Precursor제공 및 생합성 관련 유전자의 cloning을 통한 Monacolin-K 생산성 향상

이미진,정용섭,전계택 한국생물공학회 2008 KSBB Journal Vol.23 No.6

Monacolin-K is a strong anti-hypercholesterolemic agent produced by Monascus sp. via polyketide pathway. High-yielding mutants of monacolin-K were developed through rational screening strategies adopted based on understanding of monacolin-K biosynthetic pathway. Through the experiments for investigating various amino acids as putative precursors for the monacolin-K biosynthesis, it was found that production level of monacolin-K was remarkably increased when optimum amount of cysteine was supplemented into the production medium. We suggested that these phenomena might be related to the special roles of SAM (S-adenosyl methionine), a putative methyl group donor in the biosynthetic pathway of monacolin-K, demonstrating close interrelationship between SAM-synthesizing primary metabolism and monacolin-K synthesizing secondary metabolism. Namely, increase in the intracellular amount of SAM derived from the putative precursor, cysteine which was extracellularly supplemented into the production medium might contribute to the significant enhancement in the monacolin-K biosynthetic capability of the highly mutated producers. On the basis of these assumptions derived from the above fermentation results, we decided to construct efficient expression vectors harboring SAM synthetase gene (metK) cloned from A. nidulans, with the hope that increased intracellular level of SAM could lead to further enhancement in the monacolin-K production through overcoming a rate-limiting step associated with monacolin-K biosynthesis. Hence, in order to overcome the plausible rate-limiting step associated with monacolin-K biosynthesis by increasing intracellular level of SAM, we transformed the producer mutants with an efficient expression vector harboring gpdA promoter of the producer microorganism, and metK gene. Notably, from the resulting various transformants, we were able to screen a very high-yielding transformant which showed approximately 3.3 fold higher monacolin-K productivity than the parallel nontransformed mutants in shake flask cultures performed under the identical fermentation conditions.

Monascus 배양물의 첨가급여가 산란계의 난황과 계육 및 혈청의 콜레스테롤 함량에 미치는 영향

김상인,함영훈,이규호,Kim, Sang-In,Ham, Yeong-Hun,Lee, Kyu-Ho 한국가금학회 2003 韓國家禽學會誌 Vol.30 No.4

Monacolin-K가 0.6% 들어있는 Monascus 배양물을 시판 산란계 시료에 각각 0.00%, 2.67%, 5.33% 및 8.00%씩 추가로 첨가하여, 1일 1사당 사료섭취량이 125g일 경우 1일1수당 Monacolin-K 섭취량이 각각 0, 20, 40 및 60mg이 되도록 하는 4개 처리를 두었고, 총 180수의 산란계를 공시하여 10주간 사양시험을 실시한 결과 산란율과 평균 난중은 처리간에 유의적인 차이가 없었으나, 사료섭취량과 산란량 kg당 사료 요구율은 Monascus 배양물의 첨가수준이 증가할수록 감소하였다. 전 기간 평균 혈청 콜레스테롤 함량은 Monascus 배양물의 첨가수준이 증가할수록 감소하는 경향이었으나, 유의적인 차이는 없었다. 난황의 콜레스테롤 함량은 Monascus 배양물 첨가급여 4~5주 후부터 유의적으로 감소하였고(P<0.05), 사양시험 종료시 대퇴부근육의 콜레스테롤 함량도 유의적으로 감소하였다(P<0.05). This experiment was carried out to study the effect of dietary Monascus culture on the cholesterol contents of egg yolk, muscle and serum of layers with 180 Isa-Brown laying hens for 10 weeks. Control group(C) was fed the commercial laying hen diet and 2.67(T1), 5.33(T2) and 8.00(T3)% of Monascus culture which contained 0.6% monacolin-k added to control diet so as to supply the monacolin-k 20(T1), 40(T2) and 60(T3) mg respectively, per hen-day with 125g diet. Hen-day egg production and average egg weight were not affected by the dietary Monascus culture, but feed intake and feed conversion per kg egg were significantly decreased(P<0.05) as the dietary Monascus culture increased. Cholesterol contents of egg yolk measured 4~5 weeks after feeding the Monascus culture and those of thigh meat measured at the end of experiment were significantly decreased(P<0.05) as the dietary Monascus culture increased. Average cholesterol contents of serum showed a trend to decrease as the dietary Monascus culture increased without significant difference.

