KR20140063599A - 피키아 시페리이 세포 및 그의 용도 - Google Patents
피키아 시페리이 세포 및 그의 용도 Download PDFInfo
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- KR20140063599A KR20140063599A KR1020147003745A KR20147003745A KR20140063599A KR 20140063599 A KR20140063599 A KR 20140063599A KR 1020147003745 A KR1020147003745 A KR 1020147003745A KR 20147003745 A KR20147003745 A KR 20147003745A KR 20140063599 A KR20140063599 A KR 20140063599A
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Abstract
본 발명은 유전자 조작에 의해 변형된 피키아 시페리이(Pichia ciferrii) 세포, 그의 용도, 및 스핑고이드 염기 및 스핑고지질을 제조하는 방법에 관한 것이다.
Description
본 발명은 유전자 변형된 피키아 시페리이(Pichia ciferrii) 세포, 그의 용도, 및 스핑고이드 염기 및 스핑고지질을 제조하는 방법에 관한 것이다.
1960년대 초 이래로 피키아 시페리이는 스핑고이드 염기 및 스핑고지질의 제조에 사용되어 왔다 (문헌 [Wickerham et al. 1960, J Bacteriol. 80, 484-91] 참조).
항상 스핑고이드 염기 및 스핑고지질의 야생형 균주 수율을 개선할 만한 가치가 있다.
본 발명의 목적은 스핑고이드 염기 및 스핑고지질에 관하여 증가한 생산성을 보유하는 이용가능한 피키아 시페리이 세포를 제조하는 것이다.
놀랍게도 본 발명자들은 이하에서 기재하는 감소한 특이적 효소 활성을 보유하는 세포가 본 발명의 목적을 달성할 수 있음을 발견하였다.
따라서 본 발명은 야생형과 비교하여 본원 특허청구범위 제1항에 기재한 바와 같은 효소의 감소한 활성을 보유하는 유전자 변형된 피키아 시페리이 세포를 기재한다.
또한 본 발명은 전술한 세포의 용도, 및 스핑고이드 염기 및 스핑고지질을 제조하는 방법에 관한 것이다.
본 발명의 하나의 이점은 본 발명에 따른 세포가 높은 세포 밀도로 증식할 수 있다는 것이다.
본 발명의 또 다른 이점은 세포를 적당한 영양 배지에서 증식시킬 때 세포가 현저히 증가한 역가의 아세틸화 스핑고이드 염기를 생성한다는 것이다.
본 발명의 추가 이점은 본래 유전자형으로의 복귀를 금지하는 균주의 높은 유전적 안정성이다. 상기 높은 유전적 안정성은, 유지시키고자 하는 도태압이 존재하지 않으므로, 항생물질의 부재하에서 배양을 또한 가능케 해준다.
본 발명의 추가 이점은 저렴하고 재생가능한 원료로부터 스핑고이드 염기의 생물공학적이고 환경친화적인 생산에 세포를 사용하는 가능성이다.
본 발명은 피키아 시페리이 세포가 그의 야생형과 비교하여,
A) 서열 1, 서열 3, 서열 5, 서열 7, 서열 9, 서열 11, 및
B) 서열 1, 서열 3, 서열 5, 서열 7, 서열 9, 서열 11의 서열 중 어느 것과 적어도 80%, 특히 바람직하게는 적어도 90%, 추가로 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 동일한 서열
로 이루어진 2개 군 A) 및 B)로부터 선택된 인트론-무함유(intron-free) 핵산 서열에 의하여 코딩되는 적어도 1종의 효소의 감소한 활성을 보유함을 특징으로 하는, 피키아 시페리이 세포에 관한 것이다.
이와 관련해서, A)군은 본 발명에 따라 바람직한 핵산 서열 군이다.
세포의 "야생형"이란 본 발명과 관련하여 바람직하게는, 본 발명에 따른 세포가 특정된 핵산 서열에 의하여 코딩된 효소의 활성에 영향을 미치는 요소(예를 들어, 상응하는 효소를 코딩하는 특정된 핵산 서열을 포함하는 유전자 또는 상응하는 유전자에 존재하고 특정된 핵산 서열에 기능적으로 연결된 프로모터)의 변형을 통해 발달되는 모 균주를 의미한다.
서열 1 또는 3에 의하여 또는 서열 1 또는 3과 적어도 80%, 특히 바람직하게는 적어도 90%, 추가로 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 동일한 서열에 의하여 코딩된 효소에 관하여 용어 "효소의 활성"이란 항상 "5,10-메틸렌테트라히드로폴레이트 + L-글리신 + H2O <=> 테트라히드로폴레이트 + L-세린" 반응을 촉매하는 효소 활성을 의미하는 것으로 이해된다.
당해 활성은 바람직하게는 문헌 [Schluepen, 2003]에 기재된 방법으로 측정된다.
서열 5에 의하여 또는 서열 5와 적어도 80%, 특히 바람직하게는 적어도 90%, 추가로 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 동일한 서열에 의하여 코딩된 효소에 관하여 용어 "효소의 활성"이란 항상 "L-세린 <=> 피루베이트 + NH3 " 반응을 촉매하는 효소 활성을 의미하는 것으로 이해된다.
당해 활성은 바람직하게는 문헌 [Ramos and Wiame, Eur J Biochem. 1982 Apr;123(3):571-6]에 기재된 방법으로 측정된다.
서열 7에 의하여 또는 서열 7과 적어도 80%, 특히 바람직하게는 적어도 90%, 추가로 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 동일한 서열에 의하여 코딩된 효소에 관하여 용어 "효소의 활성"이란 항상 "ATP + 스핑가닌 <=> ADP + 스핑가닌 1-포스페이트" 반응을 촉매하는 효소 활성을 의미하는 것으로 이해된다.
당해 활성은 바람직하게는 문헌 [Lanterman and Saba, Biochem J. 1998 Jun 1;332 (Pt 2):525-31]에 기재된 방법으로 측정된다.
서열 9에 의하여 또는 서열 9와 적어도 80%, 특히 바람직하게는 적어도 90%, 추가로 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 동일한 서열에 의하여 코딩된 효소에 관하여 용어 "효소의 활성"이란 항상 "스핑가닌 1-포스페이트 <=> 포스포에탄올아민 + 팔미트알데히드" 반응을 촉매하는 효소 활성을 의미하는 것으로 이해된다.
당해 활성은 바람직하게는 문헌 [Van Veldhoven and Mannaerts, J Biol Chem. 1991 Jul 5;266(19):12502-7]에 기재된 방법으로 측정된다.
서열 11에 의하여 또는 서열 11과 적어도 80%, 특히 바람직하게는 적어도 90%, 추가로 바람직하게는 적어도 95%, 가장 바람직하게는 적어도 99% 동일한 서열에 의하여 코딩된 효소에 관하여 용어 "효소의 활성"이란 해당 효소의 발현율 수준, 특히 세포내 농도를 의미하는 것으로 이해된다. 이는 후술되는 2-D 겔 기술 또는 웨스턴 블롯 방법으로 측정된다.
용어 "그의 야생형과 비교하여 감소한 활성"이란 바람직하게는, 야생형 활성을 기준으로 적어도 50%만큼, 특히 바람직하게는 적어도 90%만큼, 추가로 바람직하게는 적어도 99.9%만큼, 추가로 한층 더 바람직하게는 적어도 99.99%만큼, 가장 바람직하게는 적어도 99.999%만큼 감소한 활성을 의미한다.
본 발명에 따른 세포의 그의 야생형과 비교한 특정 활성의 감소는 가능하면 동등한 세포수/농도, 예를 들어 배지, 가스처리, 교반과 같은 동일한 조건하에서 증식시킨 세포를 사용함으로써 활성을 측정하는 전술한 방법으로 측정한다.
언급한 서열에 관하여 "뉴클레오티드 동일성"은 공지 방법의 도움을 빌려 측정될 수 있다. 일반적으로, 특별한 필요조건을 고려에 넣는 알고리즘을 갖춘 특수 컴퓨터 프로그램이 사용된다.
동일성을 측정하는 바람직한 방법은 맨 먼저 비교하고자 하는 서열간의 최대 일치를 생성한다. 동일성 측정을 위한 컴퓨터 프로그램으로는
- GAP (문헌 [Deveroy, J. et al., Nucleic Acid Research 12 (1984), page 387, Genetics Computer Group University of Wisconsin, Medicine (Wi)]), 및
- BLASTP, BLASTN 및 FASTA (문헌 [Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410])을 포함한 GCG 프로그램 패키지를 포함하며 그들에 한정되지 않는다. BLAST 프로그램은 미국 국립생물공학정보센터 (National Center For Biotechnology Information, NCBI)로부터 및 그 밖의 정보원 (BLAST manual, Altschul S. et al., NCBI NLM NIH Bethesda ND 22894; 및 Altschul S. et al., 상기 참조)으로부터 입수할 수 있다.
공지의 스미스-워터맨(Smith-Waterman) 알고리즘 역시 뉴클레오티드 동일성의 측정에 사용될 수 있다.
BLASTN 프로그램 (문헌 [Altschul, S. et al., Journal of Molecular Biology 215 (1990), pages 403-410]) 사용시 "뉴클레오티드 동일성" 측정을 위한 바람직한 파라미터는 다음과 같다:
기대 역치: 10
단어 길이: 28
매치 스코어: 1
미스매치 스코어: -2
갭 코스트: 선형
상기 파라미터는 뉴클레오티드 서열 비교에서의 디폴트 파라미터이다.
GAP 프로그램은 상기 파라미터와 또한 사용될 수 있다.
상기 알고리즘에 따른 80%의 동일성은 본 발명과 관련하여 80% 동일성을 의미한다. 보다 높은 동일성에도 동일하게 적용된다.
용어 "인트론-무함유 핵산 서열에 의하여 코딩되는"은 본원에서 명시하는 서열을 수반하는 서열 비교는 비교하고자 하는 핵산 서열이 있을지 모르는 인트론을 사전에 제거시킬 것을 요구함을 명백히 표명한다.
달리 명시하지 않는 한, 모든 백분율(%)은 질량 백분율이다.
본 발명에 따라 바람직한 세포는 효소 활성의 감소가 상기에 특정된 핵산 서열 군 A) 및 B)로부터 선택된 임의의 서열을 포함하는 적어도 1종의 유전자를 변형시킴으로써 달성되며, 여기서 변형은 유전자 내로의 외래 DNA 삽입, 유전자의 적어도 일부 결실, 유전자 서열에서의 점 돌연변이, 및 RNA 간섭의 영향에의 유전자 노출, 또는 외래 DNA, 특히 프로모터 영역의 외래 DNA에 의한 유전자의 일부 치환을 포함하는, 바람직하게는 그들로 이루어진 군으로부터 선택됨을 특징으로 하다.
외래 DNA는 이와 관련하여 유전자에 대해 "외래(foregin)"이고 (유기체에 대해서는 외래가 아닌) 임의의 DNA 서열을 의미하는 것으로 이해되며, 즉 심지어는 피키아 시페리이 내생성 DNA 서열조차도 이와 관련하여 "외래 DNA"로서 작용할 수 있다.
이와 관련하여, 유전자는 선택 마커 유전자의 삽입에 의하여 붕괴되는 것이 특히 바람직하고, 따라서 외래 DNA는 선택 마커 유전자, 특히 예를 들어 문헌 [Schorsch et al., 2009; Current Genetics (2009), 55(4), 381-389]에 기재된 바와 같이 스트렙토미세스 노우르세이(Streptomyces noursei) nat1 유전자를 코딩하는 서열을 포함하며, 그 서열은 바람직하게는 피키아 PDA1 프로모터의 서열과 피키아 TEF 터미네이터의 서열에 의하여 플랭킹되는 선택 마커 유전자이며, 스트렙토미세스 노우르세이 nat1 유전자를 코딩하는 서열은 바람직하게는 피키아 시페리이에 대하여 코돈-최적화되며, 상기 삽입은 바람직하게는 유전자좌 내로의 상동 재조합에 의하여 달성된다.
이와 관련하여, 선택 마커 유전자를 추가 기능성에 의하여 연장시키는 것이 유리할 수 있으며 이는 유전자로부터 후속 제거를 가능케 해주며, 이러한 과정은 예를 들어 유기체에 대해 외래인 재조합 시스템, 예컨대 Cre/loxP 시스템 또는 FRT(플립파제 인식 표적) 시스템, 또는 유기체 자신의 상동 재조합 시스템에 의해 달성될 수 있다.
본 발명에 따르면 세포가 그의 야생형과 비교하여, 하기 인트론-무함유 핵산 서열에 의하여 코딩되는 효소의 감소한 활성의 조합을 갖는 것인 바람직하다:
서열 1 또는 그의 B군 유사체;
서열 3 또는 그의 B군 유사체;
서열 5 또는 그의 B군 유사체;
서열 7 또는 그의 B군 유사체;
서열 9 또는 그의 B군 유사체;
서열 11 또는 그의 B군 유사체;
서열 1 또는 그의 B군 유사체 및 서열 3 또는 그의 B군 유사체;
서열 1 또는 그의 B군 유사체 및 서열 5 또는 그의 B군 유사체;
서열 3 또는 그의 B군 유사체 및 서열 5 또는 그의 B군 유사체;
서열 1 또는 그의 B군 유사체 및 서열 3 또는 그의 B군 유사체 및 서열 5 또는 그의 B군 유사체;
서열 1 또는 그의 B군 유사체 및 서열 3 또는 그의 B군 유사체 및 서열 5 또는 그의 B군 유사체 및 서열 7 또는 그의 B군 유사체;
서열 1 또는 그의 B군 유사체 및 서열 3 또는 그의 B군 유사체 및 서열 5 또는 그의 B군 유사체 및 서열 11 또는 그의 B군 유사체;
서열 1 또는 그의 B군 유사체 및 서열 3 또는 그의 B군 유사체 및 서열 5 또는 그의 B군 유사체 및 서열 7 또는 그의 B군 유사체 및 서열 11 또는 그의 B군 유사체.
상기 열거된 조합과 관련하여, A군의 멤버에 의하여 코딩된 효소 활성의 감소가 바람직하다.
본 발명에 따라 바람직한 세포는 피키아 시페리이 세포가 피키아 시페리이 NRRL Y-1031 F-60-10, WO 95/12683의 실시예에 개시된 피키아 시페리이 균주, 및 문헌 [Schorsch et al., 2009, Curr Genet. 55, 381-9]에 기재된 균주 피키아 시페리이 CS.PCΔPro2로 이루어진 군으로부터 선택된 균주로부터 유래함을 특징으로 한다.
본 발명에 따라 바람직한 세포는 세포가 그의 야생형과 비교하여, 세린과 팔미토일-CoA의 반응을 촉매하여 3-케토스핑가닌을 생성하는 효소 E1, 특히 세린 팔미토일 트랜스페라제, 특히 서열 13 및/또는 서열 15에 의하여 코딩되는 것, 및 스핑가닌에서 피토스핑고신으로의 반응을 촉매하는 효소 E2, 특히 스핑가닌 C4-히드록실라제, 특히 서열 17에 의하여 코딩되는 것으로부터 선택되는 적어도 1종의 효소의 증가한 효소 활성을 보유함을 특징으로 한다.
효소 E1에 관하여 용어 "효소의 활성"은 항상 "팔미토일-CoA + L-세린 <=> CoA + 3-데히드로-D-스핑가닌 + CO2 " 반응을 촉매하는 효소 활성을 의미하는 것으로 이해된다.
당해 활성은 바람직하게는 문헌 [Zweerink et al., J Biol Chem. 1992 Dec 15;267(35):25032-8]에 기재된 방법으로 측정된다.
효소 E2에 관하여 용어 "효소의 활성"은 항상 "스핑가닌 + NADPH + H+ + O2 <=> 피토스핑고신 + NADP+ + H2O" 반응을 촉매하는 효소 활성을 의미하는 것으로 이해된다.
당해 활성은 바람직하게는 문헌 [Grilley et al., J Biol Chem. 1998 May 1;273(18):11062-8]에 기재된 방법으로 측정된다.
본 발명과 관련하여 상기에서 및 하기 주석에서 사용되는 용어 "효소의 증가한 활성"은 바람직하게는 증가한 세포내 활성을 의미하는 것으로 이해된다.
세포내 효소 활성의 증가와 관련한 하기 주석은 효소 E1 내지 E2의 활성 증가에 및 필요에 따라 활성을 증가시킬 수 있는 하기에서 특정된 모든 효소에 양측 모두 적용된다.
원칙적으로, 효소 활성의 증가는 효소를 코딩하는 유전자 서열(들)의 카피수를 증가시킴으로써, 강력한 프로모터를 사용함으로써, 유전자의 코돈 사용빈도를 변경함으로써, 다양한 방식으로 mRNA의 또는 효소의 반감기를 증가시킴으로써, 유전자 발현의 조절을 변형함으로써, 또는 증가한 활성을 보유하는 상응하는 효소를 코딩하는 유전자 또는 대립유전자를 사용함으로써, 및 필요에 따라 이들 수단을 조합함으로써 달성될 수 있다. 본 발명에 따라 유전자 변형된 세포는 예를 들어, 목적 유전자, 당해 유전자의 대립유전자 또는 그의 일부 및 그 유전자가 발현될 수 있게 해주는 프로모터를 함유하는 벡터를 가지고서 형질전환, 형질도입, 접합 또는 이들 방법의 조합에 의하여 생성된다. 이종 발현은 특히 유전자 또는 대립유전자를 세포의 염색체 또는 염색체외에서 복제하는 벡터 내로 통합시킴으로써 달성된다.
세포내 효소 활성을 증가시키기 위한 옵션에 관한 개요는 본원에 참고로 포함되고 개시내용이 세포내 효소 활성을 증가시키기 위한 옵션에 관련한 본 발명의 개시내용의 일부를 형성하는 DE-A-100 31 999에 예로서 피루베이트 카르복실라제에 대하여 제공되어 있다.
상기에서 특정된 효소 또는 유전자 및 하기에서 특정된 모든 효소 또는 유전자의 발현은 1차원 및 2차원 단백질 겔 분획화 및 적당한 평가 소프트웨어를 사용하는 겔내 단백질 농도의 후속 광학적 확인의 도움을 빌려 검출가능하다.
