CN114752516B - 一种生产甲基酮的重组酿酒酵母及其构建方法和应用 - Google Patents
一种生产甲基酮的重组酿酒酵母及其构建方法和应用 Download PDFInfo
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- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
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Abstract
本发明公开一种生产甲基酮的重组酿酒酵母及其构建方法与应用。本发明提供的重组菌在酿酒酵母中对β‑酮酰辅酶A硫解酶基因进行敲除或失活,或降低该基因的表达或活性,可以产生甲基酮。配合过表达内源酰基辅酶A氧化酶基因,或(3R)‑羟烷基‑辅酶A脱氢酶/2‑烯酰辅酶A水合酶2基因,或酰基辅酶A硫酯酶基因,或脂酰辅酶A转运蛋白编码基因,或外源β‑酮酸脱羧酶基因中的一种或多种,大大提高产生甲基酮的能力。进一步配合过表达外源氧化酶基因,或水合酶基因,或脱氢酶基因,或硫酯酶基因,或脱羧酶基因中的一种或多种,可进一步提高产生甲基酮的能力。本发明通过实验证明获得的重组菌具有较大实际应用价值。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种生产甲基酮的重组酿酒酵母及其构建方法与应用。
背景技术
甲基酮是一类甲基直接与羰基相连的化合物的统称,包括丙酮,2-丁酮,2-戊酮,2-己酮,2-庚酮,2-辛酮,2-壬酮,2-癸酮,2-十一烷酮,2-十二烷酮,2-十三烷酮,2-十四烷酮,2-十五烷酮,2-十六烷酮,2-十七烷酮,2-十八烷酮,2-十九烷酮,2-二十烷酮,等等。甲基酮具有多种重要的天然和商业作用,其在植物中可以充当信息素和天然杀虫剂,而商业化生产的甲基酮可作为香料,为精油提供香气,并在奶酪和其他乳制品中起到调味剂的作用,也可作为化学合成中间体或者柴油调和剂。
利用合成生物学原理,通过对脂肪酸β-氧化途径进行遗传改造,已经在多种微生物中实现了甲基酮的重组生产。2012年Goh等( Goh EB, Baidoo EE, Keasling JD,Beller HR. Engineering of bacterial methyl ketone synthesis for biofuels.Appl Environ Microbiol 2012,78(1):70-80.)在工程化的大肠杆菌DH1菌株中对β-氧化途径进行了重新设计,过表达了来源于Micrococcus luteus的酰基辅酶A氧化酶基因ACO,大肠杆菌自身编码脂肪酸氧化复合体α亚基的FadB基因以及编码天然硫酯酶的FadM基因,证明了利用葡萄糖可以生产链长为C11-C15的甲基酮。2013年(Müller J, MacEachran D,Burd H, Sathitsuksanoh N, Bi C, Yeh YC, Lee TS, Hillson NJ, Chhabra SR,Singer SW, Beller HR. Engineering of Ralstoniaeutropha H16 for autotrophicand heterotrophic production of methyl ketones. Appl Environ Microbiol 2013,79(14):4433-4439.),该甲基酮合成途径被引入到Ralstoniaeutropha H16菌株中,首次进行了自养型细菌以CO2作为唯一碳源生产甲基酮的报道。2018年,Hanko等人(Hanko EKR,Denby CM, Sànchez I Nogué V, Lin W, Ramirez KJ, Singer CA, Beckham GT,Keasling JD. Engineering β-oxidation in Yarrowialipolytica for methyl ketoneproduction. MetabEng 2018, 48:52-62.)将同样的甲基酮合成途径导入解脂耶氏酵母中,使其在过氧化物酶体中表达,实现了C13-C23甲基酮在真菌中的重组生产。
酿酒酵母作为模式菌株,遗传背景清晰,可操作性强,具有良好的鲁棒性和稳定性。与原核生物相比,酿酒酵母具有蛋白质翻译后修饰功能,并且具有过氧化物酶体、内质网、高尔基体等细胞器,可以为目标产物的合成提供合适的内环境,实现亚细胞代谢工程改造。但针对酿酒酵母生产甲基酮的研究不多,例如文献(Zhu Z, Zhou YJ, Krivoruchko A,Grininger M, Zhao ZK, Nielsen J. Expanding the product portfolio of fungaltype I fatty acid synthases. Nat Chem Biol 2017, 13(4):360-362.),文中主要是对I型脂肪酸合成酶进行改造,通过删除或融合不同来源的结构域,拓宽产物谱,其中获得的酿酒酵母甲基酮的产量最高为10 μg/g DCW,主要包括2-十一烷酮,2-十三烷酮和2-十五烷酮。
发明内容
本发明研究表明,在酿酒酵母中敲除β-酮酰辅酶A硫解酶基因POT1,可以生产甲基酮;尤其是研究表明,强化过氧化物酶体途径,即过表达(优选为内源的)酰基辅酶A氧化酶基因POX1,(3R)-羟烷基-辅酶A脱氢酶/2-烯酰辅酶A水合酶2基因POX2,酰基辅酶A硫酯酶基因TES1,脂酰辅酶A转运蛋白编码基因PXA1/PXA2和外源β-酮酸脱羧酶基因ADC,可以提高甲基酮产量;同时引入细胞质途径即过表达外源酰基辅酶A氧化酶基因ACO,烯酰辅酶A水合酶基因HTD,3-羟烷基-辅酶A脱氢酶基因KR,酰基辅酶A硫酯酶基因FadM和β-酮酸脱羧酶基因MKS1,可进一步提高甲基酮产量。其中,本发明筛选了不同来源的酶基因,重新设计组合了一条新的细胞质途径。其中HTD和KR的使用是本发明发现的。MKS1在大肠杆菌中也是没有作用的,但是本发明发现其在酿酒酵母中的脱羧作用很明显,能显著提高甲基酮产量。由此完成本发明。
因此,本发明的目的是提供生产甲基酮的酿酒酵母重组菌株及其构建方法。
具体来说,本发明提供一种生产甲基酮的重组酿酒酵母的构建方法,其特征在于,在酿酒酵母中对β-酮酰辅酶A硫解酶基因POT1进行敲除或失活,或降低该基因的表达或活性。
优选地,进一步在所述酿酒酵母中过表达内源酰基辅酶A氧化酶基因POX1,或(3R)-羟烷基-辅酶A脱氢酶/2-烯酰辅酶A水合酶2基因POX2,或酰基辅酶A硫酯酶基因TES1,或脂酰辅酶A转运蛋白编码基因PXA1/PXA2,或外源β-酮酸脱羧酶基因ADC中的一种或多种。优选地,所述外源β-酮酸脱羧酶基因来源于Clostridium acetobutylicum,更优选地其核苷酸序列如SEQ ID NO:1所示。
更优选地,进一步在所述酿酒酵母中过表达外源酰基辅酶A氧化酶基因ACO,或烯酰辅酶A水合酶基因HTD,或3-羟烷基-辅酶A脱氢酶基因KR,或酰基辅酶A硫酯酶基因FadM,或β-酮酸脱羧酶基因MKS1中的一种或多种。
优选地,所述外源酰基辅酶A氧化酶基因ACO来源于Micrococcus luteus,更优选地其核苷酸序列如SEQ ID NO:2所示;
所述外源烯酰辅酶A水合酶基因HTD的氨基酸序列包括如SEQ ID NO:7所示的氨基酸序列;
所述外源3-羟烷基-辅酶A脱氢酶基因KR的氨基酸序列包括如SEQ ID NO:8所示的氨基酸序列。
所述外源烯酰辅酶A水合酶基因HTD来源于Yarrowialipolytica,更优选地其核苷酸序列如SEQ ID NO:3所示;
所述外源3-羟烷基-辅酶A脱氢酶基因KR来源于Yarrowialipolytica,更优选地其核苷酸序列如SEQ ID NO:4所示。
另外优选地,进一步将所述酿酒酵母中的半乳糖调节基因GAL80进行敲除或失活,或降低该基因的表达或活性。
在一个具体实施方式中,所述酿酒酵母的出发菌株的基因型为MATa MAL2-8cSUC2 ura3-52。
在另一个具体实施方式中,所述酿酒酵母的出发菌株的基因型为CEN.PK113-5D。
本发明还提供所述的构建方法获得的生产甲基酮的重组酿酒酵母。
本发明进一步所述的重组酿酒酵母在生产甲基酮中的应用。
本发明提供的重组菌在酿酒酵母中对β-酮酰辅酶A硫解酶基因POT1进行敲除或失活,或降低该基因的表达或活性,可以产生甲基酮。进一步配合过表达酰基辅酶A氧化酶基因POX1,或(3R)-羟烷基-辅酶A脱氢酶/2-烯酰辅酶A水合酶2基因POX2,或酰基辅酶A硫酯酶基因TES1,或脂酰辅酶A转运蛋白编码基因PXA1/PXA2,或外源β-酮酸脱羧酶基因ADC中的一种或多种,大大提高产生甲基酮的能力。本发明通过实验证明表明所获得的重组菌具有较大实际应用价值。
附图说明
图1为重组菌的甲基酮产量。
具体实施方式
实施例一、DNA片段及gRNA质粒的构建
本发明中所使用的PCR扩增体系及扩增程序如下:
PCR扩增体系:使用PrimeSTAR HS DNA polymerase配置扩增体系(TAKARA公司),扩增体系为:5×PS Buffer 10μL,dNTP Mix 4μL,引物F和R各1μL,DNA模板1μL,HS聚合酶(2.5U/μL) 0.5μL,补加蒸馏水至总体积50μL。
PCR扩增程序:98℃预变性1分钟(1个循环);98℃变性10秒、55℃退火5秒、72℃延伸x分钟(30个循环);72℃延伸10分钟(1个循环)。PrimeSTAR HS DNA polymerase的扩增速率为1kb/分钟,故x视片段长度而定。
一、Cas9整合元件的构建
