CN114921482A - 一种用于微生物合成乙醇的融合基因、蛋白及表达方法 - Google Patents
一种用于微生物合成乙醇的融合基因、蛋白及表达方法 Download PDFInfo
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Abstract
本发明属于基因工程技术领域，公开了一种用于微生物合成乙醇的融合基因、蛋白及表达方法，所述用于微生物合成乙醇的融合基因核苷酸序列为SEQ IDNO：1；所述用于微生物合成乙醇的融合基因表达的蛋白的氨基酸序列为SEQ IDNO：2；所述融合表达载体的构建方法包括：利用一种含有30个碱基的linker去融合丙酮酸脱羧酶基因(pdc基因)和乙醇脱氢酶基因(slr1192基因)，构建得到1个融合表达载体pET‑Fus1sp；所述含有30个碱基的linker核苷酸序列为SEQ ID：7；pdc基因来自运动发酵单胞菌；slr1192基因来自集胞藻PCC6803。本发明通过两个外源基因融合，提高两个外源基因的协同表达和表达一致性。
Description
技术领域
本发明属于基因工程技术领域，尤其涉及一种用于微生物合成乙醇的融合基因、蛋白及表达方法。
背景技术
目前，随着化石能源的逐渐枯竭和由于使用化石能源导致环境问题的日益严重，人们对可再生清洁能源的需求持续增加。生物乙醇作为一种可再生清洁燃料用在现有的内燃机上目前已经实现商业化应用。然而，目前生物乙醇主要是通过(耕种)粮食作物发酵得到。这种生物乙醇生产方式被广泛认为造成全球粮食价格飞涨，是非可持续生产方式。近年来以非粮食作物为核心的纤维素乙醇也得到了大力发展。但是由于降解纤维素生物质的纤维素酶生产成本太高，目前纤维素乙醇还没有进入大范围应用阶段。
微生物合成乙醇被认为是一种可持续和环境友好的生产方式。为了实现乙醇在微生物中的合成，人们在微生物中通过基因同组插入外源基因构建了异源生物合成乙醇途径。这一异源生物合成乙醇途径主要包括丙酮酸脱羧酶基因和乙醇脱氢酶基因。由于这两个基因在微生物中的表达量和时空时序不一致导致生物合成乙醇产量依然较低。为了使得这两个外源基因的表达量和时空时序一致，一种方法就是将两个外源基因融合起来构建融合表达基因从而提高两个外源基因的协同表达和表达一致性。
通过上述分析，现有技术存在的问题及缺陷为：
(1)目前生物乙醇主要是通过(耕种)粮食作物发酵得到，该生物乙醇的生产方法需要消耗大量的粮食作物，是非可持续生产方式。
(2)以非粮食作物为核心的纤维素乙醇的生产方法中，由于降解纤维素生物质的纤维素酶生产成本太高，目前纤维素乙醇还没有进入大范围应用阶段。
(3)现有的微生物合成乙醇的方法中，通过基因同组插入外源基因构建异源生物合成乙醇途径，但由于两个基因在微生物中的表达量和时空时序不一致，导致生物合成乙醇产量依然较低。
发明内容
针对现有技术存在的问题，本发明提供了一种用于微生物合成乙醇的融合基因、蛋白及表达方法。
本发明是这样实现的，一种用于微生物合成乙醇的融合基因。
进一步，所述用于微生物合成乙醇的融合基因核苷酸序列为SEQ ID NO：1。
本发明的另一目的在于提供一种应用所述的用于微生物合成乙醇的融合基因表达的蛋白。
进一步，所述用于微生物合成乙醇的融合基因表达的蛋白的氨基酸序列为SEQ IDNO：2。
本发明的另一目的在于提供一种应用所述的用于微生物合成乙醇的融合基因构建得到的融合表达载体。
本发明的另一目的在于提供一种应用所述的融合表达载体的构建方法，所述融合表达载体的构建方法包括：
1)以运动发酵单胞菌的基因组为模板，以Fus1sp_pdc_rev和Fus1sp_pdc_fwd为引物扩增pdc基因，Fus1sp_pdc_rev和Fus1sp_pdc_fwd引物序列分别为SEQ ID NO：3和SEQ IDNO：4。
2)以集胞藻PCC6803的基因组为模板，以Fus1sp_VEC_rev和Fus1sp_VEC_fwd为引物扩增集胞藻PCC6803基因组中的slr1192基因，Fus1sp_VEC_rev和Fus1sp_VEC_fwd引物序列分别为SEQ ID NO：5和SEQ ID NO：6。
3)以上述扩增得到的pdc基因和slr1192基因互为引物和模板进行常规分子克隆，然后再加入Fus1sp_pdc_rev和Fus1sp_VEC_fwd引物进行分子克隆，构建得到pdc和slr1192基因连接在一起的融合基因。该融合基因含有一个包含30个碱基的linker，其核苷酸序列为SEQ ID NO：7。
4)将上述融合基因和pET-28a载体用相同的内切酶酶切后再用连接酶连接构建得到融合表达载体pET-Fus1sp。
进一步，pdc基因来自运动发酵单胞菌。
进一步，slr1192基因来自集胞藻PCC6803。
本发明的另一目的在于提供一种所述的用于微生物合成乙醇的融合基因在微生物合成乙醇中的应用。
结合上述的技术方案和解决的技术问题，请从以下几方面分析本发明所要保护的技术方案所具备的优点及积极效果为：
第一、针对上述现有技术存在的技术问题以及解决该问题的难度，紧密结合本发明的所要保护的技术方案以及研发过程中结果和数据等，详细、深刻地分析本发明技术方案如何解决的技术问题，解决问题之后带来的一些具备创造性的技术效果。具体描述如下：