효소 활성의 증가가 전적으로 상응하는 유전자의 발현 증가에 기초하는 경우, 상기 효소 활성의 증가는 단순히 야생형 및 유전자 변형 세포의 1차원 또는 2차원 단백질 분획화를 비교함으로써 정량될 수 있다. 박테리아의 경우에 단백질 겔을 제조하고 단백질을 확인하는 통상적인 방법은 문헌 [Hermann et al., Electrophoresis, 22: 1712.23 (2001)]에 기재된 절차이다. 단백질 농도는 검출하고자 하는 단백질에 특이적인 항체와의 웨스턴 블롯 하이브리드화 (문헌 [Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989]) 및 농도 측정을 위한 적당한 소프트웨어를 사용하는 후속 광학적 평가 (문헌 [Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647])에 의하여 또한 분석될 수 있다.
본 발명에 따르면 세포가 그의 야생형과 비교하여
서열 1 또는 그의 B군 유사체 및 서열 3 또는 그의 B군 유사체 및 서열 5 또는 그의 B군 유사체의 조합으로, 또는
서열 1 또는 그의 B군 유사체 및 서열 3 또는 그의 B군 유사체 및 서열 5 또는 그의 B군 유사체 및 서열 7 또는 그의 B군 유사체 및 서열 11 또는 그의 B군 유사체의 조합으로 인트론-무함유 핵산 서열에 의하여 코딩된 효소의 감소한 활성의 조합을 가지는, 그의 야생형과 비교하여 효소 E1의 증가한 효소 활성을 가지는 세포가 바람직하다.
본 발명에 따르면 세포가 그의 야생형과 비교하여 인트론-무함유 핵산 서열:
서열 1 또는 그의 B군 유사체 및 서열 3 또는 그의 B군 유사체 및 서열 5 또는 그의 B군 유사체 및 서열 7 또는 그의 B군 유사체 및 서열 11 또는 그의 B군 유사체에 의하여 코딩된 효소의 감소한 활성의 조합을 가지는, 그의 야생형과 비교하여 효소 E1 및 E2의 증가한 효소 활성을 가지는 세포가 바람직하다.
하나의 별법 실시양태에서, 본 발명에 따른 피키아 시페리이 세포는 예컨대 WO2006048458 및 WO2007131720에 기재되고 추가로 본 발명에 따른 당해 세포에 관하여 상기 기재한 효소 활성의 변화를 보이는 것들이다.
본 발명의 목적 달성에의 추가 기여는 스핑고이드 염기 및 스핑고지질을 생성하는 본 발명에 따른 세포를 사용함으로써 이루어진다.
본 발명과 관련하여 용어 "스핑고이드 염기"는 피토스핑고신, 스핑고신, 스핑가디에닌, 6-히드록시스핑고신 및 스핑가닌(디히드로스핑고신), 또한 아세틸화 형태로, 예컨대 예를 들어 테트라아세틸피토스핑고신, 트리아세틸피토스핑고신, 디아세틸피토스핑고신, O-아세틸피토스핑고신, 트리아세틸스핑가닌, 디아세틸스핑가닌, O-아세틸스핑가닌, 트리아세틸스핑고신, 디아세틸스핑고신, O-아세틸스핑고신, 테트라아세틸-6-히드록시스핑고신, 트리아세틸-6-히드록시스핑고신, 디아세틸-6-히드록시스핑고신, O-아세틸-6-히드록시스핑고신, 트리아세틸스핑가디에닌, 디아세틸스핑가디에닌, O-아세틸스핑가디에닌을 의미하는 것으로 이해된다.
본 발명과 관련하여 용어 "스핑고지질"은 아미드 결합을 통해 지방산에 공유결합한 스핑고이드 염기를 포함하는 화합물을 의미하는 것으로 이해된다. 지방산은 포화되거나 또는 단일불포화- 또는 다불포화될 수 있다. 지방산 측쇄는 길이가 다양할 수 있다. 지방산 측쇄는 히드록시기와 같은 관능기를 또한 보유할 수 있다. 스핑고지질로는 예를 들어, 피토세라미드, 세라미드 및 디히드로세라미드, 및 좀더 복잡한 글루코실세라미드(세레브로시드) 및 이노시톨 포스포릴세라미드, 만노실이노시톨 포스포릴세라미드 및 만노실디이노시톨 포스포릴세라미드가 포함된다. 여기서 스핑고지질은 아미드 결합을 통해 아세틸 라디칼에 결합한 스핑고이드 염기, 예컨대 예를 들어 N-아세틸피토스핑고신, N-아세틸스핑가닌, N-아세틸스핑고신, N-아세틸-6-히드록시스핑고신을 또한 포함한다. 이들 화합물은 단쇄형 세라미드라는 용어로도 알려져 있다.
피토스핑고신, 스핑고신, 스핑가디에닌, 6-히드록시스핑고신, 스핑가닌(디히드로스핑고신), 테트라아세틸피토스핑고신(TAPS), 트리아세틸피토스핑고신, 디아세틸피토스핑고신, O-아세틸피토스핑고신, N-아세틸피토스핑고신, 트리아세틸스핑가닌(TriASa), 디아세틸스핑가닌, O-아세틸스핑가닌, N-아세틸스핑가닌, 트리아세틸스핑고신(TriASo), 디아세틸스핑고신, O-아세틸스핑고신, N-아세틸스핑고신, 테트라아세틸-6-히드록시스핑고신, 트리아세틸-6-히드록시스핑고신, 디아세틸-6-히드록시스핑고신, O-아세틸-6-히드록시스핑고신, N-아세틸-6-히드록시스핑고신, 트리아세틸스핑가디에닌, 디아세틸스핑가디에닌, 및 O-아세틸스핑가디에닌으로 이루어진 군으로부터 선택된 스핑고이드 염기 및 스핑고지질을 제조하기 위한 본 발명에 따른 세포의 용도가 특히 유리하다. 테트라아세틸피토스핑고신(TAPS)을 제조하기 위한 본 발명에 따른 세포의 용도가 매우 특히 바람직하다.
본 발명에 따라 바람직한 용도는 본 발명에 따르면 전술한 바와 같은 본 발명에 따라 바람직한 세포가 사용됨을 특징으로 한다.
특히 전술한 스핑고신과 스핑가닌 유도체의 제조에 사용되는 피키아 시페리이 세포는 예컨대 WO2006048458 및 WO2007131720에 기재되고 추가로 본 발명에 따른 당해 세포에 관하여 상기 기재한 효소 활성의 변화를 보이는 것들이다.
본 발명의 목적 달성에의 추가 기여는 본 발명에 따른 앞서 기재한 세포를 제조하는 방법에 의하여 이루어지며, 상기 방법은
I) 피키아 시페리이 세포를 제공하는 단계, 및
II) 특허청구범위 제1항에 특정된 핵산 서열 군 A) 및 B)로부터 선택된 임의의 서열을 포함하는 적어도 1종의 유전자를,
그 유전자 내로의 외래 DNA, 특히 선택 마커 유전자를 코딩하는 DNA, 바람직하게는 흔적을 남기지 않으면서 제거될 수 있고 표적 유전자에 결실을 남기는 DNA의 삽입,
유전자의 적어도 일부 결실,
유전자 서열에서의 점 돌연변이,
RNA 간섭의 영향에의 유전자 노출, 및
외래 DNA, 특히 프로모터 영역의 외래 DNA에 의한 유전자의 일부 치환에 의하여 변형시키는 단계를 포함한다.
본 발명의 목적 달성에의 추가 기여는 스핑고이드 염기 및 스핑고지질을 제조하는 방법에 의하여 이루어지며, 상기 방법은
a) 본 발명에 따른 세포를, 탄소원을 포함하는 배지와 접촉시키는 단계,
b) 세포가 상기 탄소원으로부터 스핑고이드 염기 및 스핑고지질을 생성할 수 있게 해주는 조건하에서 세포를 배양하는 단계, 및
c) 임의로, 생성된 스핑고이드 염기 및 스핑고지질을 단리하는 단계를 포함한다.
본 발명에 따라 바람직한 방법은 본 발명에 따라 바람직한 것으로 상기에서 특정한 세포를 사용한다.
사용될 수 있는 탄소원은 탄수화물, 예컨대 예를 들어 글루코스, 프룩토스, 글리세롤, 수크로스, 말토스, 당밀, 또는 그 밖의 알콜, 예컨대 예를 들어 에탄올, 및 유기산, 예컨대 예를 들어 아세테이트이다. 사용될 수 있는 질소원은 예를 들어 암모니아, 황산암모늄, 질산암모늄, 염화암모늄, 유기 질소 화합물(예컨대, 효모 추출물, 맥아 추출물, 펩톤, 옥수수침지액)이다. 무기 화합물, 예컨대 예를 들어 인산염, 마그네슘염, 칼륨염, 아연염, 철염 등이 또한 사용될 수 있다.
피키아 시페리이에 대한 적합한 배양 조건은 예를 들어 WO2006048458 및 WO2007131720로부터 당업자에게 공지되어 있다.
본 발명에 따른 방법은 테트라아세틸피토스핑고신(TAPS)의 제조에 특히 적합하다.
이하에 열거한 실시예는 예로서 본 발명을 설명하며, 상기 예에서 특정된 실시양태는 본 발명을 제한하는 것으로 의도되지 않으며, 발명의 용도 범위는 전체의 상세한 설명과 특허청구범위에 따른다.
하기 도면은 실시예의 일부이다:
도 1: 유전자 결실 카세트의 설계 원리
도 2: 과발현 카세트의 설계 원리
도 1: 유전자 결실 카세트의 설계 원리
도 2: 과발현 카세트의 설계 원리
실시예
유전자 결실 카세트의 구축
달리 명시하지 않는 한, 유전자 결실은 문헌 [Rothstein 1983, Methods Enzymol 101: 202-211]에 기재된 바와 같이 고전적인 "원스텝 유전자 치환"에 의하여 수행하였다.
결실 카세트를 생체내 클로닝에 의하여 구축하였으며, 그에 따라 궁극적으로 결실 카세트의 PCR-기반 증폭을 위한 주형으로 사용되는 플라스미드가 생성된다. 이어서 이들 PCR 산물을 특정 유전자의 결실을 목적으로 하는 피키아 시페리이에 형질전환시켰다.
플라스미드 p426HXT7-6HIS (문헌 [Hamacher et al., 2000; Microbiology 148, 2783-8])을 셔틀 벡터로 사용하여 결실 카세트를 구축하였다. p426HXT7-6HIS을 먼저 BamHI 및 EcoRI로 절단하였으며, 그에 따라 5.69 kb 단편이 생성되며 후속 클로닝 단계를 위한 골격으로 사용하였다. 처음에, 각각의 피키아 시페리이 결실 카세트에 대하여 PCR에 의하여 3개의 중첩하는 DNA 단편을 생성하였다: 중심부(nat1 저항성 카세트) (문헌 [Schorsch et al., Curr Genet. 2009 Aug;55(4):381-9] 참조)로서, 나중에 다시 제거될 수 있는 우점하는 clonNAT 마커, 결실시키고자 하는 ORF의 5'-비번역 영역(프로모터 영역, PR)을 나타내며 clonNAT 마커 단편의 개시점까지의 중첩부를 갖는 약 500 bp 길이의 제2 단편, 및 결실시키고자 하는 ORF의 3'-비번역 영역(터미네이터 영역, TR)을 나타내며 clonNAT 마커 단편의 말단까지의 중첩부를 갖는 약 500 bp 길이의 제3 단편.
피키아 시페리이 야생형 게놈 DNA로부터 결실시키고자 하는 유전자의 프로모터 영역(PR)과 터미네이터 영역(TR)을 PCR에 의하여 증폭시킴으로써 각각의 결실 카세트를 구축하였으며, 각각의 경우에 유전자-특이적 프라이머를 사용하였다. 이를 위하여, 프라이머쌍, 즉 PR에 대해 P1/P2 및 TR에 대해 P3/P4를 각각의 경우에 사용하였다. 프라이머는 5' 말단에 융합시키고자 하는 DNA 요소와 중첩하는 약 30-35 bp 길이의 영역을 갖도록 선택하였다:
중심 단편(nat1 저항성 카세트, 서열 19)을 각각의 경우에 프라이머쌍 LPNTL.fw(TGGCGCTTCGTACCACTGGGTAAC)와 LPNTL.rv(GAAATTAATACGACTCACTATAGG)을 사용하여 증폭하였으며, 플라스미드 pCS.LoxP.nat1 (문헌 [Schorsch et al., 2009; Curr. Genet. 55, 381-9])이 주형으로 사용된다(모든 프라이머 서열은 5'→3' 배향으로 나타내었다).
프라이머쌍 P1/P2, P3/P4 및 LPNTL.fw/LPNTL.rv의 PCR 산물을, BamHI 및 EcoRI로 소화시켜 사전에 선형화한 p426HXT7-6HIS 플라스미드와 함께 사카로미세스 세레비지아에(S. cerevisiae) 균주 K26에 형질전환시켰다. PCR 산물과 선형화 벡터를 상동 재조합에 의하여 생체내에서 함께 연결하여, 선형화 벡터를 재-환형화하여 사카로미세스 세레비지아에에서 증식될 수 있게 하였다. 수득된 형질전환체를 clonNAT를 함유한 YEPD 플레이트상에서 마커 유전자(nat1)에 의하여 선택하였으며, 그들의 DNA를 단리하여 이. 콜라이(E. coli)에 형질전환시키고, 그로부터 재-단리한 플라스미드를 제한지도 작성 또는 서열분석에 의하여 확인하였다. 달리 명시하지 않는 한, 프라이머쌍 426L.fw(GCTTCCGGCTCCTATGTTG, 서열 23)와 426R.rv(ACCCTATGCGGTGTGAAATAC, 서열 24) 또는 HXT7(GCCAATACTTCACAATGTTCGAATC, 서열 25)와 CYC(CGTGAATGTAAGCGTGACATAAC, 서열 26)을 사용하여 결실 카세트를 증폭하였다.
명확을 기하기 위하여 도 1을 참조하기 바란다.
다중 유전자를 연속하여 결실시키기 위하여, 각각의 결실 후에 마커 구제를 수행하였다. 이는 이전에 기재된 바와 같은 플라스미드 pCS.opt.Cre(서열 20) (문헌 [Schorsch et al., Curr Genet. 2009 Aug;55(4):381-9])을 가지고 형질전환에 의하여 달성되었다. 유전자 결실은 형질전환체의 게놈 DNA를 주형으로 사용하여 PCR 분석에 의하여 확인하였다.
서열 1, 서열 3, 서열 5, 서열 7, 서열 9 및 서열 11의 서열을 갖는 유전자의 특정 유전자 결실 카세트를 하기 표에 열거된 프라이머를 사용하여 구축하였다. 서열의 각각에 대하여, 열거된 처음 두 프라이머(SH11와 SH12 또는 SH21와 SH22 또는 C1와 C2 또는 HXT7-LCB4.fw와 LCB4.HXT7.rv 또는 HXT7-DPL1.fw와 DPL1.rv2 또는 ORM-426L.fw와 ORM-LPNTL.rv)를 각각의 경우에 PR의 증폭을 위해 사용하였으며, 열거된 그 다음 두 프라이머(SH13와 SH14 또는 SH23와 SH24 또는 C3와 C4 또는 LCB4.rv와 LCB4.fw 또는 DPL1.fw2와 CYC-DPL1.rv 또는 ORM-LPNTL.fw2와 ORM-426R.rv)는 TR의 증폭을 위해 사용된다. 각각의 경우에 열거된 마지막 두 프라이머(SHMT1.pop-in.fw와 SHMT1.veri.rv 또는 SHMT2.pop-in.fw와 SHMT2.veri.rv 또는 CHA1.pop-in.fw와 CHA1.veri.rv 또는 LCB4.pop-in.fw와 LCB4.veri.rv 또는 DPL1.pop-in.fw와 DPL1.veri.rv 또는 ORM1.pop-in.fw와 ORM.veri.rv)를 통합 또는 야생형 대립유전자의 검출에 사용하였다.
과발현 카세트의 구축
원칙적으로 결실 카세트에 대하여 사용된 방법과 동일한 방법으로 과발현 카세트를 구축하였다. 그러나 과발현 카세트의 경우에는, PcTDH3 또는 PcENO1 프로모터의 단편을 나타내는 부가적인 제4 PCR 산물을 생성하였다(프로모터 단편, PF). 이 산물을 나중에 생체내에서 nat1 저항성 카세트 및 제3 PCR 단편(이 경우에 과발현시키고자 하는 ORF의 개시부와의 중첩부를 보유하였다)에 연결하였다(도 2 참조).
유전자 산물 서열 13의 과발현을 위해, 피키아 시페리이 내 천연 프로모터를 PcENO1 -584-1(서열 21) 프로모터 단편으로 치환하였다. 이와는 대조적으로, 유전자 산물 서열 15 및 서열 17의 과발현을 위해서는 피키아 시페리이 내 그 특정 천연 프로모터를 PcTDH3 -420-1 프로모터 단편(서열 22)으로 치환하였다.
원칙적으로, 3종류의 상이한 유전자-특이적 프라이머쌍을 특정 과발현 카세트의 구축에 사용하였다. 프라이머는 5' 말단에 융합시키고자 하는 DNA 요소와 중첩하는 약 30-35 bp 길이의 영역을 보유하도록 선택하였다.
nat1 저항성 카세트는 각각의 경우에 프라이머쌍 LPNTL.fw와 LPNTL.rv을 사용하여 증폭하였으며, 플라스미드 pCS.LoxP.nat1 (문헌 [Schorsch et al., 2009; Curr. Genet. 55, 381-9])을 주형으로 사용한다. 프라이머쌍 P5/P6, P7/P8, P9/P10 및 LPNTL.fw/LPNTL.rv의 PCR 산물을, HpaI 및 NgoMIV로 소화시켜 사전에 선형화한 p426HXT7-6HIS 플라스미드와 함께 사카로미세스 세레비지아에 균주 K26에 형질전환시켰다. PCR 산물과 선형화 벡터를 상동 재조합에 의하여 생체내에서 함께 연결하여, 선형화 벡터를 재-환형화하여 사카로미세스 세레비지아에에서 증식될 수 있게 하였다. 수득된 형질전환체를 clonNAT를 함유한 YEPD 플레이트상에서 마커 유전자(nat1)에 의하여 선택하였으며, 그들의 DNA를 단리하여 이. 콜라이에 형질전환시키고, 그로부터 재-단리한 플라스미드를 제한지도 작성 또는 서열분석에 의하여 확인하였다. 각각의 경우에 프라이머쌍 "426L.fw & 426R.rv"을 사용하여 과발현 카세트를 증폭하였다.
