MptriA, an Acetyltransferase Gene Involved in Pigment

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Bioactive Constituents, Metabolites, and Functions

MptriA, an acetyltransferase gene involved in pigment biosynthesis in M. purpureus YY-1 Bin Liang, Xin-jun Du, Ping Li, Chanchan Sun, and Shuo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00661 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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Journal of Agricultural and Food Chemistry

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Title:

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MptriA, an acetyltransferase gene involved in pigment biosynthesis in M.

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purpureus YY-1

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Authors:

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Bin Lianga, Xinjun Dua, Ping Lia, Chanchan Suna,Shuo Wang a, *

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Affiliation:

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a

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Technology), Ministry of Education, Tianjin 300457, China

Key Laboratory of Food Nutrition and Safety (Tianjin University of Science &

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* Corresponding Author:

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Shuo Wang

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Key Laboratory of Food Nutrition and Safety (Tianjin University of Science &

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Technology), Ministry of Education, Tianjin 300457, China

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Tel: 86-22-60912484

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Fax: 86-22-60912484

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Monascus pigments (Mps) have been used as food colorants for several centuries in

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Asian countries. MptriA is a putative acetyltransferase gene involved in the MPs biosynthesis.

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In order to analyze the function of MptriA, an MptriA disruption strain (∆MptriA) and a

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complementation strain (∆MptriA::MptriA) were successfully obtained In addition to the loss

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of color, the disruption of MptriA had little effect on the phenotypes during growth on four

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different medium. The ∆MptriA strain showed decreased pigment and citrinin production

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during the liquid-fermentation process. Transcriptional analysis showed that the expression of

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several genes involved in the synthesis of pigments and citrinin was down-regulated in

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∆MptriA. These results demonstrated that the role of MptriA was to transfer an acyl group to

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the pyranoquinone structure of the polyketide chromophore during Monascus pigment

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biosynthesis and to influence the citrinin biosynthesis pathway. This study contributes to the

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exploration of pigment biosynthesis in M. purpureus.

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KEYWORDS: M. purpureus; MptriA gene; Pigments; Disruption; Complementation

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INTRODUCTION

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As significant traditional edible fungi, Monascus species have been used in food,

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medicine and industry for more than one thousand years, and more than one billion people

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consume Monascus-fermented products as part of their daily diet1, 2, 3. One of the most famous

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Monascus-fermented products, red fermented rice, has been used extensively as a natural food

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colorant, folk medicine, fermentation starter in East and Southeast Asia4, 5, 6, 7. Meanwhile,

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previous reports have shown that Monascus species can produce various natural and

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functional secondary metabolites, such as Monascus pigments (Mps), monacolin K, and

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γ-aminobutyric acid (GABA)8-10. Therefore, the utility of Monascus species has attracted the

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attention of many research teams.

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Among secondary metabolites of Monascus spp., pigments used as food additives for

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several centuries in Asian countries4, have been supposed as polyketides11, 12. Pigments

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produced by Monascus species can be divided into three major groups: red pigments

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(monascorubramine

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rubropunctatin), and yellow pigments (monascin and ankaflavin)13. To date, at least 90 kinds

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of Mps have been identified1, 14, and many showed multifarious biological activities such as

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preventing hypertension15, and lowering cholesterol16, hypolipidemic effects17, and

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anti-obesity18, 19, anti-tumor20, and anti-cancer activities21. Therefore, it is important to select a

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high pigment-producing strain using molecular biological methods and optimize the

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fermentation conditions to improve pigment production.

and

rubropunctamine),

orange

pigments

(monascorubrin

and

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Analysis of the genomes of M. pilosus, M. purpureus, and M. ruber via bioinformatics

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and RT-PCR showed that the Mps gene cluster contains a minimum of 16 genes, namely,

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MpigA (nonreducing polyketide synthase, NR-PKS), MpigB (transcription factor), MpigC

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(dehydrogenase), MpigD (3-O-transacetylase), MpigE (dehydrogenase), MpigF (monoamine

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oxidase), MpigG (oxidoreductase), MpigH (dehydrogenase), MpigI (transcription factor),

