CN113265430A - 重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法 - Google Patents
重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法 Download PDFInfo
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Abstract
本发明公开了重组大肠杆菌基于纤维素一步发酵产1,2,4‑丁三醇的方法,先构建利用木糖生产1,2,4‑丁三醇的重组大肠杆菌,将重组大肠杆菌制得的种子液接种至含有纤维素及纤维素酶的缓冲液中发酵产1,2,4‑丁三醇。现有技术中直接以葡萄糖为碳源,1,2,4‑丁三醇产量为2.9g/L,木糖至1,2,4‑丁三醇的转化率为0.29g/g。本发明以纤维素作为碳源,1,2,4‑丁三醇产量为3.52g/L,木糖至1,2,4‑丁三醇的转化率为0.352g/g。纤维素作为碳源可以保证在发酵过程中连续提供葡萄糖,操作简便,提升底物利用率,且许多废弃农业物如玉米芯等中含有大量纤维素,为后续废弃物利用提供方向。
Description
技术领域
本发明属于生物技术领域,具体涉及重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法。
背景技术
1,2,4-丁三醇(1,2,4-butanetriol 简称 BT),是一种非天然、高价值的 C4 平台化合物,是一种重要的商品化学品。它最重要的应用是生产1,2,4-丁三醇三硝酸盐(BTTN),这是一种在推进剂和炸药配方中的高能增塑剂,有可能在军事领域取代传统的硝酸甘油。目前,利用 NaBH4还原苹果酸及其衍生物是工业上合成 BT 的主要方法。但化学法合成BT反应所需条件苛刻,且收率较低,反应后难以分离纯化,较高的成本和复杂的操作使化学法合成 BT难以应用于大规模生产。而利用生物法合成BT反应条件温和,操作简便,成本低廉,在大规模生产上具有显著优势。BT的生物合成涉及四步酶反应:D-木糖在木糖脱氢酶的催化下转化为木糖酸,木糖酸在木糖酸脱水酶的催化下转化为D-3-脱氧甘油戊酮糖酸,D-3-脱氧甘油戊酮糖酸在2-酮酸脱羧酶的催化下转化为D-3,4-二羟基丁醛,D-3,4-二羟基丁醛在醛还原酶催化下转化为1,2,4-丁三醇。BT的生物合成涉及两种方法:一种是大量培养表达上述四种酶的微生物,培养结束后收集细胞,利用收集的细胞将木糖转化为BT。这种方法具有转化率较高的优势,但过程中步骤繁琐,不利于大规模生产。一种是在培养表达上述四种酶的微生物的过程中,加入底物木糖,直接生产BT。利用微生物直接发酵生产BT成本低廉,过程中操作简便,有利于大规模生产。
大肠杆菌的培养过程一般需要添加葡萄糖作为碳源,以支持其生长,葡萄糖在大肠杆菌中代谢产生的能量亦有利于酶的表达。由于重组大肠杆菌中BT的合成过程涉及到的酶较多,因此葡萄糖的添加必不可少。
纤维素是由葡萄糖组成的大分子多糖,是植物细胞壁的主要成分,是自然界中分布最广、含量最多的一种多糖,许多农业废弃物如玉米芯等都含有大量的纤维素。如何有效利用废弃生物质纤维素作为有利资源,是一项重要的研究课题。
发明内容
针对现有技术的不足,本发明提供了一种重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,该方法使用纤维素代替葡萄糖作为碳源,降低了1,2,4-丁三醇的发酵成本,优化培养条件,提升了1,2,4-丁三醇的转化率,展示了使用可再生资源作为碳源的潜力,污染小、且对环境友好;且含有纤维素的可再生资源重组而廉价,使用纤维素作为碳源具有巨大的经济优势。
为了解决上述技术问题,本发明采取的技术方案为:
重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,构建表达木糖脱氢酶xylb、木糖酸脱水酶kdcA、酮酸脱羧酶yjhG和醛还原酶adhp的重组大肠杆菌,制备重组大肠杆菌的种子液,并接种至含有纤维素、纤维素酶、酵母粉、蛋白胨、氯化钠、木糖的柠檬酸钠缓冲液中发酵生产1,2,4-丁三醇。
作为改进的是,所述木糖脱氢酶基因xylb的核苷酸序列如SEQ ID NO.1所示:
ATGTCTTCTGCTATCTACCCGTCTCTGAAAGGTAAACGTGTTGTTATCACCGGTGGTGGTTCTGGTATCGGTGCTGGTCTGACCGCTGGTTTCGCTCGTCAGGGTGCTGAAGTTATCTTCCTGGACATCGCTGACGAAGACTCTCGTGCTCTGGAAGCTGAACTGGCTGGTTCTCCGATCCCGCCGGTTTACAAACGTTGCGACCTGATGAACCTGGAAGCTATCAAAGCTGTTTTCGCTGAAATCGGTGACGTTGACGTTCTGGTTAACAACGCTGGTAACGACGACCGTCACAAACTGGCTGACGTTACCGGTGCTTACTGGGACGAACGTATCAACGTTAACCTGCGTCACATGCTGTTCTGCACCCAGGCTGTTGCTCCGGGTATGAAAAAACGTGGTGGTGGTGCTGTTATCAACTTCGGTTCTATCTCTTGGCACCTGGGTCTGGAAGACCTGGTTCTGTACGAAACCGCTAAAGCTGGTATCGAAGGTATGACCCGTGCTCTGGCTCGTGAACTGGGTCCGGACGACATCCGTGTTACCTGCGTTGTTCCGGGTAACGTTAAAACCAAACGTCAGGAAAAATGGTACACCCCGGAAGGTGAAGCTCAGATCGTTGCTGCTCAGTGCCTGAAAGGTCGTATCGTTCCGGAAAACGTTGCTGCTCTGGTTCTGTTCCTGGCTTCTGACGACGCTTCTCTGTGCACCGGTCACGAATACTGGATCGACGCTGGTTGGCGT
木糖酸脱水酶kdcA的核苷酸序列如SEQ ID NO.2所示:
ATGTATACAGTAGGAGATTACCTGTTAGACCGATTACACGAGTTGGGAATTGAAGAAATTTTTGGAGTTCCTGGTGACTATAACTTACAATTTTTAGATCAAATTATTTCACGCGAAGATATGAAATGGATTGGAAATGCTAATGAATTAAATGCTTCTTATATGGCTGATGGTTATGCTCGTACTAAAAAAGCTGCCGCATTTCTCACCACATTTGGAGTCGGCGAATTGAGTGCGATCAATGGACTGGCAGGAAGTTATGCCGAAAATTTACCAGTAGTAGAAATTGTTGGTTCACCAACTTCAAAAGTACAAAATGACGGAAAATTTGTCCATCATACACTAGCAGATGGTGATTTTAAACACTTTATGAAGATGCATGAACCTGTTACAGCAGCGCGGACTTTACTGACAGCAGAAAATGCCACATATGAAATTGACCGAGTACTTTCTCAATTACTAAAAGAAAGAAAACCAGTCTATATTAACTTACCAGTCGATGTTGCTGCAGCAAAAGCAGAGAAGCCTGCATTATCTTTAGAAAAAGAAAGCTCTACAACAAATACAACTGAACAAGTGATTTTGAGTAAGATTGAAGAAAGTTTGAAAAATGCCCAAAAACCAGTAGTGATTGCAGGACACGAAGTAATTAGTTTTGGTTTAGAAAAAACGGTAACTCAGTTTGTTTCAGAAACAAAACTACCGATTACGACACTAAATTTTGGTAAAAGTGCTGTTGATGAATCTTTGCCCTCATTTTTAGGAATATATAACGGGAAACTTTCAGAAATCAGTCTTAAAAATTTTGTGGAGTCCGCAGACTTTATCCTAATGCTTGGAGTGAAGCTTACGGACCGCTCAACAGGTGCATTCACACATCATTTAGATGAAAATAAAATGATTTCACTAAACATAGATGAAGGAATAATTTTCAATAAAGTGGTAGAAGATTTTGATTTTAGAGCAGTGGTTTCTTCTTTATCAGAATTAAAAGGAATAGAATATGAAGGACAATATATTGATAAGCAATATGAAGAATTTATTCCATCAAGTGCTCCCTTATCACAAGACCGTCTATGGCAGGCAGTTGAAAGTTTGACTCAAAGCAATGAAACAATCGTTGCTGAACAAGGAACCTCATTTTTTGGAGCTTCAACAATTTTCTTAAAATCAAATAGTCGTTTTATTGGACAACCTTTATGGGGTTCTATTGGATATACTTTTCCAGCGGCTTTAGGAAGCCAAATTGCGGATAAAGAGAGCAGACACCTTTTATTTATTGGTGATGGTTCACTTCAACTTACCGTACAAGAATTAGGACTATCAATCAGAGAAAAACTCAATCCAATTTGTTTTATCATAAATAATGATGGTTATACAGTTGAAAGAGAAATCCACGGACCTACTCAAAGTTATAACGACATTCCAATGTGGAATTACTCGAAATTACCAGAAACATTTGGAGCAACAGAAGATCGTGTAGTATCAAAAATTGTTAGAACAGAGAATGAATTTGTGTCTGTCATGAAAGAAGCCCAAGCAGATGTCAATAGAATGTATTGGATAGAACTAGTTTTGGAAAAAGAAGATGCGCCAAAATTACTGAAAAAAATGGGTAAATTATTTGCTGAGCAAAATAAATAA
酮酸脱羧酶基因yjhG的核苷酸序列如SEQ ID NO.3所示:
ATGTCTGTTCGCAATATTTTTGCTGACGAGAGCCACGATATTTACACCGTCAGAACGCACGCCGATGGCCCGGACGGCGAACTCCCATTAACCGCAGAGATGCTTATCAACCGCCCGAGCGGGGATCTGTTCGGTATGACCATGAATGCCGGAATGGGTTGGTCTCCGGACGAGCTGGATCGGGACGGTATTTTACTGCTCAGTACACTCGGTGGCTTACGCGGCGCAGACGGTAAACCCGTGGCGCTGGCGTTGCACCAGGGGCATTACGAACTGGACATCCAGATGAAAGCGGCGGCCGAGGTTATTAAAGCCAACCATGCCCTGCCCTATGCCGTGTACGTCTCCGATCCTTGTGACGGGCGTACTCAGGGTACAACGGGGATGTTTGATTCGCTACCATACCGAAATGACGCATCGATGGTAATGCGCCGCCTTATTCGCTCTCTGCCCGACGCGAAAGCAGTTATTGGTGTGGCGAGTTGCGATAAGGGGCTTCCGGCCACCATGATGGCACTCGCCGCGCAGCACAACATCGCAACCGTGCTGGTCCCCGGCGGCGCGACGCTGCCCGCAAAGGATGGAGAAGACAACGGCAAGGTGCAAACCATTGGCGCACGCTTCGCCAATGGCGAATTATCTCTACAGGACGCACGCCGTGCGGGCTGTAAAGCCTGTGCCTCTTCCGGCGGCGGCTGTCAATTTTTGGGCACTGCCGGGACATCTCAGGTGGTGGCCGAAGGATTGGGACTGGCAATCCCACATTCAGCCCTGGCCCCTTCCGGTGAGCCTGTGTGGCGGGAGATCGCCAGAGCTTCCGCGCGAGCTGCGCTGAACCTGAGTCAAAAAGGCATCACCACCCGGGAAATTCTCACCGATAAAGCGATAGAGAATGCGATGACGGTCCATGCCGCGTTCGGTGGTTCAACAAACCTGCTGTTACACATCCCGGCAATTGCTCACCAGGCAGGTTGCCATATCCCGACCGTTGATGACTGGATCCGCATCAACAAGCGCGTGCCCCGACTGGTGAGCGTACTGCCTAATGGCCCGGTTTATCATCCAACGGTCAATGCCTTTATGGCAGGTGGTGTGCCGGAAGTCATGTTGCATCTGCGCAGCCTCGGATTGTTGCATGAAGACGTTATGACGGTTACCGGCAGCACGCTGAAAGAAAACCTCGACTGGTGGGAGCACTCCGAACGGCGTCAGCGGTTCAAGCAACTCCTGCTCGATCAGGAACAAATCAACGCTGACGAAGTGATCATGTCTCCGCAGCAAGCAAAAGCGCGCGGATTAACCTCAACTATCACCTTCCCGGTGGGCAATATTGCGCCAGAAGGTTCGGTGATCAAATCCACCGCCATTGACCCCTCGATGATTGATGAGCAAGGTATCTATTACCATAAAGGTGTGGCGAAGGTTTATCTGTCCGAGAAAAGTGCGATTTACGATATCAAACATGACAAGATCAAGGCGGGCGATATTCTGGTCATTATTGGCGTTGGACCTTCAGGTACAGGGATGGAAGAAACCTACCAGGTTACCAGTGCCCTGAAGCATCTGTCATACGGTAAGCATGTTTCGTTAATCACCGATGCACGTTTCTCGGGCGTTTCTACTGGCGCGTGCATCGGCCATGTGGGGCCAGAAGCGCTGGCCGGAGGCCCCATCGGTAAATTACGCACCGGGGATTTAATTGAAATTAAAATTGATTGTCGCGAGCTTCACGGCGAAGTCAATTTCCTCGGAACCCGTAGCGATGAACAATTACCTTCACAGGAGGAGGCAACTGCAATATTAAATGCCAGACCCAGCCATCAGGATTTACTTCCCGATCCTGAATTGCCAGATGATACCCGGCTATGGGCAATGCTTCAGGCCGTGAGTGGTGGGACATGGACCGGTTGTATTTATGATGTAAACAAAATTGGCGCGGCTTTGCGCGATTTTATGAATAAAAACTGA
醛还原酶adhp基因的核苷酸序列如SEQ ID NO.4所示:
ATGAAGGCTGCAGTTGTTACGAAGGATCATCATGTTGACGTTACGTATAAAACACTGCGCTCACTGAAACATGGCGAAGCCCTGCTGAAAATGGAGTGTTGTGGTGTATGTCATACCGATCTTCATGTTAAGAATGGCGATTTTGGTGACAAAACCGGCGTAATTCTGGGCCATGAAGGCATCGGTGTGGTGGCAGAAGTGGGTCCAGGTGTCACCTCATTAAAACCAGGCGATCGTGCCAGCGTGGCGTGGTTCTACGAAGGATGCGGTCATTGCGAATACTGTAACAGTGGTAACGAAACGCTCTGCCGTTCAGTTAAAAATGCCGGATACAGCGTTGATGGCGGGATGGCGGAAGAGTGCATCGTGGTCGCCGATTACGCGGTAAAAGTGCCAGATGGTCTGGACTCGGCGGCGGCCAGCAGCATTACCTGTGCGGGAGTCACCACCTACAAAGCCGTTAAGCTGTCAAAAATTCGTCCAGGGCAGTGGATTGCTATCTACGGTCTTGGCGGTCTGGGTAACCTCGCCCTGCAATACGCGAAGAATGTCTTTAACGCCAAAGTGATCGCCATTGATGTCAATGATGAGCAGTTAAAACTGGCAACCGAAATGGGCGCAGATTTAGCGATTAACTCACACACCGAAGACGCCGCCAAAATTGTGCAGGAGAAAACTGGTGGCGCTCACGCTGCGGTGGTAACAGCGGTAGCTAAAGCTGCGTTTAACTCGGCAGTTGATGCTGTCCGTGCAGGCGGTCGTGTTGTGGCTGTCGGTCTACCGCCGGAGTCTATGAGCCTGGATATCCCACGTCTTGTGCTGGATGGTATTGAAGTGGTCGGTTCGCTGGTCGGCACGCGCCAGGATTTAACTGAAGCCTTCCAGTTTGCCGCCGAAGGTAAAGTGGTGCCGAAAGTCGCCCTGCGTCCGTTAGCGGACATCAACACCATCTTTACTGAGATGGAAGAAGGCAAAATCCGTGGCCGCATGGTGATTGATTTCCGTCACTAA。
作为改进的是,将重组大肠杆菌制得的种子液按4%的体积比的接种量接种。
作为改进的是,所述发酵为37℃下培养至OD600=2时加入IPTG至终浓度为1 mmol/L,加入IPTG 后在33℃培养66h至发酵结束。
作为改进的是,重组大肠杆菌的构建方法包括如下步骤:
(1)构建重组质粒pTRC99a-xylb-kdcA和重组质粒PCWJ-yjhG-adhp;
(2)再将重组质粒转化宿主菌,所述的宿主菌为大肠Trans 1 T1,得到产D-1,2,4丁三醇的重组大肠杆菌。
作为改进的是,所述柠檬酸钠缓冲液中各组分的含量为:5g/L酵母粉,10g/L蛋白胨,10g/L纤维素,15FPU纤维素酶、10g/L氯化钠、10g/L木糖;溶剂为0.5 mol/L柠檬酸钠缓冲液。
进一步改进的是,0.5 mol/L柠檬酸钠缓冲液配制方法为分别配制0.5 mol/L柠檬酸溶液和0.5 mol/L柠檬酸钠溶液,再用0.5 mol/L柠檬酸溶液加入0.5 mol/L柠檬酸钠溶液调节pH至6,即可。
有益效果:
现有技术通常直接使用葡萄糖作为碳源,一次性添加过多的葡萄糖会导致短时间内大肠杆菌利用过多葡萄糖,消耗大量氧气,而溶氧不足会导致大肠杆菌产生乙酸,抑制酶的表达和菌体生长;而少量多次添加葡萄糖虽有利于丁三醇的生成和菌株生长,但操作繁琐,过程中易染菌。针对上述问题的不足,本发明重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,使用纤维素代替葡萄糖作为碳源,可以实现同步糖化发酵,即纤维素在纤维素酶催化下不断水解为葡萄糖,水解出的葡萄糖不断被菌株吸收利用。在整个发酵过程中,既满足菌株对葡萄糖的需求,又简化了过程,无需人工添加葡萄糖,节省人工成本,且解除了纤维素酶水解过程中的产物抑制,提高了纤维素利用率,真正意义上实现了一步法连续化催化发酵产1,2,4-丁三醇。
附图说明
图1为不同浓度葡萄糖下重组大肠杆菌的生长状况;
图2 为不同浓度葡萄糖发酵产丁三醇的情况;
图3为重组大肠杆菌利用纤维素作为碳源发酵生产1,2,4-丁三醇的情况;
图4为纯纤维素水解积累的葡萄糖和接菌之后葡萄糖积累的发酵情况对比图。
具体实施方案
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的内容仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
以下实施例中如无特别说明,所用酶及质粒均为购买所得。
实施例1:含YjhG基因菌株的构建
(1)利用YjhG基因5’端和3’端引物(本领域人员均可按照常规设计,即能得到)引入酶切位点(NcoI和HindⅢ),对YjhG基因和pCWJ质粒进行双酶切,然后将YjhG基因连接到pCWJ载体上;
(2)将上述重组载体转入大肠杆菌Trans 1T1的感受态细胞(全式金生物技术有限公司)里,涂布在带有50mg/L氯霉抗性的LB平板,37℃过夜培养;
(3)挑取平板上生长的单菌落,转接到含有50mg/L氯霉素抗性的LB培养基里,然后提取质粒,再经限制性内切酶Spe I和Kpn I进行酶切验证,最后得到了重组质粒pCWJ-YjhG。
参考上述方法,继续制备重组质粒PCWJ-yjhG-adhp和pTRC99a-xylb-kdcA。
实施例2:重组大肠杆菌利用葡萄糖作为碳源发酵生产1,2,4-丁三醇
将含有重组质粒pTRC99a-xylb-kdcA和重组质粒PCWJ-yjhG-adhp的重组大肠杆菌在LB平板(链霉抗性和氨苄抗性)上划线活化,挑取单菌落,接入5 mL LB培养基中,37℃,200rpm培养7-10 h。
取一组500ml三角瓶,分别装入50ml含有不同浓度葡萄糖(0-15g/L)的发酵培养基中,再分别将重组大肠杆菌制得的种子液按4%的体积比的接种量接种到发酵培养基中,37℃下培养至OD600=2时加入IPTG至终浓度为1 mmol/L,加入IPTG 后在33℃培养66h至发酵结束,每隔12 h取一次样,测定OD600和1,2,4-丁三醇产量。其见图1与图2所示,由图可见,适当添加葡萄糖(即碳源)有利于菌体的生长和丁三醇产量的提高。但一次添加过多葡萄糖不仅不利于菌株生长,且不生成丁三醇。
其中,发酵培养基的配方如下:5g/L酵母粉,10g/L蛋白胨,10g/L氯化钠,0-15 g/L葡萄糖,10g/L木糖。
实施例3:重组大肠杆菌利用纤维素作为碳源发酵生产1,2,4-丁三醇
将含有重组质粒pTRC99a-xylb-kdcA和重组质粒PCWJ-yjhG-adhp的重组大肠杆菌在LB平板(链霉抗性和氨苄抗性)上划线活化,挑取单菌落,接入5 mL LB培养基中,37℃,200 rpm培养7-10 h。
取500ml三角瓶装入50ml的缓冲液,再将重组大肠杆菌制得的种子液按4%的体积比的接种量接种到缓冲液中,所述缓冲液为含有纤维素、纤维素酶、酵母粉、蛋白胨、氯化钠、木糖的柠檬酸钠缓冲液,37℃下培养至OD600=2时加入IPTG至终浓度为1 mmol/L,加入IPTG 后在33℃培养66h至发酵结束,每隔12 h取一次样,测定1,2,4-丁三醇产量及葡萄糖浓度。发酵66h后测得1,2,4-丁三醇浓度为3.52g/L,木糖至1,2,4-丁三醇的转化率为0.352g/g,直接以葡萄糖为碳源,10g/L木糖可转化为2.9g/L 1,2,4-丁三醇,木糖至1,2,4-丁三醇的转化率为0.29g/g,产量比使用葡萄糖为碳源的实施例提高21.37%,见图3。发酵过程前期培养基中葡萄糖含量很低,后期葡萄糖开始积累,证明纤维素连续水解葡萄糖且水解出的葡萄糖被有效利用。