명확을 기하기 위하여 도 2를 참조하기 바란다.
다중 유전자의 조합 과발현을 위하여, 또는 1종 이상의 표적 유전자의 과발현을 하나 이상의 유전자 결실과 조합하기 위하여, 각각의 단계(표적 유전자의 결실 또는 과발현 카세트의 염색체 통합) 후에 마커 구제를 수행하였다. 이는 이전에 기재된 바와 같은 플라스미드 pCS.opt.Cre (문헌 [Schorsch et al., Curr Genet. 2009 Aug;55(4):381-9])을 가지고 형질전환에 의하여 달성하였다. 과발현 카세트의 통합은 형질전환체의 게놈 DNA를 주형으로 사용하여 PCR 분석에 의하여 확인하였다.
서열 13, 서열 15 및 서열 17의 서열에 의하여 코딩된 효소에 대한 특정 과발현 카세트는 하기 표에 열거된 프라이머를 사용하여 구축하였다. 서열의 각각에 대하여, 열거된 처음 두 프라이머(LCB1.426L.fw와 LCB1.LPNTL.rv 또는 LCB2-426L.fw와 LCB2-LPNTL.rv 또는 SYR2oe.426L와 SYR2oe.LPNTL.rv)를 각각의 경우에 PR의 증폭을 위해 사용하였다. 열거된 그 다음 두 프라이머(P-ENO.LPNTL.fw와 LCB1.P-ENO.rv 또는 TDH3-LPNTL.fw와 P-TDH3.rv 또는 TDH3-LPNTL.fw와 P-TDH3.rv)는 특정 PcENO1 -584-1 또는 PcTDH3 -420-1 프로모터 단편의 증폭을 위해 사용하였다. 열거된 그 다음 두 프라이머(P-ENO.LCB1.fw와 LCB1.426R.rv 또는 LCB2.P-TDH3.fw와 LCB2-426R.rv 또는 SYR2oe.P-TDH3.fw와 SYR2oe.426R)는 각각의 경우에 과발현시키고자 하는 표적 유전자의 5'-ORF 단편의 증폭을 위해 사용하였다. 각각의 경우에 열거된 마지막 두 프라이머(P-ENO.veri.rv와 LCB1uee.veri.rv 또는 P-TDH3.pop.fw와 LCB2uee.veri.rv 또는 P-TDH3.pop.fw와 SYR2oe.veri.rv)는 통합 또는 야생형 대립유전자의 검출에 사용하였다.
유전자 변형 균주에 의한
아세틸화
스핑고이드
염기의 생성
아세틸화 스핑고이드 염기의 증가한 역가는 하기 유전자 변형에 의하여 달성되었다:
하기의 표는 진탕 플라스크에서 정지기로의 증식 후 상이한 재조합 피키아 시페리이 균주의 아세틸화 스핑고이드 염기(테트라아세틸피토스핑고신, TAPS 및 임의로는 트리아세틸스핑가닌, TriASa)의 역가를 보여준다.
세부사항(사용되는 배지, 성장조건, 추출, HPLC 분석에 의한 정량)은 문헌 [Schorsch et al., Curr Genet. 2009 Aug;55(4):381-9]에 기재되어 있다. 본 출원에 사용되는 균주는 상기 참조문헌에서 명시한 피키아 시페리이 CS.PCΔPro2에 해당하며, 이는 이하에서 "CS"로도 약칭한다.
먼저, 다양한 유전자의 결실이 아세틸화 스핑고이드 염기의 생성에 미치는 영향을 조사하였다. 결과를 하기 표에 나타내었다. 개별적으로, 특히 PcSHM2의 결실이 아세틸화 스핑고이드 염기의 생성을 현저히 증가시키는 것으로 나타났다. 이러한 효과는 PcSHM1 결실과의 조합에 의하여 추가 증진되었다. PcCHA1의 부가적인 결실에 의하여 추가 증진이 달성되었다. cha1 shm1 shm2의 관련 유전자형을 보유한 당해 균주는 64 mg의 TAPS * g-1(CDW) + 3 mg의 TriASa * g-1(CDW)의 단연 최고 역가를 산출하였다.
다음, 효소 활성 증진을 위한 다양한 유전자 변형의 영향을 균주 CS.CSS(cha1 shm1 shm2)의 배경에서 조사하였다. 이러한 목적을 위하여, 개별적 및 선택된 조합 둘 모두에 의한 하기 유전자 변형을 균주 CS.CSS에서 수행하였다:
PcLCB4, 서열 7의 결실
PcDPL1, 서열 9의 결실
PcORM12, 서열 11의 결실
PcLCB1, 서열 13의 과발현
PcLCB2, 서열 15의 과발현
PcSYR2, 서열 17의 과발현.
또한, PcLCB4 및 PcDPL1 결실의 영향을 또한 단독으로, 즉 cha1 shm1 shm2 유전자형과의 조합 없이 역점을 두어 다루었다.
상가 효과 또는 상승작용 효과를 달성하기 위하여, 스핑고이드 염기 생성을 촉진하는 다수의 유전자 변형을 단일 균주에서 상이한 방식으로 조합하였다. 하기 유전자형을 보유한 균주가 여기서 최적인 것으로 판명되었다:
cha1 shm1 shm2 lcb4 orm12 TDH3p : LCB2 ENO1p : LCB1 TDH3p : SYR2.
당해 균주는 진탕 플라스크에서 199 mg의 TAPS * g-1(CDW) (+ 12 mg의 트리아세틸스핑가닌(TriASa) * g-1(CDW))의 역가를 생성한 반면에, CS 참조 균주는 겨우 21 mg의 TAPS * g-1(CDW)을 생성하였다.
결과를 하기 표에 나타내었다.
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atg cca tac gct tta cca gaa tct cac aga caa tta gtc gaa ggt cat 48
Met Pro Tyr Ala Leu Pro Glu Ser His Arg Gln Leu Val Glu Gly His
1 5 10 15
tta aaa gat acc gat cca gaa gtt gaa caa atc att aaa gat gaa att 96
Leu Lys Asp Thr Asp Pro Glu Val Glu Gln Ile Ile Lys Asp Glu Ile
20 25 30
gaa cgt caa aga cat tca atc gtc tta att gca tca gaa aat ttc act 144
Glu Arg Gln Arg His Ser Ile Val Leu Ile Ala Ser Glu Asn Phe Thr
35 40 45
tca act gct gtt ttc gat gct tta gga act cca atg tgt aat aaa tat 192
Ser Thr Ala Val Phe Asp Ala Leu Gly Thr Pro Met Cys Asn Lys Tyr
50 55 60
tct gaa ggt tat cca ggt gca aga tat tat ggt ggt aat gaa cat att 240
Ser Glu Gly Tyr Pro Gly Ala Arg Tyr Tyr Gly Gly Asn Glu His Ile
65 70 75 80
gat aga att gaa atc tta tgt caa gaa aga gct tta aaa gct ttt aat 288
Asp Arg Ile Glu Ile Leu Cys Gln Glu Arg Ala Leu Lys Ala Phe Asn
85 90 95
atc act tct gat aaa tgg ggg gtt aat gtt caa act ctt tct ggg tct 336
Ile Thr Ser Asp Lys Trp Gly Val Asn Val Gln Thr Leu Ser Gly Ser
100 105 110
cct gct aat tta caa gtt tat caa gct att atg aaa cct cat gaa aga 384
Pro Ala Asn Leu Gln Val Tyr Gln Ala Ile Met Lys Pro His Glu Arg
115 120 125
tta atg ggt ctt gat tta cct cat ggt ggt cat tta tct cat ggt tat 432
Leu Met Gly Leu Asp Leu Pro His Gly Gly His Leu Ser His Gly Tyr
130 135 140
caa act gat act aga aaa atc tct gct gtt tca act tat ttt gaa act 480
Gln Thr Asp Thr Arg Lys Ile Ser Ala Val Ser Thr Tyr Phe Glu Thr
145 150 155 160
atg cct tat aga gtt gat tta gaa act ggt att att gat tat gat acc 528
Met Pro Tyr Arg Val Asp Leu Glu Thr Gly Ile Ile Asp Tyr Asp Thr
165 170 175
tta gaa aag aat gcc tta tta ttc aga cct aag gtc ctt gtt gct ggt 576
Leu Glu Lys Asn Ala Leu Leu Phe Arg Pro Lys Val Leu Val Ala Gly
180 185 190
act tct gct tat tgt aga tta att gat tat aaa aga atg aga gaa att 624
Thr Ser Ala Tyr Cys Arg Leu Ile Asp Tyr Lys Arg Met Arg Glu Ile
195 200 205
gct gat aaa gtt ggt gct tat tta gtt gtt gat atg gct cat att tca 672
Ala Asp Lys Val Gly Ala Tyr Leu Val Val Asp Met Ala His Ile Ser
210 215 220
ggt tta atc gct gct ggt gtt atc cca tct cca ttt gaa tat gct gat 720
Gly Leu Ile Ala Ala Gly Val Ile Pro Ser Pro Phe Glu Tyr Ala Asp
225 230 235 240
att gtc act aca act aca cat aaa tcc cta aga ggt cca aga ggt gcc 768
Ile Val Thr Thr Thr Thr His Lys Ser Leu Arg Gly Pro Arg Gly Ala
245 250 255
atg att ttc ttt aga aga ggt gtt aga tca att aac gct aaa act ggt 816
Met Ile Phe Phe Arg Arg Gly Val Arg Ser Ile Asn Ala Lys Thr Gly
260 265 270
gct gaa att aaa tat gat tta gaa aat cca att aat ttc tca gtt ttc 864
Ala Glu Ile Lys Tyr Asp Leu Glu Asn Pro Ile Asn Phe Ser Val Phe
275 280 285
cca ggt cat caa ggt ggt cca cat aat cat act att acc gcg tta gca 912
Pro Gly His Gln Gly Gly Pro His Asn His Thr Ile Thr Ala Leu Ala
290 295 300
aca gca tta aaa caa gct tca act cca gaa ttt aaa caa tat caa gaa 960
Thr Ala Leu Lys Gln Ala Ser Thr Pro Glu Phe Lys Gln Tyr Gln Glu
305 310 315 320
caa gtt tta aaa aat gct aaa gct tta gaa gaa gaa ttc tta aaa tta 1008
Gln Val Leu Lys Asn Ala Lys Ala Leu Glu Glu Glu Phe Leu Lys Leu
325 330 335
tct tat aaa tta gtt tca aat ggt act gat tct cat atg gtt tta gtt 1056
Ser Tyr Lys Leu Val Ser Asn Gly Thr Asp Ser His Met Val Leu Val
340 345 350
tca tta aaa gat aaa ggt atc gat ggt gca aga att gaa acc gtt tgt 1104
Ser Leu Lys Asp Lys Gly Ile Asp Gly Ala Arg Ile Glu Thr Val Cys
355 360 365
gaa aac ata aac att gcc tta aac aaa aac tca atc cca ggt gat aaa 1152
Glu Asn Ile Asn Ile Ala Leu Asn Lys Asn Ser Ile Pro Gly Asp Lys
370 375 380
tcc gct ctt gtg cca ggt ggt att aga att ggt gca cca gca atg tct 1200
Ser Ala Leu Val Pro Gly Gly Ile Arg Ile Gly Ala Pro Ala Met Ser
385 390 395 400
aca aga ggt ctt ggt gaa gaa gat ttt aaa aaa att gca cat tat att 1248
Thr Arg Gly Leu Gly Glu Glu Asp Phe Lys Lys Ile Ala His Tyr Ile
405 410 415
gat tgg tct gtt caa tat gct aaa aaa att caa agt gaa tta cca aaa 1296
Asp Trp Ser Val Gln Tyr Ala Lys Lys Ile Gln Ser Glu Leu Pro Lys
420 425 430
gaa gct aat aga tta aaa gat ttt aaa gct aag att gct caa ggt tct 1344
Glu Ala Asn Arg Leu Lys Asp Phe Lys Ala Lys Ile Ala Gln Gly Ser
435 440 445
gat gaa tta act aaa act aag aat gaa att tat gaa tgg gct ggt gaa 1392
Asp Glu Leu Thr Lys Thr Lys Asn Glu Ile Tyr Glu Trp Ala Gly Glu
450 455 460
ttc cca tta tct gtt taa 1410
Phe Pro Leu Ser Val
465
<210> 4
<211> 469
<212> PRT
<213> Pichia ciferrii
<400> 4
Met Pro Tyr Ala Leu Pro Glu Ser His Arg Gln Leu Val Glu Gly His
1 5 10 15
Leu Lys Asp Thr Asp Pro Glu Val Glu Gln Ile Ile Lys Asp Glu Ile
20 25 30
Glu Arg Gln Arg His Ser Ile Val Leu Ile Ala Ser Glu Asn Phe Thr
35 40 45
Ser Thr Ala Val Phe Asp Ala Leu Gly Thr Pro Met Cys Asn Lys Tyr
50 55 60
Ser Glu Gly Tyr Pro Gly Ala Arg Tyr Tyr Gly Gly Asn Glu His Ile
65 70 75 80
Asp Arg Ile Glu Ile Leu Cys Gln Glu Arg Ala Leu Lys Ala Phe Asn
85 90 95
Ile Thr Ser Asp Lys Trp Gly Val Asn Val Gln Thr Leu Ser Gly Ser
100 105 110
Pro Ala Asn Leu Gln Val Tyr Gln Ala Ile Met Lys Pro His Glu Arg
115 120 125
Leu Met Gly Leu Asp Leu Pro His Gly Gly His Leu Ser His Gly Tyr
130 135 140
Gln Thr Asp Thr Arg Lys Ile Ser Ala Val Ser Thr Tyr Phe Glu Thr
145 150 155 160
Met Pro Tyr Arg Val Asp Leu Glu Thr Gly Ile Ile Asp Tyr Asp Thr
165 170 175
Leu Glu Lys Asn Ala Leu Leu Phe Arg Pro Lys Val Leu Val Ala Gly
180 185 190
Thr Ser Ala Tyr Cys Arg Leu Ile Asp Tyr Lys Arg Met Arg Glu Ile
195 200 205
Ala Asp Lys Val Gly Ala Tyr Leu Val Val Asp Met Ala His Ile Ser
210 215 220
Gly Leu Ile Ala Ala Gly Val Ile Pro Ser Pro Phe Glu Tyr Ala Asp
225 230 235 240
Ile Val Thr Thr Thr Thr His Lys Ser Leu Arg Gly Pro Arg Gly Ala
245 250 255
Met Ile Phe Phe Arg Arg Gly Val Arg Ser Ile Asn Ala Lys Thr Gly
260 265 270
Ala Glu Ile Lys Tyr Asp Leu Glu Asn Pro Ile Asn Phe Ser Val Phe
275 280 285
Pro Gly His Gln Gly Gly Pro His Asn His Thr Ile Thr Ala Leu Ala
290 295 300
Thr Ala Leu Lys Gln Ala Ser Thr Pro Glu Phe Lys Gln Tyr Gln Glu
305 310 315 320
Gln Val Leu Lys Asn Ala Lys Ala Leu Glu Glu Glu Phe Leu Lys Leu
325 330 335
Ser Tyr Lys Leu Val Ser Asn Gly Thr Asp Ser His Met Val Leu Val
340 345 350
Ser Leu Lys Asp Lys Gly Ile Asp Gly Ala Arg Ile Glu Thr Val Cys
355 360 365
Glu Asn Ile Asn Ile Ala Leu Asn Lys Asn Ser Ile Pro Gly Asp Lys
370 375 380
Ser Ala Leu Val Pro Gly Gly Ile Arg Ile Gly Ala Pro Ala Met Ser
385 390 395 400
Thr Arg Gly Leu Gly Glu Glu Asp Phe Lys Lys Ile Ala His Tyr Ile
405 410 415
Asp Trp Ser Val Gln Tyr Ala Lys Lys Ile Gln Ser Glu Leu Pro Lys
420 425 430
Glu Ala Asn Arg Leu Lys Asp Phe Lys Ala Lys Ile Ala Gln Gly Ser
435 440 445
Asp Glu Leu Thr Lys Thr Lys Asn Glu Ile Tyr Glu Trp Ala Gly Glu
450 455 460
Phe Pro Leu Ser Val
465
<210> 5
<211> 1029
<212> DNA
<213> Pichia ciferrii
<220>
<221> CDS
<222> (1)..(1029)
<400> 5
atg aca atc aca aaa gat cat aaa gtc cca tac atc aag act cca tta 48
Met Thr Ile Thr Lys Asp His Lys Val Pro Tyr Ile Lys Thr Pro Leu
1 5 10 15
gtt gat tgt aaa gaa cta tca gaa caa tca cca tgt aga ata ttc cta 96
Val Asp Cys Lys Glu Leu Ser Glu Gln Ser Pro Cys Arg Ile Phe Leu
20 25 30
aag caa gaa ttc att caa cca tcg ggt tct tac aaa ata cgt gga ctt 144
Lys Gln Glu Phe Ile Gln Pro Ser Gly Ser Tyr Lys Ile Arg Gly Leu
35 40 45
tca aat tta att aga act tca att gaa gaa att aaa tca aat cct aat 192
Ser Asn Leu Ile Arg Thr Ser Ile Glu Glu Ile Lys Ser Asn Pro Asn
50 55 60
aat ttg ggt aaa aca att cat gtt tat gct gct tct ggt ggt aat gct 240
Asn Leu Gly Lys Thr Ile His Val Tyr Ala Ala Ser Gly Gly Asn Ala
65 70 75 80
ggt aat gct gtc tct tgt gct tct caa ttt tat gga tta gaa tca aca 288
Gly Asn Ala Val Ser Cys Ala Ser Gln Phe Tyr Gly Leu Glu Ser Thr
85 90 95
gtt gtt ata cca aaa gct aca agt gat aaa atg aag caa aaa atc ttt 336
Val Val Ile Pro Lys Ala Thr Ser Asp Lys Met Lys Gln Lys Ile Phe
100 105 110
aaa aat gga tca aaa ata att gtt caa ggt gaa act att ggt gaa gct 384
Lys Asn Gly Ser Lys Ile Ile Val Gln Gly Glu Thr Ile Gly Glu Ala
115 120 125
gca att tat tta aaa gat gtc tta atc cct tca tta gat gat tct att 432
Ala Ile Tyr Leu Lys Asp Val Leu Ile Pro Ser Leu Asp Asp Ser Ile
130 135 140
ata cct atc tat tgt cat cct tat gat atc cca gct ata tgg cat ggt 480
Ile Pro Ile Tyr Cys His Pro Tyr Asp Ile Pro Ala Ile Trp His Gly
145 150 155 160
cat tct tct att ata gat gaa att gtt gat caa ttg gcc tct tca aat 528
His Ser Ser Ile Ile Asp Glu Ile Val Asp Gln Leu Ala Ser Ser Asn
165 170 175
gaa tta tca aaa tta aaa ggt att gtt tgt tca att ggt ggt ggt gga 576
Glu Leu Ser Lys Leu Lys Gly Ile Val Cys Ser Ile Gly Gly Gly Gly
180 185 190
ctt tat aat ggt tta gtt caa ggt tta caa aga aat caa tta tca aaa 624
Leu Tyr Asn Gly Leu Val Gln Gly Leu Gln Arg Asn Gln Leu Ser Lys
195 200 205
att cca ata atg act tta gaa aca gat act tgt cca act ttc cat gaa 672
Ile Pro Ile Met Thr Leu Glu Thr Asp Thr Cys Pro Thr Phe His Glu
210 215 220
tct att aaa gca caa aaa caa gta ttc att aaa aaa acc aat aca att 720
Ser Ile Lys Ala Gln Lys Gln Val Phe Ile Lys Lys Thr Asn Thr Ile
225 230 235 240
gca att tct tta gct tgt cct tat gtc tct ttg aaa act ctt gaa tat 768
Ala Ile Ser Leu Ala Cys Pro Tyr Val Ser Leu Lys Thr Leu Glu Tyr
245 250 255
tat aat tct cac aag act aag aat tta tta gtt agt gat tct gat gct 816
Tyr Asn Ser His Lys Thr Lys Asn Leu Leu Val Ser Asp Ser Asp Ala
260 265 270
gca aat tct tgt tta aat ttt gca aat gaa ttt aat att ata gtg gaa 864
Ala Asn Ser Cys Leu Asn Phe Ala Asn Glu Phe Asn Ile Ile Val Glu
275 280 285
cct gct tgt gga gtt gct ttg tgc agt gtt tat aat aat ttg att caa 912
Pro Ala Cys Gly Val Ala Leu Cys Ser Val Tyr Asn Asn Leu Ile Gln
