CN115975832B - 甲酸脱氢酶在提高微生物发酵菌株对纤维素水解液中甲酸和乙酸抗性中的应用 - Google Patents
甲酸脱氢酶在提高微生物发酵菌株对纤维素水解液中甲酸和乙酸抗性中的应用 Download PDFInfo
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Abstract
本发明公开了甲酸脱氢酶在提高微生物发酵菌株对纤维素水解液中甲酸和乙酸抗性中的应用，属于生物技术领域。本发明利用氨基酸序列如SEQ ID NO.3所示的甲酸脱氢酶提高了微生物乙醇发酵菌株对纤维素水解液中甲酸和乙酸抗性的方法，在微生物中过表达甲酸脱氢酶，以提高甲酸脱氢酶活性，进而改善了甲酸和乙酸的抗性，在使用纤维素水解液作为底物进行发酵时，可免去发酵前脱毒的步骤，简化了工艺，节约了成本。
Description
技术领域
本发明属于生物技术领域，尤其涉及甲酸脱氢酶在提高微生物发酵菌株对纤维素水解液中甲酸和乙酸抗性中的应用。
背景技术
纤维素原料来源广泛、价格低廉，有效转化纤维素原料还有利于农村经济，被广泛研究为生产生物燃料和生物基化学品。木质纤维素原料需要进行预处理过程来获得微生物可直接利用的糖类，但是预处理之后会产生多种抑制物对细胞生长代谢有毒害作用，影响后续的发酵，抑制物主要包括：弱酸(甲酸和乙酸等)、呋喃类(糠醛和5-羟甲基糠醛等)、酚类化合物(香草醛和4-水杨酸等)，预处理的方法不同，水解液中的抑制物含量也有所不同。
乙酸是纤维素水解液中含量最高的抑制物之一，浓度为1.0g/L～15.0g/L[Wei N,Enhanced biofuel production through coupled acetic acid and xyloseconsumption by engineered yeast[J].Nature Communication,2013,4:2580-2587.]，其通过释放H+导致细胞内环境酸化，为了保持胞内环境稳定，细胞通过消耗ATP将乙酸排到胞外，造成能量损失致使细胞活性下降。乙酸的存在还会引起微生物细胞的氧化应激反应，产生大量ROS，引起细胞损伤。
甲酸在纤维素水解液中的浓度较低为1.4g/L左右，但是对酵母的毒性较乙酸强，甲酸pKa＝3.75小于乙酸的pKa＝4.75，且甲酸的分子更小更易于扩散，所以具有更强的毒性。由于胞内的pH较高，未解离的甲酸可以从培养基中扩散到整个质膜表面，从而降低胞质pH，影响细胞活性[Oshoma CE，Screening of Non-Saccharomyces cerevisiaeStrainsfor Tolerance to Formic Acid in BioethanolFermentation[J].PLoS One,2015，10(8),e0135626.]。所以提高微生物发酵菌株对纤维素水解液中主要抑制物(甲酸和乙酸)的耐受能力，对以纤维素水解液为底物生产的生物乙醇、生物油脂及其他生物基化学品，具有重要意义。
甲酸脱氢酶是通过催化甲酸的氧化产生二氧化碳，并产生还原性NADH和ATP，其是已知的，广泛的应用在腐胺的生成(专利申请号201780017515.4)、辅酶的还原(专利申请号201711230338.6)等反应中，但是对提高乙酸的胁迫能力方面，目前并无报道。
发明内容
为了解决上述技术问题，本发明提供了甲酸脱氢酶在提高微生物发酵菌株对纤维素水解液中甲酸和乙酸抗性中的应用。
为达上述目的，本发明采用如下的技术方案：
甲酸脱氢酶在提高微生物发酵菌株对纤维素水解液中甲酸和乙酸抗性中的应用，所述应用是通过在微生物发酵菌株中过表达氨基酸序列如SEQ ID NO.3所示的甲酸脱氢酶来实现的。
优选地，所述微生物发酵菌株选自大肠杆菌、毕赤酵母、运动假单胞菌、马克斯克鲁维酵母、假丝酵母或Spathaspora passalidarum。
更优选地，通过构建表达甲酸脱氢酶基因序列的重组表达载体，并转化至微生物发酵菌株中，实现微生物菌株过表达甲酸脱氢酶。
更优选地，所述表达载体选自天然或重组质粒、黏粒、病毒或噬菌体。
更优选地，利用序列如SEQ ID NO.1所示的上游引物和SEQ ID NO.2所示的下游引物PCR扩增序列SEQ ID NO.3对应的基因片段，后构建表达载体。
