CN113755420A - 一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用 - Google Patents
一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用 Download PDFInfo
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Abstract
本发明公开了一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用。本发明的基因工程菌是一种包含pSIP409‑nox‑alsS质粒并在敲除α‑乙酰乳酸脱羧酶基因(alsD)的干酪乳杆菌上表达的干酪乳杆菌，所述的pSIP409‑nox‑alsS质粒能够表达NADH氧化酶和α‑乙酰乳酸合成酶，不能够表达α‑乙酰乳酸脱羧酶。本发明的基因工程菌较野生型干酪乳杆菌具有更高的酶活，且在发酵酸奶中的丁二酮和乙偶姻的产量分别是野生型菌株TCS的2.38倍、11.19倍，本发明为干酪乳杆菌生物合成丁二酮和乙偶姻提供了新的思路，为今后优化酸奶风味生产提供了理论依据，具有广阔的应用前景。
Description
技术领域
本发明涉及一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用，属于生物工程技术领域。
背景技术
丁二酮和乙偶姻是酸奶中公认的关键香气物质，它们为酸奶贡献了牛奶和黄油的味道，这对于酸奶的典型风味是必不可少的。丁二酮和乙偶姻常用作各种乳制品、咖啡、酒类等食品的增香剂，用于增加产品的风味，也用于各种香精配方中。目前丁二酮和乙偶姻多作为食品添加剂生产，在酸奶发酵过程中很少作为风味化合物生产。而目前有通过内源生成而非外源添加来增强食品风味的趋势，更加符合当代社会对绿色天然食品的要求。因此现采用微生物发酵法，利用内源增香方式通过基因工程的手段来促进丁二酮和乙偶姻的积累。
由于酸奶的风味主要是由乳酸菌代谢产生的，目前常用于食品的乳酸菌主要集中于双歧杆菌属(Bifidobacterium)、链球菌属(Streptococcus)、肠球菌属(Enterococcus)、乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)等，这些菌属在酸奶生产过程中已显示出具有生产丁二酮的潜力。在我们前期的研究中，我们鉴定了一株具有较高丁二酮生产能力的菌株-干酪乳杆菌TCS(Lacticaseibacillus casei TCS)(专利申请号：CN201810414559.7)。它可以作为亲本菌株进行遗传改造和辅助培养，以此来提高酸奶中的奶香味。
目前，对菌株的单基因遗传操作技术已经比较完善，但随着代谢工程研究的深入，经常需要同时在一个宿主菌中改造多个基因以实现对细胞的综合调节，从而提高目标代谢产物的产量。利用基因工程手段实现多个基因在同一个宿主菌中表达的技术称为共表达，其转化策略包括利用含单基因的载体进行多重转化，或利用含多基因载体一次共表达多个基因。这种基因工程的内源增香方式可以更好的提高代谢产物的含量，可以提高干酪乳杆菌在酸奶生产中丁二酮和乙偶姻的产量。
发明内容
本发明所要解决的技术问题是：针对目前传统发酵剂菌种嗜热链球菌、保加利亚乳杆菌发酵酸奶的丁二酮和乙偶姻的产量比较低，难以满足酸奶风味的需求，提供一种用于提高酸奶中丁二酮和乙偶姻产量的基因工程菌。
为了解决上述问题，本发明提供了一种提高酸奶中丁二酮和乙偶姻产量的基因工程菌，是一种将pSIP409-nox-alsS质粒导入敲除了alsD基因的干酪乳杆菌中所得的工程菌，所述的工程菌中包含pSIP409-nox-alsS质粒，不包含alsD基因，所述的pSIP409-nox-alsS质粒能够表达NADH氧化酶和α-乙酰乳酸合成酶。
优选地，所述的pSIP409-nox-alsS质粒包含串联表达的nox基因和alsS基因。
本发明还提供了一种细胞，为一种包含pSIP409-nox-alsS质粒的E.coli DH5α感受态细胞，所述的pSIP409-nox-alsS质粒能够表达NADH氧化酶和α-乙酰乳酸合成酶。
本发明还提供了一种细胞，为一种敲除alsD基因的干酪乳杆菌感受态细胞。
本发明还提供了上述的提高酸奶中丁二酮和乙偶姻产量的基因工程菌在酸奶和/或酸奶制品的发酵中的应用。
与现有技术相比，本发明的有益效果在于：
1.本发明主要是对NADH氧化酶基因(nox)、α-乙酰乳酸合成酶基因(alsS)和α-乙酰乳酸脱羧酶基因(alsD)三个基因进行研究，采用过表达和基因敲除策略，通过多个基因共表达的基因修饰手段来确定这些基因对丁二酮和乙偶姻产量的影响；本发明为干酪乳杆菌生物合成丁二酮和乙偶姻提供了新的思路，为今后优化酸奶风味生产提供了理论依据；
