Xylan 대사 유전자를 가진 미니 효모인공염색체의 가공 및 Mitotic Stability 분석

강다인,김연희 한국미생물·생명공학회 2022 한국미생물·생명공학회지 Vol.50 No.3

In this study, yeast artificial chromosome Insert (YAC) harboring genes which related xylan metabolism was constructed by using chromosome manipulation technique. For efficient chromosome manipulation, each splitting fragment (DNA module) required for splitting process was prepared and these DNA modules were transformed into Saccharomyces cerevisiae strain YKY164. By two-rounds chromosome splitting, yeast chromosome VII (1,124 kb) was split 887 kb-YAC, 45 kb-mini YAC and 198 kb-YAC and YKY183 strain containing 18 chromosomes was constructed. Splitting efficiency for chromosome manipulation was 50- 78% and expression level of foreign genes on 45 kb-mini YAC and enzyme activity were indistinguishable from that of the YKY164 strain. Furthermore, xylan-degraded products by recombinant enzymes were confirmed and mini-yeast artificial chromosome maintained stable mitotic stability without chromosome loss during 160 generations. 본 연구에서는 염색체가공기술을 이용하여 xylan으로부터다양한 대사산물을 생산할 수 있는 유전자를 도입한 효모인공염색체를 구축하였다. 효율적인 염색체가공기술인 PCS법을 이용하기 위해 염색체 splitting에 필요한 splitting fragment (DNA module)를 각각 제작하였고, xylan 대사에관여하는 유전자군을 가진 YKY164 균주에 형질전환하였다. 두번의 염색체 splitting에 의해 1,124 kb의 효모 7번염색체는887 kb-YAC, 45 kb-mini YAC와 198 kb-YAC로 가공되었으며, 총 18개의 염색체를 가진 YKY183 균주를 구축하였다. 염색체가공을 위한 splitting efficiency는 50−78%였으며, 45 kb-mini YAC 상에 있는 외래유전자의 발현 및 효소활성은 염색체가공 전과 비교하여 유의미한 변화 및 저하는 관찰되지 않았다. 또한 생산된 재조합효소에 의한 xylan의 분해산물을 확인하였으며, 160 generation 동안 미니 효모인공염색체는 염색체의 결실없이 안정적인 mitotic stability를유지하였다.

반복적 염색체 삽입 형질전환에 의한 Saccharomyces diastaticus로의 외래 α-Amylase 유전자의 도입

김근 수원대학교 기초과학연구소 1995 基礎科學論文集 Vol.4 No.-

To overcome the unstable mitotic stability of heterologous α-amylase gene located on the episomal plasmid, two linearized integrating vectors, YIpMS△R(LEU2) and YIpMS△R(LEU2/URA3) were constructed and used for integrative and repetitive integrative transformation of Saccharomyces diastaticus secreting glucoamylase. The resulting tranformants showed increased amylolytic activities and mitotic stabilities compared to the original recipient. The repetitively integrated transformant showed higher amylolytic activity and mitotic stability of α-amylase gene than the once integrated transformant. The highest mitotic stability of repeated integrative transformant was 99.2% after 100 generations of cell-multiplication while the mitotic stabilities of episomal transformant and once integrative transformant were 0.0 and 73.3%, respectively.

전분발효 효모에서의 외래 $\alpha$-Amylase 유전자의 세포분열시 안정성 증진

김정희,김근,최영길,Kim, Jung-Hee,Kim, Keun,Choi, Yong-Keel 한국미생물학회 1994 미생물학회지 Vol.32 No.4

To develop a yeast strain which stably secretes both $\alpha$-amylase and glucoamylase and therefore is able to convert starch directly to ethanol, a mouse salivary $\alpha$-amylase cDNA gene with a yeast alcohol dehydrogenase I promoter has been introduced into the cell of a Saccharomyces diactaticus hybrid strain secreting only glucoamylase. To secrete both enzymes more stably without loss of the $\alpha$-amylase gene during a cell-multiplication, an integrating plasmid vector containing $\alpha$-amylase gene was constructed and introduced into the yeast cell. The results showed that the linearized form of the integrating vector was superior in the transformation efficiency and the rate of the expression of the $\alpha$-amylase gene than the circular type of the vector. The yeast transformant having a linearized plasmid vector exhibited higher mitotic stability than the yeast transformant habouring episomat plasmid vector. The transformant containing the linearized vector producing both $\alpha$-amylase and glucoamylase exhibited 2-3 times more amylolytic activity than the original untransformed strain secreting only glucoamylase. ${\alpha}$-Amylase와 glucoamylase를 동시에 안정하게 분비하여 전분을 일단계로 직접 에탄올로 발효시킬수 있는 효모 균주를 개발하기 위하여 glucoamylase를 분비하는 Saccharomyces diastaticus hybrid 균주에 쥐의 침샘 유래의 ${\alpha}$-amylase cDNA 유전자를 plasmid vector를 이용하여 도입하였다. 이 균주로부터 효소생산에 필요한 유전자를 잃어버림이 없이 안정하게 분비할 수 있도록 하기 위하여 $\alpha$-amylase 유전자를 효모의 염색체에 삽입시키기 위한 integrating plasmid vector인 YIpMS$\Delta$R(LEU2)를 제작하였다. 이 vector의 효모형질전환에 있어 원형(circular)상태와 제한 효소 XbaI으로 처리된 직선화된(linearized) 상태의 두가지 형태를 비교한 결과 형질전환 효율에서나 형질전환체내의 $\alpha$-amylase 유전자 보유정도가 모두 직선화된 형태의 경우가 원형상태의 경우보다 높았다. Linearized vecotr를 가진 효모 형질전환체에서의 유전자 발현 안정도는 세포분열을 거듭할수록 episomal vecotr에 의한 효모 형질전환체에서의 발현 안정도보다 우수하게 나타났다. 또한 이 linearized vector를 가진 형질전환체는 $\alpha$-amylase와 glucoamylase를 동시에 분비하여 glucoamylase만 분비하는 원균주보다 2배 이상의 전분분해력을 보였다.