更优选地，所述PCR扩增条件为：98℃预变性5min；98℃15s，55℃15s，72℃60s，30循环；72℃延伸7min。
与现有技术相比，本发明具有如下有益效果：
本发明利用甲酸脱氢酶提高微生物乙醇发酵菌株对纤维素水解液中甲酸和乙酸抗性的方法。在微生物中过表达甲酸脱氢酶，提高甲酸脱氢酶活性，能够提高甲酸和乙酸的抗性，以便在使用纤维素水解液作为底物进行发酵时，免去发酵前脱毒的步骤，简化了工艺，节约了成本。
附图说明
实施例2中的图1A和图1B是在酿酒酵母中过表达甲酸脱氢酶后，重组菌株与对照经10倍浓度稀释后，在4g/L乙酸(图1A)和1g/L甲酸(图1B)的固体培养基中的生长情况比较。从结果可以看出，过表达甲酸脱氢酶，可显著提高乙酸的抗性，提高幅度为2个数量级。也可提高甲酸的抗性，提高幅度为1个数量级。
实施例3中的图2A和图2B是在酿酒酵母中过表达甲酸脱氢酶后，重组菌株与对照在4g/L乙酸的液体培养基中的葡萄糖消耗和生物量情况。从结果可以看出，过表达甲酸脱氢酶后，在较高浓度乙酸存在的情况下，重组菌的生长速度和消耗葡萄糖的速率都高于对照菌株。
实施例4中的图3A和图3B是在酿酒酵母中过表达甲酸脱氢酶后，重组菌株与对照在1g/L甲酸的液体培养基中的葡萄糖消耗和生物量情况。从结果可以看出，过表达甲酸脱氢酶后，在较高浓度甲酸存在的情况下，重组菌经过延迟期后，出现了生长和葡萄糖的消耗，但对照菌株在这种情况下被甲酸完全抑制并死亡。
具体实施方式
实施例1
本实施例提供菌株S.cerevisiae-FDH的构建过程，具体步骤如下：
1)以酿酒酵母模式菌株S288C的基因组为模板进行PCR扩增，获得Fdh基因片段，上下游引物核苷酸序列分别如SEQ ID NO.1，SEQ ID NO.2所示：
上游引物：attg cggccgct atgtcgaagggaaaggttttg(SEQ ID NO.1)
下游引物：acgcgc gtcgac ttatttcttctgtccataag(SEQ ID NO.2)
PCR扩增所用试剂盒为HS DNAPolymerase(Code No.:R010A)，购于宝生物工程(大连)有限公司，PCR反应按照试剂盒说明书进行。
PCR扩增条件为：98℃预变性5min；98℃15s，55℃15s，72℃60s，30循环；72℃延伸7min；
2)获得的Fdh基因片段使用限制性核酸内切酶Not I和Sal I进行双酶切，再用连接酶进行连接到pRS424上(购于淼灵质粒平台)，得到重组质粒pRS424-FDH；
3)将构建好的重组质粒pRS424-FDH转化入酿酒酵母S288C中，获得过表达甲酸脱氢酶的重组菌株S.cerevisiae-FDH；
筛选培养基为：SD-Trp培养基：YNB 6.7g/L，葡萄糖20g/L，不含Trp的氨基酸补充溶液。
其中，甲酸脱氢酶(Fdh)的氨基酸序列为(SEQ ID NO.3)：
Mskgkvllvlyeggkhaeeqekllgcienelgirnfieeqgyelvttidkdpeptstvdrelkdaeivittpffpayisrnriaeapnlklcvtagvgsdhvdleaanerkitvtevtgsnvvsvaehvmatilvlirnyngghqqaingewdiagvakneydledkiistvgagrigyrvlerlvafnpkkllyydyqelpaeainrlneasklfngrgdivqrvekledmvaqsdvvtincplhkdsrglfnkklishmkdgaylvntargaicvaedvaeavksgklagyggdvwdkqpapkdhpwrtmdnkdhvgnamtvhisgtsldaqkryaqgvknilnsyfskkfdyrpqdiivqngsyatraygqkk。
实施例2
本实施例在含甲酸和乙酸的平板上进行生长情况的考察，具体过程如下：
1)活化培养基的制备：活化培养基：YNB 6.7g/L，葡萄糖10g/L，不含Trp的氨基酸补充溶液。
2)菌株的活化：取冰箱保藏的实施例1中制备的菌株S.cerevisiae-FDH和原始菌株S.cerevisiae S288C接种至活化培养基中，置于摇床中30℃，150rpm培养24h。
3)种子培养基的制备：种子培养基：YNB 6.7g/L，葡萄糖20g/L，不含Trp的氨基酸补充溶液。
