Feasibility and transcriptomic analysis of betalain production by biomembrane surface fermentation of Penicillium novae-zelandiae
© The Author(s) 2018
Received: 25 October 2017
Accepted: 19 December 2017
Published: 8 January 2018
In this study, a biomembrane surface fermentation was used to produce red pigments of Penicillium novae-zelandiae, and the significant improvement in pigment production by the addition of 0.4 g/L of tyrosine demonstrated that the red pigments probably contained betalain. Therefore, one red pigment was purified, and identified as 2-decarboxybetanin by high-resolution mass spectrometry (MS) and MS/MS analysis. Transcriptomic analysis revealed the differentially expressed genes and metabolic profile of P. novae-zelandiae in response to different cultivations and exhibited the complete biosynthetic pathway of 2-decarboxybetanin in P. novae-zelandiae. Betalains are important water-soluble nitrogen-containing food coloring agents, obtained mainly from beetroot by chemical extraction. This paper is the first report about the production of betalain by microbial fermentation, and results exhibit the possible use of fungal fermentation in future 2-decarboxybetanin production.
Pigments, including synthetic and natural pigments, are widely used in food, brewing, and cosmetic industries. Synthetic pigments were extensively used in the past because of their bright color, high stability, and low cost. However, most of them are harmful to human health in varying degrees, and many people interpret the content of chemical pigments as a contaminant. Therefore, synthetic pigments have been prohibited or strictly limited in many countries. Natural pigments hold advantages over synthetic pigments in terms of nutritious and pharmacological functions (Arad and Yaron 1992). With the continuous improvements in the quality of life, the demand for natural pigments is growing rapidly. In Japan, China, and the United States, the numbers of natural pigments allowed are 97, 48, and 30, respectively (Mapari et al. 2005, 2010).
Betalains are a group of water-soluble plant pigments that are used as food colorants. They are ammonium derivatives of betalamic acid and present in considerably high amounts in certain foodstuffs, including red beets, prickly pear fruits, and Amaranthus seeds (Strack et al. 2003). These pigments have no toxic effects on the human body; as important commercial color additives, no upper limit is necessary for the recommended daily intake of betalains (Delgado-Vargas et al. 2000). More interestingly, very few pharmacological applications of betalains exist. Recently, they have received attention because they show antiviral, antioxidant, and antimicrobial activities (Manohar et al. 2017; Sreekanth et al. 2007; Kanner et al. 2001). Beet roots currently represent the main commercial source of betalains. Thus, the high pigment content in beetroots is crucial. Recent efforts are centered around the betalain content in red beets through selective breeding, because the average pigment content in beets is approximately 130 mg/100 g fresh weight (Delgado-Vargas et al. 2000; Sekiguchi et al. 2013). Betalains can be extracted from plant roots with pure water, at room (or reduced) temperature. But the use of methanol or ethanol solutions in most cases is necessary to achieve complete extraction. Chemical extraction is characterized by high production cost and serious environmental pollution. Agricultural fields used for planting beets are diminishing with the decrease in arable land, and the cost associated with overcoming of environmental pollution is unsustainable for manufacturing enterprises because betalains are inexpensive products. Therefore, the search for a novel production method with low cost and high productivity has become an important issue for betalain production (Pavlov et al. 2005; Moreno et al. 2008).
In this study, biomembrane surface fermentation was used to produce the potential red pigments of Penicillium novae-zelandiae (Wang et al. 2011). A betalain, 2-decarboxybetanin, was separated from the pigments and then identified by high-resolution mass spectrometry. This work aimed to (i) investigate the feasibility of the production of betalain by fungal fermentation and (ii) reveal the biosynthetic pathway of 2-decarboxybetanin in P. novae-zelandiae by RNA sequencing.
Materials and methods
Microorganism and chemicals
Penicillium novae-zelandiae HSD07B (CCTCCM2012198) was obtained from the Henan Province Engineering Laboratory for Bioconversion Technology of Functional Microbes, Henan Normal University, Xinxiang, China. The fungus was stored on potato dextrose agar (PDA) plate at − 20 °C before use. All the chemicals used were of spectral or analytical grade unless otherwise stated.