290 295 300
aaa aat att gaa ttt ttt gat gat tta aaa tct gat gat att gtg gtt 960
Lys Asn Ile Glu Phe Phe Asp Asp Leu Lys Ser Asp Asp Ile Val Val
305 310 315 320
att att gtt tgt ggt ggg agt tca aca acc gtt caa gat tta aca aat 1008
Ile Ile Val Cys Gly Gly Ser Ser Thr Thr Val Gln Asp Leu Thr Asn
325 330 335
tat aaa cta ctc tat cat tag 1029
Tyr Lys Leu Leu Tyr His
340
<210> 6
<211> 342
<212> PRT
<213> Pichia ciferrii
<400> 6
Met Thr Ile Thr Lys Asp His Lys Val Pro Tyr Ile Lys Thr Pro Leu
1 5 10 15
Val Asp Cys Lys Glu Leu Ser Glu Gln Ser Pro Cys Arg Ile Phe Leu
20 25 30
Lys Gln Glu Phe Ile Gln Pro Ser Gly Ser Tyr Lys Ile Arg Gly Leu
35 40 45
Ser Asn Leu Ile Arg Thr Ser Ile Glu Glu Ile Lys Ser Asn Pro Asn
50 55 60
Asn Leu Gly Lys Thr Ile His Val Tyr Ala Ala Ser Gly Gly Asn Ala
65 70 75 80
Gly Asn Ala Val Ser Cys Ala Ser Gln Phe Tyr Gly Leu Glu Ser Thr
85 90 95
Val Val Ile Pro Lys Ala Thr Ser Asp Lys Met Lys Gln Lys Ile Phe
100 105 110
Lys Asn Gly Ser Lys Ile Ile Val Gln Gly Glu Thr Ile Gly Glu Ala
115 120 125
Ala Ile Tyr Leu Lys Asp Val Leu Ile Pro Ser Leu Asp Asp Ser Ile
130 135 140
Ile Pro Ile Tyr Cys His Pro Tyr Asp Ile Pro Ala Ile Trp His Gly
145 150 155 160
His Ser Ser Ile Ile Asp Glu Ile Val Asp Gln Leu Ala Ser Ser Asn
165 170 175
Glu Leu Ser Lys Leu Lys Gly Ile Val Cys Ser Ile Gly Gly Gly Gly
180 185 190
Leu Tyr Asn Gly Leu Val Gln Gly Leu Gln Arg Asn Gln Leu Ser Lys
195 200 205
Ile Pro Ile Met Thr Leu Glu Thr Asp Thr Cys Pro Thr Phe His Glu
210 215 220
Ser Ile Lys Ala Gln Lys Gln Val Phe Ile Lys Lys Thr Asn Thr Ile
225 230 235 240
Ala Ile Ser Leu Ala Cys Pro Tyr Val Ser Leu Lys Thr Leu Glu Tyr
245 250 255
Tyr Asn Ser His Lys Thr Lys Asn Leu Leu Val Ser Asp Ser Asp Ala
260 265 270
Ala Asn Ser Cys Leu Asn Phe Ala Asn Glu Phe Asn Ile Ile Val Glu
275 280 285
Pro Ala Cys Gly Val Ala Leu Cys Ser Val Tyr Asn Asn Leu Ile Gln
290 295 300
Lys Asn Ile Glu Phe Phe Asp Asp Leu Lys Ser Asp Asp Ile Val Val
305 310 315 320
Ile Ile Val Cys Gly Gly Ser Ser Thr Thr Val Gln Asp Leu Thr Asn
325 330 335
Tyr Lys Leu Leu Tyr His
340
<210> 7
<211> 1530
<212> DNA
<213> Pichia ciferrii
<220>
<221> CDS
<222> (1)..(1530)
<400> 7
atg ccg agt ttt gac tct caa aga att aaa ctg atg gat aca gta tca 48
Met Pro Ser Phe Asp Ser Gln Arg Ile Lys Leu Met Asp Thr Val Ser
1 5 10 15
gta aac ccc cca aga gcc atc att ggt gat aca ggt atc atc att aaa 96
Val Asn Pro Pro Arg Ala Ile Ile Gly Asp Thr Gly Ile Ile Ile Lys
20 25 30
gat caa tct tca ttc tat tac aat caa cat gat aac gct tca ttt tca 144
Asp Gln Ser Ser Phe Tyr Tyr Asn Gln His Asp Asn Ala Ser Phe Ser
35 40 45
tct tgc tta agt tgc tct tca tca aac tcg aat ggt acg gtg aaa tct 192
Ser Cys Leu Ser Cys Ser Ser Ser Asn Ser Asn Gly Thr Val Lys Ser
50 55 60
tca ggt cca aaa cat att cca ttt gtt gat ata ttg agt gtt cga tat 240
Ser Gly Pro Lys His Ile Pro Phe Val Asp Ile Leu Ser Val Arg Tyr
65 70 75 80
ata aat gaa aat aat gaa tct tta tta gaa gct gga agt tcc act gtg 288
Ile Asn Glu Asn Asn Glu Ser Leu Leu Glu Ala Gly Ser Ser Thr Val
85 90 95
act agt gat gaa cct gat gtc gaa gtg gta ttt gtt aga caa aag ggt 336
Thr Ser Asp Glu Pro Asp Val Glu Val Val Phe Val Arg Gln Lys Gly
100 105 110
aaa act ctt gta cca act cca ata ata tta tca ata gat act tta ggt 384
Lys Thr Leu Val Pro Thr Pro Ile Ile Leu Ser Ile Asp Thr Leu Gly
115 120 125
cat gat gat gtt gta caa gaa att tgg aga tta agt tat caa gga aca 432
His Asp Asp Val Val Gln Glu Ile Trp Arg Leu Ser Tyr Gln Gly Thr
130 135 140
aaa cca aga aaa tca ata tta gtt ctt gtt aat cca cat ggt ggg aaa 480
Lys Pro Arg Lys Ser Ile Leu Val Leu Val Asn Pro His Gly Gly Lys
145 150 155 160
ggt aaa gct ata aat tca ttc tta act caa tca aaa cct gta tta att 528
Gly Lys Ala Ile Asn Ser Phe Leu Thr Gln Ser Lys Pro Val Leu Ile
165 170 175
ggt gct caa gct tct gtt gaa gtt aga cat act caa tat tat caa cat 576
Gly Ala Gln Ala Ser Val Glu Val Arg His Thr Gln Tyr Tyr Gln His
180 185 190
gct aca gat att gca cgc act ttg aat att gat aaa tat gat ata att 624
Ala Thr Asp Ile Ala Arg Thr Leu Asn Ile Asp Lys Tyr Asp Ile Ile
195 200 205
gca tgt gct tca ggt gat ggt gtc cca cat gaa gtc ttg aat gga ttt 672
Ala Cys Ala Ser Gly Asp Gly Val Pro His Glu Val Leu Asn Gly Phe
210 215 220
tat caa aga tct gat aga gct gaa gct ttc aat aag att aca ata act 720
Tyr Gln Arg Ser Asp Arg Ala Glu Ala Phe Asn Lys Ile Thr Ile Thr
225 230 235 240
caa tta cca tgt ggt tca ggt aat gca atg agt gaa tca tgt cat ggt 768
Gln Leu Pro Cys Gly Ser Gly Asn Ala Met Ser Glu Ser Cys His Gly
245 250 255
aca aat aat cca agt ttt gcc gct cta tca tta ttg aaa tca agt acg 816
Thr Asn Asn Pro Ser Phe Ala Ala Leu Ser Leu Leu Lys Ser Ser Thr
260 265 270
gta aat tta gat tta atg gct tgt aca caa ggt gat aaa act tat gtt 864
Val Asn Leu Asp Leu Met Ala Cys Thr Gln Gly Asp Lys Thr Tyr Val
275 280 285
tca ttc tta agt caa act gtc ggt gtt ata gca gat tct gat att ggt 912
Ser Phe Leu Ser Gln Thr Val Gly Val Ile Ala Asp Ser Asp Ile Gly
290 295 300
act gaa gca ctt aga tgg tta ggt cct tca aga ttt gaa tta ggt gtt 960
Thr Glu Ala Leu Arg Trp Leu Gly Pro Ser Arg Phe Glu Leu Gly Val
305 310 315 320
gct tat aaa gtt tta tca aga tca aga tat cca tgt gat ata tct gtt 1008
Ala Tyr Lys Val Leu Ser Arg Ser Arg Tyr Pro Cys Asp Ile Ser Val
325 330 335
aaa tat gct gca aaa tcg aaa aat gaa tta aga caa cat ttt gat gaa 1056
Lys Tyr Ala Ala Lys Ser Lys Asn Glu Leu Arg Gln His Phe Asp Glu
340 345 350
cat tcc act att gtt tca aca aaa gat atc caa ata act gaa gat act 1104
His Ser Thr Ile Val Ser Thr Lys Asp Ile Gln Ile Thr Glu Asp Thr
355 360 365
tat aat tta aaa tat gat cca aat ggt cca ata cct gat gat tgg gaa 1152
Tyr Asn Leu Lys Tyr Asp Pro Asn Gly Pro Ile Pro Asp Asp Trp Glu
370 375 380
gag att gat aaa gat ctt tca gaa aat tta ggt att ttc tat aca ggt 1200
Glu Ile Asp Lys Asp Leu Ser Glu Asn Leu Gly Ile Phe Tyr Thr Gly
385 390 395 400
aaa atg cca tat att gca aaa gat gtt caa ttt ttc cct gca gct tta 1248
Lys Met Pro Tyr Ile Ala Lys Asp Val Gln Phe Phe Pro Ala Ala Leu
405 410 415
cca aat gat ggt act ttt gat tta gtt ata aca gat gct cgt aca agt 1296
Pro Asn Asp Gly Thr Phe Asp Leu Val Ile Thr Asp Ala Arg Thr Ser
420 425 430
ata gca cgt atg gca cca act tta tta tca tta gat caa ggt tct cat 1344
Ile Ala Arg Met Ala Pro Thr Leu Leu Ser Leu Asp Gln Gly Ser His
435 440 445
gtt tta caa cca gaa gtt caa cat tct aaa ata ata gca tat aga tta 1392
Val Leu Gln Pro Glu Val Gln His Ser Lys Ile Ile Ala Tyr Arg Leu
450 455 460
act cca aag cag caa cat ggt tat tta agt gtt gat ggt gaa agt tat 1440
Thr Pro Lys Gln Gln His Gly Tyr Leu Ser Val Asp Gly Glu Ser Tyr
465 470 475 480
cca ttt gaa act att caa gtt gaa att cta ccc ggt gct gca aag act 1488
Pro Phe Glu Thr Ile Gln Val Glu Ile Leu Pro Gly Ala Ala Lys Thr
485 490 495
tta cta aga aat ggt act tat gtt gaa aca aat ttt tat tga 1530
Leu Leu Arg Asn Gly Thr Tyr Val Glu Thr Asn Phe Tyr
500 505
<210> 8
<211> 509
<212> PRT
<213> Pichia ciferrii
<400> 8
Met Pro Ser Phe Asp Ser Gln Arg Ile Lys Leu Met Asp Thr Val Ser
1 5 10 15
Val Asn Pro Pro Arg Ala Ile Ile Gly Asp Thr Gly Ile Ile Ile Lys
20 25 30
Asp Gln Ser Ser Phe Tyr Tyr Asn Gln His Asp Asn Ala Ser Phe Ser
35 40 45
Ser Cys Leu Ser Cys Ser Ser Ser Asn Ser Asn Gly Thr Val Lys Ser
50 55 60
Ser Gly Pro Lys His Ile Pro Phe Val Asp Ile Leu Ser Val Arg Tyr
65 70 75 80
Ile Asn Glu Asn Asn Glu Ser Leu Leu Glu Ala Gly Ser Ser Thr Val
85 90 95
Thr Ser Asp Glu Pro Asp Val Glu Val Val Phe Val Arg Gln Lys Gly
100 105 110
Lys Thr Leu Val Pro Thr Pro Ile Ile Leu Ser Ile Asp Thr Leu Gly
115 120 125
His Asp Asp Val Val Gln Glu Ile Trp Arg Leu Ser Tyr Gln Gly Thr
130 135 140
Lys Pro Arg Lys Ser Ile Leu Val Leu Val Asn Pro His Gly Gly Lys
145 150 155 160
Gly Lys Ala Ile Asn Ser Phe Leu Thr Gln Ser Lys Pro Val Leu Ile
165 170 175
Gly Ala Gln Ala Ser Val Glu Val Arg His Thr Gln Tyr Tyr Gln His
180 185 190
Ala Thr Asp Ile Ala Arg Thr Leu Asn Ile Asp Lys Tyr Asp Ile Ile
195 200 205
Ala Cys Ala Ser Gly Asp Gly Val Pro His Glu Val Leu Asn Gly Phe
210 215 220
Tyr Gln Arg Ser Asp Arg Ala Glu Ala Phe Asn Lys Ile Thr Ile Thr
225 230 235 240
Gln Leu Pro Cys Gly Ser Gly Asn Ala Met Ser Glu Ser Cys His Gly
245 250 255
Thr Asn Asn Pro Ser Phe Ala Ala Leu Ser Leu Leu Lys Ser Ser Thr
260 265 270
Val Asn Leu Asp Leu Met Ala Cys Thr Gln Gly Asp Lys Thr Tyr Val
275 280 285
Ser Phe Leu Ser Gln Thr Val Gly Val Ile Ala Asp Ser Asp Ile Gly
290 295 300
Thr Glu Ala Leu Arg Trp Leu Gly Pro Ser Arg Phe Glu Leu Gly Val
305 310 315 320
Ala Tyr Lys Val Leu Ser Arg Ser Arg Tyr Pro Cys Asp Ile Ser Val
325 330 335
Lys Tyr Ala Ala Lys Ser Lys Asn Glu Leu Arg Gln His Phe Asp Glu
340 345 350
His Ser Thr Ile Val Ser Thr Lys Asp Ile Gln Ile Thr Glu Asp Thr
355 360 365
Tyr Asn Leu Lys Tyr Asp Pro Asn Gly Pro Ile Pro Asp Asp Trp Glu
370 375 380
Glu Ile Asp Lys Asp Leu Ser Glu Asn Leu Gly Ile Phe Tyr Thr Gly
385 390 395 400
Lys Met Pro Tyr Ile Ala Lys Asp Val Gln Phe Phe Pro Ala Ala Leu
405 410 415
Pro Asn Asp Gly Thr Phe Asp Leu Val Ile Thr Asp Ala Arg Thr Ser
420 425 430
Ile Ala Arg Met Ala Pro Thr Leu Leu Ser Leu Asp Gln Gly Ser His
435 440 445
Val Leu Gln Pro Glu Val Gln His Ser Lys Ile Ile Ala Tyr Arg Leu
450 455 460
Thr Pro Lys Gln Gln His Gly Tyr Leu Ser Val Asp Gly Glu Ser Tyr
465 470 475 480
Pro Phe Glu Thr Ile Gln Val Glu Ile Leu Pro Gly Ala Ala Lys Thr
485 490 495
Leu Leu Arg Asn Gly Thr Tyr Val Glu Thr Asn Phe Tyr
500 505
<210> 9
<211> 1515
<212> DNA
<213> Pichia ciferrii
<220>
<221> CDS
<222> (1)..(1515)
<400> 9
ttg gcg gtt aac atc act ggt tat ggg tta atc ggc tac tta aag atc 48
Met Ala Val Asn Ile Thr Gly Tyr Gly Leu Ile Gly Tyr Leu Lys Ile
1 5 10 15
gta tat aat gaa tta gca aaa gct gta ttc aga aca ttt tta tcc tta 96
Val Tyr Asn Glu Leu Ala Lys Ala Val Phe Arg Thr Phe Leu Ser Leu
20 25 30
cca ttt gtt aaa agt aaa gtt gat tca gaa gtt aga gaa aat ttg gac 144
Pro Phe Val Lys Ser Lys Val Asp Ser Glu Val Arg Glu Asn Leu Asp
35 40 45
aaa tta gaa gat tct tta att gtc aaa aca cca aat gtt caa gat ttc 192
Lys Leu Glu Asp Ser Leu Ile Val Lys Thr Pro Asn Val Gln Asp Phe
50 55 60
caa tca ata cca aca act ggt tta tca gat gat agc att tta gac tta 240
Gln Ser Ile Pro Thr Thr Gly Leu Ser Asp Asp Ser Ile Leu Asp Leu
65 70 75 80
ttg caa aaa cta caa aat tta aaa cat tca gat tgg caa ggt ggt aaa 288
Leu Gln Lys Leu Gln Asn Leu Lys His Ser Asp Trp Gln Gly Gly Lys
85 90 95
gtc tca ggt gct gtt tac cat ggt ggt gat gat att att aag atc caa 336
Val Ser Gly Ala Val Tyr His Gly Gly Asp Asp Ile Ile Lys Ile Gln
100 105 110
tct gat gct ttc aaa gtc ttt tgt gtt gct aat caa tta cat cca gac 384
Ser Asp Ala Phe Lys Val Phe Cys Val Ala Asn Gln Leu His Pro Asp
115 120 125
gtt ttc cca ggt gtt cgt aaa atg gaa gct gaa gtt gtt gca atg act 432
Val Phe Pro Gly Val Arg Lys Met Glu Ala Glu Val Val Ala Met Thr
130 135 140
ttg aaa tta ttc aat gca cca gaa tca ggt gtt ggt ggt acc agc tca 480
Leu Lys Leu Phe Asn Ala Pro Glu Ser Gly Val Gly Gly Thr Ser Ser
145 150 155 160
ggt ggt act gaa tcc tta tta ttg gct tgt ctt tct gct aaa gaa tat 528
Gly Gly Thr Glu Ser Leu Leu Leu Ala Cys Leu Ser Ala Lys Glu Tyr
165 170 175
ggt aaa cgt cat aaa ggt att gtt gaa cca gaa att att att cca gaa 576
Gly Lys Arg His Lys Gly Ile Val Glu Pro Glu Ile Ile Ile Pro Glu
180 185 190
act gca cat gct ggt ttt gat aaa gct ggt tat tat ttt ggt atg aaa 624
Thr Ala His Ala Gly Phe Asp Lys Ala Gly Tyr Tyr Phe Gly Met Lys
195 200 205
gtc cat cat gtt cca tta gat cca aag acc tat aaa gtt gat tta ggg 672
Val His His Val Pro Leu Asp Pro Lys Thr Tyr Lys Val Asp Leu Gly
210 215 220
aaa tta aag aga tta atc aat aaa aac act gtt tta tta gct ggt tct 720
Lys Leu Lys Arg Leu Ile Asn Lys Asn Thr Val Leu Leu Ala Gly Ser
225 230 235 240
gca cca aat ttc cca cat ggt atc att gat gat att gaa tct att ggt 768
Ala Pro Asn Phe Pro His Gly Ile Ile Asp Asp Ile Glu Ser Ile Gly
245 250 255
gct cta ggt caa aaa tat aat atc cca gtt cat gtt gat tgt tgt tta 816
Ala Leu Gly Gln Lys Tyr Asn Ile Pro Val His Val Asp Cys Cys Leu
