRC004).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
Footnotes
Chuannan Long and Jingjing Cui contributed equally.
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