CN103974966A - 藻类去饱和酶 - Google Patents

藻类去饱和酶 Download PDF

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CN103974966A
CN103974966A CN201280031865.3A CN201280031865A CN103974966A CN 103974966 A CN103974966 A CN 103974966A CN 201280031865 A CN201280031865 A CN 201280031865A CN 103974966 A CN103974966 A CN 103974966A
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desaturase
sequence
nucleotide sequence
acid
seq
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O·基利安
B·维克
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Aurora Biofuels Inc
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)

Abstract

本文提供了编码具有去饱和酶活性的多肽的示例性经分离核苷酸序列，所述去饱和酶利用脂肪酸作为底物。

Description

藻类去饱和酶
相关申请的交叉引用
本申请要求2011年4月28日提交的标题为“Desaturases（去饱和酶）”的美国临时专利序列号61/480,353的权益和优先权，其通过引用纳入本文。
本申请涉及2009年10月19日提交的标题为“Homologous Recombination in anAlgal Nuclear Genome（藻类核基因组的同源重组）”的美国非临时专利申请序列号第12/581,812，其通过引用纳入本文。
本申请涉及2009年6月8日提交的标题为“VCP-Based Vectors for Algal CellTransformation（用于藻类细胞转化的基于VCP的载体）”的美国非临时专利申请序列号12/480,635号，其通过引用纳入本文。
本申请涉及2009年6月8日提交的标题为“Transformation of Algal Cells（藻类细胞转化）”的美国非临时专利申请序列号12/480,611，其通过引用纳入本文。
关于序列表
本申请与所附序列表一起提交，其通过引用纳入本文。
发明背景
技术领域
本发明涉及分子生物学，更具体而言，涉及藻类去饱和酶。
发明内容
编码具有去饱和酶活性的多肽的经分离核苷酸序列，所述去饱和酶用脂肪酸作为底物。
附图简要说明
图1为示例性去饱和酶的列表。
图2显示了编码去饱和酶3的核苷酸序列(SEQ ID NO:1)。
图3显示了编码去饱和酶5的核苷酸序列(SEQ ID NO:2)。
图4显示了编码去饱和酶7的核苷酸序列(SEQ ID NO:3)。
图5显示了编码去饱和酶9的核苷酸序列(SEQ ID NO:4)。
图6显示了编码去饱和酶10的核苷酸序列(SEQ ID NO:5)。
图7显示了编码去饱和酶3的氨基酸序列(SEQ ID NO:6)。
图8显示了编码去饱和酶5的氨基酸序列(SEQ ID NO:7)。
图9显示了编码去饱和酶7的氨基酸序列(SEQ ID NO:8)。
图10显示了编码去饱和酶9的氨基酸序列(SEQ ID NO:9)。
图11显示了编码去饱和酶10的氨基酸序列(SEQ ID NO:10)。
发明详述
脂肪酸是具有长脂族尾部（链）的羧酸，可以是饱和或不饱和的。饱和脂肪酸是通常具有12～24个碳原子并且不具有双键的长链羧酸。不饱和脂肪酸在碳原子间有一个或多个双键。大部分天然存在的脂肪酸具有偶数碳原子链，范围为4～28。脂肪酸去饱和酶是去除脂肪酸中两个氢原子从而产生碳/碳双键的酶。这些去饱和酶分为Δ去饱和酶，表示双键在脂肪酸羧基的固定位点产生（例如，Δ9（“Δ9”）去饱和酶在离羧基端的第9位点产生双键），或分为ω去饱和酶（例如，ω3“ω3”去饱和酶，其在离脂肪酸甲基端的第三个和第四个碳之间产生双键）。
本文提供了编码具有去饱和酶活性的多肽的经分离核苷酸序列，所述去饱和酶用脂肪酸作为底物。
发明人对微拟球藻属（genus Nannochloropsis）的整个基因组进行测序并鉴定了涉及脂肪酸代谢的基因。他们鉴定了多种去饱和酶，包括命名为去饱和酶3（“desat3”）、去饱和酶5（“desat5”）、去饱和酶7（“desat7”）、去饱和酶9（“desat9”）和去饱和酶10（“desat10”）示例性去饱和酶。