Cas9及标记基因KanMX均为人工合成的序列,已按照酿酒酵母的密码子偏好性进行优化,Cas9(含核定位信号序列)的序列同GenBank: KY825143.1 (Range2647-6783),KanMX序列同GenBank: MN555460.1 (Range3324-4133)。启动子PTEF和终止子TTEF和TCYC均扩增自酿酒酵母基因组,其在GenBank中的序列分别为CP020138.1 (Range700422-700824),CP014738.1 (Range114659-114923)和CP063267.1(Range660625-660861)。
(1)Cas9表达盒的构建
以酿酒酵母的基因组为模板进行PCR,分别扩增PTEF和TCYC片段。同时,用引物Cas9-F和Cas9-R扩增Cas9基因。利用引物PTEF-F和TCYC-R进行重叠PCR,将片段PTEF、TCYC和Cas9进行融合,获得Cas9表达盒PTEF-Cas9-TCYC。所用引物如下:
PTEF-F:atagcttcaaaatgtttctactccttttttactcttcc
PTEF-R:ctagaaaacttagattagattgctatgctttctttctaatg
TCYC-F:gtcatgtaattagttatgtcacgcttacattcac
TCYC-R:agagtaaaaaaggagtagaaacattttgaagctatcttcgagcgtcccaaaaccttctc
Cas9-F:cattagaaagaaagcatagcaatctaatctaagttttctagatggacaagaagtactccattgggc
Cas9-R:gtgaatgtaagcgtgacataactaattacatgactcacaccttcctcttcttcttgggg。
(2)KanMX表达盒的构建
以CEN.PK113-5D基因组为模板进行PCR,分别扩增PTEF和TTEF片段。用引物KanMX-F和KanMX-R扩增KanMX基因。利用引物PTEF-F和TTEF-R进行重叠PCR,将片段PTEF、TTEF和KanMX进行融合,获得KanMX表达盒PTEF-KanMX-TTEF。所用引物如下:
TTEF-F:tcagtactgacaataaaaagattcttgttttcaag
TTEF-R:attaagggttctcgagagctcgttttc
KanMX-F:cattagaaagaaagcatagcaatctaatctaagttttctagatgggtaaggaaaagactcacgtttc
KanMX-R:cttgaaaacaagaatctttttattgtcagtactgattagaaaaactcatcgagcatcaaatgaaactg
(3)Cas9整合元件的构建
以酿酒酵母CEN.PK113-5D基因组为模板,扩增X-2位点上游序列X-2-UP(GenBank:CP020132.1, Range 194931-195600)和下游序列X-2-DW (GenBank: CP020132.1, Range195613-196019),利用引物X-2-UP-F和X-2-DW-R进行重叠PCR,将X-2-UP、X-2-DW与Cas9表达盒和KanMX表达盒进行融合,获得Cas9整合元件X-2-UP-PTEF-Cas9-TCYC-PTEF-KanMX-TTEF-X-2-DW。
扩增X-2-UP所用引物
X-2-UP-F:ggtgcaggggcagaaaatgc
X-2-UP-R:gaagagtaaaaaaggagtagaaacattttgaagctatacgtgaccacttcgagagcaag
扩增X-2-DW所用引物
X-2-DW-F:gaaaacgagctctcgagaacccttaatctgcataatcggcctcacagagg
X-2-DW-R:agaaaagtagtgaggacaggcttaattgagc
二、POT1敲除片段的构建
以酿酒酵母CEN.PK113-5D基因组为模板,扩增POT1基因ORF区上游序列POT1-UP(GenBank: CP020131.1, Range 41525-41874)和下游序列POT1-DW (GenBank:CP020131.1, Range 39921-40270),随后利用引物POT1-UP-F和POT1-DW-R进行重叠PCR,将POT1-UP和POT1-DW进行融合,获得敲除POT1所需的同源修复片段Δpot1。
扩增POT1-UP所用的引物
POT1-UP-F:ttttgtgtcacgatcatcatcactatgtaatcttc
POT1-UP-R:tgcttttatgtagtgttatattcactctgtactcag
扩增POT1-DW所用的引物
POT1-DW-F:gtgaatataacactacataaaagcattttaatacttgataatagttaatattctccctttttattatgc
POT1-DW-R:tatggctatcgaatctcccccatg
三、GAL80敲除片段的构建
以酿酒酵母基因组为模板,扩增GAL80基因ORF区上游序列GAL80-UP (GenBank:CP020135.1, Range 171137-171637)和下游序列GAL80-DW(GenBank: CP020135.1, Range172946-173271),随后利用引物GAL80-UP-F和GAL80-DW-R进行重叠PCR,将GAL80-UP和GAL80-DW进行融合,获得敲除GAL80所需的同源修复片段Δgal80。
扩增GAL80-UP所用的引物
GAL80-UP-F:ccagatggaatcccttccatagagagaagg
GAL80-UP-R:caagcacagggcaagatgcttgacgggagtggaaagaacgggaaac
扩增GAL80-DW所用的引物
GAL80-DW-F:gtttcccgttctttccactcccgtcaagcatcttgccctgtgcttg
GAL80-DW-R:cgggtatgaagatcaggaacgc
四、POX1和POX2表达元件的构建
以酿酒酵母CEN.PK113-5D基因组为模板,分别扩增POX1和POX2基因CDS区及其下游终止子序列POX1-TPOX1(GenBank: CP020129.1, Range 108324-110790)和POX2-TPOX2(GenBank: CP020133.1, Range 454085-457060);同时扩增双向启动子PGAL1-GAL10(GenBank: CP020124.1, Range 278744-279411),利用引物POX1-TPOX1-R和POX2-TPOX2-R进行重叠PCR,将POX1-TPOX1、POX2-TPOX2和启动子PGAL1-GAL10进行融合,获得长片段TPOX1-POX1-PGAL1-GAL10-POX2-TPOX2。与此同时,扩增IntA位点上游序列IntA-UP(GenBank: CP020132.1,Range 242421-242777)和下游序列IntA-DW (GenBank: CP020132.1, Range 242778-243122)。随后利用引物IntA-UP-F和IntA-DW-R进行重叠PCR,将IntA-UP和IntA-DW与长片段TPOX1-POX1-PGAL1-GAL10-POX2-TPOX2进行融合,获得POX1和POX2表达元件IntA-UP-TPOX1-POX1-PGAL1-GAL10-POX2-TPOX2-IntA-DW。
扩增POX1-TPOX1序列所用引物
POX1-TPOX1-F:atgacgagacgtactactattaatcccgattc
POX1-TPOX1-R:atttttttactactatacattggtaaatgtagtcatgtcattg
扩增POX2-TPOX2序列所用引物
POX2-TPOX2-F:atgcctggaaatttatccttcaaagatagag
POX2-TPOX2-R:tgactatctctctcttcatagtcttcccatc
扩增PGAL1-GAL10序列所用引物
PGAL1-GAL10-F:cgaatcgggattaatagtagtacgtctcgtcatttatattgaattttcaaaaattcttactttttttttggatggacgc
PGAL1-GAL10-R:ctctatctttgaaggataaatttccaggcattatagttttttctccttgacgttaaagtatagaggtatattaac
扩增IntA-UP序列所用引物
IntA-UP-F:cgattgctccactcataagaggc
IntA-UP-R:catgactacatttaccaatgtatagtagtaaaaaaatgctgctcttgaatggcgacag
扩增IntA-DW序列所用引物
IntA-DW-F:gatgggaagactatgaagagagagatagtcaaacaggcatgggaagattcgc
IntA-DW-R:ctttcacagggttctctttgatcacctc
五、TES1表达元件的构建
以酿酒酵母CEN.PK113-5D基因组为模板,扩增TES1基因CDS区(GenBank:CP020132.1, Range 473150-474199),同时扩增GAL7上游启动子序列PGAL7(GenBank:CP020124.1, Range275919-276643)和下游终止子序列TGAL7(GenBank: CP020124.1,Range274316-274817)。然后,利用引物PGAL7-F和TGAL7-R进行重叠PCR,将TES1,PGAL7和TGAL7进行融合,获得TES1表达元件PGAL7-TES1-TGAL7。
扩增TES1序列所用引物
TES1-F:gataaaaaaaaacagttgaatattccctcaaaaatgagtgcttccaaaatggccatgtc
TES1-R:gaaaaaatatgatatgaatgaatattccactttcttttcagaacttggctcgaatgtctcg
扩增PGAL7序列所用引物
PGAL7-F:tttgccagcttactatccttcttgaaaatatgc