260 265 270
ggt tca ttt att gtc tct tat atg gaa aaa gca ggt tat gaa tta cca 864
Gly Ser Phe Ile Val Ser Tyr Met Glu Lys Ala Gly Tyr Glu Leu Pro
275 280 285
cct ttt gac ttt aga gtt cct ggt gtc act tca att tct tgt gat acc 912
Pro Phe Asp Phe Arg Val Pro Gly Val Thr Ser Ile Ser Cys Asp Thr
290 295 300
cac aaa tac ggg ttt gca cca aaa ggt tct tca ata atc atg tat cgt 960
His Lys Tyr Gly Phe Ala Pro Lys Gly Ser Ser Ile Ile Met Tyr Arg
305 310 315 320
aat aat gct ctt aga gaa gca caa tat tat gtt aat gtt gac tgg gtt 1008
Asn Asn Ala Leu Arg Glu Ala Gln Tyr Tyr Val Asn Val Asp Trp Val
325 330 335
ggt ggt atc tat ggc tca cca act tta gct ggt agt aga cca ggt gct 1056
Gly Gly Ile Tyr Gly Ser Pro Thr Leu Ala Gly Ser Arg Pro Gly Ala
340 345 350
atc att gtt ggt tgt tgg gca acc ttg atc aag att ggt gat gaa ggt 1104
Ile Ile Val Gly Cys Trp Ala Thr Leu Ile Lys Ile Gly Asp Glu Gly
355 360 365
tac aag aaa tca tgt aaa gat att gtt gga gct gca aga aaa ttg aaa 1152
Tyr Lys Lys Ser Cys Lys Asp Ile Val Gly Ala Ala Arg Lys Leu Lys
370 375 380
tta aga att caa aaa gaa ata cca gaa tta gaa atc att ggt gat cca 1200
Leu Arg Ile Gln Lys Glu Ile Pro Glu Leu Glu Ile Ile Gly Asp Pro
385 390 395 400
tta act tca gtt att tca ttc aaa tct gaa aaa att aat att tat gaa 1248
Leu Thr Ser Val Ile Ser Phe Lys Ser Glu Lys Ile Asn Ile Tyr Glu
405 410 415
tta tca gat ctc ttg agt tct aag gga tgg cac tta agt gca ttg caa 1296
Leu Ser Asp Leu Leu Ser Ser Lys Gly Trp His Leu Ser Ala Leu Gln
420 425 430
aag cca gca gct tta cat ctt gca gtc act aga tta tca gtt cca gtt 1344
Lys Pro Ala Ala Leu His Leu Ala Val Thr Arg Leu Ser Val Pro Val
435 440 445
att gat gaa tta gtt gat gaa ttg aaa aca gct gtt cac aaa ttg aga 1392
Ile Asp Glu Leu Val Asp Glu Leu Lys Thr Ala Val His Lys Leu Arg
450 455 460
gat tca tct gct gct aaa ggt gat act gct gca tta tac ggt gtc gct 1440
Asp Ser Ser Ala Ala Lys Gly Asp Thr Ala Ala Leu Tyr Gly Val Ala
465 470 475 480
ggt agt gtt tcc acc act ggt gtt gtt gat cgt tta gtt gtt gga ttc 1488
Gly Ser Val Ser Thr Thr Gly Val Val Asp Arg Leu Val Val Gly Phe
485 490 495
tta gat aca cta tac aaa acc aaa taa 1515
Leu Asp Thr Leu Tyr Lys Thr Lys
500
<210> 10
<211> 504
<212> PRT
<213> Pichia ciferrii
<400> 10
Met Ala Val Asn Ile Thr Gly Tyr Gly Leu Ile Gly Tyr Leu Lys Ile
1 5 10 15
Val Tyr Asn Glu Leu Ala Lys Ala Val Phe Arg Thr Phe Leu Ser Leu
20 25 30
Pro Phe Val Lys Ser Lys Val Asp Ser Glu Val Arg Glu Asn Leu Asp
35 40 45
Lys Leu Glu Asp Ser Leu Ile Val Lys Thr Pro Asn Val Gln Asp Phe
50 55 60
Gln Ser Ile Pro Thr Thr Gly Leu Ser Asp Asp Ser Ile Leu Asp Leu
65 70 75 80
Leu Gln Lys Leu Gln Asn Leu Lys His Ser Asp Trp Gln Gly Gly Lys
85 90 95
Val Ser Gly Ala Val Tyr His Gly Gly Asp Asp Ile Ile Lys Ile Gln
100 105 110
Ser Asp Ala Phe Lys Val Phe Cys Val Ala Asn Gln Leu His Pro Asp
115 120 125
Val Phe Pro Gly Val Arg Lys Met Glu Ala Glu Val Val Ala Met Thr
130 135 140
Leu Lys Leu Phe Asn Ala Pro Glu Ser Gly Val Gly Gly Thr Ser Ser
145 150 155 160
Gly Gly Thr Glu Ser Leu Leu Leu Ala Cys Leu Ser Ala Lys Glu Tyr
165 170 175
Gly Lys Arg His Lys Gly Ile Val Glu Pro Glu Ile Ile Ile Pro Glu
180 185 190
Thr Ala His Ala Gly Phe Asp Lys Ala Gly Tyr Tyr Phe Gly Met Lys
195 200 205
Val His His Val Pro Leu Asp Pro Lys Thr Tyr Lys Val Asp Leu Gly
210 215 220
Lys Leu Lys Arg Leu Ile Asn Lys Asn Thr Val Leu Leu Ala Gly Ser
225 230 235 240
Ala Pro Asn Phe Pro His Gly Ile Ile Asp Asp Ile Glu Ser Ile Gly
245 250 255
Ala Leu Gly Gln Lys Tyr Asn Ile Pro Val His Val Asp Cys Cys Leu
260 265 270
Gly Ser Phe Ile Val Ser Tyr Met Glu Lys Ala Gly Tyr Glu Leu Pro
275 280 285
Pro Phe Asp Phe Arg Val Pro Gly Val Thr Ser Ile Ser Cys Asp Thr
290 295 300
His Lys Tyr Gly Phe Ala Pro Lys Gly Ser Ser Ile Ile Met Tyr Arg
305 310 315 320
Asn Asn Ala Leu Arg Glu Ala Gln Tyr Tyr Val Asn Val Asp Trp Val
325 330 335
Gly Gly Ile Tyr Gly Ser Pro Thr Leu Ala Gly Ser Arg Pro Gly Ala
340 345 350
Ile Ile Val Gly Cys Trp Ala Thr Leu Ile Lys Ile Gly Asp Glu Gly
355 360 365
Tyr Lys Lys Ser Cys Lys Asp Ile Val Gly Ala Ala Arg Lys Leu Lys
370 375 380
Leu Arg Ile Gln Lys Glu Ile Pro Glu Leu Glu Ile Ile Gly Asp Pro
385 390 395 400
Leu Thr Ser Val Ile Ser Phe Lys Ser Glu Lys Ile Asn Ile Tyr Glu
405 410 415
Leu Ser Asp Leu Leu Ser Ser Lys Gly Trp His Leu Ser Ala Leu Gln
420 425 430
Lys Pro Ala Ala Leu His Leu Ala Val Thr Arg Leu Ser Val Pro Val
435 440 445
Ile Asp Glu Leu Val Asp Glu Leu Lys Thr Ala Val His Lys Leu Arg
450 455 460
Asp Ser Ser Ala Ala Lys Gly Asp Thr Ala Ala Leu Tyr Gly Val Ala
465 470 475 480
Gly Ser Val Ser Thr Thr Gly Val Val Asp Arg Leu Val Val Gly Phe
485 490 495
Leu Asp Thr Leu Tyr Lys Thr Lys
500
<210> 11
<211> 576
<212> DNA
<213> Pichia ciferrii
<220>
<221> CDS
<222> (1)..(576)
<400> 11
atg act aca aca cat gaa cca att tct gtt gat gga tca tta tca cca 48
Met Thr Thr Thr His Glu Pro Ile Ser Val Asp Gly Ser Leu Ser Pro
1 5 10 15
aat tca aat aca aat aat aat aat caa cat cgt cgt cgt tca tca tca 96
Asn Ser Asn Thr Asn Asn Asn Asn Gln His Arg Arg Arg Ser Ser Ser
20 25 30
ata att tct cat gtt gaa cct gaa act ttt gaa gaa aaa att gat caa 144
Ile Ile Ser His Val Glu Pro Glu Thr Phe Glu Glu Lys Ile Asp Gln
35 40 45
gat tca aca cca aat tta aat gca aat tgg gtt cat tca aaa ggt gct 192
Asp Ser Thr Pro Asn Leu Asn Ala Asn Trp Val His Ser Lys Gly Ala
50 55 60
tgg tta gtt cat att gtt att ata tta tta tta aaa att ttc ttt gat 240
Trp Leu Val His Ile Val Ile Ile Leu Leu Leu Lys Ile Phe Phe Asp
65 70 75 80
tta ata cct ggt tta tca aat gaa att agt tgg tca tta aca aat gct 288
Leu Ile Pro Gly Leu Ser Asn Glu Ile Ser Trp Ser Leu Thr Asn Ala
85 90 95
aca tat gtt att ggt tca tat att atg ttt cat tta gtt aaa ggt acg 336
Thr Tyr Val Ile Gly Ser Tyr Ile Met Phe His Leu Val Lys Gly Thr
100 105 110
cca ttt gaa ttt aat tca ggt gct tat gat aat tta aca atg tgg gaa 384
Pro Phe Glu Phe Asn Ser Gly Ala Tyr Asp Asn Leu Thr Met Trp Glu
115 120 125
caa tta gat gag gga gat ttt tat aca cca agt aaa aaa ttc tta gtt 432
Gln Leu Asp Glu Gly Asp Phe Tyr Thr Pro Ser Lys Lys Phe Leu Val
130 135 140
ggt gta cca att tgg tta ttt ctt tgt tca act cat tat agt cat tat 480
Gly Val Pro Ile Trp Leu Phe Leu Cys Ser Thr His Tyr Ser His Tyr
145 150 155 160
gat tta aaa tta ttt att ata aat tta tta att tgt gct gtt ggt gtt 528
Asp Leu Lys Leu Phe Ile Ile Asn Leu Leu Ile Cys Ala Val Gly Val
165 170 175
gta cca aaa att cca att ttt gat cgt tta aga att aca ttt ttt taa 576
Val Pro Lys Ile Pro Ile Phe Asp Arg Leu Arg Ile Thr Phe Phe
180 185 190
<210> 12
<211> 191
<212> PRT
<213> Pichia ciferrii
<400> 12
Met Thr Thr Thr His Glu Pro Ile Ser Val Asp Gly Ser Leu Ser Pro
1 5 10 15
Asn Ser Asn Thr Asn Asn Asn Asn Gln His Arg Arg Arg Ser Ser Ser
20 25 30
Ile Ile Ser His Val Glu Pro Glu Thr Phe Glu Glu Lys Ile Asp Gln
35 40 45
Asp Ser Thr Pro Asn Leu Asn Ala Asn Trp Val His Ser Lys Gly Ala
50 55 60
Trp Leu Val His Ile Val Ile Ile Leu Leu Leu Lys Ile Phe Phe Asp
65 70 75 80
Leu Ile Pro Gly Leu Ser Asn Glu Ile Ser Trp Ser Leu Thr Asn Ala
85 90 95
Thr Tyr Val Ile Gly Ser Tyr Ile Met Phe His Leu Val Lys Gly Thr
100 105 110
Pro Phe Glu Phe Asn Ser Gly Ala Tyr Asp Asn Leu Thr Met Trp Glu
115 120 125
Gln Leu Asp Glu Gly Asp Phe Tyr Thr Pro Ser Lys Lys Phe Leu Val
130 135 140
Gly Val Pro Ile Trp Leu Phe Leu Cys Ser Thr His Tyr Ser His Tyr
145 150 155 160
Asp Leu Lys Leu Phe Ile Ile Asn Leu Leu Ile Cys Ala Val Gly Val
165 170 175
Val Pro Lys Ile Pro Ile Phe Asp Arg Leu Arg Ile Thr Phe Phe
180 185 190
<210> 13
<211> 1713
<212> DNA
<213> Pichia ciferrii
<220>
<221> CDS
<222> (1)..(1713)
<400> 13
atg aac gtc act gct aca act ata aca act tca aca aca aca att gca 48
Met Asn Val Thr Ala Thr Thr Ile Thr Thr Ser Thr Thr Thr Ile Ala
1 5 10 15
tta caa gat att tgg aat aca act tct gat gtt gtt tct cgt tat tta 96
Leu Gln Asp Ile Trp Asn Thr Thr Ser Asp Val Val Ser Arg Tyr Leu
20 25 30
ttc att ata tta aat tat att gaa tta ata cct ggt ggt tca att tta 144
Phe Ile Ile Leu Asn Tyr Ile Glu Leu Ile Pro Gly Gly Ser Ile Leu
35 40 45
gtt cgt tat ata aaa tct tct cat aaa aat gat cca att aga act tta 192
Val Arg Tyr Ile Lys Ser Ser His Lys Asn Asp Pro Ile Arg Thr Leu
50 55 60
ttt gaa att gct tta ttt att ttt gca att aga tat ttt act aca gca 240
Phe Glu Ile Ala Leu Phe Ile Phe Ala Ile Arg Tyr Phe Thr Thr Ala
65 70 75 80
aaa tat gaa aga tct aaa aaa gat cat att aaa ttg aaa aat tct gaa 288
Lys Tyr Glu Arg Ser Lys Lys Asp His Ile Lys Leu Lys Asn Ser Glu
85 90 95
att gat gaa tta att gat gat tgg atg ccg gaa cct tta gtt ttg gat 336
Ile Asp Glu Leu Ile Asp Asp Trp Met Pro Glu Pro Leu Val Leu Asp
100 105 110
att agt cca aag gaa caa tgg caa tta aat tca att cca att gtt aaa 384
Ile Ser Pro Lys Glu Gln Trp Gln Leu Asn Ser Ile Pro Ile Val Lys
115 120 125
ggt cca ata gat act aaa gtg aac cta gtt ggt gaa gaa ggt gac ttt 432
Gly Pro Ile Asp Thr Lys Val Asn Leu Val Gly Glu Glu Gly Asp Phe
130 135 140
tta aat ttt gct tct tca aat ttt tta aat ttt ggt att aat cca att 480
Leu Asn Phe Ala Ser Ser Asn Phe Leu Asn Phe Gly Ile Asn Pro Ile
145 150 155 160
gtt aaa aat gaa tgt aaa aaa att att cat agt aat ggt gtt ggt gct 528
Val Lys Asn Glu Cys Lys Lys Ile Ile His Ser Asn Gly Val Gly Ala
165 170 175
tgt ggt cca cca aat ttt tat ggt aat caa gat att cat att aaa tta 576
Cys Gly Pro Pro Asn Phe Tyr Gly Asn Gln Asp Ile His Ile Lys Leu
180 185 190
gaa aat gat tta gca aaa ttt ttc gaa gtt ggt ggt gct gta tta tat 624
Glu Asn Asp Leu Ala Lys Phe Phe Glu Val Gly Gly Ala Val Leu Tyr
195 200 205
ggt caa gat ttt tgt act gca ggt tca gtt tta cca agt ttt tta aaa 672
Gly Gln Asp Phe Cys Thr Ala Gly Ser Val Leu Pro Ser Phe Leu Lys
210 215 220
aga ggt gat ttt gtt att gct gat gct tca tca aat gtt gca att caa 720
Arg Gly Asp Phe Val Ile Ala Asp Ala Ser Ser Asn Val Ala Ile Gln
225 230 235 240
aaa gct tta caa tta tca aga tgt gaa att tat tgg ttt aat cat aat 768
Lys Ala Leu Gln Leu Ser Arg Cys Glu Ile Tyr Trp Phe Asn His Asn
245 250 255
gat ttg gat cat tta gaa gaa att tta att gat tta caa aaa aat att 816
Asp Leu Asp His Leu Glu Glu Ile Leu Ile Asp Leu Gln Lys Asn Ile
260 265 270
ttt aaa ttt gaa aaa cca att tca aga aaa ttt att gtt act gaa ggt 864
Phe Lys Phe Glu Lys Pro Ile Ser Arg Lys Phe Ile Val Thr Glu Gly
275 280 285
att ttt gca aat aaa ggt gat tca cca tat tta cca aga tta att gaa 912
Ile Phe Ala Asn Lys Gly Asp Ser Pro Tyr Leu Pro Arg Leu Ile Glu
290 295 300
tta aag aaa aaa ttt aaa ttt aga tta ttt ttg gat gaa tct tta tct 960
Leu Lys Lys Lys Phe Lys Phe Arg Leu Phe Leu Asp Glu Ser Leu Ser
305 310 315 320
tta ggt gtt tta ggt aaa tct ggt aaa ggt tta gct gaa cat tat aat 1008
Leu Gly Val Leu Gly Lys Ser Gly Lys Gly Leu Ala Glu His Tyr Asn
325 330 335
att aaa aga tca gaa att gat gta act ata agt tca atg gct aat tca 1056
Ile Lys Arg Ser Glu Ile Asp Val Thr Ile Ser Ser Met Ala Asn Ser
340 345 350
ttc tct tct tca ggt gct ttt tgt att ggt gat aaa gtt atg act tat 1104
Phe Ser Ser Ser Gly Ala Phe Cys Ile Gly Asp Lys Val Met Thr Tyr
355 360 365
cat caa aga att ggt tca atg gct tat tgt ttt agt gct tca tta cct 1152
His Gln Arg Ile Gly Ser Met Ala Tyr Cys Phe Ser Ala Ser Leu Pro
370 375 380
gct tat gtt gca aga gct aca tca gtt gca tta aga tta tta act gat 1200
Ala Tyr Val Ala Arg Ala Thr Ser Val Ala Leu Arg Leu Leu Thr Asp
385 390 395 400
tct caa gat tcc cag ggt gaa tca tca att gta aaa aaa tta caa tca 1248
Ser Gln Asp Ser Gln Gly Glu Ser Ser Ile Val Lys Lys Leu Gln Ser
405 410 415
aat aat tat caa tta ttt aat tta ttt aat aaa gat aga aaa tta agt 1296
Asn Asn Tyr Gln Leu Phe Asn Leu Phe Asn Lys Asp Arg Lys Leu Ser
420 425 430
aaa tat tta aaa att ata tca aat gaa att tca cca att tta cat ttt 1344
Lys Tyr Leu Lys Ile Ile Ser Asn Glu Ile Ser Pro Ile Leu His Phe
435 440 445
gaa att aat tca gat tta aga aaa ctt tta aat ttc cca att agt tat 1392
Glu Ile Asn Ser Asp Leu Arg Lys Leu Leu Asn Phe Pro Ile Ser Tyr
450 455 460
aca ggt aaa gga tca gaa att gaa tat aaa aat aaa aaa gga att tct 1440
Thr Gly Lys Gly Ser Glu Ile Glu Tyr Lys Asn Lys Lys Gly Ile Ser