홍국균접종 쌀배아 섭취가 중등도 비만 초등학생의 체지방 및 혈중지질의 개선에 미치는 영향

강순아,최영숙,박동기,임융호,권순주 대한지역사회영양학회 2005 대한지역사회영양학회지 Vol.10 No.5

The purpose of this study was to investigate the effect of Monacolin-inoculated rice embryo on the body fat and serum lipid profiles in 61 obese elementary school students. The subjects divided into two groups 31 obese children had the experimental diet (rice embryo inoculated Monacolin) and 30 obese children had the control diet three times a day after meals. The mean age, height, weight, and BMI of 61 subjects were 10.5 ± 0.5 years, 143.6 ± 6.8 cm, 55.0 ± 8.9 kg, and 26.1 ± 3.9, respectively. The changes of body fat mass (kg) in experimental group and control group after 6 weeks were -0.60 kg and -0.03 kg, respectively. The changes of body fat (%) in experimental group and control group after 6 weeks were -1.44% and -0.25%, respectively. These changes of body fat in experimental group were significantly higher than in control group (p < 0.05). The change of total-cholesterol in experimental group and control group during 6 weeks were -17.52 mg/dL and -1.70 mg/dL, respectively. The change of LDL-cholesterol in experimental group and control group during 6 weeks were -17.06 mg/dL and -2.80 mg/dL, respectively. The change of triglyceride in experimental group and control group after 6 weeks were -9.58 mg/dL and 11.67 mg/dL. Total-cholesterol, LDL- cholesterol and triglyceride of experimental group after 6 weeks significantly decreased compared to control group (p < 0.05). After experimental diet (6 weeks), total-cholesterol was negatively correlated with body water contents, soft lean mass and fat free mass (p < 0.05). Triglyceride showed a significantly positive correlation with body weight and body fat mass, however, it was negatively correlated with fat free mass (p < 0.05). HDL-cholesterol showed a significantly positive correlation with fat free mass (p < 0.05). These results show that Monacolin-inoculated rice embryo is effective in decreasing body fat and blood lipid in obese children. (Korean J Community Nutrition 10(5): 565 ~ 573, 2005)

Chinese Red Yeast Rice Versus Lovastatin Effects on Prostate Cancer Cells With and Without Androgen Receptor Overexpression

Mee Young Hong,Navindra P. Seeram,Yanjun Zhang,David Heber 한국식품영양과학회 2008 Journal of medicinal food Vol.11 No.4

Chinese red yeast rice (RYR), a food herb made by fermenting Monascus purpureus Went yeast on white rice, contains a mixture of eight different monacolins that inhibit cholesterogenesis and also red pigments with antioxidant properties. Monacolin K (MK) is identical to lovastatin (LV). Both LV and RYR contain statins, which could inhibit de novo cholesterogenesis, which is critical to the growth of tumor cells. Dysregulation of the cholesterol biosynthetic pathway has been demonstrated during progression to androgen independence in xenograft models, and it has been proposed that cholesterogenesis and androgen receptor (AR) up-regulation are essential to androgen-independent cell survival. This study was designed to examine the differences between the effects of RYR and LV on androgen-dependent LNCaP cells and androgen-independent cells overexpressing AR (LNCaP-AR). RYR showed more potent inhibition effect on prostate cancer cell growth compared to LV. Both the pigment and monacolin-enriched fractions purified from RYR inhibited proliferation (P < .001) to a lesser extent than intact RYR. While mevalonate, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), restored proliferation in LV-treated cells, it failed to do so in RYR-treated cells. Expression of the HMGCR gene was up-regulated by LV (P < .001) but not RYR in both LNCaP and LNCaP-AR cells. These results suggest that the RYR matrix beyond MK alone may be bioactive in inhibiting androgen-dependent and -independent prostate cancer growth. In vivo studies are needed to further establish the potential advantages of RYR over LV in prostate cancer chemoprevention and in the prevention of the emergence of androgen independence.

