Kluyveromyces marxianus에 의한 유청막걸리 품질특성

김수환,허창기,김소망,조인경,김용두 한국식품저장유통학회 2015 한국식품저장유통학회지 Vol.22 No.4

유청을 막걸리 제조시 용수로 사용하기 위해 효모에 따른 유당 분해능과 ethanol 생성능력을 측정하고, 유청 첨가 비율에 따른 막걸리를 제조하여 총산, pH, 환원당, ethanol 및 관능평가 등 품질특성을 확인한 결과는 다음과 같다. 유당 분해능은 K. marxianus KCCM 35455과 K. marxianus KCCM 50700이 가장 뛰어난 유당 분해능을 보였으며, ethanol 생성능력은 K. marxianus KCCM 50700 균주의 ethanol 함량이 가장 높은 것으로 나타났다. 따라서 유당 분해능과 ethanol 생성능이 우수했던 K. marxianus KCCM 50700의 효모를 선정하여 유청 막걸리 제조 연구를 진행하였다. 유청 첨가비율에 따른 막걸리의 발효과정 중 총산은 K. marxianus KCCM 50700으로 담금한 시료구의 경우 S. cerevisiae로 담금한 막걸리의 산도보다 비교적 낮은 경향을 보였으며, pH는 K. marxianus KCCM 50700로 담금한 시료구의 경우 S. cerevisiae로 담금한 막걸리의 pH보다 비교적 높은 경향을 보였다. 환원당 함량은 S. cerevisiae 시료구에서는 유청 첨가비율이 높아짐에 따라 환원당 함량이 높아졌으며, K. marxianus KCCM 50700 시료구에서는 큰 함량 차이가 나타내지 않았다. ethanol 함량 변화는 K. marxianus KCCM 50700로 담금한 시료구가 S. cerevisiae로 담금한 시료구 보다 높게 나타났다. 관능평가는 향, 색, 맛 및 종합기호도면에서 K. marxianus KCCM 50700로 담금한 유청 100% 첨가 시료구가 높은 기호도를 보였다.

Kluyveromyces marxianus var. marxianus IFO 1735에 의한 Inulin Fructotransferase의 생산 및 이용에 관한 연구

김재근,坂井石夫 한국산업미생물학회 1997 한국미생물·생명공학회지 Vol.25 No.3

Iunlin을 di-D-fructofuranose 1,2' : 2,3' dianhydride (DFAⅢ)로 전환시키는 inulin fructotransferase (inulaseⅡ)를 생산하는 균주로 Kluyveromyces marxianus var. marxianus를 선별하였다. 공시균의 inulaseⅡ의 생성을 위한 최적 배지조성은 1.0%의 sucrose 및 0.75%의 YNB이었으며 최적 배양조건은 초기 pH, 배양온도 및 배지량은 각각 4.7, 30℃ 및 140 ㎖이었다. 상기 최적 조건으로 36시간 배양하였을 때 효소 생산량이 가장 높아 배양액 ㎖당 약 4.2 unit를 나타내었다. 본 효소의 이용성을 검토한 결과 돼지감자 추출액을 이용한 천연배지의 경우 상기 최적 배양조건에서 48시간 배양으로 DFAⅢ 생산량이 가장 높아 배양액 ㎖당 13.25 ㎎을 나타내었다. Kluyveromyces marxianus var. marxianus isolated as an inulin-assimilating microorganism produces inulin fructotransferase (inulaseⅡ) which catalyses the conversion of inulin into di-D-fructofuranose 1, 2' : 2, 3' dianhydrde (DFAⅢ). The DFA produced by the organism was isolated by using active carbon column, and identified as DFAⅢ by high performance liguid chromatography. The culture medium giving maximum inulaseⅡ producton was found to consist of 1% sucrose and 0.75% yeast nitrogen base (YNB). The inulaseⅡ production was induced by inulin or sucrose as a carbon source and increased by addition of YNB as a nitrogen source. Optimal initial pH of the culture medium, culture temperature and medium volume for the enzyme production were pH 4.7, 30℃ and 140 ㎖, respectively. Under the optimal conditions described above, the enzyme activity in the culture supernatant reached 4.2 units/㎖ after cultivation for 36 h. The DGAⅢ was accumulated at 13.25 ㎎/㎖ after 48 h of culture in the Jerusalem artichoke tuber medium.