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MpigJ (fatty acid synthase, α subunit), MpigK (fatty acid synthase, β subunit), MpigL

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(ankyrin), MpigM (P450-monooxygenase), MpigN/O (monooxygenase), MpigP (unknown

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function), and MpigQ (transporter)3,

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themselves to studying the Mps biosynthesis pathway, several steps and the identities of

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related enzymes remain unclear or controversial1, 3. Mps biosynthesis is believed to follow a

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polyketide pathway, in which the PKS genes have been shown to be extremely important to

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the biosynthetic pathways of Mps, owning to targeted inactivation of MpPKS5 in M.

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purpureus or pksPT in M. ruber gave rise to loss of pigment24, 25. In addition to PKS, several

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genes involved in pigment biosynthesis have been investigated. Xie, et al.26 identified a

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pigment biosynthesis regulatory gene (pigR) in M. ruber M7, which upregulated pigment

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production. Targeted deletion of mrflbA, Mgb1 and Mgg1 resulted in phenotypic alterations

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such as decreased vegetative growth and asexual sporulation and altered citrinin and pigment

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production27, 28. Liu, et al.29 obtained an MpigE (as well as mppC in M. purpureus) gene

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deletion strain (∆MpigE), which yielded only four kinds of yellow pigment and very few red

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pigments but had no influence on citrinin. Liu, Zhou, Yi and Zhao23 reported that a mutant d

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in which an approximately 30-kb region of the pigment gene cluster from M. ruber M7 was

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deleted could induce the accumulation of high levels of M7PKS-1, which has been previously

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shown to be an initial intermediate of Mps. Balakrishnan, et al.30 discovered a reductase gene,

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mppE, that controls the biosynthesis of the yellow pigments, ankaflavin and monascin in the

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. Even though many researchers have devoted

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azaphilone polyketide pathway. Although many scientists3, 25, 31, 32 have contributed to the

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prediction of parts of the synthetic pathway of Mps, the identities of related genes involved in

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pigment biosynthesis remain unclear or controversial, which limits the practical industrial

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application of Monascus.

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In our previous studies3, transcriptional differences in M. purpureus YY-1 grown in

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different medium on the eighth day of growth indicated that MptriA is upregulated when M.

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purpureus is grown in rice medium (high-yield pigment states). Therefore, we predicted that

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the MptriA gene plays a very significant role in the production of pigments. Previous reports

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have shown that MptriA homologs are found in two relevant azaphilone biosynthetic gene

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clusters: azaD and cazE, which are involved in the biosynthesis of azanigerones and

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chaetoviridin, respectively33, 34. In this study, we constructed a putative acetyltransferase

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MptriA gene-deletion mutant of M. purpureus YY-1 and its revertant strain to investigate the

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role of MptriA, and the results revealed that ∆MptriA caused little phenotypic alterations in

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addition to colors and played a vital role in the production of some secondary metabolites,

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such as pigments and citrinin. This work will guide further exploration of the function of

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MptriA in the biosynthetic pathways of pigments in M. purpureus.

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MATERIALS AND METHODS

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Fungal strains, culture medium, and growth conditions. M. purpureus YY-1 obtained

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from Gutian Shenghua Monascus Ltd. of Ningde City (Fujian Province, China) was used for

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the generation of the ∆MptriA strain3. The ∆MptriA strain was used to generate the

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∆MptriA::MptriA strain. For phenotypic characterization, four kinds of medium were used,

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namely, potato dextrose agar medium (PDA), malt extract agar medium (MA), Czapek yeast

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extract agar medium (CYA), and glycerol nitrate agar medium (25%) (G25N)35. For

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sporulation, M. purpureus YY-1 was grown on COM medium (30 g of glucose, 3 g of peptone,

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0.5 g of KH2PO4, 0.5 g of MgSO4 per liter, pH 5.5-6.0). For total DNA extraction and to

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screen the constructed strains, MA medium supplemented with the appropriate antibiotic was

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used. All strains were maintained on MA slants at 28°C. Minimal medium (MM), induction

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medium

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tumefaciens-mediated transformation (ATMT)36. Luria-Bertani medium (LB), supplemented

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with antibiotic when necessary, was used to cultivate Escherichia coli for propagating

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plasmids. M. purpureus YY-1 and its derivatives and A. tumefaciens were grown at 28°C.