Effectiveness and Safety of Novel Nutraceutical Formulation Added to Ezetimibe in Statin-Intolerant Hypercholesterolemic Subjects with Moderate-to-High Cardiovascular Risk

Alberto Mazza,Mariaceleste Nicoletti,Salvatore Lenti,Gioia Torin,Gianluca Rigatelli,Marzia Pellizzato,Andrea Fratter 한국식품영양과학회 2021 Journal of medicinal food Vol.24 No.1

The effectiveness of statins in the primary and secondary prevention of cardiovascular (CV) diseases has been widely proven. However, the onset of adverse events associated with their use prevents to achieve the therapeutic targets recommended by the guidelines (GL) for the management of dyslipidemia. In the event of statin intolerance, the GL recommend to use bile acid sequestrants, fibrates, and ezetimibe in monotherapy, but their benefits in improving lipid pattern are quite modest. This study aims at evaluating the effectiveness and safety of a nutraceutical compound (NC) associated with ezetimibe (EZE) on the lipid profile in statin-intolerant patients with moderate-to-high CV risk. Ninety-six statin-intolerant hypertensive and hypercholesterolemic subjects treated pharmacologically with EZE 10 mg daily were randomized in open label (n = 48) to take for 3 months a NC containing Monacolin-K (MK), Berberine Hydrochloride (BC), t-Resveratrol (RES), Quercetin (QUER), and Chromium (CH) in the form of a gastro-resistant tablet that improves enteric bioaccessibility and bioavailability of these substances. The control group (n = 48) took only EZE in monotherapy at the same dosage; both groups followed a standardized lipid-lowering diet. The total serum cholesterol (TC), low density lipoprotein cholesterol (LDLC), high density lipoprotein cholesterol (HDLC), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine phosphokinase (CPK) levels were compared at the follow-up in both groups using Student's t-test. TC and LDL levels reduced in both groups, but were lower in the group treated with EZE + NC (−25.9% vs. −15%, P < .05 and −38.7% vs. −21.0%, P < .05, respectively). No changes were observed in either group regarding a decrease in TG (−9.4% vs. −11.7%, NS) and an increase in HDLC (+4.2% vs. +1.1%, NS). The AST, ALT, and CPK levels increased in the group treated with the EZE + NC compared to the control group, but were still within the acceptable range. There was no difference concerning the lipid-lowering treatment between gender, and no patient withdrew from the study. In the short term, the EZE + NC combination therapy is well tolerated and effective in improving TC and LDLC levels in statin-intolerant patients with moderate-to-high CV risk.