465 470 475 480
gat aaa ttt gtt gaa tca ttt aat tat gaa aat tta att ttt caa aaa 1488
Asp Lys Phe Val Glu Ser Phe Asn Tyr Glu Asn Leu Ile Phe Gln Lys
485 490 495
att ata aat tta tcc aag aaa caa ggt att tta ata aca aga tca att 1536
Ile Ile Asn Leu Ser Lys Lys Gln Gly Ile Leu Ile Thr Arg Ser Ile
500 505 510
ttt aca att gaa caa gaa gct ctg cct ctg att cca aat tta aaa att 1584
Phe Thr Ile Glu Gln Glu Ala Leu Pro Leu Ile Pro Asn Leu Lys Ile
515 520 525
cat tca aat gtt gat ttt act aag gat gaa att gaa aaa gtt tat aaa 1632
His Ser Asn Val Asp Phe Thr Lys Asp Glu Ile Glu Lys Val Tyr Lys
530 535 540
att gtt tcc aaa gta att tta gat gtt ttt gaa aat tta act gtt gaa 1680
Ile Val Ser Lys Val Ile Leu Asp Val Phe Glu Asn Leu Thr Val Glu
545 550 555 560
tca tta tca tta tta act gaa gaa gtt att taa 1713
Ser Leu Ser Leu Leu Thr Glu Glu Val Ile
565 570
<210> 14
<211> 570
<212> PRT
<213> Pichia ciferrii
<400> 14
Met Asn Val Thr Ala Thr Thr Ile Thr Thr Ser Thr Thr Thr Ile Ala
1 5 10 15
Leu Gln Asp Ile Trp Asn Thr Thr Ser Asp Val Val Ser Arg Tyr Leu
20 25 30
Phe Ile Ile Leu Asn Tyr Ile Glu Leu Ile Pro Gly Gly Ser Ile Leu
35 40 45
Val Arg Tyr Ile Lys Ser Ser His Lys Asn Asp Pro Ile Arg Thr Leu
50 55 60
Phe Glu Ile Ala Leu Phe Ile Phe Ala Ile Arg Tyr Phe Thr Thr Ala
65 70 75 80
Lys Tyr Glu Arg Ser Lys Lys Asp His Ile Lys Leu Lys Asn Ser Glu
85 90 95
Ile Asp Glu Leu Ile Asp Asp Trp Met Pro Glu Pro Leu Val Leu Asp
100 105 110
Ile Ser Pro Lys Glu Gln Trp Gln Leu Asn Ser Ile Pro Ile Val Lys
115 120 125
Gly Pro Ile Asp Thr Lys Val Asn Leu Val Gly Glu Glu Gly Asp Phe
130 135 140
Leu Asn Phe Ala Ser Ser Asn Phe Leu Asn Phe Gly Ile Asn Pro Ile
145 150 155 160
Val Lys Asn Glu Cys Lys Lys Ile Ile His Ser Asn Gly Val Gly Ala
165 170 175
Cys Gly Pro Pro Asn Phe Tyr Gly Asn Gln Asp Ile His Ile Lys Leu
180 185 190
Glu Asn Asp Leu Ala Lys Phe Phe Glu Val Gly Gly Ala Val Leu Tyr
195 200 205
Gly Gln Asp Phe Cys Thr Ala Gly Ser Val Leu Pro Ser Phe Leu Lys
210 215 220
Arg Gly Asp Phe Val Ile Ala Asp Ala Ser Ser Asn Val Ala Ile Gln
225 230 235 240
Lys Ala Leu Gln Leu Ser Arg Cys Glu Ile Tyr Trp Phe Asn His Asn
245 250 255
Asp Leu Asp His Leu Glu Glu Ile Leu Ile Asp Leu Gln Lys Asn Ile
260 265 270
Phe Lys Phe Glu Lys Pro Ile Ser Arg Lys Phe Ile Val Thr Glu Gly
275 280 285
Ile Phe Ala Asn Lys Gly Asp Ser Pro Tyr Leu Pro Arg Leu Ile Glu
290 295 300
Leu Lys Lys Lys Phe Lys Phe Arg Leu Phe Leu Asp Glu Ser Leu Ser
305 310 315 320
Leu Gly Val Leu Gly Lys Ser Gly Lys Gly Leu Ala Glu His Tyr Asn
325 330 335
Ile Lys Arg Ser Glu Ile Asp Val Thr Ile Ser Ser Met Ala Asn Ser
340 345 350
Phe Ser Ser Ser Gly Ala Phe Cys Ile Gly Asp Lys Val Met Thr Tyr
355 360 365
His Gln Arg Ile Gly Ser Met Ala Tyr Cys Phe Ser Ala Ser Leu Pro
370 375 380
Ala Tyr Val Ala Arg Ala Thr Ser Val Ala Leu Arg Leu Leu Thr Asp
385 390 395 400
Ser Gln Asp Ser Gln Gly Glu Ser Ser Ile Val Lys Lys Leu Gln Ser
405 410 415
Asn Asn Tyr Gln Leu Phe Asn Leu Phe Asn Lys Asp Arg Lys Leu Ser
420 425 430
Lys Tyr Leu Lys Ile Ile Ser Asn Glu Ile Ser Pro Ile Leu His Phe
435 440 445
Glu Ile Asn Ser Asp Leu Arg Lys Leu Leu Asn Phe Pro Ile Ser Tyr
450 455 460
Thr Gly Lys Gly Ser Glu Ile Glu Tyr Lys Asn Lys Lys Gly Ile Ser
465 470 475 480
Asp Lys Phe Val Glu Ser Phe Asn Tyr Glu Asn Leu Ile Phe Gln Lys
485 490 495
Ile Ile Asn Leu Ser Lys Lys Gln Gly Ile Leu Ile Thr Arg Ser Ile
500 505 510
Phe Thr Ile Glu Gln Glu Ala Leu Pro Leu Ile Pro Asn Leu Lys Ile
515 520 525
His Ser Asn Val Asp Phe Thr Lys Asp Glu Ile Glu Lys Val Tyr Lys
530 535 540
Ile Val Ser Lys Val Ile Leu Asp Val Phe Glu Asn Leu Thr Val Glu
545 550 555 560
Ser Leu Ser Leu Leu Thr Glu Glu Val Ile
565 570
<210> 15
<211> 1689
<212> DNA
<213> Pichia ciferrii
<220>
<221> CDS
<222> (1)..(1689)
<400> 15
atg tca ttg gta ata cct caa ata gat cta tca ggt ctt tcc atc gaa 48
Met Ser Leu Val Ile Pro Gln Ile Asp Leu Ser Gly Leu Ser Ile Glu
1 5 10 15
gac aag aaa caa aat gaa ttc ggt gct cta act tca aat gaa tat cgt 96
Asp Lys Lys Gln Asn Glu Phe Gly Ala Leu Thr Ser Asn Glu Tyr Arg
20 25 30
tac aaa aca att tca aga cag ggg aaa cca tta cct gat cca att gaa 144
Tyr Lys Thr Ile Ser Arg Gln Gly Lys Pro Leu Pro Asp Pro Ile Glu
35 40 45
gat gaa cca cca tat cat gtc ctt ttc atc act tat tta aac tat tta 192
Asp Glu Pro Pro Tyr His Val Leu Phe Ile Thr Tyr Leu Asn Tyr Leu
50 55 60
atc ttg att atc gtt ggt cat att aaa gat ttc aca ggt att ctg ttc 240
Ile Leu Ile Ile Val Gly His Ile Lys Asp Phe Thr Gly Ile Leu Phe
65 70 75 80
aac cca aaa aat tac caa gat tta tta gaa caa aat ggc ctt gct cca 288
Asn Pro Lys Asn Tyr Gln Asp Leu Leu Glu Gln Asn Gly Leu Ala Pro
85 90 95
tgg tat aat aaa ttt gaa agt ttt tat att cgt cgt atg aaa caa aaa 336
Trp Tyr Asn Lys Phe Glu Ser Phe Tyr Ile Arg Arg Met Lys Gln Lys
100 105 110
att gat gat tgt ttt gca aga cca act tgt ggt gtc cca ggt aga tta 384
Ile Asp Asp Cys Phe Ala Arg Pro Thr Cys Gly Val Pro Gly Arg Leu
115 120 125
atc act tgt att gat cgt gat gct cat gat tat aat tca tat ttt agt 432
Ile Thr Cys Ile Asp Arg Asp Ala His Asp Tyr Asn Ser Tyr Phe Ser
130 135 140
tat cct ggt act act tca act tgt tta aat tta tca tca tat aat tat 480
Tyr Pro Gly Thr Thr Ser Thr Cys Leu Asn Leu Ser Ser Tyr Asn Tyr
145 150 155 160
ttg ggg ttt gca caa tct gaa ggg gca tgt act caa gcc gct tta gaa 528
Leu Gly Phe Ala Gln Ser Glu Gly Ala Cys Thr Gln Ala Ala Leu Glu
165 170 175
att ttg gat tat tat ggt gtt ggt tct ggt ggt cca aga aat gtt att 576
Ile Leu Asp Tyr Tyr Gly Val Gly Ser Gly Gly Pro Arg Asn Val Ile
180 185 190
ggt act act gat tta cat tta aaa act gaa aaa act ata gca aaa ttt 624
Gly Thr Thr Asp Leu His Leu Lys Thr Glu Lys Thr Ile Ala Lys Phe
195 200 205
att ggt aaa gat gat tca atc tta ttt tca atg ggg tat gca aca aat 672
Ile Gly Lys Asp Asp Ser Ile Leu Phe Ser Met Gly Tyr Ala Thr Asn
210 215 220
gca agt tta ttt agt tct tta ttg gat aag aaa tca ctt gtt att tct 720
Ala Ser Leu Phe Ser Ser Leu Leu Asp Lys Lys Ser Leu Val Ile Ser
225 230 235 240
gat gaa tta aat cat gct tca att aga act ggt gtt aga tta tct ggt 768
Asp Glu Leu Asn His Ala Ser Ile Arg Thr Gly Val Arg Leu Ser Gly
245 250 255
tct aca gtt aaa act ttc cct cat aat aat atg att gcc ttg gaa aaa 816
Ser Thr Val Lys Thr Phe Pro His Asn Asn Met Ile Ala Leu Glu Lys
260 265 270
att ctt aga gaa caa att tct caa ggt caa cca aga tct cat cgt cca 864
Ile Leu Arg Glu Gln Ile Ser Gln Gly Gln Pro Arg Ser His Arg Pro
275 280 285
tgg aaa aaa atc att gtt gca gtt gaa ggg ctt tat tca atg gag ggt 912
Trp Lys Lys Ile Ile Val Ala Val Glu Gly Leu Tyr Ser Met Glu Gly
290 295 300
aca atg gca aat tta cct gca tta att gaa tta aga aga aaa tat aaa 960
Thr Met Ala Asn Leu Pro Ala Leu Ile Glu Leu Arg Arg Lys Tyr Lys
305 310 315 320
ttt aat tta ttt gtt gat gaa gct cat tca att ggt gct att ggt cca 1008
Phe Asn Leu Phe Val Asp Glu Ala His Ser Ile Gly Ala Ile Gly Pro
325 330 335
tca ggt cgt ggt gtt tgt gat tat ttt ggt ata gat ccc tca aat gtt 1056
Ser Gly Arg Gly Val Cys Asp Tyr Phe Gly Ile Asp Pro Ser Asn Val
340 345 350
gat tta tta atg ggg act tta act aaa tca ttt ggt gct gca ggt ggt 1104
Asp Leu Leu Met Gly Thr Leu Thr Lys Ser Phe Gly Ala Ala Gly Gly
355 360 365
tat att gct ggt tca caa caa att ata aat cgt tta aaa tta aat att 1152
Tyr Ile Ala Gly Ser Gln Gln Ile Ile Asn Arg Leu Lys Leu Asn Ile
370 375 380
aat tca caa aat tat gca gaa tct atc cct gca cct gtt ttg gca caa 1200
Asn Ser Gln Asn Tyr Ala Glu Ser Ile Pro Ala Pro Val Leu Ala Gln
385 390 395 400
att att tct tcg tta aat atc atc tcg ggt gat tta aat cct ggt gaa 1248
Ile Ile Ser Ser Leu Asn Ile Ile Ser Gly Asp Leu Asn Pro Gly Glu
405 410 415
ggt tcg gaa aga tta gaa aga att gct ttt aat tca cgt tat tta aga 1296
Gly Ser Glu Arg Leu Glu Arg Ile Ala Phe Asn Ser Arg Tyr Leu Arg
420 425 430
tta ggt tta caa aga tta ggt ttt atc gta tac gga gtt gat gat tca 1344
Leu Gly Leu Gln Arg Leu Gly Phe Ile Val Tyr Gly Val Asp Asp Ser
435 440 445
cca gtg att cca tta tta tta ttc gcc cca gcc aaa atg cca gca ttt 1392
Pro Val Ile Pro Leu Leu Leu Phe Ala Pro Ala Lys Met Pro Ala Phe
450 455 460
tca cgt atg cta tat caa aga aaa att gca gtt gtt gtt gtt gga tac 1440
Ser Arg Met Leu Tyr Gln Arg Lys Ile Ala Val Val Val Val Gly Tyr
465 470 475 480
ccg gca act cca ctg act tca tca aga gtt cgt ctt tgt gtt tct gca 1488
Pro Ala Thr Pro Leu Thr Ser Ser Arg Val Arg Leu Cys Val Ser Ala
485 490 495
tct tta aca aaa gaa gat att gat tat ctt tta cgt cat tta tcc gag 1536
Ser Leu Thr Lys Glu Asp Ile Asp Tyr Leu Leu Arg His Leu Ser Glu
500 505 510
gtg ggt gat aaa tta ttt tta aaa ttt agt tct ggt att gct ggt ggt 1584
Val Gly Asp Lys Leu Phe Leu Lys Phe Ser Ser Gly Ile Ala Gly Gly
515 520 525
tct tta gat ggt tca cca cca aga tgg aat att gaa gat gtt ttg aaa 1632
Ser Leu Asp Gly Ser Pro Pro Arg Trp Asn Ile Glu Asp Val Leu Lys
530 535 540
gag act cca aag gat tgt aaa gaa tct aaa tat ttt att gca act gca 1680
Glu Thr Pro Lys Asp Cys Lys Glu Ser Lys Tyr Phe Ile Ala Thr Ala
545 550 555 560
aat aat tga 1689
Asn Asn
<210> 16
<211> 562
<212> PRT
<213> Pichia ciferrii
<400> 16
Met Ser Leu Val Ile Pro Gln Ile Asp Leu Ser Gly Leu Ser Ile Glu
1 5 10 15
Asp Lys Lys Gln Asn Glu Phe Gly Ala Leu Thr Ser Asn Glu Tyr Arg
20 25 30
Tyr Lys Thr Ile Ser Arg Gln Gly Lys Pro Leu Pro Asp Pro Ile Glu
35 40 45
Asp Glu Pro Pro Tyr His Val Leu Phe Ile Thr Tyr Leu Asn Tyr Leu
50 55 60
Ile Leu Ile Ile Val Gly His Ile Lys Asp Phe Thr Gly Ile Leu Phe
65 70 75 80
Asn Pro Lys Asn Tyr Gln Asp Leu Leu Glu Gln Asn Gly Leu Ala Pro
85 90 95
Trp Tyr Asn Lys Phe Glu Ser Phe Tyr Ile Arg Arg Met Lys Gln Lys
100 105 110
Ile Asp Asp Cys Phe Ala Arg Pro Thr Cys Gly Val Pro Gly Arg Leu
115 120 125
Ile Thr Cys Ile Asp Arg Asp Ala His Asp Tyr Asn Ser Tyr Phe Ser
130 135 140
Tyr Pro Gly Thr Thr Ser Thr Cys Leu Asn Leu Ser Ser Tyr Asn Tyr
145 150 155 160
Leu Gly Phe Ala Gln Ser Glu Gly Ala Cys Thr Gln Ala Ala Leu Glu
165 170 175
Ile Leu Asp Tyr Tyr Gly Val Gly Ser Gly Gly Pro Arg Asn Val Ile
180 185 190
Gly Thr Thr Asp Leu His Leu Lys Thr Glu Lys Thr Ile Ala Lys Phe
195 200 205
Ile Gly Lys Asp Asp Ser Ile Leu Phe Ser Met Gly Tyr Ala Thr Asn
210 215 220
Ala Ser Leu Phe Ser Ser Leu Leu Asp Lys Lys Ser Leu Val Ile Ser
225 230 235 240
Asp Glu Leu Asn His Ala Ser Ile Arg Thr Gly Val Arg Leu Ser Gly
245 250 255
Ser Thr Val Lys Thr Phe Pro His Asn Asn Met Ile Ala Leu Glu Lys
260 265 270
Ile Leu Arg Glu Gln Ile Ser Gln Gly Gln Pro Arg Ser His Arg Pro
275 280 285
Trp Lys Lys Ile Ile Val Ala Val Glu Gly Leu Tyr Ser Met Glu Gly
290 295 300
Thr Met Ala Asn Leu Pro Ala Leu Ile Glu Leu Arg Arg Lys Tyr Lys
305 310 315 320
Phe Asn Leu Phe Val Asp Glu Ala His Ser Ile Gly Ala Ile Gly Pro
325 330 335
Ser Gly Arg Gly Val Cys Asp Tyr Phe Gly Ile Asp Pro Ser Asn Val
340 345 350
Asp Leu Leu Met Gly Thr Leu Thr Lys Ser Phe Gly Ala Ala Gly Gly
355 360 365
Tyr Ile Ala Gly Ser Gln Gln Ile Ile Asn Arg Leu Lys Leu Asn Ile
370 375 380
Asn Ser Gln Asn Tyr Ala Glu Ser Ile Pro Ala Pro Val Leu Ala Gln
385 390 395 400
Ile Ile Ser Ser Leu Asn Ile Ile Ser Gly Asp Leu Asn Pro Gly Glu
405 410 415
Gly Ser Glu Arg Leu Glu Arg Ile Ala Phe Asn Ser Arg Tyr Leu Arg
420 425 430
Leu Gly Leu Gln Arg Leu Gly Phe Ile Val Tyr Gly Val Asp Asp Ser
435 440 445
Pro Val Ile Pro Leu Leu Leu Phe Ala Pro Ala Lys Met Pro Ala Phe
450 455 460
Ser Arg Met Leu Tyr Gln Arg Lys Ile Ala Val Val Val Val Gly Tyr
465 470 475 480
Pro Ala Thr Pro Leu Thr Ser Ser Arg Val Arg Leu Cys Val Ser Ala
485 490 495
Ser Leu Thr Lys Glu Asp Ile Asp Tyr Leu Leu Arg His Leu Ser Glu
500 505 510
Val Gly Asp Lys Leu Phe Leu Lys Phe Ser Ser Gly Ile Ala Gly Gly
515 520 525
Ser Leu Asp Gly Ser Pro Pro Arg Trp Asn Ile Glu Asp Val Leu Lys
530 535 540
Glu Thr Pro Lys Asp Cys Lys Glu Ser Lys Tyr Phe Ile Ala Thr Ala
545 550 555 560
Asn Asn
<210> 17
<211> 978
<212> DNA
<213> Pichia ciferrii
<220>
<221> CDS
<222> (1)..(978)
<400> 17
atg agc tct cat cag ttt ttg atc aac caa aca act ttg gcg gct cca 48