本发明利用一个包含30个碱基的linker去融合来自运动发酵单胞菌的丙酮酸脱羧酶基因(pdc基因)和来自集胞藻PCC6803的乙醇脱氢酶基因(slr1192基因)，最终构建得到了融合表达载体：pET-Fus1sp。
第二，把技术方案看做一个整体或者从产品的角度，本发明所要保护的技术方案具备的技术效果和优点，具体描述如下：
本发明通过将两个外源基因融合起来构建融合表达基因，从而提高两个外源基因的协同表达和表达一致性。
第三，作为本发明的权利要求的创造性辅助证据，还体现在以下几个重要方面：
本发明的技术方案转化后的预期收益和商业价值为：本发明提出的融合表达基因在微生物中的表达量和表达一致性得到了提高，从而提高了微生物合成乙醇的产量，具有较大的商业价值。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对本发明实施例中所需要使用的附图做简单的介绍，显而易见地，下面所描述的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明实施例1提供的基因扩增结果。
图2是本发明实施例2提供的融合基因表达载体图谱。
图3是本发明实施例3提供的融合基因编码蛋白的表达结果。
图4是本发明实施例4提供的微生物合成乙醇的产量比较。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
针对现有技术存在的问题，本发明提供了一种用于微生物合成乙醇的融合基因、蛋白及表达方法，下面结合附图对本发明作详细的描述。
一、解释说明实施例。为了使本领域技术人员充分了解本发明如何具体实现，该部分是对权利要求技术方案进行展开说明的解释说明实施例。
本发明实施例提供的用于微生物合成乙醇的融合基因的核苷酸序列为SEQ IDNO：1。
1 atgggcagca gccatcatca tcatcatcac agcagcggcatgattaaagc
51 ctacgctgcc ctggaagcca acggaaaact ccaacccttt gaatacgacc
101 ccggtgccct gggtgctaat gaggtggaga ttgaggtgca gtattgtggg
151 gtgtgccaca gtgatttgtc catgattaat aacgaatggg gcatttccaa
201 ttacccccta gtgccgggtc atgaggtggt gggtactgtg gccgccatgg
251 gcgaaggggt gaaccatgtt gaggtggggg atttagtggg gctgggttgg
301 cattcgggct actgcatgac ctgccatagt tgtttatctg gctaccacaa
351 cctttgtgcc acggcggaat cgaccattgt gggccactac ggtggctttg
401 gcgatcgggt tcgggccaag ggagtcagcg tggtgaaatt acctaaaggc
451 attgacctag ccagtgccgg gccccttttc tgtggaggaa ttaccgtttt
501 cagtcctatg gtggaactga gtttaaagcc cactgcaaaa gtggcagtga
551 tcggcattgg gggcttgggc catttagcgg tgcaatttct ccgggcctgg
601 ggctgtgaag tgactgcctt tacctccagt gccaggaagc aaacggaagt
651 gttggaattg ggcgctcacc acatactaga ttccaccaat ccagaggcga
701 tcgccagtgc ggaaggcaaa tttgactata ttatctccac tgtgaacctg
751 aagcttgact ggaacttata catcagcacc ctggcgcccc agggacattt
801 ccactttgtt ggggtggtgt tggagccttt ggatctaaat ctttttcccc
851 ttttgatggg acaacgctcc gtttctgcct ccccagtggg tagtcccgcc
901 accattgcca ccatgttgga ctttgctgtg cgccatgaca ttaaacccgt
951 ggtggaacaa tttagctttg atcagatcaa cgaggcgatc gcccatctag
1001 aaagcggcaa agcccattat cgggtagtgc tcagccatag taaaaatagc
1051 agcggcctgg tgccgcgcgg cagccatatg agttatactg tcggtaccta
1101 tttagcggag cggcttgtcc agattggtct caagcatcac ttcgcagtcg
1151 cgggcgacta caacctcgtc cttcttgaca acctgctttt gaacaaaaac