Shake-flask cultivation and biomembrane surface cultivation
Effect of amino acids on red pigment production
The effects of 12 amino acids, namely, Glutamic acid (Glu), Aspartic acid (Asp), Arginine (Arg), Proline (Pro), Valine (Val), Isoleucine (Ile), Glycine (Gly), Alanine (Ala), Serine (Ser), Lysine (Lys), Histidine (His), and Tyrosine (Tyr), on pigment production were evaluated. Amino acids at a dosage of 0.5 g/L were added to the modified Czapek Dox liquid medium, and the fermentation process was the same as that of biomembrane surface cultivation described previously. Another Tyr dosage test was also conducted. In this test, we added 0, 0.2, 0.4, 0.6, 0.8, and 1.0 g/L of Tyr to 500 mL flasks containing 200 mL of modified Czapek Dox liquid medium. Biomembrane surface cultivation was conducted at 28 °C in a thermostat shaker. During the tests, CV and the concentration of red pigment were measured daily.
Identification of pigment component
The fermentation broth was filtered using filter paper (Grade 1:11 µm, Whatman, UK). The cell-free filtrate was mixed with ethanol (filtrate:ethanol = 1:1.5), and the mixture was centrifuged at 2600g for 10 min. The supernatant was dried in a rotary evaporator at 50 °C, and the crude pigment was mixed with 100 mL of petroleum to remove hydrophobic substances. The remaining red pigment was used as a sample for silica gel (200 mesh) column chromatography eluted with 1-butanol: ethanol: petroleum ether (2:2:6, V/V). A collected component of red pigment was analyzed by silica gel thin-layer chromatography (TLC) using 1-butanol: ethanol: water (3:5:2) as mobile phase. A high-resolution electrospray mass spectrometer (MicrOTOF-Q II, Bruker Daltonics Corporation, USA) was operated in negative ion mode for mass spectrometer analysis. The capillary voltage was set at 4500 V with an end plate offset potential of 500 V. Data were collected from 50 to 1000 m/z with an acquisition rate of 1 spectrum per second. The dry gas was set to 1.2 L/min at 120 °C with a nebulization gas pressure of 0.4 bar. In addition, HPLC-MS/MS analysis was conducted on an Esquire plus 3000 (Bruker Daltonics, Billerica, MA, USA) ion trap mass spectrometer with an electrospray interface (ESI) utilizing HPLC eluted with the mobile phase consisted of 25 mM NH4OAc as well as 25 mM NH4OH in water, and acetonitrile (90:10). The analysis was conducted in positive-ion mode and operated according to defined conditions: nitrogen gas temperature, 320 °C; drying gas flow rate, 7 L/min; capillary voltage, 4500 V; nebulizing pressure, 27 psi. Mass spectra were recorded using the full scan mode in the range of 200–800 Daltons.
Sample preparation, RNA extraction, and RNA sequencing
All samples, including the cultures from biomembrane surface cultivation at hour 36 (T1) and hour 96 (T2) and from shake cultivation at hour 36 (Ck1) and hour 96 (Ck2), were prepared. These samples were immediately frozen in liquid nitrogen and then stored at − 80 °C until RNA isolation. Total RNA was extracted using a Trizol reagent according to the manufacturer’s protocol (Invitrogen, China) and then treated with DNase to remove DNA contamination. The yield and purity of RNA sample were checked using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific, USA) at 260 and 280 nm. The integrity of all RNA samples was assessed by 1.0% agarose gel. The mRNA from total RNA was isolated and enriched using oligo (dT) magnetic beads (Illumina, CA, USA). Subsequently, mRNA was fragmented to short fragments to be used as templates for random hexamer-primed synthesis of first-strand cDNA by fragmentation buffer. Second-strand cDNA was synthesized using buffer, dNTPs, RNase H, and DNA polymerase I. A paired-end cDNA library was synthesized using a Genomic Sample Preparation Kit (Illumina, CA, USA) according to the manufacturer’s instructions. Short fragments were purified with a QIAQuick1 polymerase chain reaction (PCR) extraction kit (Qiagen, Germany) and eluted in 10 µL of elution buffer. An Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and ABI Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) were used to examine the quality and quantify the sample library (Firon et al. 2013). Finally, cDNA libraries were sequenced on an Illumina HiSeq™ 2500 (Novogene, Beijing, China).