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Fungal spores and A. tumefaciens were co-cultured at 24°C for 3 days. E. coli DH5α was

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grown at 37°C for routine cloning. All strains and plasmids used in this study are listed in

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Table 1.

(IM)

and

co-cultivation

medium

(CM)

were

used

for

Agrobacterium

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DNA extraction. Fungal genomic DNA was isolated from mycelia grown on cellophane

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membranes covering MA plates using the cetyltrimethylammonium bromide (CTAB) method

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described by Shao, et al.37.

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Cloning and analysis of the MptriA gene. A pair of primers, MptriA-F/MptriA-R

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(Table 2), was designed to amplify the MptriA gene. PCR was carried out to amplify the

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MptriA gene from the genome of M. purpureus YY-13, and the protocol was as follows: initial

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denaturation at 94°C for 2 min; 30 amplification cycles of 98°C for 10 s, 55°C for 30 s, and

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68°C for 2.5 min; and a final extension step at 72°C for 10 min; a TProfessional thermal

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cycler (Biometra, Germany) was used for the PCR. The amino acid sequence encoded by

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MptriA

was

predicted

using

SoftBerry's

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FGENESH

program

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(http://linux1.softberry.com/berry.phtml), and the MptriA functional regions were analyzed

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using the Pfam 30.0 program (http://pfam.xfam.org/). The homology of the deduced amino

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acid sequence was analyzed using the BLASTP program on the NCBI web site

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(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

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∆MptriA strain construction. To construct the MptriA disruption mutant, the 5′ and 3′

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flanking regions (2355 bp and 2297 bp, respectively) of the MptriA gene were amplified with

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the primer pairs triA5-F/triA5-R and triA3-F/triA3-R using KOD-FX DNA polymerase

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(Toyobo, Japan) (Table 2). The PCR products of the 5’ and 3’ flanking regions were purified

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and cloned into the pEASY-Blunt vector (Transgen, China) to generate pEBTL and pEBTR,

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respectively. Successful cloning of the inserts into the resulting plasmids was verified by

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sequencing. Then, pEBTL was digested with KpnI and ApaI and ligated into the

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corresponding sites of pAg1-H3 (a vector containing the hygromycin phosphotransferase

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gene hph) to generate pAgHL. Then, both pEBTR and pAgHL were digested with AscI and

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SbfI and ligated with T4 DNA ligase to generate the plasmid pAgHLR. A 2-kb SbfI-digested

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DNA fragment containing the neomycin phosphotransferase resistance gene (neo) from the

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plasmid pAgHN was inserted into the corresponding sites of pAgHLR, yielding pAgHNLR.

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The plasmid pAgHNLR was transformed into A. tumefaciens AGL-1 via ATMT as described

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previously38 with the exception that cellophane was used instead of nitrocellulose membrane

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during the co-culture phase. The A. tumefaciens AGL-1 clones containing pAgHNLR were

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incubated for transformation with M. purpureus YY-1 to yield the fungal transformants. All

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the fungal transformants were selected on MA plates supplemented with 200 µg/mL

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hygromycin B and 500 µg/mL cefotaxime. Hygromycin-resistant and neomycin-sensitive

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strains were selected, and ∆MptriA was confirmed by PCR analysis using the internal primers

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YtriA1-F/YtriA1-R, external the outer primers YtriA2-F/YtriA2-R and cross-validation

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primers YtriA3-F/YtriA3-R, and YtriA3-F/YtriA3-R (Table 2).

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Complementation of ∆MptriA with MptriA of M. purpureus YY-1. To further verify

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whether all the differences exhibited by the ∆MptriA strain were caused by the disruption of

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MptriA, this gene was complemented. For complementation, the entire MptriA gene along

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with a 760-bp upstream region containing the putative promoter region of the gene and a

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599-bp downstream region was amplified from wild-type M. purpureus YY-1 with the

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primers pair triA-F/triA-R and inserted into pEASY-Blunt to generate pEBtriA (Table 2).