1201 atggagcagg tttattgctg taacgaactg aactgcggtt tcagtgcaga
1251 aggttatgct cgtgccaaag gcgcagcagc agccgtcgtt acctacagcg
1301 tcggtgcgct ttccgcattt gatgctatcg gtggcgccta tgcagaaaac
1351 cttccggtta tcctgatctc cggtgctccg aacaacaatg atcacgctgc
1401 tggtcacgtg ttgcatcacg ctcttggcaa aaccgactat cactatcagt
1451 tggaaatggc caagaacatc acggccgcag ctgaagcgat ttacacccca
1501 gaagaagctc cggctaaaat cgatcacgtg attaaaactg ctcttcgtga
1551 gaagaagccg gtttatctcg aaatcgcttg caacattgct tccatgccct
1601 gcgccgctcc tggaccggca agcgcattgt tcaatgacga agccagcgac
1651 gaagcttctt tgaatgcagc ggttgaagaa accctgaaat tcatcgccaa
1701 ccgcgacaaa gttgccgtcc tcgtcggcag caagctgcgc gcagctggtg
1751ctgaagaagc tgctgtcaaa tttgctgatg ctctcggtgg cgcagttgct
1801accatggctg ctgcaaaaag cttcttccca gaagaaaacc cgcattacat
1851cggtacctca tggggtgaag tcagctatcc gggcgttgaa aagacgatga
1901aagaagccga tgcggttatc gctctggctc ctgtcttcaa cgactactcc
1951accactggtt ggacggatat tcctgatcct aagaaactgg ttctcgctga
2001accgcgttct gtcgtcgtta acggcgttcg cttccccagc gttcatctga
2051aagactatct gacccgtttg gctcagaaag tttccaagaa aaccggtgct
2101ttggacttct tcaaatccct caatgcaggt gaactgaaga aagccgctcc
2151ggctgatccg agtgctccgt tggtcaacgc agaaatcgcc cgtcaggtcg
2201aagctcttct gaccccgaac acgacggtta ttgctgaaac cggtgactct
2251tggttcaatg ctcagcgcat gaagctcccg aacggtgctc gcgttgaata
2301tgaaatgcag tggggtcaca tcggttggtc cgttcctgcc gccttcggtt
2351atgccgtcgg tgctccggaa cgtcgcaaca tcctcatggt tggtgatggt
2401tccttccagc tgacggctca ggaagtcgct cagatggttc gcctgaaact
2451gccggttatc atcttcttga tcaataacta tggttacacc atcgaagtta
2501tgatccatga tggtccgtac aacaacatca agaactggga ttatgccggt
2551ctgatggaag tgttcaacgg taacggtggt tatgacagcg gtgctggtaa
2601aggcctgaag gctaaaaccg gtggcgaact ggcagaagct atcaaggttg
2651ctctggcaaa caccgacggc ccaaccctga tcgaatgctt catcggtcgt
2701gaagactgca ctgaagaatt ggtcaaatgg ggtaagcgcg ttgctgccgc
2751caacagccgt aagcctgtta acaagctcct ctactag
本发明实施例提供的用于微生物合成乙醇的融合基因表达的蛋白的氨基酸序列为SEQ ID NO：2。
1 M G S S H H H H H H S S G M I K A Y A A
21 L E A N G K L Q P F E Y D P G A L G A N
41 E V E I E V Q Y C G V C H S D L S M I N
61 N E W G I S N Y P L V P G H E V V G T V
81 A A M G E G V N H V E V G D L V G L G W
101 H S G Y C M T C H S C L S G Y H N L C A
121 T A E S T I V G H Y G G F G D R V R A K
141 G V S V V K L P K G I D L A S A G P L F
