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Overexpression of Monacolin K Biosynthesis Genes in the Monascus purpureus Azaphilone Polyketide Pathway Chan Zhang, Jian Liang, Anan Zhang, Shuai Hao, Han Zhang, Qianqian Zhu, Baoguo Sun, and Chengtao Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05524 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019
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Journal of Agricultural and Food Chemistry
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Overexpression of Monacolin K Biosynthesis Genes in the Monascus purpureus
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Azaphilone Polyketide Pathway
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Chan Zhanga,b,∗ , Jian Lianga, Anan Zhanga, Shuai Haoa,b, Han Zhanga, Qianqian Zhua, Baoguo
4
Suna,b, and Chengtao Wanga,b,∗
5
a
6
& Business University (BTBU),Beijing 100048,China.
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b
8
Business University (BTBU), Beijing 100048, China.
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∗ Corresponding author:
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology
Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology &
10
Chan Zhang & Chengtao Wang: Beijing Advanced Innovation Center for Food Nutrition and
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Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing
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Technology & Business University, No. 11 Fucheng Road, Haidian District, Beijing, 100048,
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China.
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E-mail address:
[email protected] (Chan Zhang)
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[email protected] (Chengtao Wang).
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ABSTRACT
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Monascus purpureus is one of the important food and drug microbial resources
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through the production of a variety of secondary metabolites, including monacolin K,
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a well-recognized cholesterol-lowering agent. However, the high production costs and
21
naturally low contents of monacolin K have restricted its large-scale production. Thus,
22
in this study we sought to improve the production of monacolin K in M. purpureus
23
through overexpression of four genes (mokC, mokD, mokE, and mokI). Four
24
overexpression
25
conversion, which resulted in a 234.3%, 220.8%, 89.5%, and 10% increase in the yield
26
of monacolin K, respectively. The overexpression strains showed clear changes to the
27
mycelium surface with obvious folds and the spores with depressions, whereas the
28
pBC5 mycelium had a more full structure with a flatter surface. Further investigation
29
of these strains can provide the theoretical basis and technical support for the
30
development of functional Monascus varieties.
strains were successfully constructed by protoplast electric shock
31 32 33
KEYWORDS: Monacolin K; Gene overexpression; Monascus purpureus; mokC;
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mokD; mokE; mokI
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INTRODUCTION
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Monascus spp. are one of the medicinal and edible filamentous fungi in East
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Asian countries1, have long played a significant role in local life and culture, such as
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medicine, wine, fermented bean curd and food coloring industries, and have received
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attention worldwide owing to their diverse products, including abundant beneficial
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metabolites2. Monascus species produce various secondary metabolites, including
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monacolins, pigments, γ-aminobutyric acid, and citrinin3-5.
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In 1979, Endo6 first isolated an active substance from the fermentation broth of
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Monascus ruber, which strongly inhibited cholesterol synthesis and named it
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monacolin K. Monacolin K can block the activity of 3-hydroxy-3-methyl glutaryl
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coenzyme A (HMG-CoA) reductase as a competitive inhibitor7 in cholesterol
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biosynthesis. Thus, monacolin K shows great potential for broad clinical application;
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indeed, it is currently considered as one of the most effective drugs available for the
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treatment of hyperlipidemia8. In addition, monacolin K has been reported to prevent
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Alzheimer's disease, stroke, and also has ability to regulate kidney immunity9.
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Manzoni10 first identified the biosynthetic pathway of lovastatin in Aspergillus
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terreus in 2002. In brief, lovastatin biosynthesis is catalyzed by lovastatin nonaketide
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synthase (LNKS) using acetyl-CoA and malonyl-CoA as substrates under the action
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of the formation of polyketone compounds11. In 2008, Chen et al.12 screened a gene
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cluster containing monacolin K synthase (mokA-mokI) in the Monascus pilosus
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genome and through a series of experiments determined high similarity to the
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lovastatin gene cluster in A. terreus (lovB-lovI).
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With respect to industrial production, Monascus fermentation largely relies on
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the Monascus strain used, and thus identifying or engineering Monascus with
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excellent performance and high production is a critical factor for further industrial
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development13 toward achieving economic benefits for the Monascus industry14. With
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continuous progress in mold transformation methods, direct seeding can now be
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achieved by the knockout and overexpression of specific genes in Monascus15.