2.本发明的基因工程菌较野生型干酪乳杆菌TCS具有更高的酶活(NADH氧化酶和α-乙酰乳酸合成酶酶活)，本发明的基因工程菌在发酵酸奶中的丁二酮和乙偶姻的产量分别是野生型菌株TCS的2.38倍、11.19倍。
附图说明
图1为丁二酮和乙偶姻的生物合成途径(a)和工程菌的重组质粒图谱(b)；
图2为重组质粒pSIP409-nox-alsS导入敲除alsD基因的干酪乳杆菌验证产物，其中图中的M表示DNA Marker；
图3为野生型菌和工程菌发酵酸奶中丁二酮和乙偶姻的含量。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
实施例中所用的干酪乳杆菌来自实验室前期鉴定的一株具有较高丁二酮生产能力的菌株-干酪乳杆菌TCS(Lacticaseibacillus casei TCS)，其公开在申请号为CN201810414559.7的专利中。
实施例1
产丁二酮和乙偶姻工程菌的构建：
1.表达载体pSIP409-nox-alsS的构建
根据变异链球菌Streptococcus mutans的nox基因序列和干酪乳杆菌TCS全基因组中的alsS基因序列，为了在单一载体中共表达nox和alsS基因，在alsS基因的5'端增加一个核糖体结合位点，利用Premier 5.0和Vector NTI软件设计引物nox-F、nox-R和alsS-F、alsS-R，并利用一步法克隆试剂盒将nox和alsS片段连接到载体pSIP409上。其中在引物nox-F的5'端引入NcoI酶切位点，在引物alsS-R的5’端引入XhoI酶切位点。PCR产物经纯化后与同样酶切位点双酶切的表达载体pSIP409连接，转化E.coli DH5α感受态细胞(VazymeBiotech Co.,Ltd.,Nanjing,China)，挑取转化子，经酶切验证获得pSIP409-nox-alsS，其序列如SEQ ID NO：1所示。
2.干酪乳杆菌感受态细胞的制备
将利用CRISPR基因编辑系统构建的敲除α-乙酰乳酸脱羧酶基因(alsD)的干酪乳杆菌进行活化(具体构建方法和活化方法参照论文“干酪乳杆菌中双乙酰的合成机制初步解析及应用，刘晗”中的方法)，将活化好的干酪乳杆菌菌液按合适比例接种到100mL的MRS液体培养基中，于37℃下静置培养至OD600值为0.5-0.8左右；将培养液在冰上冰浴20min，于4℃，4,000g，离心5min收集菌体，再用预冷的电击缓冲液聚乙二醇1500缓慢吹打菌体，冰浴10min，同等条件下离心收集菌体；重复该步骤4-5次；将菌体小心悬浮在1/100体积的聚乙二醇1500缓冲液中，于-80℃保存备用。
3.构建在敲除α-乙酰乳酸脱羧酶基因(alsD)的干酪乳杆菌上共表达nox和alsS基因的工程菌并鉴定
将带有nox和alsS基因的表达质粒pSIP409-nox-alsS电转化到敲除alsD基因的干酪乳杆菌感受态细胞上，涂布在带有红霉素抗性的MRS平板上，于37℃倒置培养48-72h，挑取单菌落进行菌落PCR和质粒提取验证，分别用引物403-F、403-R和nox-R、alsS-F通过PCR验证重组质粒pSIP409-nox-alsS是否成功导入干酪乳杆菌中。重组菌株经测序验证，如图2的泳道得出一个大小2-3kb的条带，符合nox片段(1374bp)和alsS片段(1524bp)之和，成功构建出将nox和alsS基因串联表达在敲除alsD基因的干酪乳杆菌并命名重组工程菌为TCS-ΔalsD-nox-alsS，该工程菌的重组质粒图谱如图1b所示。
以上所涉及的引物序列如表1所示。
表1引物序列
实施例2
利用野生型菌TCS和工程菌TCS-ΔalsD-nox-alsS发酵酸奶：
将待用的TCS、TCS-ΔalsD-nox-alsS菌株分别接种于MRS肉汤中，37℃常规培养12～16h，于室温下6000×g离心10min收集菌体，无菌水洗涤2次后用等体积的无菌水重悬，悬浮液被用作发酵剂待用。用12％(w/v)脱脂奶粉制备酸奶，95℃灭菌10min，待灭菌脱脂乳冷却至40℃左右，将上述得到的菌悬液按照4％(v/v)的比例接种于灭菌脱脂乳中，并对诱导型重组菌TCS-ΔalsD-nox-alsS中加入终浓度为25ng/mL的IP-673进行诱导。之后在37℃下静置发酵至pH约为4.6，再置于4℃冰箱后熟12-24h，即得到酸奶样品。
实施例3
工程菌的酶活测定：
对野生菌株TCS和工程菌TCS-ΔalsD-nox-alsS菌株进行酶活测定，分别测定其中的NADH氧化酶和α-乙酰乳酸合成酶酶活含量。
(1)NADH氧化酶酶活测定：采用NADH氧化酶活性检测试剂盒(Solarbio Science&Technology，北京，中国)测定NOX活性。酶活力单位(U)定义为反应体系中每1万个细菌在反应体系中每分钟A600变化0.01定义为一个酶活力单位。