161 C G G I T V F S P M V E L S L K P T A K
181 V A V I G I G G L G H L A V Q F L R A W
201 G C E V T A F T S S A R K Q T E V L E L
221 G A H H I L D S T N P E A I A S A E G K
241 F D Y I I S T V N L K L D W N L Y I S T
261 L A P Q G H F H F V G V V L E P L D L N
281 L F P L L M G Q R S V S A S P V G S P A
301 T I A T M L D F A V R H D I K P V V E Q
321 F S F D Q I N E A I A H L E S G K A H Y
341 R V V L S H S K N S S G L V P R G S H M
361 S Y T V G T Y L A E R L V Q I G L K H H
381 F A V A G D Y N L V L L D N L L L N K N
401 M E Q V Y C C N E L N C G F S A E G Y A
421 R A K G A A A A V V T Y S V G A L S A F
441 D A I G G A Y A E N L P V I L I S G A P
461 N N N D H A A G H V L H H A L G K T D Y
481 H Y Q L E M A K N I T A A A E A I Y T P
501 E E A P A K I D H V I K T A L R E K K P
521 V Y L E I A C N I A S M P C A A P G P A
541 S A L F N D E A S D E A S L N A A V E E
561 T L K F I A N R D K V A V L V G S K L R
581 A A G A E E A A V K F A D A L G G A V A
601 T M A A A K S F F P E E N P H Y I G T S
621 W G E V S Y P G V E K T M K E A D A V I
641 A L A P V F N D Y S T T G W T D I P D P6
61 K K L V L A E P R S V V V N G V R F P S
681 V H L K D Y L T R L A Q K V S K K T G A
701 L D F F K S L N A G E L K K A A P A D P
721 S A P L V N A E I A R Q V E A L L T P N
741 T T V I A E T G D S W F N A Q R M K L P
761 N G A R V E Y E M Q W G H I G W S V P A
781 A F G Y A V G A P E R R N I L M V G D G
801 S F Q L T A Q E V A Q M V R L K L P V I
821 I F L I N N Y G Y T I E V M I H D G P Y
841 N N I K N W D Y A G L M E V F N G N G G
861 Y D S G A G K G L K A K T G G E L A E A
881 I KV A L A N T D G P T L I E C F I G R
901 E D C T E E L V K W G K R V A A A N S R
921 K P V N K L L Y*
本发明实施例提供的融合表达载体的构建方法包括：利用一个含有30个碱基的linker去融合丙酮酸脱羧基因(pdc基因)和乙醇脱氢酶基因(slr1192基因)，构建得到1个融合表达载体pET-Fus1sp。
本发明实施例提供的pdc基因来自运动发酵单胞菌，slr1192基因来自集胞藻PCC6803。
本发明实施例提供的融合基因的构建方法，其步骤包括：
1)以运动发酵单胞菌的基因组为模板，以Fus1sp_pdc_rev和Fus1sp_pdc_fwd为引物扩增pdc基因，Fus1sp_pdc_rev和Fus1sp_pdc_fwd引物序列分别为SEQ ID NO：3和SEQ IDNO：4。
SEQ ID NO:3
ggtggtggtg ctagtagagg agcttgttaa c
SEQ ID NO:4
agcagcggcc tggtgccgcg cggcagccat atgagttata ctgtcggtac
2)以集胞藻PCC6803的基因组为模板，以Fus1sp_VEC_rev和Fus1sp_VEC_fwd为引物扩增集胞藻PCC6803基因组中的slr1192基因，Fus1sp_VEC_rev和Fus1sp_VEC_fwd引物序列分别为SEQ ID NO：5和SEQ ID NO：6。