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Through continuous exploration and efforts, gene overexpression and functional
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validation have been successfully achieved for the Monascus protein-coding gene
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mokH16, and the transcription factors LaeA and MpLaeA17,18. In 2013, Lee et al.17
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overexpressed MpLaeA in M.pilosus, monacolin K and pigment production increased
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significantly. In 2016, Liu et al.18 knocked out the MrLaeA gene in M. ruber, resulting
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in a decrease in the secondary metabolite production, especially monascus and citrinin.
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Therefore, it was speculated that the regulatory factors encoded by laeA, which also
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have a regulatory effect on the synthesis of monacolin K in Monascus. This previous
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work has provided a good foundation for the transformation of Monascus. Based on
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this technology, in the present study, we aimed to optimize the production of
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monacolin K in M. purpureus by modifying the targeted genes in the biosynthesis
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cluster. In order to explore the regulation of gene clusters on monacolin K
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biosynthesis in M. purpureus and to achieve the high yield of monacolin K, our study
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intended to use the laboratory-preserved M. purpureus M1 (strain No. CGMCCC 3.0568)
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as the starting strain, through the four genes (mokC, mokD, mokE, and mokI) were
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overexpressed to construct monacolin K high-yield strains.
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MATERIALS AND METHODS
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Fungal Strain and Culture Conditions M. purpureus M1, a stable producer of
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monacolin K, was obtained from the Chinese General Microbiological Culture
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Collection Center (strain No. CGMCCC 3.0568). M. purpureus M1 was maintained on
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potato dextrose agar for 5 days at 30°C and cultured with 50 mL seed medium
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containing (per liter): 30 g glucose, 15 g soybean powder, 1 g MgSO4·7H2O, 2 g
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KH2PO4, 70 g glycerol, 2 g NaNO3,and 10 g peptone at a neutral pH. Then the seed
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solution was incubated on fermentation medium (20 g/L rice powder, 1 g/L
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MgSO4·7H2O, 2 g/L ZnSO4·7H2O, 2.5 g/L KH2PO4, 90 g/L glycerol, 5 g/L NaNO3, and
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10 g/L peptone) for 12 days at 25°C with 150 rpm.
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Overexpression of mokC, mokD, mokE, and mokI Gene overexpression is a widely
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used technique in the molecular biology of filamentous fungi. To engineer a
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high-yield monacolin K Monascus strain, mokC, mokD, mokE, and mokI from M.
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purpureus M1 were overexpressed in M1. Gene fragments of mokC (1575 bp), mokD
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(792 bp), mokE (1083 bp), and mokI (1815 bp) were amplified by polymerase chain
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reaction (PCR) respectively using specific primers (Table 1) by Invitrogen (Shanghai,
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China), and cloned in the restriction sites of the pBC-hygro plasmid (Miaoling
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Bioscience & Technology Ltd. Co., Wuhan, China). The resulting pBC-hygro
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derivatives were designated pBC-hygro-mokC, pBC-hygro-mokD, pBC-hygro-mokE,
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and pBC-hygro-mokI, respectively (Fig. 1). The ligation products were transformed
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into Escherichia coli DH5α competent cells, and then the pBC-hygro plasmids were
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cultured, selected, and verified. Recombinant plasmid size verification, using
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pBC-hygro-mokC as an example, The ligated PCR product fragment of pBC-Hygro
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plasmid and mokC gene was double digested with QuickCut Sma I and QuickCut Not
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I, and the digested product was purified and ligated into E. coli competent DH5α at 34
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µg/mL chloramphenicol. The positive transformants were screened on the plates, and
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the plasmid was extracted. The recombinant plasmid was detected by electrophoresis
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with the original plasmid. As shown in Fig.1 (b), the length of the recombinant
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plasmid was about 8400 bp, which was consistent with the expected size.
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Overexpression constructs were introduced into M. purpureus through
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protoplast electric shock conversion19, 20 under the following conditions: voltage of 3
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kV/cm, pulse time of 4 ms, capacitance of 25 µF, and resistance of 400 Ω (Fig. 2a).