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Successful cloning of the insert into the resulting plasmid was verified by sequencing. Then,

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the plasmids pEBtriA and pAgHN were digested with SacI and KpnI, and the 2727-bp DNA

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fragment containing the intact MptriA was inserted into the corresponding sites of pAgHN to

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generate pAgHNtriA. Finally, the plasmid pAgHNtriA was transformed into A. tumefaciens

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AGL-1; and then the A. tumefaciens AGL-1 clones containing pAgHNtriA were incubated for

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transformation with the ∆MptriA strain by ATMT, as described previously, to yield the

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MptriA-complementation strain (∆MptriA::MptriA) by ATMT as described previously38.

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Transformants were selected on MA plates supplemented with 20 µg/mL neomycin and 500

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µg/mL cefotaxime at 28ºC. Neomycin-resistant strains were selected. The complementation

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was confirmed by PCR amplification with the primer pairs YtriA1-F/YtriA1-R and

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Neo-F/Neo-R (Table 2).

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Southern hybridization analysis. To further verify the homologous recombination

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events, Southern hybridization analysis was conducted. For Southern blot assays, the DIG

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High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany) was used

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according to the manufacturer’s protocol. The DNA (20 µg) of the M. purpureus YY-1,

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putative ∆MptriA and ∆MptriA::MptriA strains were digested with XhoI. Probe 1 and probe 2

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were amplified via PCR with the primer pairs ProtriA-F/ProtriA-R and Prohph-F/Prohph-R

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(Table 2), respectively. Probe 1 and probe 2 were used to verify the MptriA disruptant, and the

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∆MptriA::MptriA strain was confirmed with probe 1.

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RNA isolation and complementary DNA preparation. Total RNA of M. purpureus

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YY-1, ∆MptriA, and ∆MptriA::MptriA was isolated from mycelia after 48 h of cultivation

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using the RNeasy® Plant Mini Kit (QIAGEN, Germany) according to the manufacturer’s

173

protocol. RNA concentration was determined by measuring the absorbance at 260 and 280 nm

174

(A260/A280), and RNA integrity was verified by visualization on 1% agarose gels. RNA

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samples were stored at −80°C. For reverse transcription, total RNA (390 ng) was added to a

176

20 µL mixture derived from the PrimeScript™ RT Reagent Kit (Takara, Japan), and the

177

reaction conditions followed the manufacturer’s protocol.

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Quantitative real-time PCR analysis. The changes in mRNA levels obtained by

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RNA-seq were further validated by quantitative real-time PCR (qRT-PCR) of the MptriA gene.

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Each reaction (20 µL) contained 10 µL of SYBR Premix Ex Taq II, 0.8 µL of 10 µM forward

181

primer, 0.8 µL of 10 µM reverse primer, 0.4 µL of ROX Reference Dye II (Takara, Japan), 2

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µL of template cDNA, and 6 µL of ddH2O. All real-time PCRs were performed using the

183

Mastercycler ep realplex system (Eppendorf, Germany) with the following steps (two-step

184

PCR amplification, standard procedure): 30 s at 95°C, 40 cycles of 5 s at 95°C, and 34 s at

185

60°C. Sample melting curves were assessed to evaluate the specificity of the amplification.

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186

GAPDH was used as the reference gene29. The primers used in this part are listed in Table 2.

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Samples were analyzed in triplicate, and the experiments were repeated at least three times.

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MP and Citrinin analysis. Three kinds of COM medium (100 mL) with appropriate

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antibiotics were fermented by the wild-type strain M. purpureus YY-1, ∆MptriA or

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∆MptriA::MptriA. After agitation with 170 rpm for 40 h at 30°C, 20 mL of the fermentation

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mixtures of the above three strains were inoculated into 200 mL of RM medium (20 g of rice

192

powder, 20 g of glucose, 20 g of peptone, 2 g of NaNO3, 1.5 g of KH2PO4, 1 g of MgSO4 per

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liter) with the appropriate antibiotics. The fermentation was carried out at 28ºC, and samples

194

were taken every other day from the 2nd day to the 18th day of fermentation to measure MP

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and citrinin production. In addition, samples were harvested from 2 d to 8 d for RNA isolation

196

and transcriptional analysis by real-time PCR.