Met Ser Ser His Gln Phe Leu Ile Asn Gln Thr Thr Leu Ala Ala Pro
1 5 10 15
cct gtt cat ttg gtg gag aaa cca agt ttg att aat ggc ata ccg gat 96
Pro Val His Leu Val Glu Lys Pro Ser Leu Ile Asn Gly Ile Pro Asp
20 25 30
aac att tta gcc ttg att gca cct gtt ata gct tat tat tca tat tca 144
Asn Ile Leu Ala Leu Ile Ala Pro Val Ile Ala Tyr Tyr Ser Tyr Ser
35 40 45
gga ttt ttc tat gtg att gat act tta gaa att gca gaa ctt tat aga 192
Gly Phe Phe Tyr Val Ile Asp Thr Leu Glu Ile Ala Glu Leu Tyr Arg
50 55 60
att cat cca cct gaa gaa gtt agt tca aga aat aaa gct aca aaa ttt 240
Ile His Pro Pro Glu Glu Val Ser Ser Arg Asn Lys Ala Thr Lys Phe
65 70 75 80
gat gtt tta aaa gat gtt gtt tta caa cat ttt ata cag agt gtt gtt 288
Asp Val Leu Lys Asp Val Val Leu Gln His Phe Ile Gln Ser Val Val
85 90 95
ggt tat atc ttt aca tat ttt gat cca att caa tat act ggt gat gaa 336
Gly Tyr Ile Phe Thr Tyr Phe Asp Pro Ile Gln Tyr Thr Gly Asp Glu
100 105 110
gaa tat caa gct tgg aaa tta caa caa act tta cca ttt tta cca ttt 384
Glu Tyr Gln Ala Trp Lys Leu Gln Gln Thr Leu Pro Phe Leu Pro Phe
115 120 125
gat gtt gca tat tat tgg aat atg tat ggt tgg agt tgt ttg aaa att 432
Asp Val Ala Tyr Tyr Trp Asn Met Tyr Gly Trp Ser Cys Leu Lys Ile
130 135 140
ggt ctt gca ttt tta att att gat tca tgg caa tat tgg tta cat aga 480
Gly Leu Ala Phe Leu Ile Ile Asp Ser Trp Gln Tyr Trp Leu His Arg
145 150 155 160
att atg cat tta aac aag aca tta tac aaa aga ttc cat tca aga cat 528
Ile Met His Leu Asn Lys Thr Leu Tyr Lys Arg Phe His Ser Arg His
165 170 175
cat cgt ctt tat gtc cca tat gct ttt ggt gct tta tat aat gat cca 576
His Arg Leu Tyr Val Pro Tyr Ala Phe Gly Ala Leu Tyr Asn Asp Pro
180 185 190
ttt gaa ggg ttt tta ttg gat acc tta ggt acc ggt att gct gca att 624
Phe Glu Gly Phe Leu Leu Asp Thr Leu Gly Thr Gly Ile Ala Ala Ile
195 200 205
gtt act caa tta act cca aga gaa tct att gtt tta tat aca ttt tca 672
Val Thr Gln Leu Thr Pro Arg Glu Ser Ile Val Leu Tyr Thr Phe Ser
210 215 220
act ttg aaa act gtt gat gat cat tgt ggt tat tca tta cct tat gat 720
Thr Leu Lys Thr Val Asp Asp His Cys Gly Tyr Ser Leu Pro Tyr Asp
225 230 235 240
cct ttc caa att ttg ttc cca aat aac tca att tat cat gat att cat 768
Pro Phe Gln Ile Leu Phe Pro Asn Asn Ser Ile Tyr His Asp Ile His
245 250 255
cat caa caa ttt ggt atc aag acc aat ttt tca caa cct ttc ttt aca 816
His Gln Gln Phe Gly Ile Lys Thr Asn Phe Ser Gln Pro Phe Phe Thr
260 265 270
cat tgg gat gtt ttc agt aat aca aga tat aaa gaa att gat gaa tac 864
His Trp Asp Val Phe Ser Asn Thr Arg Tyr Lys Glu Ile Asp Glu Tyr
275 280 285
aga gaa aag caa aaa gct att aca att gcc aaa tat aaa gag ttt tta 912
Arg Glu Lys Gln Lys Ala Ile Thr Ile Ala Lys Tyr Lys Glu Phe Leu
290 295 300
cat gat cgt gaa att gca aaa caa aag aag aag gct gaa att tat aaa 960
His Asp Arg Glu Ile Ala Lys Gln Lys Lys Lys Ala Glu Ile Tyr Lys
305 310 315 320
gat aag aaa act gat tga 978
Asp Lys Lys Thr Asp
325
<210> 18
<211> 325
<212> PRT
<213> Pichia ciferrii
<400> 18
Met Ser Ser His Gln Phe Leu Ile Asn Gln Thr Thr Leu Ala Ala Pro
1 5 10 15
Pro Val His Leu Val Glu Lys Pro Ser Leu Ile Asn Gly Ile Pro Asp
20 25 30
Asn Ile Leu Ala Leu Ile Ala Pro Val Ile Ala Tyr Tyr Ser Tyr Ser
35 40 45
Gly Phe Phe Tyr Val Ile Asp Thr Leu Glu Ile Ala Glu Leu Tyr Arg
50 55 60
Ile His Pro Pro Glu Glu Val Ser Ser Arg Asn Lys Ala Thr Lys Phe
65 70 75 80
Asp Val Leu Lys Asp Val Val Leu Gln His Phe Ile Gln Ser Val Val
85 90 95
Gly Tyr Ile Phe Thr Tyr Phe Asp Pro Ile Gln Tyr Thr Gly Asp Glu
100 105 110
Glu Tyr Gln Ala Trp Lys Leu Gln Gln Thr Leu Pro Phe Leu Pro Phe
115 120 125
Asp Val Ala Tyr Tyr Trp Asn Met Tyr Gly Trp Ser Cys Leu Lys Ile
130 135 140
Gly Leu Ala Phe Leu Ile Ile Asp Ser Trp Gln Tyr Trp Leu His Arg
145 150 155 160
Ile Met His Leu Asn Lys Thr Leu Tyr Lys Arg Phe His Ser Arg His
165 170 175
His Arg Leu Tyr Val Pro Tyr Ala Phe Gly Ala Leu Tyr Asn Asp Pro
180 185 190
Phe Glu Gly Phe Leu Leu Asp Thr Leu Gly Thr Gly Ile Ala Ala Ile
195 200 205
Val Thr Gln Leu Thr Pro Arg Glu Ser Ile Val Leu Tyr Thr Phe Ser
210 215 220
Thr Leu Lys Thr Val Asp Asp His Cys Gly Tyr Ser Leu Pro Tyr Asp
225 230 235 240
Pro Phe Gln Ile Leu Phe Pro Asn Asn Ser Ile Tyr His Asp Ile His
245 250 255
His Gln Gln Phe Gly Ile Lys Thr Asn Phe Ser Gln Pro Phe Phe Thr
260 265 270
His Trp Asp Val Phe Ser Asn Thr Arg Tyr Lys Glu Ile Asp Glu Tyr
275 280 285
Arg Glu Lys Gln Lys Ala Ile Thr Ile Ala Lys Tyr Lys Glu Phe Leu
290 295 300
His Asp Arg Glu Ile Ala Lys Gln Lys Lys Lys Ala Glu Ile Tyr Lys
305 310 315 320
Asp Lys Lys Thr Asp
325
<210> 19
<211> 1681
<212> DNA
<213> Artificial
<220>
<223> Kassette
<400> 19
gaaattaata cgactcacta tagggagacc ggcagatccg cggccgcata ggccactagt 60
ggatctgata tcatcgatga attcgagctc ataacttcgt atagcataca ttatacgaag 120
ttattcgaca ctggatggcg gcgttagtat cgaatcgaca gcagtatagc gaccagcatt 180
cacatacgat tgacgcatga tattactttc tgcgcactta acttcgcatc tgggcagatg 240
atgtcgaggc gaaaaaaaat ataaatcacg ctaacatttg attaaaatag aacaactaca 300
atataaaaaa actatacaaa tgacaagttc ttgaaaacaa gaatcttttt attgtcagta 360
ctgattatta tggacatggc attgacatat ataaagcttg ttcaccatct gaagcagtac 420
catcatataa agcagtatct aaaccacata atgtgaaacc cattcttcta taagcatgaa 480
tagctggagc attaacatta gtaacttcta accataaatg accagcacca cgttctctag 540
caaattcagt agctaaaccc attaaagctc taccaacacc atgacctcta tgttctggag 600
caacttcaat atcttcaaca gttaatcttc tattccaacc tgaatatgaa acaacaacaa 660
aaccagctaa gtctccgtca tcaccataag caacaaaagt tcttgaatct ggatcaccat 720
cttcaccgtc atcagactcg tcatctgatt catcatctgg aaaaacttta gttaatggtg 780
gatcaactgg aacttctctt aaagtaaaac catcaccagt agcagtaact ctaaaaacag 840
tatcagtagt aaatgaacca tctaaagctt caatagcttc agcatcacct ggaactgaag 900
ttctatatct ataagcagta tcatctaaag tagtacccat tgataataaa gttgattttg 960
aagtttggaa agtagtttct ggaacttcta attcataacc ttcaaatgat gaagctggta 1020
agttaatagt gacaatctgt gtgaaaacgg gttagtaatt aaacattgtc tagtgtttcc 1080
cactgatttg gattgaaaat ttggtgattt gtggttgtat agatctaaat cttgattgtc 1140
ccctatcttt cctagatatc aaaaaaacaa tcacaaaaca atcaaagaac caattaaaat 1200
ccaatcaatt catctccatt atccacaatt catcatcgat ccaaaaatat aataacaatc 1260
tacttacttc atcatcttgg ttggcttcag tggccatagt tctggcaact ctttgagttg 1320
atctcaaaga agttgtgttt gaaagaggac gaacaatatt cttcaacatc atctttgtat 1380
agtagtctga actcctccgg gaaagtttag ttgtgttgaa tatttagttg aaaatggggg 1440
agaattgcaa acctctaata aaagttgaat acttctacta ttttcaaacc aaacaaatta 1500
tcaattgaat gtattattga attttgaatt caaaatcgat aaatttactt ttcgtttttt 1560
cgcatcaggt gtttgaaaat ggccggtgcg tcgcgaaccg ggcaaattta gagcacaata 1620
acttcgtata gcatacatta tacgaagtta tctgcaggtt acccagtggt acgaagcgcc 1680
a 1681
<210> 20
<211> 3872
<212> DNA
<213> Artificial
<220>
<223> Kassette
<400> 20
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 60
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 120
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 180
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 240
gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 300
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 360
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 420
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 480
tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 540
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 600
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 660
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 720
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 780
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 840
cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 900
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 960
ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1020
agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1080
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 1140
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 1200
cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 1260
gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 1320
tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 1380
tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 1440
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 1500
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 1560
cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 1620
aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 1680
ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 1740
tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 1800
ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 1860
acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgctg 1920
tgctctaaat ttgcccggtt cgcgacgcac cggccatttt caaacacctg atgcgaaaaa 1980
acgaaaagta aatttatcga ttttgaattc aaaattcaat aatacattca attgataatt 2040
tgtttggttt gaaaatagta gaagtattca acttttatta gaggtttgca attctccccc 2100
attttcaact aaatattcaa cacaactaaa ctttcccgga ggagttcaga ctactataca 2160
aagatgatgt tgaagaatat tgttcgtcct ctttcaaaca caacttcttt gagatcaact 2220
caaagagttg ccagaactat ggccactgaa gccaaccaag atgatgaagt aagtagattg 2280
ttattatatt tttggatcga tgatgaattg tggataatgg agatgaattg attggatttt 2340
aattggttct ttgattgttt tgtgattgtt tttttgatat ctaggaaaga taggggacaa 2400
tcaagattta gatctataca accacaaatc accaaatttt caatccaaat cagtgggaaa 2460
cactagacaa tgtttaatta ctaacccgtt ttcacacaga ttgtcactat taacttacca 2520
gcttcatcat ttgaaggtta tgaattagaa gttccagaaa ctactttcca aacttcaaaa 2580
tcaactttat tatcaatgtc aaatttatta actgttcatc aaaatttacc agctttacca 2640
gttgatgcta cttcagatga agttagaaaa aatttaatgg atatgtttag agatagacaa 2700
gctttttcag aacatacttg gaaaatgtta ttatcagttt gtagatcatg ggctgcttgg 2760
tgtaaattaa ataatagaaa atggtttcca gctgaaccag aagatgttag agattactta 2820
ttatatttac aagctagagg tttagctgtt aaaactattc aacaacactt aggacaatta 2880
aatatgttac atcgtagatc aggtttacca agaccatcag attcaaatgc tgtttcatta 2940
gttatgagaa gaattagaaa agaaaatgtt gatgctggtg aaagagctaa acaagcttta 3000
gcttttgaaa gaactgattt tgatcaagtt agatcattaa tggaaaattc agatagatgt 3060
caagatatta gaaacttagc ttttttaggt attgcttata atactttatt aagaattgct 3120
gaaattgcta gaattagagt taaagatatt tcaagaactg atggtggtag aatgttaatt 3180
catattggta gaactaaaac tttagtttca actgctggtg ttgaaaaagc tttatcatta 3240
ggtgttacta aattagttga aagatggatt tcagtttcag gtgttgctga tgatccaaat 3300
aattacttat tctgtagagt tagaaaaaat ggtgttgctg ctccatcagc tacttcacaa 3360
ttatcaacta gagctttaga aggtattttt gaagctactc atcgtttaat ctatggtgct 3420
aaagatgatt caggtcaaag atacttagct tggagtggac attcagctag agttggtgct 3480
gctagagata tggctagagc tggtgtttca attccagaaa ttatgcaagc tggaggatgg 3540
actaatgtta atattgttat gaattatatt agaaacttag attcagaaac tggtgctatg 3600
gttcgtttat tagaagatgg tgattaatca gtactgacaa taaaaagatt cttgttttca 3660
agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta 3720
gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc 3780
agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc 3840
gatactaacg ccgccatcca gtgtcgagca tg 3872
<210> 21
<211> 584
<212> DNA
<213> Pichia ciferrii
<400> 21
cagatcaaac cacatcatga gcttcaattg ataaacatga gaacatgaga ttccaattct 60
ttaacgttgt gcgtggcttg acggatccta tatacgctaa cacgctaaac gctaaacgtc 120
aagacgagaa ccaacccgca tcttgccatt gcaaggccaa ttcaagagat gttttctgga 180
taattagtgt aaagtgttca attgtgctcg aggaatccaa ccattataac ctcatccttt 240
tgagaacaat agatttggta cttattgtta taaattctat cgcaacttgt cctgtctaac 300
ggtgggaaat tggcatcacc tggtgatgtt ttggccaaca cctgagccat tacccgctgc 360
ttctcagcac catattttgt taaaccactt gatctcacca tacaacgaca ccaccgggta 420
ccacacttgc gttgggcaga aattctcaat tcgccaattc caattgtagt ataaatacat 480
catttattcc cttttatcag aaactataag taataataga agaattcttt tttcctttct 540
ctatcaattg tactcaattc gataaaacat atacacatta caca 584
<210> 22
<211> 420
<212> DNA
<213> Pichia ciferrii
<400> 22
cggaccgtta attaccaaca atctcaattg tacaacatag tgttaaaaca ggataacttg 60
atgattatat gtgatattaa gttcaaacaa gtaccaataa atagataatt aatagctcta 120
taatatatca tttaattgaa ttaatatcaa tagttgttgt ttaattatcc ctagttttct 180