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The strains receiving the pBC-hygro plasmid were screened on hygromycin plates of
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different concentrations to obtain hygromycin-resistant strains. To verify the genetic
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stability of the different transformants, the transformants introduced with the
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recombinant plasmids were passaged on the hygromycin selection (10 µg/mL
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hygromycin) plate for five generations, and 10 single colonies were randomly selected
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for further analysis (Fig. 2b).
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Monacolin K Production The supernatants of fermentation medium were analyzed
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by high-performance liquid chromatography (HPLC) fitted with a InertsilODS-3 C18
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column at 25°C (5 µm, 150 × 4.6 mm) after filtration of the supernatant through a
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0.45-µm filter. The HPLC parameters were as follows: the mobile phase, 0.1%
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H3PO4/methanol (1:3, v/v); flow rate, 1 mL/min; and detection by ultraviolet
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spectroscopy at a wavelength of 237 nm21-23. A standard monacolin K compound was
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used to confirm the HPLC analysis.
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Reverse Transcription-Quantitative PCR (RT-qPCR) Analysis of Monacolin K
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Biosynthetic Gene Clusters The Monascus mycelia were obtained when the
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monacolin K concentration peaked. Total mycelial RNA was extracted by RNAprep
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Pure Plant Kit (Tiangen-bio, Beijing, China), and first-strand cDNA was synthesised
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by FastQuant RT Kit (with gDNase; Tiangen-bio, Beijing, China), with the FQ-RT
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Primer Mix. RT-qPCR was conducted to monitor gene expression levels using the
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SYBR Green PCR master mix (Tiangen-bio, Beijing, China). Primers for mokA-mokI
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(GenBank accession no. DQ176595.1) and GAPDH (GenBank accession no.
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HQ123044.1) were designed by Beacon Designer 8 software to amplify a portion of
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the nine genes (Table 1).
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RT-qPCR was performed using a CFX96 Real-Time PCR Detection System
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version (Bio-Rad, Hercules, CA, USA) as previously described24,25 with the following
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amplification program: 95 °C for 15 min, followed by a three-step PCR (40 cycles of
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denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, and extension at 72 °C for
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30 s). Amplification was performed using Super Real Pre Mix Plus (SYBR Green) for
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detection of the fluorophore SYBR green with fluorescein. Relative expression levels
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were calculated by the 2-∆∆Ct method24. All values were normalised using the
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housekeeping reference expression level of the GAPDH gene. RT-qPCR was carried
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out in triplicate for each sample.
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Scanning Electron Microscopy (SEM) of the Monascus Mycelium SEM was used
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to observe the morphological differences in mycelia of the different strains. For SEM,
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the mycelium cells of samples were fixed in 25% glutaraldehyde solution in
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phosphate-buffered saline (PBS)
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solution in PBS buffer, pH 7.2. The cells were dehydrated with different
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concentrations of ethanol(30%,50%,70%,80%,90%,100%) in distilled water,
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being left for 10min at each stage, and centrifuged at 12, 000 rpm for 5 min. The cells
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were replaced with isoamyl acetate-ethanol solution(1:1, v:v), and the cells were
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resuspended in each solvent for 10 min, centrifuged at 12, 000 rpm for 5 min. The
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samples were added a certain volume of hexamethyl disilazane, and were dried to a
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powder at 60˚C. After primary fixation, the mycelia were coated with gold palladium
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for 2 min. Photomicrographs were then acquired using a VEGA 3LMU/LMH SEM
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(TESCAN, Brno, Czech Republic).
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RESULTS AND DISCUSSION
buffer (12h, 25°C), and rinsed with 0.1M H3PO4
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Monacolin K, also known as lovastatin, is involved in cholesterol biosynthesis,
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and can suppress the activity of HMG-CoA reductase as a competitive inhibitor15, 26.
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The monacolin K biosynthetic gene clusters in Monascus have gained substantial
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research attention, and the strategies for synthesizing the bioactivity of monacolin K
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are achieved through regulation of gene clusters13. The monacolin K biosynthetic gene
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cluster was identified according to similarities with lovastatin synthetic genes in
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Aspergillus, and nine genes (mokA-mokI) were determined to be associated with
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monacolin K synthesis27,28. The mokA-deficient mutant strain in M. pilosus
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BCRC38072 cannot produce monacolin K, indicating that mokA encodes the
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polyketide synthase responsible for monacolin K biosynthesis in this strain. In
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addition, the mokB-deficient mutant of M. pilosus NBRC4480 cannot produce
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monacolin K, but rather accumulates monacolin J, indicating that mokB is responsible
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for synthesis of the diketide side chain of monacolin K. Overexpression of the mokH
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gene in M. pilosus results in significantly higher monacolin K production than that
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detected in wild-type strains, indicating that mokH positively regulates monacolin K
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production.