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MP production was detected by following the method described by the Chinese

198

standards (GB 1816.15-2015) with slight modification. The supernatant was removed by

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centrifugation from the fermentation broth, and the fungal mycelia were washed twice with

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sterile water and then dried at 60°C in a hot air oven until a constant weight was attained.

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Dried mycelia (0.02 g) were treated with 10 mL of 70% ethanol and heated at 60°C in water

202

for 1 h. After filtration, the absorbance values of the red, orange, and yellow pigments were

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determined at the specific wavelengths 505 nm, 465 nm and 410 nm, respectively, using a 50

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Bio UV-Visible spectrophotometer (Cary Varian, USA); 70% ethanol was used as a negative

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control. Pigment yield was expressed as absorbance at the λmax of the pigment per g of dry M.

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purpureus YY-1 mycelia39.

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To detect citrinin production, HPLC was performed following the method described by

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the Chinese standards (GB/T 5009.222-2008) with slight modification. After filtration and

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drying as described above, 0.01 g of dried mycelia was extracted with 1 mL of

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acetate/formic acid (TEF, 7:3:1 by volume) buffer. The prepared samples were filtered

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through a 0.2-µm filter and analyzed by HPLC using a reverse-phase C18 column (5 µm, 4.6

212

mm×250 mm). The HPLC parameters were as follows: mobile phase, 75% (v/v)

213

acetonitrile/25% (v/v) water (pH 2.5, adjusted by orthophosphoric acid); column temperature,

214

28°C; flow rate, 1.0 mL/min. The elution was monitored using a fluorescence detector at an

215

emission wavelength of 500 nm and an excitation wavelength of 331 nm. A citrinin standard

216

(Sigma, USA) was used to verify the HPLC analysis.

toluene/ethyl

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Transcriptome sequencing and analysis. Vegetative hyphae of M.purpureus YY-1 and

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∆MptriA were harvested from 8-d liquid RM cultures. There were three biological replicates

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for each strain. Transcriptome sequencing and analysis were performed by Novogene

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Bioinformatics Technology Co., Ltd. (Beijing, China). Briefly, Total RNA was extracted using

221

TRIzol reagent according to the manufacturer’s protocol. RNA purity was checked using a

222

NanoPhotometer® spectrophotometer (Implen, USA). RNA concentration and integrity were

223

measured using the Qubit® RNA Assay Kit with a Qubit® 2.0 Fluorometer (Life Technologies,

224

USA) and the RNA Nano 6000 Assay Kit with a Bioanalyzer 2100 system (Agilent

225

Technologies, USA). Then, mRNA was purified from total RNA using poly-T oligo-attached

226

magnetic beads, and fragmentation was carried out using NEBNext First Strand Synthesis

227

Reaction Buffer. The obtained mRNA fragments were used as templates to synthesize

228

first-strand cDNA with random hexamer primers, and then, the second-strand cDNA was

229

synthesized using DNA polymerase I and RNase H. After adenylation of the 3’ ends of the

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DNA fragments, NEBNext Adaptor was ligated to prepare the samples for hybridization.

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cDNA that were 150-200 bp in length were preferentially size-selected and purified with the

232

AMPure XP system (Beckman Coulter, USA). After PCR amplification, the library

233

preparations were sequenced on an Illumina HiseqTM 2000 sequencer (Illumina, USA), and

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125 bp/150-bp paired-end reads were generated. The index of the reference genome was built

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using Bowtie v2.2.3, and paired-end clean reads were aligned to the reference genome using

236

TopHat v2.0.12. Gene expression was calculated using the FPKM (expected fragments per kb

237

of transcript per million mapped reads) method with HTSeq v0.6.1, and differentially

238

expressed genes were selected based on P-value

MptriA, an Acetyltransferase Gene Involved in Pigment

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