PGAL7-R:ttttgagggaatattcaactgtttttttttatcatgttg
扩增TGAL7序列所用引物
TGAL7-F:aaagaaagtggaatattcattcatatcatattttttctattaactgcc
TGAL7-R:cgacaatgagccttgctgcaac
六、ADC表达元件的构建
ADC为来源于Clostridium acetobutylicum的乙酰乙酰盐脱羧酶编码基因,按照酿酒酵母的密码子偏好性进行优化,其序列如下(SEQ ID NO:1):atgttgaaagacgaagtcataaagcaaatatccacaccattgacaagtccagcattcccaagaggtccttacaagttccataatagagaatacttcaacatcgtttacagaactgatatggatgcattgagaaaagttgttccagaaccattggaaatcgatgaaccattagttagattcgaaatcatggcaatgcatgatacttcaggtttaggttgttatacagaatcaggtcaagctattccagtttcttttaatggtgttaagggtgactatttgcatatgatgtacttagataacgaaccagctattgcagttggtagagaattgtcagcttatccaaagaaattgggttacccaaagttgttcgttgattctgatactttggttggtacattggattacggtaaattgagagttgctactgcaacaatgggttacaagcataaggctttggatgcaaacgaagctaaggatcaaatctgtagaccaaactacatgttgaagatcatcccaaactacgatggttctccaagaatctgtgaattgattaatgcaaagattactgatgttacagttcatgaagcttggactggtccaacaagattgcaattgttcgatcatgctatggcaccattgaacgatttgccagttaaggaaatcgtttcttcatctcatattttagcagacattattttaccaagagcagaagtcatctacgattatttgaagtccaagttgtga。
扩增ADC序列所用引物
ADC-F:gattaacataataaaaaaaataattctttcataatgttgaaagacgaagtcataaagc
ADC-R:gcgtctcacttcaaacgcatcacaacttggacttcaaataatcg。
以人工合成的质粒为模板,利用引物ADC-F和ADC-R进行PCR,扩增ADC序列;同时以酿酒酵母CEN.PK113-5D基因组为模板,扩增GAL2启动子PGAL2(GenBank: CP020134.1,Range 289546-290250)和终止子序列TGAL2(GenBank: CP020134.1, Range 291976-292319),利用引物PGAL2-F和TGAL2-R进行重叠PCR,将ADC,PGAL2和TGAL2进行融合,获得ADC表达元件PGAL2-ADC-TGAL2。
扩增PGAL2所用引物
PGAL2-F:ctgtactaatccaaggaggtttacggacc
PGAL2-R:tatgaaagaattattttttttattatgttaatcttgtgtttacttaactattac
扩增TGAL2所用引物
TGAL2-F:tgcgtttgaagtgagacgctcc
TGAL2-R:gaccccccagagataagtctgg。
七、PXA1表达元件的构建
以酿酒酵母CEN.PK113-5D基因组为模板,分别扩增PXA1基因及其终止子序列PXA1-TPXA1(GenBank: CP020138.1, Range 273493-276399),JEN1启动子序列PJEN1(GenBank: CP020133.1, Range 21282-22238)及IntB位点上游序列IntB-UP(GenBank:CP020133.1, Range 93660-93988)和下游序列IntB-DW(GenBank: CP020133.1, Range93989-94360),通过重叠PCR技术,利用引物IntB-UP-F和IntB-DW-R将上述片段进行融合,获得PXA1表达元件IntB-UP-PJEN1-PXA1-TPXA1-IntB-DW。
扩增PXA1-TPXA1所用的引物
PXA1-F:caaagagattaaatactgctactgaaaatatgtcaacaacattagcagcaccagc
PXA1-R:cccaatcctcaaaaataaaatggcccttcagttttttgttttacagtatatgtttccatgaaaagg
扩增PJEN1所用的引物
PJEN1-F:cattgtcttccattcagagtctaatcgaacg
PJEN1-R:attttcagtagcagtatttaatctctttg
扩增IntB-UP序列所用的引物
IntB-UP-F:gagatctaccacccaaccggc
IntB-UP-R:caaaattagagacatatcgtagaaatcagacgc
扩增IntB-DW序列所用的引物
IntB-DW-F:gaagggccattttatttttgaggattggg
IntB-DW-R:gagcaggagccaatagtagtcgg。
八、PXA2表达元件的构建
以酿酒酵母CEN.PK113-5D基因组为模板,分别扩增PXA2基因及其终止子序列PXA2-TPXA2(GenBank: CP020133.1, Range 85913-88795),JEN1启动子序列PJEN1及IntC位点上游序列IntC-UP(GenBank: CP020134.1, Range 830290-830742)和下游序列IntC-DW(GenBank: CP020134.1, Range 830743-831160)。然后通过重叠PCR技术,用引物IntC-UP-F和IntC-DW-R将上述片段进行融合,获得PXA2表达元件IntC-UP-PJEN1-PXA2-TPXA2-IntC-DW。
PJEN1序列及扩增该片段所用引物见(7)PXA1表达元件的构建部分。
扩增PXA2-TPXA2所用的引物
PXA2-F:caaagagattaaatactgctactgaaaatatgatctcaacagcttctgcattttatcag
PXA2-R:ctataaaacttggttcactttataaaaaatggaaagttctcaggc
扩增IntC-UP所用的引物
IntC-UP-F:cctgacagttggaccaacgcaac
IntC-UP-R:cgttcgattagactctgaatggaagacaatgaacggggaaggaatagtacaaagttgg
扩增IntC-DW所用的引物
IntC-DW-F:tactcaattcttgaagccaatttgtacaattcc
IntC-DW-R:ctggaggaatagccgctccc。
九、ACO表达元件的构建
ACO为来源于Micrococcus luteus的酰基辅酶A氧化酶基因,按照酿酒酵母的密码子偏好性进行优化,其序列如下(SEQ ID NO:2):atgaccgtccacgaaaagttagcaccacaatccccaacacactccaccgaagttccaaccgatgtagccgaaatagcaccagaaagaccaactccaggttctttggatgctgcagctttagaagaagctttgttaggtagatgggcagctgaaagaagagaatcaagagaattggctaaagatccagcattatggagagatccattgttgggtatggatgaacatagagctagagttttgagacaattaggtgttttggttgaaagaaatgctgttcatagagcatttccaagagaatttggtggtgaagataatcatggtggtaatatttctgcatttggtgacttggttttagctgatccatcattgcaaattaaagctggtgttcaatggggtttattttcttcagcaattttgcatttgggtactgctgaacatcatagaagatggttgccaggtgctatggatttgtctgttccaggtgcttttgcaatgacagaaattggtcatggttctgatgttgcttcaattgcaactacagctacttacgatgaagcaacacaagaattcgttatccatactccttttaaaggtgcttggaaagattacttgggtaatgcagctttacatggtagagcagctactgtttttgcacaattgattacacaaggtgttaaccatggtgttcattgtttctacgttccaatcagagatgaaaaaggtgcatttttgccaggtgttggtggtgaagatgatggtttgaaaggtggtttaaacggtatcgataacggtagattgcatttcactcaagttagaatcccaagaacaaatttgttgaacagatacggtgacgttgcagaagatggtacatactcttcaccaattgcttctccaggtagaagatttttcactatgttgggtacattggttcaaggtagagtttctttgtcattagcagctactacagcatcatttttgggtttacatggtgctttagcatacgctgaacaaagaagacaattcaatgcttcagatccacaaagagaagaagttttgttggattaccaaaaccatcaaagaagattgatcgatagattggcaagagcttacgcagatgctttcgcatcaaacgaattggttgttaagttcgatgatgttttctctggtagatcagatactgatgttgatagacaagaattggaaacattggcagctgcagttaaaccattaactacatggcatgctttagatactttgcaagaagcaagagaagcttgtggtggtgctggtttcttggcagaaaatagagttacacaaatgagagctgatttggatgtttacgttactttcgaaggtgacaacacagttttgttgcaattggttggtaaaagattgttgactgattactctaaggaatttggtagattaaatgttggtgctgtttctagatacgttgttcatcaagcatcagatgctattcatagagctggtttgcataaagcagttcaatcagttgctgatggtggttctgaaagaagatcagcaaattggtttaaagatccagctgttcaacatgaattgttgactgaaagagttagagcaaagactgctgatgttgcaggtacattatctggtgctcgtggtaaaggtcaagctgcacaagctgaagcttttaatacaagacaacatgaattgatcgaagctgcaagaaatcatggtgaattgttacaatgggaagcttttactagagcattggaaggtatcacagatgaaactacaaagactgttttgacatggttgagagatttgttcgcattgagattgatcgaagatgatttgggttggtttgttgctcatggtagagtttcttcacaaagagctagagcattaagaggttacgttaatagattggctgaaagattaagaccatttgcattggaattagttgaagcttttggtttggaaccagaacatttgagaatggctgttgcaactgatgcagaaacacaaagacaagaagaagctcatgcatggtttacagctagaagagctgcaggtgaagaaccagaagatgaaaaagcagtaagagcaagagaaaaagcagcaagaggtagaagaggttga。
扩增ACO所用引物
ACO-F:atgaccgtccacgaaaagttagc
ACO-R:tcaacctcttctacctcttgctgc
以人工合成的质粒为模板,PCR扩增ACO序列;同时以酿酒酵母CEN.PK113-5D基因组为模板,扩增GAL1启动子区PGAL1(GenBank: CP020124.1, Range 279023-279411)和终止子序列TGAL1(GenBank: CP020124.1, Range 280999-281348),利用引物PGAL1-F和TGAL1-R进行重叠PCR,将ACO,PGAL1和TGAL1进行融合,获得ACO表达元件PGAL1-ACO-TGAL1。
扩增PGAL1所用引物如下:
PGAL1-F:tcttcaccggtcgcgttcc
PGAL1-R:gctaacttttcgtggacggtcattatagttttttctccttgacgttaaagtatagaggtatattaac
扩增TGAL1所用引物如下:
TGAL1-F:gcagcaagaggtagaagaggttgagtatacttcttttttttactttgttcagaacaacttctc
TGAL1-R:cgtcagttggcaacttgccaag。
十、HTD表达元件的构建
烯酰辅酶A水合酶基因HTD来源于解脂耶氏酵母,其核苷酸序列如下(SEQ ID NO:3):atgcagaccacccctttctcttcctctgctcccgcccagacctttggtgacaagaagtacgagcacatcctgacctccacccccgtccccaaggtcgctctcgtcaccctcaaccggcccaaggccctcaacgccctgtgcactcctctcattaaggagctgaacgaggctctccaggctgctgatgccgaccccaccattggcgccatcgttctcaccggatccgagaagtcctttgctgccggcgccgacatcaaggagatgaaggacaagaccgtcacttccgtgctcaacgagaacttcatcgaggagtggggcaacatggccaacatcaagaaacccatcattgctgccgtcaacggctttgccctcggtggtggatgcgagcttgccatgatggccgacatcatctacgccggcgccaaggccaagtttggccagcccgagatcaagcttggtgtcattcccggagctggaggaacccagcgactcacccgagccattggcctctaccgagctaaccactacattcttaccggagagatgttcactgctcagcaggctgctgactggggtctggccgccaaggtctacgagcccgcccagctcgttgacgagtccgtcaaggctgctgctcagatcgcctcttacggccagttggctgtccaggccgccaaggcttctgtccaccagtccgctgaggtcggtctccgagccggtctcgagtttgagcgagtccgattccacggtctctttggcacccacgaccagaaggagggcatggctgcttttgccgagaagcgagagcccaacttcaagaacgagtaa。去掉信号肽后氨基酸序列如下(SEQ ID NO:7):MQTTPFSSSAPAQTFGDKKYEHILTSTPVPKVALVTLNRPKALNALCTPLIKELNEALQAADADPTIGAIVLTGSEKSFAAGADIKEMKDKTVTSVLNENFIEEWGNMANIKKPIIAAVNGFALGGGCELAMMADIIYAGAKAKFGQPEIKLGVIPGAGGTQRLTRAIGLYRANHYILTGEMFTAQQAADWGLAAKVYEPAQLVDESVKAAAQIASYGQLAVQAAKASVHQSAEVGLRAGLEFERVRFHGLFGTHDQKEGMAAFAEKREPNFKNE。
以解脂耶氏酵母菌株W29基因组为模板扩增HTD及其终止子THTD(GenBank:CP028449.1, Range 1398832-1400080);同时以酿酒酵母CEN.PK113-5D基因组为模板,扩增GAL2启动子PGAL2和IntD位点的上游序列IntD-UP(GenBank: CP020134.1, Range803152-803422)和下游序列IntD-DW(GenBank: CP020134.1, Range 803438-803816)。利用引物IntD-UP-F和IntD-DW-R进行重叠PCR,将HTD-THTD,PGAL2,IntD-UP和IntD-DW进行融合,获得HTD表达元件IntD-UP-PGAL2-HTD-THTD-IntD-DW。
PGAL2序列及扩增该片段所用引物见(6)ADC表达元件的构建部分。
扩增HTD-THTD序列(不含信号肽)所用引物
HTD-THTD-F:gtaaacacaagattaacataataaaaaaaataattctttcataatgcagaccacccctttctcttcctctg
HTD-THTD-R:acgcagtacaaggacgcgttaagaaaaatttcgagagagtcgccgatattactctaaactggactgcacattttgttaag
扩增IntD-UP序列所用引物
IntD-UP-F:gcgggccaagaactggttcc
IntD-UP-R:ccgtaaacctccttggattagtacagcataacgcgttacacggaaggagagc
扩增IntD-DW序列所用引物
IntD-DW-F:tatcggcgactctctcgaaatttttcttaac
IntD-DW-R:cggttaaaacagctgtagtgttctgg。
十一、KR表达元件的构建
3-羟烷基-辅酶A脱氢酶基因KR来源于解脂耶氏酵母,其核苷酸序列如下(SEQ IDNO:4):atgaagaaggtcgactctctctccgtaattggcgccggccagatgggtctgggaatcgctctggtggctgccaacaaggccggtctccaggtcaacctcattgacgccaaccagggcgccctggacaagggtctcaagttcatggacaagctgcttgagaaggacgttggcaaaggccgtctgaccagcgacgaggcccaggccgtgcgaggccgggtcactggtcacaccaacctgcagtctgccgtggccgacgtggacatgatcattgaggcagtccccgagatccccaagctcaagtttgacatcttccgagacctcaacgagtggacccagaaggataccatcctggccaccaacacctcgtccatctccatcaccaagattgctgctgctgccggcgctggtgccccccgagtcatttctgcccacttcatgaaccccgtgcccgtccagaagggtgtcgagatcatcaccggcctgcagacctctcccgagaccctcgccaccaccctggaggttgtgaagcgaatgggcaagatcccttccatctccaaggactcccctggcttcctggctaaccgaatcctcatgccctacatcaacgaggccatcatcaccctcgagaccggtgttggagaaaaggaggacattgacaacattctcaagaacggctgtgccatgcccatgggaccccttgctctcgccgacttcattggtctcgatacttgtctcgccatcatgcgagtgctgtacgaggacactggtgactccaagtaccgaccctccgtcctgcttaacaagtacgtcgatgctggctggctcggaaagaagtctggaaagggcttctatgactactaa。去掉信号肽后的氨基酸序列如下(SEQ ID NO:8):MKKVDSLSVIGAGQMGLGIALVAANKAGLQVNLIDANQGALDKGLKFMDKLLEKDVGKGRLTSDEAQAVRGRVTGHTNLQSAVADVDMIIEAVPEIPKLKFDIFRDLNEWTQKDTILATNTSSISITKIAAAAGAGAPRVISAHFMNPVPVQKGVEIITGLQTSPETLATTLEVVKRMGKIPSISKDSPGFLANRILMPYINEAIITLETGVGEKEDIDNILKNGCAMPMGPLALADFIGLDTCLAIMRVLYEDTGDSKYRPSVLLNKYVDAGWLGKKSGKGFYDY。