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The present study was designed to verify the effects of mokC, mokD, mokE, mokI
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in monacolin K biosynthesis in M. purpureus M1. These genes were overexpressed in
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M. purpureus M1 to construct high monacolin K yielding strains, which were used to
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explore the regulation effect of these four genes on monacolin K metabolism in M.
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purpureus. After introduction of the pBC-hygro plasmid to Monascus competent cells
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through protoplast click conversion, nine strains of Monascus (designated pBC1,
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pBC2, pBC3, pBC4, pBC5, pBC6, pBC7, pBC8, and pBC10) were obtained after five
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generations of inheritance and selection. Based on the gene transcription levels,
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metabolism, and verification results, we confirmed that the mokC, mokD, mokE and
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mokI gene overexpression strains were successfully constructed (Fig. 1).
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The RNA of Monascus pBC5 and M. purpureus M1 was extracted and
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reverse-transcribed into cDNA, using the hygroma-hygrogel-R and hygro-R primers.
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The pBC5 clearly harbor hygromycin gene (1000 bp) suggesting that the pBC hygro
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plasmid was successfully transformed into the wild-type M. purpureus (Fig. 3),
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whereas the M. purpureus M1 strain could not amplify the hygromycin gene fragment.
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This result verified that the transformant of the pBC-hygro plasmid was successfully
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constructed and determined the suitability of the pBC5 strain as a control strain in the
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subsequent analyses.
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Eight hygromycin-resistant strains were obtained by passaging of the
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mokC-overexpressing strain (Cn strain, where n indicates the strain number 1-8) for
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five generations, and were cultured at the same time as the control strain pBC5. The
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content of monacolin K in the fermentation broth was determined by HPLC. The yield
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of monacolin K in the control strain pBC5 was 72.5 mg/L (Fig. 4a), whereas that in the
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mokC overexpression strain C8 was 242.4 mg/L (Fig. 4b), representing a 234.3%
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increase. Therefore, strain C8 was used as the candidate mokC-overexpressing strain.
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Eight
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mokD-overexpressing strain (Dn) for five generations. The yield of monacolin K in
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strain D8 was 232.6 mg/L (Fig. 4c), representing a 220.8% increase compared with the
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production
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mokD-overexpressing strain. Eight hygromycin-resistant strains were obtained by
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passage of the mokE-overexpressing strain (En) for five generations. The monacolin
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K yield of strain E3 was 137.4 mg/L (Fig. 4d), representing a 89.5% increase compared
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with
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mokE-overexpressing strain. Five hygromycin-resistant strains were obtained by
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passage of the mokI-overexpressing strain (In) for five generations. The monacolin K
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yield of strain I1 was 79.7 mg/L (Fig. 4e), representing a 10% increase compared to
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that
211
mokI-overexpressing strain.
hygromycin-resistant
that
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pBC5.
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strain
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D8 was
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I1 strain
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used
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candidate
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The PCR results demonstrated that strains pBC5, C8, D8, E3, and I1 of M.
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purpureus could significantly amplify the hygromycin gene fragment of about 1000 bp,
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thus validating the successful construction of these transformants of pBC-hygro
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plasmids (Fig. 3). The expression levels of monacolin K synthesis-related genes in the
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overexpression strains were assessed on day 8 when stable overexpression was
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achieved.