Monacolin K 강화 홍국쌀 생산을 위한 균주 및 특성 연구

박지영,한상익,서우덕,나지은,심은영,남민희 韓國作物學會 2014 한국작물학회지 Vol.59 No.2

혈중 콜레스테롤 저하작용이 있는 monacolin K 고함유 홍국쌀 생산을 위해 2종의 홍국 균주와 7종의 쌀 품종을 사용하여 홍국쌀을 생산하였다. 생산된 홍국쌀의 monacolin K 함량을 분석한 결과는 다음과 같다. 1. 발효된 홍국쌀의 색은 균주에 비해 품종에 따라 차이가 크게 나는 경향이었으며, 상주찰벼가 붉은 색소 함량이 높았지만, monacolin K 생산량과 붉은 색소의 정도가 상관관계가 크다고 판단할 수 없다. 2. 홍국추출물을 UPLC로 이용하여 활성형 monacolin K와 비활성형 monacolin K가 분리 되었고, LC-MS/MS로 분자량을 확인한 결과, 활성형과 비활성형 monacolin K의 선구이온(precursor ion)은 각각 m/z 421, 405였고, 생성이온(product ion) 중 활성형은 m/z 101,319, 비활성형은 m/z 199, 285의 분자량을 확인하였다. 3. M. ruber KCTC6122과 M. ruber KCCM60141 균주를 이용하여 쌀 품종별로 생산한 홍국쌀의 monacolin K 함량을 분석한 결과, 상주찰벼가 각각 47.24, 117.03ppm으로 가장 많은 함량을 나타내었다. 4. 7종의 품종을 비교했을 때, M. ruber KCCM60141이 KCTC6122과 비교하여 monacolin K 생산량이 더 높은 균주이고, 그 중 상주찰벼를 이용하는 것이 홍국쌀 생산에 적절한 품종 선택이라고 판단된다. Red yeast rice (RYR) is the product of fermented yeast by Monascus strains on rice, and has recently become a popular dietary supplement as a traditional food in Asia. RYR contains monacolin K substances known to inhibit cholesterol synthesis as efficiently as statin drugs. To determine the optimal rice cultivar for manufacturing RYR, 7 rice cultivars (Goami, Goami2, Sangjuchalbyeo, Seolgaeng, Saegyejinmi, Yeonghojinmi and Chilbo) were fermented using two Monascus strains (M. ruber KCTC6122 and KCCM60141 of M. ruber) in this study. The monacolin K content of Sangjuchalbyeo were 47.24 ppm on KCTC6122 cultures and 117.03 ppm on KCCM60141 cultures, respectively. Other cultivars, especially Goami and Goami2, which had less content of monacolin K could not seem to ferment normally because those didn't show red color. These results imply that Sangjuchalbyeo can be optimal rice cultivar as a commercial RYR which is well fermented rice and has high content of monacolin K.

A Novel Statin Compound from Monacolin J Produced by CYP102A1- catalyzed Regioselective C-hydroxylation

Ngoc Tan CAO,Ngoc Anh NGUYEN,Chan-Mi PARK,Chul-Ho YUN 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Statins inhibit the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase), which plays a central role in the cholesterol production. They reduce cardiovascular disease and mortality in those who are at high risk of cardiovascular disease. Monacolin J is a statin compound as a lovastatin precursor. It is found in red yeast rice with various strains of the yeast Monascus purpureus. Monacolin J has a hydroxyl substituent at position C’-8 of monacolin L. This study was conducted to synthesize new statin compounds from monacolin J through bacterial CYP102A1 catalysis. Here we found the catalytic ability of CYP102A1 to catalyze regioselective hydroxylation of monacolin J. More specifically, a major product was found with a hydroxyl group attached at C-6’a position of monacolin J, which has never been reported. C-6’a-hydroxymethyl monacolin J has shown the ability to inhibit HMG-CoA reductase higher than the monacolin J substrate itself. Human liver microsomes and human CYP3A4 also showed the ability to catalyze monacolin J to produce the same product of CYP102A1-catalyzed reaction. This result will be a premise as a new platform for development of statin drug candidates.