以解脂耶氏酵母菌株W29基因组为模板扩增KR及其终止子TKR(GenBank:CP028450.1, Range 1171996-1173279);同时以酿酒酵母CEN.PK113-5D基因组为模板,扩增GAL2启动子PGAL2和IntE位点的上游序列IntE-UP(GenBank: CP020132.1, Range229773-230100)和下游序列IntE-DW (GenBank: CP020132.1, Range 230116-230373)。利用引物IntE-UP-F和IntE-DW-R进行重叠PCR,将KR-TKR,PGAL2,IntE-UP和IntE-DW进行融合,获得KR表达元件IntE-UP-PGAL2-KR-TKR-IntE-DW。
PGAL2序列及扩增该片段所用引物见(6)ADC表达元件的构建部分。
扩增KR-TKR序列所用引物
KR-TKR-F:gtaaacacaagattaacataataaaaaaaataattctttcataatgaagaaggtcgactctctctccgtaattgg
KR-TKR-R:gagctcgtccttttactagcatatcaatatccgtttcattgaaaagtggccttagtgcggttcagtattgagagttggcg
扩增IntE-UP序列所用引物
IntE-UP-F:tggaaaagctcactgtgaggttcc
IntE-UP-R:ggtccgtaaacctccttggattagtacagagtctcgtatgtcggctctcg
扩增IntE-DW序列所用引物
IntE-DW-F:ctaaggccacttttcaatgaaacggatattg
IntE-DW-R:agcgtaacagagtttcaacaacatgc。
十二、FadM表达元件的构建
FadM为来源于大肠杆菌的酰基辅酶A硫酯酶编码基因,按照酿酒酵母的密码子偏好性进行优化,其序列如下(SEQ ID NO:5):atgcaaactcaaatcaaagtcagaggttaccacttggatgtatatcaacacgtcaataatgccagatacttagaatttttagaagaagcaagatgggatggtttggaaaactctgactcatttcaatggatgacagcccataatattgctttcgttgtcgtaaacataaacatcaactacagaagaccagctgtcttgtctgatttgttgactatcacatcccaattgcaacaattgaacggtaaatccggtatcttgagtcaagtaattaccttagaacctgaaggtcaagttgtcgctgatgcattgatcaccttcgtttgtatcgacttaaagactcaaaaagccttggcattggaaggtgaattgagagaaaagttagaacaaatggtaaagtga。
扩增FadM所用的引物
FadM-F:cctctatactttaacgtcaaggagaaaaaactataatgcaaactcaaatcaaagtcagagg
FadM-R:caatactcattaaaaaactatatcaattaatttgaattaactcactttaccatttgttctaacttttctctcaattc。
以人工合成的质粒为模板,PCR扩增FadM序列;同时以酿酒酵母CEN.PK113-5D基因组为模板,扩增GAL1启动子区PGAL1(GenBank: CP020124.1, Range 278812-279411)和FBA1终止子序列TFBA1(GenBank: CP020133.1, Range 326217-326416),利用引物PGAL1-F和TFBA1-R进行重叠PCR,将FadM,PGAL1和TFBA1进行融合,获得长片段PGAL1-FadM-TFBA1。
同时以酿酒酵母CEN.PK113-5D基因组为模板,扩增IntF位点的上游序列IntF-UP(GenBank: CP020134.1, Range 839333-839646)和下游序列IntF-DW(GenBank:CP020134.1, Range 839661-839909)。利用引物IntF-UP-F和IntF-DW-R进行重叠PCR,将其与PGAL1-FadM-TFBA1进行融合,获得FadM表达元件IntF-UP-PGAL1-FadM-TFBA1-IntF-DW。
扩增PGAL1序列所用引物
PGAL1-F:acatggcattaccaccatatacatatccatatc
PGAL1-R:tatagttttttctccttgacgttaaagtatagagg
扩增TFBA1序列所用引物
TFBA1-F:gttaattcaaattaattgatatagttttttaatgagtattg
TFBA1-R:ctagttcgaatgatgaacttgcttgctgtcaaacttctgagttgccgctgaaagatgagctaggcttttgtaaaaatatc
扩增IntF-UP序列所用引物
IntF-UP-F:caaacaaggaaagggaaaatactgggtg
IntF-UP-R:ggatatgtatatggtggtaatgccatgtcaataaattcaaaccggttatagcggtctc
扩增IntF-DW序列所用引物
IntF-DW-F:cagcggcaactcagaagtttgac
IntF-DW-R:gcgtgaacaccttatataacttagcccg。
十三、MKS1表达元件的构建
MKS1为来源于Solanum habrochaites的β-酮酸脱羧酶编码基因,按照酿酒酵母的密码子偏好性进行优化,其序列如下(SEQ ID NO:6):atggaaaaatctatgtcaccattcgttaagaaacatttcgttttggttcatactgcatttcatggtgcttggtgttggtacaagatcgttgcattgatgagatcatctggtcataatgttacagctttggatttgggtgcttctggtattaatccaaagcaagcattgcaaatcccaaacttctctgattacttatcaccattgatggagtttatggcatctttgccagctaacgaaaagattatcttggttggtcatgctttaggtggtttggcaatttcaaaagctatggaaacttttccagaaaagatttctgttgcagttttcttgtcaggtttgatgccaggtccaaacatcgatgctactacagtttgtacaaaagcaggttctgctgttttgggtcaattggataactgtgttacttatgaaaacggtccaacaaatccaccaactacattgatcgcaggtccaaagttcttggctactaacgtttaccatttgtcaccaattgaagatttggctttagcaacagctttggttagaccattgtatttgtacttggcagaagatatctctaaggaagttgttttatcttcaaagagatacggttcagttaagagagtttttattgttgcaactgaaaacgatgctttgaagaaagaatttttgaaattgatgattgaaaagaatccaccagatgaagttaaggaaatcgaaggttctgatcatgttacaatgatgtcaaagccacaacaattgtttactacattgttatctattgctaataagtacaaataa。
扩增MKS1所用的引物
MKS1-F:atgataaaaaaaaacagttgaatattccctcaaaaatggaaaaatctatgtcaccattcgttaag
MKS1-R:ttatttgtacttattagcaatagataacaatgtagtaaac。
以人工合成的质粒为模板,PCR扩增MKS1序列;同时以酿酒酵母CEN.PK113-5D基因组为模板,扩增GAL7启动子区PGAL7和ADH1终止子序列TADH1(GenBank: CP020137.1, Range159411-159702),利用引物PGAL7-F和TADH1-R进行重叠PCR,将MKS1,PGAL7和TADH1进行融合,获得长片段PGAL7-MKS1-TADH1。
同时以酿酒酵母CEN.PK113-5D基因组为模板,IntG位点的上游序列IntG-UP(GenBank: CP022976.1, Range 67825-68136)和下游序列IntG-DW(GenBank:CP022976.1, Range 68151-68404)。利用引物IntG-UP-F和IntG-DW-R进行重叠PCR,将其与PGAL7-MKS1-TADH1进行融合,获得MKS1表达元件IntG-UP-PGAL7-MKS1-TADH1-IntG-DW。