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Overexpression of mokC in C8 upregulated the expression of the other genes to
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different degrees. The expression level of the mokC gene was 63.2-fold higher than
220
that in the control pBC5. The levels of mokA, mokH, and mokI were increased by
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0.1–0.6-fold, whereas the expression level of mokF was decreased by 0.9-fold. In D8,
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the expression level of the mokC gene was 40.6-fold higher than that in the control
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pBC5. The expression levels of mokD, mokH, and mokI genes were increased by
224
3.3-fold, 3.7-fold, and 1.9-fold, with smaller increase for mokA, mokE, and mokG
225
between 0.1–0.8-fold. By contrast, the expression levels of mokB and mokF were
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decreased 0.1-fold and 0.7-fold, respectively (Fig. 5b). In E3, the mokC expression
227
level showed the greatest enhancement, with a 40.6-fold increase compared to that of
228
the control. The expression levels of mokE, mokH, and mokI genes were increased by
229
3.3-fold, 3.7-fold, and 1.9-fold, although the increase for mokA, mokE, and mokG was
230
small, between 0.1–0.8-fold. By contrast, the expression levels of mokB and mokF
231
were decreased by 0.1-fold and 0.7-fold, respectively (Fig. 5c). In I1, the expression
232
level of the mokI gene was 4.1-fold higher than that in the control pBC5. the
233
expression levels of mokA, mokD, mokC, mokE, mokB, mokH, and mokG were all
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significantly higher than that in the control pBC5, with increases of 4.1-, 3.5-, 3.2-, 3.0-,
235
2.7-, 2.0-, 1.8-, and 1.6-fold, respectively (Fig. 5d).
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Therefore, the RT-qPCR analysis showed that maximal monacolin K
237
biosynthesis was reached on day 8 based previous study28, at which point most of the
238
related genes showed higher transcription in the overexpression strains.
239
The effect of monacolin K biosynthesis gene overexpression on fungal
240
morphology was assessed with SEM (5000× and 10, 000×). The mycelia of the
241
overexpression strains C8, D8, E3, I1 and the control strain pBC5 were observed for
242
structural differences. It could be seen from Fig. 6e and 6j that the pBC5 mycelium
243
was dense and mostly combined with a network connection, and it had spores on the
244
top or side of the mycelium, which was consistent with the Ji et al.29 description.
245
Under the same magnification, overexpression strains showed clear changes to
246
the mycelium surface with obvious folds and the mycelium length shorter, whereas
247
the pBC5 mycelium had a more full structure with a flatter surface (Fig. 6B). The
248
spores of the overexpression strains showed different degrees of depression, while the
249
pBC5 strain had smooth surface and no depression. Wang et al.30 have studied the
250
overexpression of the mokE gene, it was found that mokE gene had a certain influence
251
on the morphology of mycelium and spores in Monascus. It was speculated that the
252
overexpression of mokE gene promoted the length of mycelium to be shortened, and
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the network structure between hyphae to be reduced, thereby promoting the
254
production of monacolin K. This was basically consistent with the results of the
255
experiment. It suggested that overexpression of four genes had a certain effect on the
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morphological structure of Monascus31-33, which promoted the synthesis of monacolin
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K. And Lv et al.34 have studied that hyphal diameter was highly correlated with the
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biosynthesis of the Monascus yellow pigments. Many factors contributed to the
259
development of morphological form in submerged fermentation, and many cases
260
indicated that the fungal morphology could influence the productivity of fungal
261
fermentations to some extent35.
262
In summary, we successfully constructed mokC, mokD, mokE, and mokI
263
overexpression Monascus strains, which all showed substantially increased yields of
264
monacolin K, demonstrating a significant impact of these genes on monacolin K
265
anabolism and good candidates for producing high-yielding strains. Moreover, we
266
detected a folding phenomenon of parts of the mycelium surface in the overexpression
267
strains, indicating that these genes could also influence the mycelia morphology.
268
Together, these findings lay the foundation for further in-depth analysis of the
269
function of this gene cluster to uncover the complex network regulation mechanism of
270
the monacolin K synthesis pathway. Further exploration of these strains and
271
underlying mechanisms will help to achieve the industrial-scale production of
272
monacolin K toward its widespread clinical application to best exploit its
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health-promoting benefits.
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ABBREVIATIONS
275
HMG-CoA, 3-hydroxy-3-methyl glutaryl coenzyme A; LNKS, Lovastatin Nonaketide
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Synthase; PCR, Polymerase Chain Reaction; HPLC, High-Performance Liquid
277
Chromatography;
RT-qPCR,
Reverse
Transcription-Quantitative
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SEM,
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Scanning Electron Microscopy; PBS, Phosphate-Buffered Saline.