홍국균 발효가 발아현미의 Monacolin K 함량과 항산화 활성에 미치는 영향

이상훈(Sang Hoon Lee),장귀영(Gwi Yeong Jang),김민영(Min Young Kim),김신제(Shinje Kim),이연리(Yuon Ri Lee),이준수(Junsoo Lee),정헌상(Heon Sang Jeong) 한국식품영양과학회 2015 한국식품영양과학회지 Vol.44 No.8

발아 온도와 기간에 따른 발아현미 홍국배양물의 홍국색소, monacolin K, citrinin 및 페놀 화합물 생성량과 항산화 활성 변화를 조사하였다. 발아 기간이 증가함에 따라 명도는 증가하였으나 적색도와 황색도는 감소하였고, 적색(A500), 오렌지색(A470) 및 황색(A400) 색소 생성량도 동일한 경향이었다. 총 monacolin K는 현미(215.85 mg/kg)가 백미(40.41 mg/kg)보다 높았으며, 32°C에서 1일간 발아시킨 발아현미(1,263.04 mg/kg)에서 가장 높은 함량을 보였으나 발아 기간이 증가함에 따라 감소하였다. 곰팡이 독소인 citrinin은 모든 처리구에서 검출되지 않았다. 총 폴리페놀과 플라보노이드 함량은 발아 온도와 기간이 증가함에 따라 유의적으로 증가하여 37°C에서 4일간 발아시킨 발아현미에서 13.80 mg/g과 1.30 mg/g으로 가장 높았다. 전자공여능과 총 항산화력은 발아에 의해 증가되어 32°C에서 1일간 발아시킨 발아현미에서 각각 22.16 mg TE/g과 62.27 mg AAE/g으로 가장 높았으나 발아 기간이 증가함에 따라 감소하였다. 이상의 결과로부터 홍국색소와 monacolin K 생성량을 증가시키기 위한 기질로 발아현미가 유용하며, 배양에 사용된 Monascus pilosus 305-9는 citrinin을 생성하지 않고 monacolin K 생성량을 증가시켜 식품의 기능성 증진용 소재 및 균주로서의 활용이 기대된다. This study was performed to investigate the changes in monacolin K content and antioxidant activities of Monascus-fermented brown rice with different germination temperatures and periods. Brown rice was germinated at 32, 35 and 37°C for 1∼4 days, after white rice (WB), brown rice (BR), and germinated brown rice (GBR) were fermented with M. pilosus 305-9 at 30°C for 20 days. The redness, yellowness and Monascus pigments increased after germination. Total monacolin K content increased from 215.85 mg/kg of BR to 1,263.04 mg/kg of GBR (32°C/1 day), whereas monacolin K content decreased with increase in germination period. Citrinin was not detected in any of the samples. Total polyphenol (TPC) and flavonoid contents (TFC) increased with increase in germination temperature and period, whereas electron donating ability (EDA) and total antioxidant activities (TAA) decreased due to reduction of Monascus pigment content. The TPC and TFC showed the highest values (13.80 mg/g and 1.30 mg/g, respectively) in GBR (37°C/4 day), whereas EDA and TAA showed the highest values (22.16 mg Trolox equivalent/g and 62.27 mg ascorbic acid equivalent/g, respectively) in GBR (32°C/1 day). These results indicated that the optimal germination temperature and period for increasing monacolin K content and antioxidant activities was found to be at 32°C for 1 day. In addition, it was found that M. pilosus 305-9 was a useful strain for increasing monacolin K content without producing citrinin in functional foods and pharmaceutical industrial regions.