PGAL7序列及扩增PGAL7所用引物见(5)TES1表达元件的构建部分。
扩增TADH1序列所用引物
TADH1-F:gtttactacattgttatctattgctaataagtacaaataattaattaacaattcttcgccagaggtttgg
TADH1-R:tttccgtctgtacgcagcatttagcagagatttgccaatgccaagaaactccactagagcgacctcatgctatacctgag
扩增IntG-UP序列所用引物
IntG-UP-F:caggacgtctcgagcgctgatatc
IntG-UP-R:gcatattttcaagaaggatagtaagctggcaaatgcaatgggcttggtattccgtg
扩增IntG-DW序列所用引物
IntG-DW-F:tagtggagtttcttggcattggcaaatc
IntG-DW-R:gtcttctcaggttccggcttcg。
十四、gRNA质粒的构建
gRNA质粒的构建方法详见中国发明专利申请202110934151.4。本发明仅列出所用的gRNA质粒名称及其所含有的靶序列,如下:
实施例二、生产甲基酮的重组菌株的构建
本发明中涉及的培养基及配方如下:
YPD培养基,每L体积YPD培养基包含:20g蛋白胨,10g酵母提取物,20g葡萄糖,20g琼脂粉(YPD固体培养基添加)。
含卡那霉素的YPD平板,每L体积含卡那霉素的YPD培养基包含:20g蛋白胨,10g酵母提取物,20g葡萄糖,20g琼脂粉,50mg卡那霉素。
无尿嘧啶添加的SD平板,每L体积无尿嘧啶添加的SD培养基含:8g Ura minusmeida(北京泛基诺科技有限公司),20g琼脂粉,20g葡萄糖。
含5-氟乳清酸的SD平板,每L体积含5-氟乳清酸的SD含:8g Ura minus meida(北京泛基诺科技有限公司),20g琼脂粉,20g葡萄糖,尿嘧啶60mg,5-氟乳清酸1g。
Delft液体培养基,每L体积Delft液体培养基包含:20g葡萄糖,7.5g硫酸铵,0.5g七水硫酸镁,14.4g磷酸二氢钾,2ml微量金属盐母液(1L体积中含3.0g七水硫酸铁,4.5g七水硫酸锌,4.5g二水氯化钙,0.84g二水氯化锰,0.3g六水氯化钴,0.3g五水硫酸铜,0.4g二水钼酸钠,1.0g硼酸,0.1g碘化钾,19.0g乙二胺四乙酸二钠盐),1ml维他命母液(1L体积中含0.05g D-生物素,1.0g D-泛酸,1.0g维生素B1,1.0g吡哆醇,1.0g烟酸,0.2g 4-氨基苯酸,25.0g肌醇),40mg尿嘧啶。
酿酒酵母感受态细胞的制备及转化:
将出发菌单菌落于YPD培养基中30℃,250rpm过夜培养,计数过夜培养物细胞密度,以最终OD600nm=0.15的浊度转接至20mlYPD培养基。30℃,250rpm培养至OD600nm=1.2~2.4。使用无菌离心管3000 g离心5分钟,收集细胞。弃培养液,把细胞悬浮于无菌水中,再同上离心。弃水,把细胞悬浮于1mL100mM醋酸锂中,转悬浮物到无菌离心管中;高速短时沉淀细胞,去除醋酸锂;悬浮细胞到4倍体系的100mM醋酸锂中并分装获得感受态细胞。
取感受态细胞,3000 g离心15秒,弃上清,依次加入240 μL PEG(50%,w/v),36μL1.0mol/L醋酸锂,25μL鲑鱼精DNA(sigma)(2mg/mL),50 μL水和基因表达元件的混合物,剧烈振荡直至细胞完全混匀,置于30℃培养箱,温浴30分钟,随后置于42 ℃水浴20分钟;以6000-8000rpm离心15秒,除去转化混合液;吸100μL无菌水到反应管中,轻轻悬浮沉淀并涂布至相应的筛选平板。
(1)重组菌株TMK01的构建
酿酒酵母(Saccharomyces cerevisiae)菌株CEN.PK113-5D为常用的工程菌株,基因型为MATa MAL2-8c SUC2 ura3-52。
为了便于利用CRISPR/Cas9系统对菌株CEN.PK113-5D进行遗传操作,首先将Cas9编码基因整合到其染色体上。以酿酒酵母CEN.PK113-5D为出发菌株,制作感受态细胞,加入Cas9整合元件X-2-UP-PTEF-Cas9-TCYC-PTEF-KanMX-TTEF-X-2-DW进行转化,涂布至含卡那霉素的YPD平板上筛选阳性克隆,将得到的菌株命名TMK01,并保存。
(2)重组菌株TMK02的构建
以重组菌株TMK01为出发菌株,制作感受态细胞,加入gRNA质粒01及敲除POT1所需的同源修复片段Δpot1后进行转化,将转化后细胞涂布至无尿嘧啶添加的SD平板上筛选阳性克隆,然后挑选阳性克隆到含5-氟乳清酸的SD平板,平板中长起的菌落命名为TMK02,并保存。
(3)重组菌株TMK03的构建
为了避免在培养过程中添加半乳糖,并提高目的基因的表达水平,对半乳糖调节基因GAL80进行敲除。以重组菌株TMK02为出发菌株,制作感受态细胞,加入gRNA质粒02及敲除GAL80所需的同源修复片段Δgal80后进行转化,将转化后细胞涂布至无尿嘧啶添加的SD平板上筛选阳性克隆,然后挑选阳性克隆到含5-氟乳清酸的SD平板,平板中长起的菌落命名为TMK03,并保存。
(4)重组菌株TMK04的构建
以重组菌株TMK03为出发菌株,制作感受态细胞,加入gRNA质粒03及POX1和POX2表达元件IntA-UP-TPOX1-POX1-PGAL1-GAL10-POX2-TPOX2-IntA-DW后进行转化,将转化后细胞涂布至无尿嘧啶添加的SD平板上筛选阳性克隆,然后挑选阳性克隆到含5-氟乳清酸的SD平板,平板中长起的菌落命名为TMK04,并保存。
(5)重组菌株TMK05的构建
以重组菌株TMK04为出发菌株,制作感受态细胞,加入gRNA质粒04及TES1表达元件PGAL7-TES1-TGAL7和ADC表达元件PGAL2-ADC-TGAL2后进行转化,将转化后细胞涂布至无尿嘧啶添加的SD平板上筛选阳性克隆,然后挑选阳性克隆到含5-氟乳清酸的SD平板,平板中长起的菌落命名为TMK05,并保存。
(6)重组菌株TMK06的构建
以重组菌株TMK05为出发菌株,制作感受态细胞,加入gRNA质粒05及PXA1表达元件IntB-UP-PJEN1-PXA1-TPXA1-IntB-DW和PXA2表达元件IntC-UP-PJEN1-PXA2-TPXA2-IntC-DW后进行转化,将转化后细胞涂布至无尿嘧啶添加的SD平板上筛选阳性克隆,然后挑选阳性克隆到含5-氟乳清酸的SD平板,平板中长起的菌落命名为TMK06,并保存。
(7)重组菌株TMK07的构建
以重组菌株TMK06为出发菌株,制作感受态细胞,加入gRNA质粒06及ACO表达元件PGAL1-ACO-TGAL1后进行转化,将转化后细胞涂布至无尿嘧啶添加的SD平板上筛选阳性克隆,然后挑选阳性克隆到含5-氟乳清酸的SD平板,平板中长起的菌落命名为TMK07,并保存。
(8)重组菌株TMK08的构建
以重组菌株TMK07为出发菌株,制作感受态细胞,加入gRNA质粒07及HTD表达元件IntD-UP-PGAL2-HTD-THTD-IntD-DW和KR表达元件IntE-UP-PGAL2-KR-TKR-IntE-DW后进行转化,将转化后细胞涂布至无尿嘧啶添加的SD平板上筛选阳性克隆,然后挑选阳性克隆到含5-氟乳清酸的SD平板,平板中长起的菌落命名为TMK08,并保存。
(9)重组菌株TMK09的构建
以重组菌株TMK08为出发菌株,制作感受态细胞,加入gRNA质粒08及FadM表达元件IntF-UP-PGAL1-FadM-TFBA1-IntF-DW和MKS1表达元件IntG-UP-PGAL7-MKS1-TADH1-IntG-DW后进行转化,将转化后细胞涂布至无尿嘧啶添加的SD平板上筛选阳性克隆,然后挑选阳性克隆到含5-氟乳清酸的SD平板,平板中长起的菌落命名为TMK09,并保存。
实施例三、重组菌株在生产甲基酮中的应用
(1)重组菌培养及产物提取
在Delft液体培养基中分别活化重组菌株TMK02、TMK03、TMK04、TMK05、TMK06、TMK07、TMK08和TMK09。于Delft液体培养基中制备种子液(30℃,250rpm,24 h),以适当的接种量接种于含20mLDelft液体培养基的100mL三角瓶中,使转接后初始OD600nm=0.1,30℃,250rpm培养24 h,然后加入2ml含有2-十六烷酮的正十二烷,继续培养72 h。最后,收集三角瓶中的正十二烷层,于12000 rpm离心1 min,收集上层有机相备用。
(2)菌体生产甲基酮的定性定量分析
将收集的有机相物质用正己烷稀释50倍,用GC-MS检测。GC-MS测定条件:进样口温度280℃,进样体积1μL,不分流,溶剂延时4 min;色谱柱:HP-5ms(30m*0.25mM);色谱条件:50℃,2 min,30℃/min到140℃,10℃/min到300 ℃,保温1 min;MS条件:Full Scan:50-650。用甲基酮的标准品(sigma)进行定性定量。甲基酮的标准品为2-壬酮,2-十一烷酮,2-十三烷酮和2-十五烷酮的混合物,出峰时间分别为5.71 min,7.176 min,8.955 min,和10.941 min。步骤(1)收集的有机相物质相应时间有出峰,表明步骤(1)收集的有机相物质均含有甲基酮。
结果如图1所示,TMK02、TMK03、TMK04、TMK05、TMK06、TMK07、TMK08和TMK09的发酵4天时总甲基酮产量分别为2.45 mg/L,3.41 mg/L,2.26 mg/L,12.41 mg/L,13.70 mg/L,11.5 mg/L,15.29 mg/L,21.03 mg/L。