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ACKNOWLEDGEMENTS
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This work was supported by Beijing Natural Science Foundation (Grant No.
281
KZ201810011015), Beijing Nova Program (Grant No. Z181100006218021), Support
282
Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th
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Five--year Plan (Grant No. CIT&TCD201804023), National Natural Science
284
Foundation of China (Grant No. 31301411, 31571801, and 31401669), National Key
285
Research
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2016YFD0400502-02),The construct of innovation service ability--Science and
287
technology
288
2016-014213-000034), Beijing Municipal Science and Technology Project (Grant No.
289
Z171100002217019), and Beijing Excellent Talents Training Project (Grant No.
290
2016000020124G025).
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CONFLICTS OF INTEREST
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The authors declare that they have no conflicts of interest.
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SUPPORTING INFORMATION
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Supplementary data related to this article can be found at http://pubs.acs.org.
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Supplementary Table 1. Primer sequences for gene overexpression.
and
Development
achievement
Program
(Grant
transformation--Upgrade
No.
project
2016YFD0400802,
(Grant
No.
PXM
296 297
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Figure captions
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Fig. 1 Construction of pBC-hygro-mokC (a) and agarose gel electrophoresis of
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pBC-hygro-mokC(b), pBC-hygro-mokD(c), pBC-hygro-mokE(d), pBC-hygro-mokI(e)
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recombinant plasmid size verification.
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(b-e) Lane 1 indicates the DNA super helix marker; Lane 2 indicates a pBC-hygro-
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mokC (b), pBC-hygro-mokD (c), pBC-hygro-mokE (d), pBC-hygro-mokI (e)
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recombinant plasmid. Lane 3 indicates pBC-hygro original plasmid.
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Fig.2 To screen the tolerated concentration of hyacinth B and electric shock
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conditions in M. purpureus M1.(a) To screen the electric shock conditions, (b) To
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screen the tolerated concentration of hyacinth B.
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Fig. 3 PCR of the hygromycin B gene in M. purpureus pBC5 and overexpression
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strains. M: DL2000 DNA Marker; Lane 1: pBC5; Lane 2: C8; Lane 3: D8; Lane 4: E3;
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Lane 5: I1.
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Fig. 4 Monacolin K contents of
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genes.
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(a) Monacolin K content of pBC-Hygro conversion in M. purpureus.(b) Monacolin K
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content
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mokD-overexpression strains. (d) Monacolin K content of mokE-overexpression
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strains. (e) Monacolin K content of mokI-overexpression strains.
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Fig. 5 Expression of genes related to monacolin K biosynthesis (mokA-mokI) of M.
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purpureus pBC5 (control), and overexpression strains (a) C8, (b) D8, (c) E3, and (d)
of
mokC-overexpression
M. purpureus strains with overexpression four
strains.
(c)
Monacolin
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I1.
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Fig. 6 The colony and SEM images of M. purpureus overexpression strains C8, D8,
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E3, and I1, and pBC5 (control). (A) The colony morphology of five strains, (B) SEM
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images of five strains at different magnifications: 5000× (a–e) and 10, 000× (f–j). (a-e):
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C8, D8, E3, I1 and pBC5; (f-j):C8, D8, E3 I1 and pBC5. The red arrow represents the
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spores depression.
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Table 1. Primer sequences for gene overexpression Primer
Sequence
Tm (℃)
mokC-F mokC-R mokD-F mokD-R mokE-F mokE-R mokI-F mokI-R hygro-F hygro-R
AAGGAAAAAAGCGGCCGCATGACAGTTCCGACAGATACG (NotI) TCCCCCGGGTCAGAGATCTTCGTCCCGAC (SmaI) GCTCTAGAATGCGTATCCAACGCACCC (XbaI) CCCAAGCTTTCACCCAATGACTCTAGCCC (HindIII) GCTCTAGAATGACCATCACCTTCACCCTAC (XbaI) CCCAAGCTTTTACCCCAACTTCACCACAAC (HindIII) GCTCTAGAATGGCTTCCCACCAGTCTG (XbaI) CCCAAGCTTCTAGACTCGTTCATCGCGG (HindIII) ATGCCTGAACTCACCGCGACG GATAAGGAAACGGGAGCCTGC
64
Fig.1
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Fig.3
Fig. 4
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