其中,缓冲液的配方如下:5g/L酵母粉,10g/L蛋白胨,10g/L氯化钠,10g/L纤维素,25FPU纤维素酶,10g/L木糖,0.5mol/L柠檬酸钠缓冲液;其中,0.5 mol/L柠檬酸钠缓冲液配制方法为分别配制0.5 mol/L柠檬酸溶液和0.5 mol/L柠檬酸钠溶液,再用0.5 mol/L柠檬酸溶液加入0.5 mol/L柠檬酸钠溶液调节pH至6,即可。
基于纤维素发酵产丁三醇可以在发酵过程中连续提供碳源,操作简便,且纤维素大量存在于各类农业废弃物中,对利用可再生资源展现出巨大潜力。
序列表
<110> 南京工业大学
<120> 重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 744
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtcttctg ctatctaccc gtctctgaaa ggtaaacgtg ttgttatcac cggtggtggt 60
tctggtatcg gtgctggtct gaccgctggt ttcgctcgtc agggtgctga agttatcttc 120
ctggacatcg ctgacgaaga ctctcgtgct ctggaagctg aactggctgg ttctccgatc 180
ccgccggttt acaaacgttg cgacctgatg aacctggaag ctatcaaagc tgttttcgct 240
gaaatcggtg acgttgacgt tctggttaac aacgctggta acgacgaccg tcacaaactg 300
gctgacgtta ccggtgctta ctgggacgaa cgtatcaacg ttaacctgcg tcacatgctg 360
ttctgcaccc aggctgttgc tccgggtatg aaaaaacgtg gtggtggtgc tgttatcaac 420
ttcggttcta tctcttggca cctgggtctg gaagacctgg ttctgtacga aaccgctaaa 480
gctggtatcg aaggtatgac ccgtgctctg gctcgtgaac tgggtccgga cgacatccgt 540
gttacctgcg ttgttccggg taacgttaaa accaaacgtc aggaaaaatg gtacaccccg 600
gaaggtgaag ctcagatcgt tgctgctcag tgcctgaaag gtcgtatcgt tccggaaaac 660
gttgctgctc tggttctgtt cctggcttct gacgacgctt ctctgtgcac cggtcacgaa 720
tactggatcg acgctggttg gcgt 744
<210> 2
<211> 1644
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgtatacag taggagatta cctgttagac cgattacacg agttgggaat tgaagaaatt 60
tttggagttc ctggtgacta taacttacaa tttttagatc aaattatttc acgcgaagat 120
atgaaatgga ttggaaatgc taatgaatta aatgcttctt atatggctga tggttatgct 180
cgtactaaaa aagctgccgc atttctcacc acatttggag tcggcgaatt gagtgcgatc 240
aatggactgg caggaagtta tgccgaaaat ttaccagtag tagaaattgt tggttcacca 300
acttcaaaag tacaaaatga cggaaaattt gtccatcata cactagcaga tggtgatttt 360
aaacacttta tgaagatgca tgaacctgtt acagcagcgc ggactttact gacagcagaa 420
aatgccacat atgaaattga ccgagtactt tctcaattac taaaagaaag aaaaccagtc 480
tatattaact taccagtcga tgttgctgca gcaaaagcag agaagcctgc attatcttta 540
gaaaaagaaa gctctacaac aaatacaact gaacaagtga ttttgagtaa gattgaagaa 600