该实施例证明,在酿酒酵母中敲除β-酮酰辅酶A硫解酶基因POT1,可以生产甲基酮;强化过氧化物酶体途径,即过表达内源酰基辅酶A氧化酶基因POX1,(3R)-羟烷基-辅酶A脱氢酶/2-烯酰辅酶A水合酶2基因POX2,酰基辅酶A硫酯酶基因TES1,脂酰辅酶A转运蛋白编码基因PXA1/PXA2和外源β-酮酸脱羧酶基因ADC,可以提高甲基酮产量;同时引入细胞质途径,即过表达外源酰基辅酶A氧化酶基因ACO,烯酰辅酶A水合酶基因HTD,3-羟烷基-辅酶A脱氢酶基因KR,酰基辅酶A硫酯酶基因FadM和β-酮酸脱羧酶基因MKS1,可进一步提高甲基酮产量。
<110> 中国科学院天津工业生物技术研究所
<120> 一种生产甲基酮的重组酿酒酵母及其构建方法和应用
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 744
<212> DNA
<213> 人工序列(ADC基因优化核苷酸序列)
<400> 1
atgttgaaagacgaagtcataaagcaaatatccacaccattgacaagtccagcattcccaagaggtccttacaagttccataatagagaatacttcaacatcgtttacagaactgatatggatgcattgagaaaagttgttccagaaccattggaaatcgatgaaccattagttagattcgaaatcatggcaatgcatgatacttcaggtttaggttgttatacagaatcaggtcaagctattccagtttcttttaatggtgttaagggtgactatttgcatatgatgtacttagataacgaaccagctattgcagttggtagagaattgtcagcttatccaaagaaattgggttacccaaagttgttcgttgattctgatactttggttggtacattggattacggtaaattgagagttgctactgcaacaatgggttacaagcataaggctttggatgcaaacgaagctaaggatcaaatctgtagaccaaactacatgttgaagatcatcccaaactacgatggttctccaagaatctgtgaattgattaatgcaaagattactgatgttacagttcatgaagcttggactggtccaacaagattgcaattgttcgatcatgctatggcaccattgaacgatttgccagttaaggaaatcgtttcttcatctcatattttagcagacattattttaccaagagcagaagtcatctacgattatttgaagtccaagttgtga 744
<210> 2
<211> 2124
<212> DNA
<213> 人工序列(ACO基因优化核苷酸序列)
<400> 2
atgaccgtccacgaaaagttagcaccacaatccccaacacactccaccgaagttccaaccgatgtagccgaaatagcaccagaaagaccaactccaggttctttggatgctgcagctttagaagaagctttgttaggtagatgggcagctgaaagaagagaatcaagagaattggctaaagatccagcattatggagagatccattgttgggtatggatgaacatagagctagagttttgagacaattaggtgttttggttgaaagaaatgctgttcatagagcatttccaagagaatttggtggtgaagataatcatggtggtaatatttctgcatttggtgacttggttttagctgatccatcattgcaaattaaagctggtgttcaatggggtttattttcttcagcaattttgcatttgggtactgctgaacatcatagaagatggttgccaggtgctatggatttgtctgttccaggtgcttttgcaatgacagaaattggtcatggttctgatgttgcttcaattgcaactacagctacttacgatgaagcaacacaagaattcgttatccatactccttttaaaggtgcttggaaagattacttgggtaatgcagctttacatggtagagcagctactgtttttgcacaattgattacacaaggtgttaaccatggtgttcattgtttctacgttccaatcagagatgaaaaaggtgcatttttgccaggtgttggtggtgaagatgatggtttgaaaggtggtttaaacggtatcgataacggtagattgcatttcactcaagttagaatcccaagaacaaatttgttgaacagatacggtgacgttgcagaagatggtacatactcttcaccaattgcttctccaggtagaagatttttcactatgttgggtacattggttcaaggtagagtttctttgtcattagcagctactacagcatcatttttgggtttacatggtgctttagcatacgctgaacaaagaagacaattcaatgcttcagatccacaaagagaagaagttttgttggattaccaaaaccatcaaagaagattgatcgatagattggcaagagcttacgcagatgctttcgcatcaaacgaattggttgttaagttcgatgatgttttctctggtagatcagatactgatgttgatagacaagaattggaaacattggcagctgcagttaaaccattaactacatggcatgctttagatactttgcaagaagcaagagaagcttgtggtggtgctggtttcttggcagaaaatagagttacacaaatgagagctgatttggatgtttacgttactttcgaaggtgacaacacagttttgttgcaattggttggtaaaagattgttgactgattactctaaggaatttggtagattaaatgttggtgctgtttctagatacgttgttcatcaagcatcagatgctattcatagagctggtttgcataaagcagttcaatcagttgctgatggtggttctgaaagaagatcagcaaattggtttaaagatccagctgttcaacatgaattgttgactgaaagagttagagcaaagactgctgatgttgcaggtacattatctggtgctcgtggtaaaggtcaagctgcacaagctgaagcttttaatacaagacaacatgaattgatcgaagctgcaagaaatcatggtgaattgttacaatgggaagcttttactagagcattggaaggtatcacagatgaaactacaaagactgttttgacatggttgagagatttgttcgcattgagattgatcgaagatgatttgggttggtttgttgctcatggtagagtttcttcacaaagagctagagcattaagaggttacgttaatagattggctgaaagattaagaccatttgcattggaattagttgaagcttttggtttggaaccagaacatttgagaatggctgttgcaactgatgcagaaacacaaagacaagaagaagctcatgcatggtttacagctagaagagctgcaggtgaagaaccagaagatgaaaaagcagtaagagcaagagaaaaagcagcaagaggtagaagaggttga 2124
<210> 3
<211> 828
<212> DNA
<213> Yarrowialipolytica(HTD基因核苷酸序列)
<400> 3
atgcagaccacccctttctcttcctctgctcccgcccagacctttggtgacaagaagtacgagcacatcctgacctccacccccgtccccaaggtcgctctcgtcaccctcaaccggcccaaggccctcaacgccctgtgcactcctctcattaaggagctgaacgaggctctccaggctgctgatgccgaccccaccattggcgccatcgttctcaccggatccgagaagtcctttgctgccggcgccgacatcaaggagatgaaggacaagaccgtcacttccgtgctcaacgagaacttcatcgaggagtggggcaacatggccaacatcaagaaacccatcattgctgccgtcaacggctttgccctcggtggtggatgcgagcttgccatgatggccgacatcatctacgccggcgccaaggccaagtttggccagcccgagatcaagcttggtgtcattcccggagctggaggaacccagcgactcacccgagccattggcctctaccgagctaaccactacattcttaccggagagatgttcactgctcagcaggctgctgactggggtctggccgccaaggtctacgagcccgcccagctcgttgacgagtccgtcaaggctgctgctcagatcgcctcttacggccagttggctgtccaggccgccaaggcttctgtccaccagtccgctgaggtcggtctccgagccggtctcgagtttgagcgagtccgattccacggtctctttggcacccacgaccagaaggagggcatggctgcttttgccgagaagcgagagcccaacttcaagaacgagtaa828
<210> 4
<211> 861
<212> DNA
<213> Yarrowialipolytica(KR基因核苷酸序列)
<400> 4