SEQ ID NO:5
atggctgccg cgcggcacca ggccgctgct atttttactatggctgagca ctaccc
SEQ ID NO:6
taaaaattag caccaccacc accaccac
3)以上述扩增得到的pdc基因和slr1192基因互为引物和模板进行常规分子克隆，然后再加入Fus1sp_pdc_rev和Fus1sp_VEC_fwd引物进行分子克隆，构建得到pdc基因和slr1192基因连接在一起的融合基因。该融合基因含有一个包含30个碱基的linker，其核苷酸序列为SEQ ID NO：7。
SEQ ID NO:7
agcagcggcc tggtgccgcg cggcagccat
4)将上述融合基因和pET-28a载体用相同的内切酶酶切后再用连接酶连接构建得到融合表达载体pET-Fus1sp。
本发明尝试利用一个包含30个碱基的linker去融合来自运动发酵单胞菌的丙酮酸脱羧酶基因(pdc基因)和来自集胞藻PCC6803的乙醇脱氢酶基因(slr1192基因)，最终构建得到了融合表达载体：pET-Fus1sp。其中pdc基因来自运动发酵单胞菌，slr1192基因来自集胞藻PCC6803(见图1和图2)。图1中：(a)M：Marker；1-8：pdc基因克隆；(b)M：Marker；NC：空白对照；1-7：slr1192基因克隆；(c)M：Marker；NC：空白对照；1-7：融合基因
二、应用实施例。为了证明本发明的技术方案的创造性和技术价值，该部分是对权利要求技术方案进行具体产品上或相关技术上的应用实施例。
本发明的应用实施例提供了一种所述的用于微生物合成乙醇的融合基因在微生物合成乙醇中的应用。
实施例1
用于微生物合成乙醇的融合基因构建方法，其步骤包括：
(1)合成扩增基因所需的4个引物Fus1sp_pdc_rev、Fus1sp_pdc_fwd、Fus1sp_VEC_rev和Fus1sp_VEC_fwd，上述4个引物的核苷酸序列分别如SEQ ID NO：3，SEQ ID NO：4，SEQID NO：5和SEQ ID NO：6所示。
(2)使用常规PCR法扩增来自运动发酵单胞菌的pdc基因，反应体系为25μl，其中2×Taq 12.5μl，Fus1sp_pdc_rev 0.5μl，Fus1sp_pdc_fwd 0.5μl，运动发酵单胞菌基因组模板0.5μl，ddH2O 11μl；PCR反应条件为：95℃5min，95℃30s，55℃30s，72℃2min，30个循环；72℃10min。反应产物用1.5％琼脂糖凝胶电泳回收(图1a)。
(3)使用常规PCR法扩增来自集胞藻PCC6803的slr1192基因，反应体系为25μl，其中2×Taq 12.5μl，Fus1sp_VEC_rev 0.5μl，Fus1sp_VEC_fwd 0.5μl，集胞藻PCC6803基因组模板0.5μl，ddH2O 11μl；PCR反应条件为：95℃5min，95℃30s，55℃30s，72℃2min，30个循环；72℃10min。反应产物用1.5％琼脂糖凝胶电泳回收(图1b)。
(4)使用常规PCR法连接上述扩增的pdc基因和slr1192基因，反应体系为25μl，其中2×Taq 12.5μl，扩增的pdc基因产物2μl，扩增的slr1192基因产物2μl，ddH2O 8.5μl；PCR反应条件为：95℃5min，95℃30s，55℃30s，72℃2min，10个循环；72℃10min；然后加入Fus1sp_pdc_rev和Fus1sp_VEC_fwd引物各0.5μl，再次进行PCR反应；PCR反应条件为：95℃5min，95℃30s，55℃30s，72℃4min，30个循环；72℃10min。反应产物用1.0％琼脂糖凝胶电泳回收(图1c)。
实施例2
含有用于微生物合成乙醇的融合基因的表达载体的构建方法，其步骤包括：
(1)上述融合基因用NdeI和XhoI内切酶进行双酶切，反应体系为50μl，其中融合基因产物45μl，NdeI内切酶2.5μl，XhoI内切酶2.5μl；反应条件：将反应体系置于37℃，2h。酶切后的反应产物用1.0％琼脂糖凝胶电泳回收。
(2)pET28a载体用NdeI和XhoI内切酶进行双酶切，反应体系为50μl，其中融合基因产物45μl，NdeI内切酶2.5μl，XhoI内切酶2.5μl；反应条件：将反应体系置于37℃，2h。酶切后的反应产物用1.0％琼脂糖凝胶电泳回收。
(3)将上述两个回收的酶切产物用ligase酶连接后转化大肠杆菌DH5α感受态细胞。将DH5α菌液涂布于含卡那霉素(50μg/mL)的LB固体培养基上筛选阳性克隆，经过基因测序正确后获得融合表达载体pET-Fus1sp(图2)。
实施例3
含有用于微生物合成乙醇的融合基因编码蛋白的表达方法，其步骤包括：
(1)将融合表达载体pET-Fus1sp转化含有pTf16分子伴侣载体的大肠杆菌BL21 DE(3)感受态细胞。将BL21 DE(3)菌液涂布于含卡那霉素(50μg/mL)的LB固体培养基上；37℃恒温过夜培养后，挑取单克隆菌落做PCR鉴定。