Development of a rapid and reliable TaqMan probe-based real-time PCR assay for the detection and enumeration of the multifaceted yeast <i>Kluyveromyces marxianus</i> in dairy products

Kim, Dong-Hyeon,Jeong, Dana,Kang, Il-Byeong,Kim, Hyunsook,Seo, Kun-Ho Elsevier 2018 FOOD SCIENCE AND TECHNOLOGY -ZURICH- Vol.87 No.-

<P>Kluyveromyces marxianus is a multifaceted yeast used in the dairy, biotechnological, and probiotic industries, and has been implicated in the spoilage of dairy products. Currently, K. marxianus detection involves laborious and time-consuming culture methods. To develop a rapid and reliable detection method for K. marxianus in pure cultures and dairy products, we designed a novel real-time PCR primer/probe set, KM55, which targets the internal transcribed spacer (ITS) rRNA gene. In the inclusivity and exclusivity tests, 11 K. marxianus strains tested positive, while 28 closely related microorganisms tested negative; this confirmed 100% sensitivity and specificity of the assay. Further, the KM55 set enabled a linear K. marxianus detection in the range of 10(0)-10(5) colony-forming units (CFU) per reaction in phosphate-buffered saline, 10% skim milk, and commercial yogurts, with no cross-reactivity with microorganisms of kefir origin. The enumeration of K. marxianus in kefir revealed 5.1 to 6.9 logCFU ml(-1) of kefir, depending on the fermentation time, temperature, and grain-milk ratio. K. marxianus was successfully detected in artificially contaminated yogurt samples within 2 h. These results suggest that this method could be an effective and sensitive presumptive screening tool for detecting and enumerating K marxianus in dairy products. (C) 2017 Elsevier Ltd. All rights reserved.</P>

CRISPR-Cas9 system을 이용한 Kluyveromyces marxianus 17694-DH2 균주의 자일로스 소비속도 증진

권덕호,박중희,정덕열,박재범,박동민,강경곤,최서영,김수린,하석진 한국생물공학회 2019 KSBB Journal Vol.34 No.4

The third-generation gene editing technology, CRISPR-Cas9 system, is derived from the bacterial immune system. These CRISPR-Cas9 systems have recently been used to mutate yeast gene or replace cleavage sites with other DNA. In this study, CRISPR-Cas9 system was applied to delete PHO13 gene of Kluyveromyces marxianus. As a result, only one strain of three transformants, that the CRISPR-Cas9 system was applied, was confirmed partial deletion of PHO13 gene. Sequencing of the PHO13 gene revealed the deletion of cytosine at position 457, which resulted in premature termination of translation as compared to that from the parental strain. The ΔPHO13 strain, K. marxianus 17694-DH2, showed 18.29% and 21.28% improvement in xylose consumption and ethanol production from xylose, respectively, as compared to those form the parental strain.

Sulfuric Acid Hydrolysis and Detoxification of Red Alga Pterocladiella capillacea for Bioethanol Fermentation with Thermotolerant Yeast Kluyveromyces marxianus

( Chien Hui Wu ),( Wei Chen Chien ),( Han Kai Chou ),( Jung Woo Yang ),( Hong Ting Victor Lin ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.9

One-step sulfuric acid saccharification of the red alga Pterocladiella capillacea was optimized, and various detoxification methods (neutralization, overliming, and electrodialysis) of the acid hydrolysate were evaluated for fermentation with the thermotolerant yeast Kluyveromyces marxianus. A proximate composition analysis indicated that P. capillacea was rich in carbohydrates. A significant galactose recovery of 81.1 ± 5% was also achieved under the conditions of a 12% (w/v) biomass load, 5% (v/v) sulfuric acid, 121°C, and hydrolysis for 30 min. Among the various detoxification methods, electrodialysis was identified as the most suitable for fermentable sugar recovery and organic acid removal (100% reduction of formic and levulinic acids), even though it failed to reduce the amount of the inhibitor 5-HMF. As a result, K. marxianus fermentation with the electrodialyzed acid hydrolysate of P. capillacea resulted in the best ethanol levels and fermentation efficiency.