atgaagaaggtcgactctctctccgtaattggcgccggccagatgggtctgggaatcgctctggtggctgccaacaaggccggtctccaggtcaacctcattgacgccaaccagggcgccctggacaagggtctcaagttcatggacaagctgcttgagaaggacgttggcaaaggccgtctgaccagcgacgaggcccaggccgtgcgaggccgggtcactggtcacaccaacctgcagtctgccgtggccgacgtggacatgatcattgaggcagtccccgagatccccaagctcaagtttgacatcttccgagacctcaacgagtggacccagaaggataccatcctggccaccaacacctcgtccatctccatcaccaagattgctgctgctgccggcgctggtgccccccgagtcatttctgcccacttcatgaaccccgtgcccgtccagaagggtgtcgagatcatcaccggcctgcagacctctcccgagaccctcgccaccaccctggaggttgtgaagcgaatgggcaagatcccttccatctccaaggactcccctggcttcctggctaaccgaatcctcatgccctacatcaacgaggccatcatcaccctcgagaccggtgttggagaaaaggaggacattgacaacattctcaagaacggctgtgccatgcccatgggaccccttgctctcgccgacttcattggtctcgatacttgtctcgccatcatgcgagtgctgtacgaggacactggtgactccaagtaccgaccctccgtcctgcttaacaagtacgtcgatgctggctggctcggaaagaagtctggaaagggcttctatgactactaa861
<210>5
<211> 399
<212> DNA
<213> 人工序列(FadM基因优化核苷酸序列)
<400> 5
atgcaaactcaaatcaaagtcagaggttaccacttggatgtatatcaacacgtcaataatgccagatacttagaatttttagaagaagcaagatgggatggtttggaaaactctgactcatttcaatggatgacagcccataatattgctttcgttgtcgtaaacataaacatcaactacagaagaccagctgtcttgtctgatttgttgactatcacatcccaattgcaacaattgaacggtaaatccggtatcttgagtcaagtaattaccttagaacctgaaggtcaagttgtcgctgatgcattgatcaccttcgtttgtatcgacttaaagactcaaaaagccttggcattggaaggtgaattgagagaaaagttagaacaaatggtaaagtga 399
<210>6
<211>798
<212> DNA
<213> 人工序列(MKS1基因优化核苷酸序列)
<400> 6
atggaaaaatctatgtcaccattcgttaagaaacatttcgttttggttcatactgcatttcatggtgcttggtgttggtacaagatcgttgcattgatgagatcatctggtcataatgttacagctttggatttgggtgcttctggtattaatccaaagcaagcattgcaaatcccaaacttctctgattacttatcaccattgatggagtttatggcatctttgccagctaacgaaaagattatcttggttggtcatgctttaggtggtttggcaatttcaaaagctatggaaacttttccagaaaagatttctgttgcagttttcttgtcaggtttgatgccaggtccaaacatcgatgctactacagtttgtacaaaagcaggttctgctgttttgggtcaattggataactgtgttacttatgaaaacggtccaacaaatccaccaactacattgatcgcaggtccaaagttcttggctactaacgtttaccatttgtcaccaattgaagatttggctttagcaacagctttggttagaccattgtatttgtacttggcagaagatatctctaaggaagttgttttatcttcaaagagatacggttcagttaagagagtttttattgttgcaactgaaaacgatgctttgaagaaagaatttttgaaattgatgattgaaaagaatccaccagatgaagttaaggaaatcgaaggttctgatcatgttacaatgatgtcaaagccacaacaattgtttactacattgttatctattgctaataagtacaaataa 798
<210>7
<211>275
<212> PRT
<213> 人工序列(截掉信号肽的HTD序列)
<400> 7
MQTTPFSSSAPAQTFGDKKYEHILTSTPVPKVALVTLNRPKALNALCTPLIKELNEALQAADADPTIGAIVLTGSEKSFAAGADIKEMKDKTVTSVLNENFIEEWGNMANIKKPIIAAVNGFALGGGCELAMMADIIYAGAKAKFGQPEIKLGVIPGAGGTQRLTRAIGLYRANHYILTGEMFTAQQAADWGLAAKVYEPAQLVDESVKAAAQIASYGQLAVQAAKASVHQSAEVGLRAGLEFERVRFHGLFGTHDQKEGMAAFAEKREPNFKNE
<210>8
<211>286
<212> PRT
<213> 人工序列(截掉信号肽的KR序列)
<400> 8
MKKVDSLSVIGAGQMGLGIALVAANKAGLQVNLIDANQGALDKGLKFMDKLLEKDVGKGRLTSDEAQAVRGRVTGHTNLQSAVADVDMIIEAVPEIPKLKFDIFRDLNEWTQKDTILATNTSSISITKIAAAAGAGAPRVISAHFMNPVPVQKGVEIITGLQTSPETLATTLEVVKRMGKIPSISKDSPGFLANRILMPYINEAIITLETGVGEKEDIDNILKNGCAMPMGPLALADFIGLDTCLAIMRVLYEDTGDSKYRPSVLLNKYVDAGWLGKKSGKGFYDY
Claims (12)
1.一种生产甲基酮的重组酿酒酵母的构建方法,其特征在于,在酿酒酵母中对β-酮酰辅酶A硫解酶基因POT1进行敲除或失活,或降低该基因的表达或活性;进一步将所述酿酒酵母中的半乳糖调节基因GAL80进行敲除或失活,或降低该基因的表达或活性;且在所述酿酒酵母中过表达酰基辅酶A氧化酶基因POX1和(3R)-羟烷基-辅酶A脱氢酶/2-烯酰辅酶A水合酶2基因POX2,以及酰基辅酶A硫酯酶基因TES1和外源β-酮酸脱羧酶基因ADC。
2.如权利要求1所述的构建方法,其特征在于,进一步在所述酿酒酵母中过表达脂酰辅酶A转运蛋白编码基因PXA1/PXA2。
3.如权利要求2所述的构建方法,其特征在于,所述酰基辅酶A氧化酶基因POX1,或(3R)-羟烷基-辅酶A脱氢酶/2-烯酰辅酶A水合酶2基因POX2,或酰基辅酶A硫酯酶基因TES1,或脂酰辅酶A转运蛋白编码基因PXA1/PXA2的过表达是过表达内源基因;所述外源β-酮酸脱羧酶基因ADC来源于Clostridium acetobutylicum。
4.如权利要求3所述的构建方法,其特征在于,所述外源β-酮酸脱羧酶基因ADC的核苷酸序列如SEQ ID NO:1所示。
5.如权利要求1所述的构建方法,其特征在于,进一步在所述酿酒酵母中过表达外源酰基辅酶A氧化酶基因ACO,或烯酰辅酶A水合酶基因HTD,或3-羟烷基-辅酶A脱氢酶基因KR,或酰基辅酶A硫酯酶基因FadM,或β-酮酸脱羧酶基因MKS1中的一种或多种。
6.如权利要求5所述的构建方法,其特征在于,所述烯酰辅酶A水合酶基因HTD的氨基酸序列如SEQ ID NO:7所示的氨基酸序列;所述3-羟烷基-辅酶A脱氢酶基因KR的氨基酸序列如SEQ ID NO:8所示的氨基酸序列。
7.如权利要求6所述的构建方法,其特征在于,所述外源酰基辅酶A氧化酶基因ACO来源于Micrococcus luteus;
所述烯酰辅酶A水合酶基因HTD来源于Yarrowia lipolytica;
所述3-羟烷基-辅酶A脱氢酶基因KR来源于Yarrowia lipolytica;
酰基辅酶A硫酯酶基因FadM来源于大肠杆菌;
β-酮酸脱羧酶基因MKS1来源于Solanum habrochaites。
8.如权利要求7所述的构建方法,其特征在于,所述外源酰基辅酶A氧化酶基因ACO的核苷酸序列如SEQ ID NO:2所示;所述烯酰辅酶A水合酶基因的核苷酸序列如SEQ ID NO:3所示;所述3-羟烷基-辅酶A脱氢酶基因KR的核苷酸序列如SEQ ID NO:4所示;所述酰基辅酶A硫酯酶基因FadM的核苷酸序列如SEQ ID NO:5;所述β-酮酸脱羧酶基因MKS1的核苷酸序列如SEQ ID NO:6所示。
9.如权利要求1至8任一项所述的构建方法,其特征在于,所述酿酒酵母的出发菌株的基因型为MATa MAL2-8c SUC2 ura3-52。
10.如权利要求1至8任一项所述的构建方法,其特征在于,所述酿酒酵母的出发菌株的基因型为CEN.PK113-5D。
11.如权利要求1至10任一项所述的构建方法获得的生产甲基酮的重组酿酒酵母。
12.如权利要求11所述的重组酿酒酵母在生产甲基酮中的应用。
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