(2)挑取鉴定正确的单克隆菌落加入到2mL含有卡那霉素(50μg/mL)和氯霉素(25μg/mL)的LB培养液中过夜培养。然后按1％接种量将过夜培养液加入至100mL LB培养液中(含有相应的抗生素)进行菌体扩大培养。待培养液OD600至0.4～0.6时加入IPTG(终浓度0.2mmol/L)于16℃，180rpm继续培养24h。
(3)离心得到的菌体用20倍体积的binding buffer(20mmol/L Tris-HCl，pH7.5缓冲液)重悬，于冰浴中超声破碎3min(超声工作5s，间歇25s，有效功率25％)。菌体裂解液于4℃下12000rpm离心10min得到菌体上清液，分别取破碎后的全菌液，上清液和沉淀重悬液，用SDS-PAGE检测目的蛋白。蛋白表达如图3所示。
三、实施例相关效果的证据。本发明实施例在研发或者使用过程中取得了一些积极效果，和现有技术相比的确具备很大的优势，下面内容结合试验过程的数据、图表等进行描述。
实施例4
融合表达载体pET-Fus1sp用于集胞藻PCC6803生物合成乙醇的效果鉴定，其步骤包括：
(1)利用基因同源重组法将融合表达载体pET-Fus1sp转入集胞藻PCC6803基因组中，将鉴定转入成功的单菌落加入BG11液体培养基中培养。
(2)将pdc基因和slr1192基因分别通过基因同源重组法转入集胞藻PCC6803基因组中，将鉴定转入成功的单菌落加入BG11液体培养基中培养。
(3)在不同的培养阶段取出一定的培养液，测定液体培养基中的乙醇含量。结果如图4所示。结果表明，相比较于含有两个非融合表达的pdc基因和slr1192基因的集胞藻PCC6803培养液，含有融合表达基因的集胞藻PCC6803培养液中的乙醇产量在培养48h，72h和96h后分别提高了约160％,110％和87％。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，都应涵盖在本发明的保护范围之内。
序列表
<110> 李志敏，李至敏，张黎明，喻伟艳
<120> 一种用于微生物合成乙醇的融合基因、蛋白及表达方法
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gttcatctga aagactatct gacccgtttg gctcagaaag tttccaagaa aaccggtgct 2100
ttggacttct tcaaatccct caatgcaggt gaactgaaga aagccgctcc ggctgatccg 2160
agtgctccgt tggtcaacgc agaaatcgcc cgtcaggtcg aagctcttct gaccccgaac 2220
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atcttcttga tcaataacta tggttacacc atcgaagtta tgatccatga tggtccgtac 2520
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tatgacagcg gtgctggtaa aggcctgaag gctaaaaccg gtggcgaact ggcagaagct 2640
atcaaggttg ctctggcaaa caccgacggc ccaaccctga tcgaatgctt catcggtcgt 2700
gaagactgca ctgaagaatt ggtcaaatgg ggtaagcgcg ttgctgccgc caacagccgt 2760
aagcctgtta acaagctcct ctactag 2787
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Claims (10)
1.一种用于微生物合成乙醇的融合基因。其特征在于，所述用于微生物合成乙醇的融合基因核苷酸序列为SEQ ID NO：1。
2.一种应用如权利要求1所述用于微生物合成乙醇的融合基因表达的蛋白。
3.如权利要求2所述用于微生物合成乙醇的融合基因表达的蛋白，其特征在于，所述用于微生物合成乙醇的融合基因表达的蛋白的氨基酸序列为SEQ ID NO：2。
4.一种应用如权利要求1所述用于微生物合成乙醇的融合基因构建得到的融合表达载体。
5.一种应用如权利要求4所述融合表达载体的构建方法，其特征在于，所述融合表达载体的构建方法包括：
利用一个含有30个碱基的linker去融合丙酮酸脱羧酶基因和乙醇脱氢酶基因，构建得到1个融合表达载体。
6.如权利要求5所述构建融合表达载体的linker，其特征在于，所述构建融合表达载体的linker核苷酸序列为SEQ ID NO：7。
7.如权利要求5所述融合表达载体的构建方法，其特征在于，所述融合表达载体为pET-Fus1sp。
8.如权利要求5所述融合表达载体的构建方法，其特征在于，pdc基因来自运动发酵单胞菌。
9.如权利要求5所述融合表达载体的构建方法，其特征在于，slr1192基因来自集胞藻PCC6803。
10.一种如权利要求1所述用于微生物合成乙醇的融合基因在微生物合成乙醇中的应用。
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