ggttaaagtt acaccatcag atggttcacc accaatgttg ttcaaaccat ttccactcaa 240
ctgacgtttc aagaacatca cctgaaaaaa aaaaattcat cacacattgg gagaaattgg 300
gagaattgta tataaggagt tgaaatcgct aatattttta tacttctact cacttgtttt 360
aattctacat cagtatttta taatacaaaa acaaacaaac aaacaaataa ttaattaaca 420
<210> 23
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 23
gcttccggct cctatgttg 19
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 24
accctatgcg gtgtgaaata c 21
<210> 25
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 25
gccaatactt cacaatgttc gaatc 25
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 26
cgtgaatgta agcgtgacat aac 23
<210> 27
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 27
caaaaagtta acatgcatca ccatcaccat cacactaacc caactaggct cattaac 57
<210> 28
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 28
gttatctgca ggttacccag tggtacgaag cgccatcagc catttctgga tcaatttc 58
<210> 29
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 29
tgccggtctc cctatagtga gtcgtattaa tttcatccag ttccaggtga attataag 58
<210> 30
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 30
taactaatta catgactcga ggtcgacggt atcccatact atgcttggca tcttaaac 58
<210> 31
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 31
ttgatagggc aaattctcca ac 22
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 32
ttcacctgga taaccttctg 20
<210> 33
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 33
caaaaagtta acatgcatca ccatcaccat cacatgtcct tgcaggtggt attc 54
<210> 34
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 34
ttatctgcag gttacccagt ggtacgaagc gccaggtaaa gcgtatggca tgttg 55
<210> 35
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 35
ctgccggtct ccctatagtg agtcgtatta atttcgctgg tgaattccca ttatctg 57
<210> 36
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 36
taactaatta catgactcga ggtcgacggt atccataacc atctaaagca ttatagtc 58
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 37
aagtttcagc aaatggtttg ac 22
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 38
tatcttgcac ctggataacc 20
<210> 39
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 39
caaaaagtta acatgcatca ccatcaccat cacaatctaa gaggtaaagt tcaacattc 59
<210> 40
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 40
gttatctgca ggttacccag tggtacgaag cgccattggt ttgccgtgtg gattg 55
<210> 41
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 41
ctgccggtct ccctatagtg agtcgtatta atttcggagt tcaacaaccg ttcaag 56
<210> 42
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 42
taactaatta catgactcga ggtcgacggt atcatgaagt tgatgctgct ttgg 54
<210> 43
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 43
atttagaagc tagaggttca gaaag 25
<210> 44
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 44
tagaagaatg accatgccat atag 24
<210> 45
<211> 73
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 45
ttttaatttt aatcaaaaag ttaacatgca tcaccatcac catcacactc acagagtcaa 60
ctcctgtata ttc 73
<210> 46
<211> 75
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 46
tgaatgtaag cgtgacataa ctaattacat gactcgaggt cgacggtatc tctggcggta 60
ttgaactttg tggag 75
<210> 47
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 47
gttatctgca ggttacccag tggtaaagtg tatggatggg ttgaagtatg tctttatatc 60
<210> 48
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 48
acgaagttat gagctcgaat tcatcgatgc tacccggtgc tgcaaagact ttactaag 58
<210> 49
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 49
gtgaatggtt aatagtgcgc tatg 24
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 50
ctaacaaata ccacttcgac atcag 25
<210> 51
<211> 73
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 51
ttttaatttt aatcaaaaag ttaacatgca tcaccatcac catcacacct tccgtgagat 60
ttcccttgtt tac 73
<210> 52
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 52
tatacgaagt tatctgcagg ttacccagtg gtataaccca taaccagtga tgttaacc 58
<210> 53
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 53
gaagttatga gctcgaattc atcgatgacc actggtgttg ttgatcg 47
<210> 54
<211> 75
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 54
tgaatgtaag cgtgacataa ctaattacat gactcgaggt cgacggtatc cgacggtaat 60
gaggatgtaa atgag 75
<210> 55
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 55
aaacaagagc agcatgcaac ttgag 25
<210> 56
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 56
agtgacacca ggaactctaa ag 22
<210> 57
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 57
gctttacact ttatgcttcc ggctcctatg ttgaactatg tcaatatcga tcgtatg 57
<210> 58
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 58
tatctgcagg ttacccagtg gtacgaagcg ccaaacagaa attggttcat gtgttg 56
<210> 59
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 59
gccggtctcc ctatagtgag tcgtattaat ttctggtgta ccaatttggt tatttc 56
<210> 60
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 60
atatcagtta ttaccctatg cggtgtgaaa tacacaagta caacaacaac agatttag 58
<210> 61
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 61
tacccacctt tgacataatc ag 22
<210> 62
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 62
attcaaatgg cgtaccttta ac 22
<210> 63
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 63
gctttacact ttatgcttcc ggctcctatg ttgggactgc tacactccaa atatg 55
<210> 64
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 64
ttatctgcag gttacccagt ggtacgaagc gccataatag aagaaacacg tcaaatacc 59
<210> 65
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 65
gccggtctcc ctatagtgag tcgtattaat ttccagatca aaccacatca tgag 54
<210> 66
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 66
gtagcagtga cgttcattgt gtaatgtgta tatgttttat c 41
<210> 67
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 67
catatacaca ttacacaatg aacgtcactg ctacaac 37
<210> 68
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 68
atatcagtta ttaccctatg cggtgtgaaa tacacaagca ccaacaccat tac 53
<210> 69
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 69
gttgtgcgtg gcttgac 17
<210> 70
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 70
ataatacagc accaccaact tc 22
<210> 71
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 71
gctttacact ttatgcttcc ggctcctatg ttgggccatg agatgacttt gtacg 55
<210> 72
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 72
ttatctgcag gttacccagt ggtacgaagc gccagttctt gtttgaattc gcgtttg 57
<210> 73
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 73
gttatgagct cgaattcatc gatgatatca gggaccgtta attaccaaca atctc 55
<210> 74
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 74
tgttaattaa ttatttgttt gtttg 25
<210> 75
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 75
acaaacaaac aaacaaataa ttaattaaca atgtcattgg taatacctca aatag 55
<210> 76
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 76
atatcagtta ttaccctatg cggtgtgaaa tacaaagcgg cttgagtaca tgc 53
<210> 77
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 77
aactgacgtt tcaagaacat c 21
<210> 78
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 78
ataaacttgc atttgttgca tacc 24
<210> 79
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 79
gctttacact ttatgcttcc ggctcctatg ttgaaagtgt aaatagacgt catgag 56
<210> 80
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 80
ttatctgcag gttacccagt ggtacgaagc gccactgtgt actaaacgtg ataaatcc 58
<210> 81
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 81
gttatgagct cgaattcatc gatgatatca gggaccgtta attaccaaca atctc 55
<210> 82
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 82
tgttaattaa ttatttgttt gtttg 25
<210> 83
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 83
aaaacaaaca aacaaacaaa taattaatta acaatgagct ctcatcagtt tttg 54
<210> 84
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 84
atatcagtta ttaccctatg cggtgtgaaa tacaagacga tgatgtcttg aatg 54
<210> 85
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 85
aactgacgtt tcaagaacat c 21
<210> 86
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 86
agtaacaatt gcagcaatac c 21
Claims (8)
- 피키아 시페리이(Pichia ciferrii) 세포가 그의 야생형과 비교하여,
A) 서열 1, 서열 3, 서열 5, 서열 7, 서열 9, 서열 11; 및
B) 서열 1, 서열 3, 서열 5, 서열 7, 서열 9, 서열 11의 서열 중 어느 것과 적어도 80% 동일한 서열
로 이루어진 2개 군 A) 및 B)로부터 선택된 인트론-무함유 핵산 서열에 의하여 코딩되는 적어도 1종의 효소의 감소한 활성을 보유함을 특징으로 하는, 피키아 시페리이 세포. - 제1항에 있어서, 효소 활성의 감소가 제1항에 특정된 임의의 핵산 서열을 포함하는 유전자를 변형시킴으로써 달성되고, 여기서 변형은 유전자 내로의 외래 DNA 삽입, 유전자의 적어도 일부 결실, 유전자 서열에서의 점 돌연변이, RNA 간섭의 영향에의 유전자 노출, 및 외래 DNA, 특히 프로모터 영역의 외래 DNA에 의한 유전자의 일부 치환을 포함하는 군에서 선택됨을 특징으로 하는 피키아 시페리이 세포.
- 제2항에 있어서, 외래 DNA가 선택 마커 유전자, 바람직하게는 흔적을 남기지 않으면서 제거될 수 있고 표적 유전자에 결실을 남기는 선택 마커 유전자임을 특징으로 하는 피키아 시페리이 세포.
- 제1항 내지 제3항 중 어느 한 항에 있어서, 피키아 시페리이 NRRL Y-1031 F-60-10, WO 95/12683에 개시된 피키아 시페리이 균주 및 피키아 시페리이 CS.PCΔPro2로 이루어진 군으로부터 선택된 균주에서 유래함을 특징으로 하는 피키아 시페리이 세포.
- 제1항 내지 제4항 중 어느 한 항에 있어서, 야생형과 비교하여,
세린과 팔미토일-CoA의 반응을 촉매하여 3-케토스핑가닌을 생성하는 효소 E1, 특히 세린 팔미토일 트랜스페라제, 특히 서열 13 및/또는 서열 15에 의하여 코딩되는 것, 및 스핑가닌에서 피토스핑고신으로의 반응을 촉매하는 효소 E2, 특히 스핑가닌 C4-히드록실라제, 특히 서열 17에 의하여 코딩되는 것으로부터 선택되는 적어도 1종의 효소의 증가한 효소 활성을 보유함을 특징으로 하는 피키아 시페리이 세포. - 스핑고이드 염기 및 스핑고지질의 제조를 위한 제1항 내지 제5항 중 어느 한 항에 따른 세포의 용도.
- I) 피키아 시페리이 세포를 제공하는 단계; 및
II) 제1항에 특정된 핵산 서열 군 A) 및 B)로부터 선택된 임의의 서열을 포함하는 적어도 1종의 유전자를, 그 유전자 내로의 외래 DNA, 특히 선택 마커 유전자를 코딩하는 DNA의 삽입, 유전자의 적어도 일부 결실, 유전자 서열에서의 점 돌연변이, RNA 간섭의 영향에의 유전자 노출, 및 외래 DNA, 특히 프로모터 영역의 외래 DNA에 의한 유전자의 일부 치환에 의하여 변형시키는 단계
를 포함하는, 제1항 내지 제5항 중 어느 한 항에 따른 피키아 시페리이 세포를 제조하는 방법. - a) 제1항 내지 제5항 중 어느 한 항에 따른 세포를, 탄소원을 포함하는 배지와 접촉시키는 단계;
b) 세포가 상기 탄소원으로부터 스핑고이드 염기 및 스핑고지질을 생성할 수 있게 해주는 조건하에서 세포를 배양하는 단계; 및
c) 임의로, 생성된 스핑고이드 염기 및 스핑고지질을 단리하는 단계
를 포함하는, 스핑고이드 염기 및 스핑고지질을 제조하는 방법.
Applications Claiming Priority (3)
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DE102011110959A DE102011110959A1 (de) | 2011-08-18 | 2011-08-18 | Pichia ciferrii Zellen und deren Verwendung |
DE102011110959.9 | 2011-08-18 | ||
PCT/EP2012/064369 WO2013023878A1 (de) | 2011-08-18 | 2012-07-23 | Pichia ciferrii zellen und deren verwendung |
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KR20140063599A true KR20140063599A (ko) | 2014-05-27 |
KR102056651B1 KR102056651B1 (ko) | 2019-12-17 |
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KR1020147003745A KR102056651B1 (ko) | 2011-08-18 | 2012-07-23 | 피키아 시페리이 세포 및 그의 용도 |
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US (2) | US9404118B2 (ko) |
EP (1) | EP2744896B1 (ko) |
JP (1) | JP6023807B2 (ko) |
KR (1) | KR102056651B1 (ko) |
CN (1) | CN103748218B (ko) |
DE (1) | DE102011110959A1 (ko) |
WO (1) | WO2013023878A1 (ko) |
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KR102343479B1 (ko) * | 2020-06-23 | 2021-12-27 | 한국생명공학연구원 | 스핑고지질 및 스핑고이드 염기의 생산성이 향상된 피키아 시페라이 변이 균주 및 이의 제조방법 |
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ES2832555T3 (es) | 2017-03-27 | 2021-06-10 | Evonik Operations Gmbh | Procedimiento y producto para la preparación de formulaciones con contenido en ceramida |
JP2024503900A (ja) | 2021-01-20 | 2024-01-29 | 味の素株式会社 | フィトスフィンゴシンまたはフィトセラミドの製造法 |
CN115300410A (zh) * | 2022-02-23 | 2022-11-08 | 广州源燊创生物科技有限公司 | 水溶性神经酰胺的原料组成配比及其生产方法 |
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WO2006048458A2 (en) | 2004-11-05 | 2006-05-11 | Cosmoferm B.V. | Microbial strains producing sphingoid bases or derivatives thereof |
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KR102343479B1 (ko) * | 2020-06-23 | 2021-12-27 | 한국생명공학연구원 | 스핑고지질 및 스핑고이드 염기의 생산성이 향상된 피키아 시페라이 변이 균주 및 이의 제조방법 |
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DE102011110959A1 (de) | 2013-02-21 |
US20160304916A1 (en) | 2016-10-20 |
CN103748218A (zh) | 2014-04-23 |
JP2014529400A (ja) | 2014-11-13 |
KR102056651B1 (ko) | 2019-12-17 |
EP2744896A1 (de) | 2014-06-25 |
EP2744896B1 (de) | 2016-03-16 |
CN103748218B (zh) | 2016-10-12 |
US9598711B2 (en) | 2017-03-21 |
JP6023807B2 (ja) | 2016-11-09 |
US20140199736A1 (en) | 2014-07-17 |
WO2013023878A1 (de) | 2013-02-21 |
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