Deok-Ho Kwon

Isolation of Mutant Yeast Strains having Resistance to 1-ethyl-3-methylimidazolium Acetate through a Directed Evolutionary Approach

에너지 수요의 빠른 증가와 화석연료 사용으로 인한 지구 온난화로 친환경적이며, 재생 가능한 대체에너지를 필요로 한다. 차세대 청정에너지로 각광받고 있는 바이오 에너지 중 에서 가장 널리 이용되는 바이오 에탄올의 생산은 미국과 브 라질에서 90% 이상 생산하고 있다. 하지만 이러한 바이오 에탄올의 생산 원료는 옥수수 또는 사탕수수와 같은 식량자 원이 이용되기 때문에 식량문제와 곡물가격 상승에 대한 새 로운 문제를 야기하고 있다[1−4]. 이러한 문제를 해결하고자 식량자원이 아닌 폐목재, 나무 등 비식용 바이오 매스인 제 2세대 목질계 바이오 매스에 대한 연구가 많이 진행되고 있 다[2, 5]. 목질계 바이오 매스는 cellulose, hemicellulose, lignin으로 구성되어 있으며[6, 7], 그 중 cellulose와 hemicellulose는 미생물 발효에 사용될 수 있는 단당류로 가수분 해 될 수 있는 기질이기 때문에 대체 에너지원으로서 주목 을 받고 있다[7, 8]. 목질계 바이오 매스를 효과적으로 이용 하기 위해서 cellulose를 둘러싸고 있는 lignin을 제거하는 전 처리 과정이 필요하다[9]. 다양한 전처리 방법들 중 ionic liquid는 양이온과 음이온의 이온 결합으로 이루어진 이온성 염 화합물이며 비폭발성, 낮은 휘발성과 열적 안정성 외에도 효소의 우수한 반응성, 선택성, 안정성 등의 장점으로 인해 목질계 바이오 매스 전처리에 주로 사용된다[5, 10, 11]. Ionic liquid는 cellulose를 용해시킬 수 있다고 이전에 증 명된 바 있으며[12], 산이나 암모니아를 사용하는 기존의 방 법보다 ionic liquid를 이용한 전처리가 더 효과적이라고 알 려져 있다[13]. 또한 ionic liquid 처리된 cellulose의 경우 미 처리 된 것 보다 결정체가 적기 때문에 분해효소인 cellulase 와 쉽게 반응할 수 있다[14, 15]. 특히 다른 전처리 과정에서 생성되는 발효저해물질(furfural 또는 hydroxymethylfurfural) Isolation of Mutant Yeast Strains having Resistance to 1-ethyl-3-methylimidazolium Acetate through a Directed Evolutionary Approach Yoo-Jin Lee, Deok-Ho Kwon, Jae-Bum Park, and Suk-Jin Ha* Department of Bioengineering and Technology, Kangwon National University, Chuncheon 24341, Republic of Korea

Xylitol Production by Kluyveromyces marxianus 36907-FMEL1 at High Temperature was Considerably Increased through the Optimization of Agitation Conditions

Xylitol is usually used as a sugar substitute in food products because of low calorie content and used as the starting material for the production of valuable chemicals [1−4]. Xylitol is generally produced by biological methods for high production yield and environmental safety [5]. The XYL1 gene, coding for xylose reductase (XR), from Scheffersomyces stipitis has been mainly cloned and overexpressed among several reports for biological productions of xylitol by using recombinant yeast [6, 7]. Recently Kluyveromyces marxianus, a thermotolerant yeast, was engineered for xylitol production because thermotolerant capability from K. marxianus is more favored for a simultaneous saccharification and fermentation (SSF) process [8]. SSF process could enhance xylitol productivity from cellulosic biomass in practical aspect. However, non-thermotolerant yeast were not suitable for SSF process at high temperature [9, 10]. Previously, we performed a directed evolution and random mutagenesis with thermotolerant yeast K. marxianus ATCC 36907 for isolating the mutant K. marxianus 36907-FMEL1 having two fold improved xylose reductase activity as compared to the parental strain [11]. K. marxianus 36907-FMEL1 produced xylitol efficiently at 30°C, however, xylitol production was very poor at 40°C by flask fermentation experiments. As shown in Table 1, glucose consumption rate and ethanol production rate were not significantly changed by K. marxianus 36907-FMEL1, when fermentation temperatures were increased from 30°C to 40°C with 80 g/l of glucose as a sole carbon source. However, xylose consumption rate and xylitol production rate were highly reduced from when fermentation temperatures were increased from 30°C to 40°C with 80 g/l of xylose as a sole carbon source. At 40°C, relative xylose consumption and xylitol production rates were decreased to 69% and 47%, respectively, as compared to those from 30°C. Therefore it was speculated that that K. marxianus 36907-FMEL1 might possess low ATP regeneration capability from Recently, we isolated the mutant Kluyveromyces marxianus 36907-FMEL1, which demonstrated improved xylose reductase activity as compared to the parental strain, K. marxianus ATCC 36907. Effects of agitation conditions on xylitol production were verified using a bioreactor system. Under an agitation speed of 400 rpm, K. marxianus 36907-FMEL1 exhibited the highest xylitol yield (0.57 g/g) and productivity (0.64 g·l·h) at 30°C. When the fermentation temperature was increased to 40°C, interestingly, xylitol yield and productivity were found to be increased to 21% (0.64 g/g) and 58% (0.90 g·l·h), respectively, under the optimized agitation conditions.