고체평판배지에서의 홍국균의 배양, monacolin K 및 색소생산 특성

김경선,안준배,김창섭,박윤제 한국산업식품공학회 2012 산업 식품공학 Vol.16 No.4

홍국쌀 제조에 사용되는 홍국균의 고체평판배지에서의 생장 및 색소생산과 monacolin K 생산에 관한 특성에 대하여 조사하였다. 총 35개의 균주를 수집하여 PDYA, PDA, MEA 3개의 고체평판배지에서 25oC와 30oC에서 배양하였다. 대부분의 균주가 PDYA 배지에서 가장 빠른 성장을 보여주었으며, 25oC보다 30oC에서 훨씬 빠른 균사 생장을 보여주었다. 고체평판배지에서 적색계열의 색소생산량이 높은 균주는 황색계열의 색소생산량도 높았다. 그러나, 색소 생산량이 높은 균주에서는 monacolin K의 생산량이 높지 않았으며, 오히려 색소생산량이 낮은 균주에서 monacolin K의 생산량이 높았다. 또한 monacolin K를 생산하는 균주에서는 독소성분인 citrinin 역시 검출되지 않았다. 이러한 결과로부터 monacolin K 생산균주의 기초 스크리닝에 고체평판배지에서의 색소생산량이 낮은 균주를 선발하는 방법을 적용할 수 있을 것으로 판단된다. The production of color pigments and monacolin K by Monascus strains were investigated on agar plates while monitoring cell growth patterns. Thirty five different strains were collected and cultured on potato dextrose yeast extract agar (PDYA), potato dextrose agar (PDA) and malt extract agar medea (MEA) at two different incubation temperature conditions, 25oC and 30oC. The growth rates of most of strains were highest on PDYA medium, and hyphal growth was faster at 30oC than at 25oC. Both red and yellow pigments were highly produced by high pigment-producing strains on agar plate cultures. High pigment-producing strains produced less amount of monacolin K while low pigment-producing strains produced much more monacolin K. Any citrinin was not detected from the monacolin K-producing strains. These results imply that the selection of low pigment-producing strains cultured on agar plates could be applied for primary screening of Monascus strains for preparation of red mold rice.

일반 및 가공용 쌀 발아현미로 제조한 홍국쌀의 Monacolin K 함량과 항산화 성분 비교

오현아,김민영,이윤정,송명섭,이준수,정헌상 한국식품영양과학회 2018 한국식품영양과학회지 Vol.47 No.12

일반쌀 품종인 삼광과 가공용으로 개발된 한가루 품종의 발아현미를 이용하여 홍국쌀을 제조하고 홍국색소, monacolin K 함량 및 항산화 성분 변화를 조사하였다. 발아 전에 비하여 발아 후의 색소 생성량이 삼광은 발아 2일 차에 높았으며, 한가루는 발아 1일 차가 높았다. 총 monacolin K 함량은 삼광에 비해 한가루가 높았으며, 발아 1일 차에 323.45 mg/ kg으로 가장 높았지만 발아 3일 차에는 72.02 mg/kg으로 급격하게 감소하였다. 또한 발아 전 삼광과 한가루에서 총 폴리페놀 함량은 0.22 및 0.49 mg/g이었으며, 플라보노이드 함량은 0.01 및 0.24 mg/g이었지만 발아 후에 유의적으로 증가하여 발아 1일 차에 한가루는 총 폴리페놀 및 플라보노이드 함량이 각각 2.65 mg/g 및 0.56 mg/g으로 가장 높았다. 이상의 결과로부터 홍국색소와 monacolin K 생성량을 증가시키기 위한 기질로 일반미인 삼광 품종에 비해 가공용으로 개발된 한가루 품종이 우수하였으며, 발아 1일 차가 홍국쌀 배양에 가장 적합하였다. In this study, red yeast rice was prepared using germinated brown rice of Samkwang and Hangaru cultivars, and the changes in pigment, monacolin K, and antioxidant components were investigated. Pigment production was increased after germination compared to that before germination, but pigment production decreased rapidly at 3 days of germination. The total monacolin K was higher in Hangaru than in Samkwang. The highest level of monacolin K was 323.45 mg/kg in Hangaru at one day of germination, but decreased significantly to 72.02 mg/kg at 3 days of germination. In addition, the total polyphenol contents were 0.22 and 0.49 mg/g and flavonoid content were 0.01 and 0.24 mg/g for Samkwang and Hangaru before germination, respectively. The total polyphenol and flavonoid contents were highest at 2.65 mg/g and 0.56 mg/g on Hangaru at one day of germination. Hangaru, which was developed for processing, was suitable for Monascus as a substrate to increase the contents of pigment and monacolin K, and one day of germination was most suitable for the cultivation of red yeast rice.