发明人通过以下方法操作上述示例性去饱和酶基因的活性：
1.用强启动子过度表达所述去饱和酶基因。
2.通过同源重组在基因组中所述去饱和酶基因前面进行启动子取代或启动子插入。
3.通过向基因中插入转化构建体来敲除所述去饱和酶基因或通过同源重组取代部分或完整的所述去饱和酶基因。
对上述方法的示例性支持可参见2009年10月19日提交的标题为“藻类核基因组的同源重组”的美国非临时专利申请序列号12/581,812，2009年6月8日提交的标题为“用于藻类细胞转化的基于VCP的载体”的美国非临时专利申请序列号12/480,635和2009年6月8日提交的标题为“藻类细胞的转化”的美国非临时专利申请序列号12/480,611，所述专利申请都通过引用纳入本文。
因此，发明人能出于改变微拟球藻属中特定脂肪酸含量的目的操作多种示例性去饱和酶的活性。
图1为示例性去饱和酶的列表。该表包括各去饱和酶编号，标题为“去饱和酶号”，相应的序列标识号，标题为“SEQ ID NO”，去饱和酶种类，标题为“去饱和酶种类”，氮气存在或不存在的情况下去饱和酶基因调节的相对提高，标题为“氮气存在/不存在情况下的上调倍数”，第一去饱和酶底物，标题为“底物1”，所得第一产物，标题为“产物1”，第二去饱和酶底物（如果适用），标题为“底物2”，所得第二产物（如果适用），标题为“产物2”，特定突变构建体中去饱和酶基因上调或是下调（有关底物1转化为产物1的反应），标题为“突变体中基因上调/下调”，所观察到的通过突变构建体使相应底物转化为产物的反应提高或降低（相比野生型微拟球藻对照细胞），标题为“底物1向产物1的转化”，特定突变构建体中去饱和酶基因上调或是下调（有关底物2转化为产物2的反应）（如果适用），标题为“突变体中基因上调/下调”，所观察到的通过突变构建体使相应底物转化为产物的反应提高或降低（相比野生型微拟球藻对照细胞），标题为“底物2向产物2的转化”（如果适用）。
如图1所示，发明人鉴定了5个示例性去饱和酶，称为去饱和酶3(“desat3”)、去饱和酶5(“desat5”)、去饱和酶7(“desat7”)、去饱和酶9(“desat9”)和去饱和酶10(“desat10”)。各去饱和酶具有相对应的SEQ ID NO，其反映了各去饱和酶的核苷酸分子。图1中还显示了去饱和酶类型（表示相应的酶活性）。标题为“氮气存在/不存在情况下的上调倍数”的一列反映了在氮气充足(“N+”)或氮气不充足条件(“N-“)下生长的样品的全转录组比较。标题为“底物1向产物1的转化”和“底物2向产物2的转化”（如果适用）的列显示所观察到的通过突变构建体使相应底物转变为产物的反应提高或降低（相对野生型微拟球藻对照细胞）。
再来参见图1中的表格，去饱和酶3（一种Δ12去饱和酶）在氮气不存在的情况下大约上调1.5倍。去饱和酶3使棕榈油酸（Palmitolenic acid）(16:2(n-7))向6,9,12十六碳三烯酸(16:3(n-4))的转化提高大约85％（相比野生型微拟球藻对照细胞）。去饱和酶3还使油酸即顺式-9-十八烯酸(18:1(n-9))向亚油酸即全顺式-9,12-十八碳二烯酸(18:2(n-6))的转化提高大约50％（相比野生型微拟球藻对照细胞）。
如图1中表格所示，去饱和酶5也是Δ12去饱和酶。去饱和酶5在氮气存在或不存在时不受调节。去饱和酶5使油酸即顺式-9-十八烯酸(18:1(n-9))向12-十八碳二烯酸(18:2(n-6))的转化降低大约100％（相比野生型微拟球藻对照细胞）。
如图1中表格所示，去饱和酶7是ω3去饱和酶。去饱和酶7在氮气存在时上调约4.0倍。去饱和酶7的过度表达由于使更多的ARA转化为二十碳五烯酸即全顺式-5,8,11,14,17二十碳五烯酸(20:5(n-3))(“EPA”)从而降低了花生四烯酸即全-顺式-5,8,11,14-二十碳四烯酸(20:4(n-6))(“ARA”)的量（相比野生型微拟球藻对照细胞）。事实上，EPA与ARA之比从约7.5变为约22，大约提高了200％。去饱和酶7使亚油酸即全顺式-9,12-的十八碳二烯酸(18:2(n-6))向α-亚麻酸即全顺式-9,12,15-十八碳三烯酸(18:3(n-3))的转化提高大约650％（相比野生型微拟球藻对照细胞）。事实上，α-亚麻酸即全顺式-9,12,15-十八碳二烯酸(18:3(n-3))与亚麻酸即全顺式-9,12-十八碳二烯酸(18:2(n-6))之比从约2.5变为约15.8，大约提高550％。