Simultaneous integration of multiple genes into the Kluyveromyces marxianus chromosome

Heo, P.,Yang, T.J.,Chung, S.C.,Cheon, Y.,Kim, J.S.,Park, J.B.,Koo, H.M.,Cho, K.M.,Seo, J.H.,Park, J.C.,Kweon, D.H. Elsevier Science Publishers 2013 Journal of biotechnology Vol.167 No.3

While Kluyveromyces marxianus is a promising yeast strain for biotechnological applications, genetic engineering of this strain is still challenging, especially when multiple genes are to be transformed. Sequential gene integration, which takes advantage of repetitive insertion/excision of the URA3 gene as a marker, has been the best option until now, because the URA3-deletion mutant is the only precondition for this method. However, we found that the introduced gene is co-excised during the URA3 excision step for next gene introduction, resulting in a very low cumulative probability (<1.57x10<SUP>-6</SUP>% for 4 genes) of integrating all genes of interest. To overcome this extremely low probability, and to reduce labor and time, all 4 genes were simultaneously transformed. Surprisingly, the infamously high 'non-homologous end joining' activity of K. marxianus enabled simultaneous integration of all 4 genes in a single step, with a probability of 7.9%. Various K. marxianus strains could also be similarly transformed. Our finding not only reduces the labor and time required for such procedures, but also removes a number of preconditions, such as pre-made vectors, selection markers and knockout mutants, which are needed to introduce many genes into K. marxianus.

Co-expression of two heterologous lactate dehydrogenases genes in <i>Kluyveromyces marxianus</i> for <small>L</small>-lactic acid production

Lee, Jae Won,In, Jung Hoon,Park, Joon-Bum,Shin, Jonghyeok,Park, Jin Hwan,Sung, Bong Hyun,Sohn, Jung-Hoon,Seo, Jin-Ho,Park, Jin-Byoung,Kim, Soo Rin,Kweon, Dae-Hyuk Elsevier 2017 Journal of biotechnology Vol.241 No.-

<P>Lactic acid (LA) is a versatile compound used in the food, pharmaceutical, textile, leather, and chemical industries. Biological production of LA is possible by yeast strains expressing a bacterial gene encoding L-lactate dehydrogenase (LDH). Kluyveromyces marxianus is an emerging non-conventional yeast with various phenotypes of industrial interest. However, it has not been extensively studied for LA production. In this study, K. marxianus was engineered to express and co-express various heterologous LDH enzymes that were reported to have different pH optimums. Specifically, three LDH enzymes originating from Staphylococcus epidermidis (SeLDH; optimal at pH 5.6), Lactobacillus acidophilus (LaLDH; optimal at pH 5.3), and Bos taurus (BtLDH; optimal at pH 9.8) were functionally expressed individually and in combination in K. marxianus, and the resulting strains were compared in terms of LA production. A strain co-expressing SeLDH and LaLDH (KM5 La + SeLDH) produced 16.0 g/L LA, whereas the strains expressing those enzymes individually produced only 8.4 and 6.8 g/L, respectively. This co-expressing strain produced 24.0 g/LA with a yield of 0.48 gig glucose in the presence of CaCO3. Our results suggest that co-expression of LDH enzymes with different pH optimums provides sufficient LDH activity under dynamic intracellular pH conditions, leading to enhanced production of LA compared to individual expression of the LDH enzymes. (C) 2016 Elsevier B.V. All rights reserved.</P>

Lactobacillus bulgaricus와 Kluyveromyces marxianus의 혼합 스타터를 이용한 기능성 발효유의 특성

윤원호,남보라,김진만,김창한,Yoon Won-Ho,Nam Bo-Ra,Kim Jin-Man,Kim Chang-Han 한국축산식품학회 2006 한국축산식품학회지 Vol.26 No.2

