융합법을 이용한 바이오에탄올 생산에 적합한 효모균주의 구축

Construction of Yeast Strain Suitable for Bioethanol Production by Using Fusion Method

Abstract

본 연구는 에탄올내성, 내열성, ${\beta}-glucanase$ 활성 및 xylose 대사가 가능한 새로운 생물시스템을 육종하기 위해 원형질체융합(protoplast fusion)이라는 방법을 사용하여 S. cerevisiae BYK-F11 균주와 P. $stipitis{\Delta}ura$ 균주와의 genome shuffling을 시도하였다. P. $stipitis{\Delta}ura$ 균주는 URA3 유전자를 결실시켜 uracil 영양요구주로 구축되었다. Protoplast fusion을 통해 몇몇의 융합체가 선별되었고, 두 모균주인 BYK-F11 균주와 P. $stipitis{\Delta}ura$ 균주의 핵형(karyotype)를 모두 가지는 BYKPS-F8 균주가 22개의 융합체중에서 최종 선정되었다. 이어 ${\beta}-glucanase$ 활성, xylose 이용능, 에탄올내성, 내열성 및 에탄올생산성에 대한 다양한 표현형이 조사되었다. BYKPS-F8 균주는 모균주인 BYK-F11 균주가 가지는 ${\beta}-glucanase$ 활성을 가지게 되었고, P. $stipitis{\Delta}ura$ 균주가 가지는 xylose 이용능도 모균주보다 1.2배 증가되었음을 확인할 수 있었다. BYKPS-F8 균주는 $40^{\circ}C$에서 내열성을 보였으며, 8% 에탄올이 첨가된 배지에서 모균주에 비해 에탄올 내성이 증가되었음을 확인 할 수 있었다. 20 g/l의 xylose가 함유된 배지에서 72시간 배양에 의해 약 7.5 g/l의 에탄올을 생산할 수 있었으며, 260시간의 장기간의 배양에도 BYKPS-F8균주에 도입한 다형질이 안정적으로 유지됨을 확인하였다. 따라서, 본 연구에서 사용된 균주 육종방법을 통해 다형질을 가진 다른 속간의 균주 융합 및 산업적으로 유용한 생물시스템의 육종이 가능함을 확인하였다.

To construct useful yeast strain for bioethanol production, we improved yeast harboring various phenotypes by using yeast protoplast fusion method. In this study, S. cerevisiae BYK-F11 strain which have ethanol tolerance, thermotolerance and ${\beta}-glucanase$ activity and P. $stipitis{\Delta}ura$ strain which has xylose metabolism pathway were fused by genome shuffling. P. $stipitis{\Delta}ura$ strain was constructed for protoplast fusion by URA3 gene disruption, resulting in uracil auxotroph. By protoplast fusion, several fused cells were selected and BYKPS-F8 strain (fused cell) showing both karyotypes from two parent strains (S. cerevisiae BYK-F11 and P. $stipitis{\Delta}ura$ strain) among 22 fused cells was finally selected. Sequentially, various phenotypes such as ${\beta}-glucanase$ activity, xylose utility, ethanol tolerance, thermotolerance and ethanol productivity were analyzed. The BYKPS-F8 strain obtained ${\beta}-glucanase$ activity from BYK-F11 strain and 1.2 fold increased xylose utility from P. $stipitis{\Delta}ura$ strain. Also, the BYKPS-F8 strain showed thermotolerance at $40^{\circ}C$ and increased ethanol tolerance in medium containing 8% ethanol. In this fused cell, 7.5 g/l ethanol from 20 g/l xylose was produced and the multiple phenotypes were stably remained during long term cultivation (260 hr). It was proved that novel biological system (yeast strains) is easily and efficiently bred by protoplast fusion among yeasts having different genus.

Fig. 1. Confirmation of uracil auxotroph in P. stipitisΔura strain (A) and screening of fusants by auxotrophic test and analysis of β-glucanase activity (B). Host strains and fusants were streaked on to YPD, SD containing geneticin and YPD containing MUG medium for 3 days. The β-glucanase activity was detected by MUG degradation on UV illumination. WT, P. stipitis wild type strain: Δura3, P. stipitisΔura strain: S, S. cerevisiae BYK-F11 strain: P, P. stipitisΔura strain; No.8, selected fusant (BYKPS-F8).

Fig. 2 Karyotype analysis of BYKPS-F8 strain and comparison of cell growth in each strain on YPX (2%) medium. (A) Karyotype of each strain was analyzed by pulsed field gel electrophoresis (PFGE). Lane 1: S. cerevisiae BYK-F11 strain, lane 2: P. stipitisΔura strain, lane 3: BYKPS-F8 fusant. (B) Each strain was cultivated on YPX (2% xylose) at 30℃ for 48 hr. Graph bar □: S. cerevisiae BYK-F11 strain, ▨: P. stipitisΔura strain, ■: BYKPS-F8.

Fig. 3. Analysis for ethanol tolerance of BYKPS-F8 fusant. Aliquots (3 μl) of 10-fold serially diluted cell suspensions from S. cerevisiae BYK-F11, P. stipitisΔura strain and BYKPSF8 fusant were spotted on to YPD containing 8% ethanol (YPDE), then incubated for 3 days at 30℃. #1 and #2 indicate independent clone from BYKPS-F8 fusant.

Table 1. Comparison of various phenotypes in S. cerevisiae BYK-F11, P. stipitisΔura strain and BYKPS-F8 fusant