如图1中表格所示，去饱和酶9是Δ9去饱和酶。去饱和酶9在氮气不存在时上调约6.6倍。去饱和酶9使棕榈酸或十六烷酸(16:0)向棕榈油酸即顺式-9-十六烯酸(16:1(n-7))的转变大约提高25％，而使棕榈油酸即顺式-9-十六烯酸(16:1(n-7))与棕榈酸或十六烷酸(16:0)之比从约.48增加至0.6，大约提高25％。
如图1中表格所示，去饱和酶10是Δ9去饱和酶。去饱和酶9在氮气不存在时上调约5.0倍。去饱和酶9使棕榈酸或十六烷酸(16:0)向棕榈油酸即顺式-9-十六烯酸(16:1(n-7))的转变大约提高34％，而使棕榈油酸即顺式-9-十六烯酸(16:1(n-7))与棕榈酸或十六烷酸(16:0)之比从约1.02增加至1.19，大约提高16％。
图2显示了编码去饱和酶3的核苷酸序列(SEQ ID NO:1)。
发明人发现去饱和酶3在氮饥饿的情况下略微上调。发明人用包含强上游启动子的去饱和酶3制备构建体。这使16:3n4和18:2n6脂肪酸的表达量提高。
图3显示了编码去饱和酶5的核苷酸序列(SEQ ID NO:2)。
发明人发现去饱和酶5编码与Δ12去饱和酶具有高度同源性的脂肪酸去饱和酶。发明人还发现，当构建体在氮饥饿条件下生长时（请注意：在氮饥饿条件下诱导启动子），该去饱和酶基因在诱导型脲酶启动子调控下的过度表达导致18:1n9脂肪酸和具有18个或更多碳原子的多不饱和脂肪酸(“PUFA”)的表达水平更高。
在其它实验中，发明人确定Δ12位点上的去饱和步骤可能是使碳进入PUFA生物合成通路的主要瓶颈。虽然18:1n9脂肪酸在氮饥饿期间稳步上升，18:2n6脂肪酸（衍生自所述18:1n9脂肪酸的Δ12去饱和）是降低的（在所有脂肪酸的百分比基础上），因为该通路中所有脂肪酸引起二十碳五烯酸(“EPA”)生成。发明人得出结论，如果去饱和酶5基因过度表达，去饱和酶5基因增加氮饥饿期间进入PUFA生物合成通路的碳通量。
图4显示了编码去饱和酶7的核苷酸序列(SEQ ID NO:3)。
发明人制备多种构建体（启动子取代，敲除和过度表达构建体）并发现去饱和酶7基因的下调导致更低的EPA/花生四烯酸(“ARA”)比例，即更少的ARA被去饱和成为EPA。发明人在突变构建体中观察到，更少的去饱和酶7转录是由于野生型微拟球藻细胞中天然启动子的交换。发明人观察到，突变构建体相比野生型微拟球藻对照细胞具有近两倍的ARA水平和更低水平的EPA。
发明人还发现去饱和酶7基因的上调导致更高的18:3n3/18:2n6和EPA/ARA比例，即更多18:2n6转化为18:3n3，且更多ARA转化为EPA。因此，发明人观察到EPA/ARA比例几乎翻倍。
图5显示了编码去饱和酶9的核苷酸序列(SEQ ID NO:4)。
图6显示了编码去饱和酶10的核苷酸序列(SEQ ID NO:5)。
发明人发现去饱和酶9和去饱和酶10似乎都是Δ9去饱和酶，其主要作用于16:0脂肪酸或结合其它化合物的16:0脂肪酸。启动子交换实验中发明人用强启动子交换天然野生型微拟球藻启动子，揭示在氮缺乏条件下所述活性上调。因此在氮饥饿的情况下，高百分比的脂肪酸通过去饱和酶9的作用导向16:1n7脂肪酸的累积，这意味着更少的脂肪酸进入PUFA通路。发明人还用中等强度的启动子取代去饱和酶9基因的启动子，推定当细胞进入氮饥饿时其不会受到调节，目的是在饥饿期间避免碳通量进入16:1n7和18:1n7脂肪酸的生物合成并提高碳通量进入PUFA生物合成通路。发明人发现这些基因是为了提高PUFA生物合成而过度表达的极佳靶标。例如，通过在各去饱和酶基因前方取代内源启动子或插入更弱的启动子而下调这些（或其它）基因可产生更高含量的短链脂肪酸。在一些情况下也可通过在各去饱和酶基因前方插入常见强启动子实现转录的下调，可能通过改变去饱和酶基因周围的各染色质排列从而导致更低的转录水平。同样，在去饱和酶基因前方通过同源重组两侧来插入另一个启动子时，向去饱和酶基因导入点突变，这可能导致各基因产物的活性改变。
发明人还发现，在氮充足的条件下所选去饱和酶10的过度表达突变体中16:1n7脂质增加，这清楚地表明，相比相同实验中的野生型微拟球藻细胞，该突变体中的16:1n7脂肪酸增加了大约34%。
图7显示了编码去饱和酶3的氨基酸序列(SEQ ID NO:6)。
图8显示了编码去饱和酶5的氨基酸序列(SEQ ID NO:7)。
图9显示了编码去饱和酶7的氨基酸序列(SEQ ID NO:8)。
图10显示了编码去饱和酶9的氨基酸序列(SEQ ID NO:9)。
图11显示了编码去饱和酶10的氨基酸序列(SEQ ID NO:10)。
尽管以上描述了多个实施方式，但应理解，它们仅以举例方式呈现，并不构成限制。因此，优选实施方式的广度和范围不应受任何上述示例性实施方式的限制。