(2)α-乙酰乳酸合成酶酶活测定：ALS活性是根据Holtzclaw(Holtzclaw和Chapman1975)描述的方法测定的，并作了一些修改。在1mM焦磷酸硫胺、5mM MgCl2、0.1mM黄素腺嘌呤二核苷酸、100mM丙酮酸钠和300μL粗酶液中进行测定。反应在37℃下进行1h，之后立即加入200μL的3M H2SO4终止反应。60℃水浴脱羧15min后，加入8.5mL显色液(5％α-萘酚、0.5％肌酸、5％氢氧化钠、去离子水)。该混合液在37℃下孵育30min，使乙酰乳酸酸水解为乙偶姻，最后用分光光度计在520nm处测定吸光度。酶活单位定义为37℃下，在pH为7.0的反应条件下，每分钟合成1nmol乙酰乳酸所用的酶量。
表2 NADH氧化酶和α-乙酰乳酸合成酶酶活
由表2可知，经过基因工程共表达改造后的工程菌，NADH氧化酶和α-乙酰乳酸合成酶的酶活能力都比野生型菌株TCS有所提高。由此说明，基因工程的共表达改造可以有效提升丁二酮和乙偶姻合成途径中关键酶的能力，从而进一步可以提升丁二酮和乙偶姻的生产能力。
实施例4
酸奶中丁二酮和乙偶姻的检测：
利用顶空固相微萃取并借助气相色谱测定酸奶中丁二酮和乙偶姻的含量：
称取5.00g后熟24h的酸奶样品于加有转子的15mL萃取瓶中，并加入10μL 2-辛醇(内标，220mg/L)，于55℃的磁力恒温水浴锅中平衡5min，然后插入萃取头富集顶空中的挥发性物质40min；色谱条件如下：HP-INNOWAX色谱柱(60m×0.25mm×0.25mm)；进样口温度：230℃；升温程序：40℃恒温保持3min，再以4℃/min速率升温至120℃，最后以5℃/min速率升温至230℃，并保持10min；载气：氦气；流速：1mL/min；进样方式：采用不分流进样。
测试结果表明本发明构建的工程菌TCS-ΔalsD-nox-alsS发酵酸奶中丁二酮的产量达到7.28μg/kg，乙偶姻产量达到92.39μg/kg。而野生型干酪乳杆菌TCS发酵酸奶中丁二酮产量只有3.06μg/kg，乙偶姻产量为8.26μg/kg。由此对比发现，工程菌TCS-ΔalsD-nox-alsS发酵酸奶中丁二酮和乙偶姻的产量分别是野生型菌株TCS的2.38倍、11.19倍。由此可以看出当nox、alsS和alsD这3个基因共同修饰时，多种遗传修饰对风味增强具有协同作用，对酸奶中丁二酮和乙偶姻的产量提升有明显的影响。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 上海应用技术大学
<120> 一种提升酸奶中丁二酮和乙偶姻含量的基因工程菌及应用
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 8790
<212> DNA
<213> 人工合成
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cgggtggctt tttattagcc ctagaagggc ttcccacacg catttcagcg ccttagtgcc 5520
ttagtttgtg aatcataggt ggtatagtcc cgaaataccc gtctaaggaa ttgtcagata 5580
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ttctaatgtc actaacctgc cccgttagtt gaagaaggtt tttatattac agctccagat 5700
ctaccggttt aatttgaaaa ttgatattag cgtttaacag ttaaattaat acgttaataa 5760
tttttttgtc tttaaatagg gatttgaagc ataatggtgt tatagcgtac ttagctggcc 5820
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ttacggtagt gaaaacgaag ttgtcgtttt tgaccaaaat tctaatattt cattcttggg 6000
ttgtggaatg gcactttgga ttggaaaaca aatatcaggc cctcaaggtc ttttttatgc 6060
tgacaaggaa tcgttagaag caaaaggtgc taaaatttat atggaatcgc cagtgacagc 6120
cattgattat gatgctaaga gggttactgc tttggtcaat ggtcaagaac atgttgaaag 6180
ctatgagaag cttattttgg caacaggatc aacaccaatc ttaccaccta tcaaaggtgc 6240
agctatcaaa gaaggtagtc gtgattttga agcaactttg aaaaatcttc aatttgttaa 6300