Gene annotation and normalized expression levels
Raw reads were cleaned by removing adapter sequences, empty reads, and low quality sequences. For annotation analysis, unigenes were BLASTX-searched against five databases, namely, the National Center for Biotechnology Information (NCBI) nonredundant (NR) protein sequence database, the NCBI NR nucleotide sequence (NT) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology database, Swissprot, and PFAM database, using a cut-off E-value of 10−5. To eliminate the influence of different gene lengths and sequence discrepancies on expression calculations, gene expression levels based on read counts obtained by RSEM (version v1.2.15) were normalized using FPKM (fragments per kilo bases per million fragments) transformation (Li and Dewey 2011). The calculated gene expression levels were used for direct comparison among samples. Expression values were standardized across the dataset to enable the data from different genes to be combined.
Screening of differentially expressed genes (DEGs) and KEGG analysis
Using the R package DEGseq, DEGs were identified with a random sampling model on the basis of the read count for each gene at different developmental stages. False discovery rate ≤ 0.05 and absolute value of |log2Ratio| ≥ 1 were set as the threshold for significance of gene expression differences between adjacent samples. The KEGG database was used to assign and predict putative functions and pathways associated with the assembled sequences (Baba et al. 2012; Xie et al. 2011).
Quantitative real-time PCR
Primers of the selected genes and internal reference gene
Data availability and statistical analysis
The data sequenced in this study have been submitted into the NCBI Sequence Read Archive under the accession number of SRP114884. All experimental data in this work were presented as the mean ± standard error of the mean and evaluated using one-way ANOVA followed by the least significant difference test, with P < 0.01 and P < 0.05 (SPSS 16.0 for Windows).
Biomembrane surface cultivation and shake cultivation
Effect of amino acids on pigment production
Identification of a red pigment component
RNA sequencing datasets
Summary of RNA sequencing clean data
Analysis of DEGs
2-decarboxybetanin is a derivative of betanin, which is the main component of commercial betalains–beetroot pigments. This study is the first report about the production of betalain by the P. novae-zelandiae fermentation, although betalains also appear in the fruiting bodies of some higher fungi, including Amanita, Hygrocybe, and Hygrosporus (Strack et al. 1993). Using the 2-decarboxybetanin we purified as the standard substance, the yield of 2-decarboxybetanin produced by P. novae-zelandiae was evaluated. The result shows that the pigment content is 1.5 g/L, which accounts for approximately a quarter of total red pigments. The average pigment content of beets is approximately 1.3 g/kg fresh weight. Therefore, betalain productivity by microbial fermentation is higher than that by conventional method of extraction from beets because microbial fermentation is easier for industrial-scale production than beets planting. Thus, microbial fermentation for the pigment production has advantages over conventional chemical extraction in production cost and productivity (Stahmann et al. 2000). In addition, in consideration of the decrease in arable land, and cost of environmental pollution worldwide, it is feasible and potential to produce betalain by the microbial fermentation characterized by low cost and high productivity.
KEGG pathway analysis
Validation of the gene expression profile
In this work, the red-colored pigment, 2-decarboxybetanin, produced by the biomembrane surface fermentation of P. novae-zelandiae, was detected by high-resolution mass spectrometry, and transcriptomic analysis demonstrated the metabolic profile of P. novae-zelandiae in response to different cultivations and revealed the complete synthetic pathway of 2-decarboxybetanin in P. novae-zelandiae. These results suggest the possibility and feasibility of production of betalain by P. novae-zelandiae fermentation. This study is the first report about the production of betalains by microbial fermentation, and further work should focus on the improvement of 2-decarboxybetanin yield by optimization of fermentation factors or by metabolic regulation technology. In addition, the precise quantification of 2-decarboxybetanin should also be considered in the future considering that a small quantity of colorless compounds might exist in the 2-decarboxybetanin sample we purified.
WH: planning and designing of study. LY and ZK: experimentation. MY: data analysis. LP: manuscript drafting. All authors read and approved the final manuscript.
The authors are thankful to the technical support from Advanced Environmental Biotechnology Center, Nanyang Technological University, Singapore.
The authors declare that they have no competing interests.
Availability of data and materials
Consent for publication
All authors including Wang Hailei, Li Yi, Zhang Kun, Ma Yingqun, Li Ping agree to submit the work to AMB Express.
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.
This work was supported by the National Science Foundation of China (U1404301; U160411067), the Henan Province Science and Technology Program (172102110197; 162102210260), Program for Science&Technology Innovation Talents in Universities of Henan Province (18HASTIT039) and the Project for Youth Outstanding Teachers of Henan Province (2015GGJS-091).
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