Claims (19)

1.一种编码具有去饱和酶活性的多肽的经分离核苷酸序列，所述核苷酸序列与SEQ ID NO:1具有至少95%的序列相同性。
2.如权利要求1所述的经分离核苷酸序列，其特征在于，所述序列编码功能活性去饱和酶，所述去饱和酶利用脂肪酸作为底物。
3.如权利要求2所述的经分离核苷酸序列，其特征在于，所述功能活性去饱和酶包含具有SEQ ID NO:6所示序列或与所述序列至少95％相同序列的氨基酸。
4.一种编码具有去饱和酶活性的多肽的经分离核苷酸序列，所述核苷酸序列与SEQ ID NO:2具有至少95%的序列相同性。
5.如权利要求4所述的经分离核苷酸序列，其特征在于，所述序列编码功能活性去饱和酶，所述去饱和酶利用脂肪酸作为底物。
6.如权利要求5所述的经分离核苷酸序列，其特征在于，所述功能活性去饱和酶包含具有SEQ ID NO:7所示序列或与所述序列至少95％相同序列的氨基酸。
7.一种编码具有去饱和酶活性的多肽的经分离核苷酸序列，所述核苷酸序列与SEQ ID NO:3具有至少95%的序列相同性。
8.如权利要求7所述的经分离核苷酸序列，其特征在于，所述序列编码功能活性去饱和酶，所述去饱和酶利用脂肪酸作为底物。
9.如权利要求8所述的经分离核苷酸序列，其特征在于，所述功能活性去饱和酶包含具有SEQ ID NO:8所示序列或与所述序列至少95％相同序列的氨基酸。
10.一种编码具有去饱和酶活性的多肽的经分离核苷酸序列，所述核苷酸序列与SEQ ID NO:4具有至少95%的序列相同性。
11.如权利要求10所述的经分离核苷酸序列，其特征在于，所述序列编码功能活性去饱和酶，所述去饱和酶利用脂肪酸作为底物。
12.如权利要求11所述的经分离核苷酸序列，其特征在于，所述功能活性去饱和酶包含具有SEQ ID NO:9所示序列或与所述序列至少95％相同序列的氨基酸。
13.一种编码具有去饱和酶活性的多肽的经分离核苷酸序列，所述核苷酸序列与SEQ ID NO:5具有至少95%的序列相同性。
14.如权利要求13所述的经分离核苷酸序列，其特征在于，所述序列编码功能活性去饱和酶，所述去饱和酶利用脂肪酸作为底物。
15.如权利要求14所述的经分离核苷酸序列，其特征在于，所述功能活性去饱和酶包含具有SEQ ID NO:10所示序列或与所述序列至少95％相同序列的氨基酸。
16.如权利要求1所述的经分离核苷酸序列，其特征在于，所述序列来自藻类。
17.如权利要求16所述的经分离核苷酸序列，其特征在于，所述藻类是微拟球藻属。
18.如权利要求7所述的经分离核苷酸序列，其特征在于，所述序列来自藻类。
19.如权利要求18所述的经分离核苷酸序列，其特征在于，所述藻类是微拟球藻属。
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