attgtatcaa aatgcagaag atgttattaa taaattacag gataagagtc aaaatctgaa 6360
tcgtattgct gttgttggtg ctggttatat tggtgtagaa cttgctgaag cctttaaacg 6420
cctcggaaaa gaagtgattc ttattgatgt tgttgacact tgcttagctg gttattatga 6480
tcaggatctt tcagaaatga tgcgtcaaaa tttggaagat catggtattg aattagcatt 6540
cggagaaact gtcaaagcca ttgaaggtga tggtaaagtc gaacgtattg taactgataa 6600
agcgagccat gatgtggata tggttatttt agctgtcggt ttccgtccta atactgcact 6660
tggcaacgct aaactcaaaa ccttccgtaa tggtgctttc cttgttgata aaaaacaaga 6720
gacaagtatt cctgacgttt atgccatcgg cgattgcgcg actgtttatg acaacgctat 6780
taatgatacc aattatattg ccttagcttc aaacgctctt cgctcaggta ttgtagctgg 6840
tcataatgca gcagggcata aattggaatc tcttggtgtt caaggttcaa atggtatttc 6900
aatttttggt ctcaatatgg tttcaactgg gttaacgcaa gaaaaagcaa agcgttttgg 6960
ctataatcca gaagtcactg catttacaga ttttcagaag gctagtttta ttgaacatga 7020
taattatcct gttacactta aaattgtcta tgataaggat aaccgactgg ttcttggtgc 7080
acaaatggca tctaaagaag atatgtcaat gggaattcat atgttttcat tggctattca 7140
ggaaaaagtt accattgaac gtttagctct actggactat ttctttcttc ctcatttcaa 7200
tcaaccctat aattatatga ccaaagcagc attaaaagct aaatgacaag gagattttag 7260
atggcagaag agaagcgcta tggcgcggat ttgatcgtcg aaagtttaac gaatcacggc 7320
attgactatg tttttgggat tcctggagcc aaaattgacc gggtttttga aagtctggag 7380
catcctaaat cagaaaagag cccgcagcta atcgttgcgc gacatgaaca aaatgcggcg 7440
tttatggccg ctggcatcgg tcgtttgacc ggcaaaccag gggttgtcct gacaacttca 7500
ggaccgggag catcaaactt ggcaaccggc ttggtaaccg caacggctga aggtgatccg 7560
gttttggctt tatccggtca ggtcaaacgc gctgatttgc tgcgaagttc tcatcaaagc 7620
atgcgtaacg ccgatctttt tgccccgatc accaaatatg cagctgaagt tcaggaccct 7680
gataatgtga gtgaaataat cgcgaatgct tatcaagcgg ctgaatctgg caagcaaggc 7740
gcaagttttg tttctatccc gcaagatgtc acagattcgc cagtcaactc agaaccgata 7800
aaaccactgg tcgcaccaaa acttggaccg gctagtccca gtgatatgac gtatctggcg 7860
cacgccatca aggaagcgtc attgccagtg ttgttgttag gaatgcgggc atcgtcaagt 7920
gatgtaacag cagagattcg cgaattactt tcagtaacgg aactacctgt tgtggaaact 7980
ttccaaggtg ctggcatcat ttctcaccgt cagattgaca acttcttcgg ccgggttggc 8040
ctgttccgca atcaaccagg tgacatgttg ttacaacata gtgatttggt cattgctatt 8100