4)步骤2)中取活化后的菌体转接至步骤3)中制备的种子培养基中，置于摇床中30℃，150rpm培养24h。
5)鉴别培养基得制备：鉴别培养基：YNB 6.7g/L，葡萄糖20g/L，琼脂粉20g/L，不含Trp的氨基酸补充溶液，甲酸1g/L或乙酸4g/L。
6)菌种经活化、种子扩大培养后，离心收集细胞，将收集细胞用适量的无菌水重悬，使各个菌种初始OD620保持一致(≈10.0)将收集的细胞10倍梯度稀释，取8μL的菌液点于鉴别平板上，待菌液晾干后，30℃倒置培养2-3d。
实验结果如图1A和图1B所示，在1g/L甲酸的固体培养基中，过表达了甲酸脱氢酶基因的S.cerevisiae-FDH较原始菌株生长情况更好，显示出更好的甲酸耐受能力；在4g/L乙酸的固体培养基中，重组菌株S.cerevisiae-FDH也表现出了更好的生长情况。以上实验结果说明，过表达甲酸脱氢酶基因后，重组菌株S.cerevisiae-FDH不仅提高了对甲酸的耐受能力，对乙酸的耐受能力也有明显提高。
实施例3
本实施例在含有乙酸的液体培养基中试验发酵情况，具体过程如下：
1)活化培养基：YNB 6.7g/L，葡萄糖10g/L，不含Trp的氨基酸补充溶液。
2)取冰箱保藏实施例1中制备的菌株S.cerevisiae-FDH和原始菌株接种至活化培养基中，置于摇床中30℃，150rpm培养24h。
3)种子培养基的制备：种子培养基：YNB 6.7g/L，葡萄糖20g/L，不含Trp的氨基酸补充溶液。
4)取活化后的菌体转接至步骤3)中制备的种子培养基中，置于摇床中30℃，150rpm培养24h-48h。
5)发酵培养基的制备：发酵培养基：YNB 6.7g/L，葡萄糖40g/L，不含Trp的氨基酸补充溶液，乙酸4g/L。
6)菌种经活化、种子扩大培养后，离心收集细胞，收集细胞接种至添加乙酸的发酵培养基中，接种量OD 620≈0.5，30℃，150rpm发酵72-96h，每12-24h取样。
发酵结果如图2A和图2B所示，在4g/L乙酸存在条件下，过表达了甲酸脱氢酶基因的S.cerevisiae-FDH较原始菌株的葡萄糖消耗更快，在发酵进行72h后，S.cerevisiae-FDH较原始菌株多消耗了3.6g/L葡萄糖，且菌株的生长更快，在24h生物量OD620较出发菌株提高了11％，实验结果说明过表达甲酸脱氢酶后菌株S.cerevisiae-FDH对乙酸的耐受能力增强有所增强。
实施例4
本实施例在含有甲酸的液体培养基中试验发酵情况，具体步骤如下：
本实施例采用与实施例3相同的方法进行种子培养与扩大培养，区别仅在于：所添加的抑制物为1g/L甲酸。
实验结果如图3A和图3B所示，原始菌株在甲酸存在的条件下，由于较强的抑制作用，发酵过程中并无葡萄糖消耗，且生物量在逐渐降低，而重组菌株S.cerevisiae-FDH在经过延滞期后，葡萄糖快速消耗，生物量也随着葡萄糖的消耗快速生长至OD620至0.83。以上实验结果说明，过表达甲酸脱氢酶后菌株的甲酸耐性明显增强。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (5)
1.甲酸脱氢酶在提高微生物发酵菌株对纤维素水解液中甲酸和乙酸抗性中的应用，其特征在于，所述微生物发酵菌株为酿酒酵母(Saccharomycescerevisiae)模式菌株S288C；所述应用是通过在微生物发酵菌株中过表达氨基酸序列如SEQ ID NO.3所示的甲酸脱氢酶来实现的。
2.根据权利要求1所述的应用，其特征在于，通过构建表达甲酸脱氢酶基因序列的重组表达载体，并转化至微生物发酵菌株中，实现微生物菌株过表达甲酸脱氢酶。
3.根据权利要求2所述的应用，其特征在于，所述表达载体选自天然或重组质粒。
4.根据权利要求3所述的应用，其特征在于，利用序列如SEQ ID NO.1所示的上游引物和SEQ ID NO.2所示的下游引物PCR扩增序列SEQ ID NO.3对应的基因片段，构建表达载体。
5.根据权利要求4所述的应用，其特征在于，所述PCR扩增条件为：98℃预变性5min；98℃15s，55℃15s，72℃60s，30循环；72℃延伸7min。
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