Expression of Schwanniomyces occidentalis α-Amylase Gene in Saccharomyces cerevisiae var.diastaticus

Park, Jeong Nam,Shin, Dong Jun,Kim, Hee Ok,Kim, Dong Ho,Lee, HwangHee Blaise,Chun, Soon Bai,BAI, SUK 한국미생물 · 생명공학회 1999 Journal of microbiology and biotechnology Vol.9 No.5

The gene encoding Schwanniomyces occidentalis α-amylase (AMY) was introduced into Saccharomyces cerevisiae var. diastaticus which secreted only glucoamylase, by using a linearized yeast integrating vector to develop stable strains with a capability of secreting α-amylase and glucoamylase simultaneously. A dominant selectable marker, the geneticin (G418) resistance gene (Gt^r), was cloned into a vector to screen wild-type diploid transformants harboring the AMY gene. The amylolytic activities of transformants were about 3∼7 times higher than those of the recipient strains. When grown in nonselective media, the transformants with the linearized integrating vector containing the AMY gene exhibited almost all of the mitotic stability after 100 generations.

Physiological Analysis and Construction of Mini- yeast Artificial Chromosome Harboring Agar Degradation Genes

Min-Jun SEONG,Yeon-Hee KIM 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Many genes are stably maintained on chromosome compared plasmid and/or various vectors. Specially, the yeast artificial chromosome (YAC) system allows isolation of larger DNA fragments and easier modification of the cloned DNA than other systems. In present study, we attempted integration of agarase gene expression cluster into YAC. Several enzymes requiring agar/agarose metabolism were used as model enzymes, and a mini-YAC harboring two genes encoding β-agarase and gene encoding neoagarobiose hydrolase was constructed by using PCR-mediated Chromosome Splitting (PCS) technique. The three genes were stably integrated into yeast chromosome II. The chromosome II (813 kb) was successfully split to 269 kb, 38 kb containing the agarase gene expression cluster, and 523 kb split-YAC. The total agarase activity of integrated genes on the 38 kb split-mini YAC was still maintained compared to before chromosome splitting. Also, mitotic stability of mini-YAC in non-selective medium was 100% during about 160 generations. Cell growth rate, cell morphology and agar degradation ability were not distinguished from that before chromosome manipulation. As a result, the mini-YAC containing targeted gene cluster can be stably transplanted into other useful yeast strain and breeding of novel strain is also will be possible.

Manipulation of Artificial Chromosome Containing Desired Gene Cluster and Mitotic Stability Analysis in Yeast

Da-In KANG,Yeon-Hee KIM 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

In bacteria, operon is genetic regulatory system and many genes coding for functionally related proteins are clustered along the chromosome. In eukaryotic cell, many genes were regulated by various factors and functionally related proteins are located in various positions on many chromosomes. In present study, we attempted integration of several genes requiring xylan/xylose metabolism as gene cluster on same chromosome and construction of mini-artificial chromosome harboring gene cluster. The XYLP, XYLB, GRE3, and XYL2 genes encoding endoxylanase, β-xylosidase, xylose reductase, and xylitol dehydrogenase, respectively, were stably integrated into yeast chromosome VII. The chromosome VII (1124 kb) was successfully split to 887 kb, 45 kb containing the four genes expression cluster, and 198 kb split-YAC by PCS technique. Each enzyme activity of integrated genes on the 45 kb split-mini YAC was still maintained compared to before chromosome splitting. Also, mitotic stability of mini-YAC in non-selective medium was 100% during about 160 generations. Cell growth rate, cell morphology and xylan degradation ability were not distinguished from that before chromosome manipulation.