본 연구는 기능성 발효유로서 티벳산 발효유에서 분리한 효모(Kluyveromyces marxianus)와 유산균(Lactobacillus bulgaricus)의 혼합스타터를 이용한 발효유를 제조하여 배양 기간별 균수 측정, pH, 적정산도, ethanol 함량, 항암 활성에 대해 알아보았다. 배양은 $30^{\circ}C$에서 curd가 형성되는 시점에 마치게 되는데 이때의 최종 산도는 0.68, pH는 4.5 이었다. 배양 36시간 후의 균수는 K. marxianus는 $5.3{\times}10^9 CFU/mL$, L. bulgaricus는 $3.2{\times}10^9 CFU/mL$ 이었고 ethanol 함량은 0.35%까지 증가하였다. 36시간 배양하여 제조된 발효유의 항종양 활성은 HEp-2에 대해서는 86.6%, HEC-1B에 대해서는 70.3%, SW-156에 대해서는 60.4%, SK-MES-1에 대해서는 57.14%의 항종양 활성을 나타내었다. 이상의 결과 기존의 유산균 발효유에 효모를 첨가한 알코올 발효유의 제조가 가능하며, 제품의 항종양 활성의 측면에서도 높은 기능성을 나타내는 것으로 나타났다. This study was carried out to investigate characteristics of acid and alcohol fermented milk by mixed starters made by Lactobacillus bulgaricus (KCTC 3635) and Kluyveromyces marxianus (KCTC 17212) for 36 hours when the curds were formed. Final pH and titratable acidity were about 4.5 and 0.68%, respectively. The viable cell counts of lactic acid bacteria and yeast for alcohol fermented milk were increased to $3.2{\times}10^9 CFU/mL$ and $5.3{\times}10^9 CFU/mL$, respectively. The ethanol contents increased to 0.35% during fermentation. Antitumor activities of the fermented milk against tumor cell lines, such as HEp-2, HEC-1B, SW-156 and SK-MES-1 showed to 86.6, 70.3, 60.4 and 57.14%, respectively.

Direct fermentation of Jerusalem artichoke tuber powder for production of <small>L</small>-lactic acid and <small>D</small>-lactic acid by metabolically engineered <i>Kluyveromyces marxianus</i>

Bae, Jung-Hoon,Kim, Hyun-Jin,Kim, Mi-Jin,Sung, Bong Hyun,Jeon, Jae-Heung,Kim, Hyun-Soon,Jin, Yong-Su,Kweon, Dae-Hyuk,Sohn, Jung-Hoon Elsevier 2018 Journal of biotechnology Vol.266 No.-

<P>An efficient production system for optically pure L-and D-lactic acid (LA) from Jerusalem artichoke tuber powder (JAP) was developed by metabolic engineering of Kluyveromyces marxianus. To construct LA-producing strains, the ethanol fermentation pathway of K. marxianus was redirected to LA production by disruption of KmPDC1 and expression of L-and D-lactate dehydrogenase (LDH) genes derived from Lactobacillus plantarum under the control of the K. marxianus translation elongation factor 1 alpha promoter. To further increase the LA titer, the L-LA and D-LA consumption pathway of host strains was blocked by deletion of the oxidative LDH genes KmCYB2 and KmDLD1. The recombinant strains produced 130 g/L L-LA and 122 g/L D-LA by direct fermentation from 230 g/L JAP containing 140 g/L inulin, without pretreatment or nutrient supplementation. The conversion efficiency and optical purity were >> > 95% and >> >99%, respectively. This system using JAP and the inulin-assimilating yeast K. marxianus could lead to a cost-effective process for the production of LA.</P>

Biosynthesis of 2-phenylethanol from glucose with genetically engineered Kluyveromyces marxianus

Kim, T.Y.,Lee, S.W.,Oh, M.K. IPC Science and Technology Press ; Elsevier Scienc 2014 Enzyme and microbial technology Vol.61 No.-

2-Phenylethanol (2-PE) is an aromatic alcohol with a rose scent, which is used in the cosmetics, fragrance and food industries. 2-PE is produced in a few yeast strains by Ehrlich pathway. In this study, Kluyveromyces marxianus was genetically engineered for overproduction of 2-PE from glucose. About 1.0g/L of 2-PE was produced by overexpressing phenylpyruvate decarboxylase (ARO10) and alcohol dehydrogenase (ADH2) genes of Saccharomyces cerevisiae. A similar level of 2-PE was also produced from evolved K. marxianus, which was resistant to the phenylalanine analog, p-fluorophenylalanine. aroG<SUP>fbr</SUP> from Klebsiella pneumoniae encoding a feedback resistant mutant of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DHAP) synthase was overexpressed in the evolved K. marxianus. Finally, 1.3g/L of 2-PE was produced from 20g/L glucose without addition of phenylalanine in the medium.