gggtacgatc ccgttgaata tgaaccacgt aactggaatg cggatggcaa ggcgcgcatc 8160
attgtcattg atgatgttcc agctgagatt gatcataact ttcagccgga aactgaactc 8220
attggtgata tttcgcaaac gttagatatt ttggtgccat tgttacgtgg ctaccaagtt 8280
gcacctggca gcaagcgtta tttagaagac cttcaggcaa aactgcagga tagcgatgta 8340
ccgccggcaa ttgctgatca aaaagtgctt catccgctca gcattgttgc tgccttacag 8400
gagcgggtca cggatgaaat gaccgttgct gttgacgttg ggagtcatta tatttggatg 8460
gcccggcact tcagaagtta tgaaccacga catttgctgt tttccaatgg gatgcaaacg 8520
ctcggtgtcg ccttaccatg ggcgattgct gcaacgttgg ttcgcccagg gaaaaaggcg 8580
gtctcagttt ctggcgatgg cggttttctc ttttccggac aggaattaga aacagcagtg 8640
cgcctgcatg cagacttagt gcatattatt tggaatgatg gccattatga tatggtaaaa 8700
tttcaagagg aaatgaagta cggtcgtgca gctggcgttg actttggtcc ggttgacttt 8760
gtgaagtatg cagaagcatt ttgactcgag 8790
<210> 2
<211> 49
<212> DNA
<213> 人工合成
<400> 2
acaaggagat tttagccatg gctatgagta aaatcgttat tgttggagc 49
<210> 3
<211> 25
<212> DNA
<213> 人工合成
<400> 3
ccatctaaaa tctccttgtc attta 25
<210> 4
<211> 38
<212> DNA
<213> 人工合成
<400> 4
gacaaggaga ttttagatgg cagaagagaa gcgctatg 38
<210> 5
<211> 46
<212> DNA
<213> 人工合成
<400> 5
cggggtaccg aattcctcga gtcaaaatgc ttctgcatac ttcaca 46
<210> 6
<211> 22
<212> DNA
<213> 人工合成
<400> 6
gaaatacccg tctaaggaat tg 22
<210> 7
<211> 21
<212> DNA
<213> 人工合成
<400> 7
tggtcatgaa ttagtctcgg a 21
Claims (5)
1.一种提高酸奶中丁二酮和乙偶姻产量的基因工程菌，其特征在于，是一种将pSIP409-nox-alsS质粒导入敲除了alsD基因的干酪乳杆菌中所得的工程菌，所述的工程菌中包含pSIP409-nox-alsS质粒，不包含alsD基因，所述的pSIP409-nox-alsS质粒能够表达NADH氧化酶和α-乙酰乳酸合成酶。
2.如权利要求1所述的一种提高酸奶中丁二酮和乙偶姻产量的基因工程菌，其特征在于，所述的pSIP409-nox-alsS质粒包含串联表达的nox基因和alsS基因。
3.一种细胞，其特征在于，为一种包含pSIP409-nox-alsS质粒的E.coli DH5α感受态细胞，所述的pSIP409-nox-alsS质粒能够表达NADH氧化酶和α-乙酰乳酸合成酶。
4.一种细胞，其特征在于，为一种敲除alsD基因的干酪乳杆菌感受态细胞。
5.权利要求1或2所述的提高酸奶中丁二酮和乙偶姻产量的基因工程菌在酸奶和/或酸奶制品的发酵中的应用。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117491526A (zh) * | 2023-11-08 | 2024-02-02 | 山东哲成生物科技有限公司 | 一种果蔬汁乳酸发酵进程的监控方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117491526A (zh) * | 2023-11-08 | 2024-02-02 | 山东哲成生物科技有限公司 | 一种果蔬汁乳酸发酵进程的监控方法 |
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