agtttgaaaa atgcccaaaa accagtagtg attgcaggac acgaagtaat tagttttggt 660
ttagaaaaaa cggtaactca gtttgtttca gaaacaaaac taccgattac gacactaaat 720
tttggtaaaa gtgctgttga tgaatctttg ccctcatttt taggaatata taacgggaaa 780
ctttcagaaa tcagtcttaa aaattttgtg gagtccgcag actttatcct aatgcttgga 840
gtgaagctta cggaccgctc aacaggtgca ttcacacatc atttagatga aaataaaatg 900
atttcactaa acatagatga aggaataatt ttcaataaag tggtagaaga ttttgatttt 960
agagcagtgg tttcttcttt atcagaatta aaaggaatag aatatgaagg acaatatatt 1020
gataagcaat atgaagaatt tattccatca agtgctccct tatcacaaga ccgtctatgg 1080
caggcagttg aaagtttgac tcaaagcaat gaaacaatcg ttgctgaaca aggaacctca 1140
ttttttggag cttcaacaat tttcttaaaa tcaaatagtc gttttattgg acaaccttta 1200
tggggttcta ttggatatac ttttccagcg gctttaggaa gccaaattgc ggataaagag 1260
agcagacacc ttttatttat tggtgatggt tcacttcaac ttaccgtaca agaattagga 1320
ctatcaatca gagaaaaact caatccaatt tgttttatca taaataatga tggttataca 1380
gttgaaagag aaatccacgg acctactcaa agttataacg acattccaat gtggaattac 1440
tcgaaattac cagaaacatt tggagcaaca gaagatcgtg tagtatcaaa aattgttaga 1500
acagagaatg aatttgtgtc tgtcatgaaa gaagcccaag cagatgtcaa tagaatgtat 1560
tggatagaac tagttttgga aaaagaagat gcgccaaaat tactgaaaaa aatgggtaaa 1620
ttatttgctg agcaaaataa ataa 1644
<210> 3
<211> 1968
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgtctgttc gcaatatttt tgctgacgag agccacgata tttacaccgt cagaacgcac 60
gccgatggcc cggacggcga actcccatta accgcagaga tgcttatcaa ccgcccgagc 120
ggggatctgt tcggtatgac catgaatgcc ggaatgggtt ggtctccgga cgagctggat 180
cgggacggta ttttactgct cagtacactc ggtggcttac gcggcgcaga cggtaaaccc 240
gtggcgctgg cgttgcacca ggggcattac gaactggaca tccagatgaa agcggcggcc 300
gaggttatta aagccaacca tgccctgccc tatgccgtgt acgtctccga tccttgtgac 360
gggcgtactc agggtacaac ggggatgttt gattcgctac cataccgaaa tgacgcatcg 420
atggtaatgc gccgccttat tcgctctctg cccgacgcga aagcagttat tggtgtggcg 480
agttgcgata aggggcttcc ggccaccatg atggcactcg ccgcgcagca caacatcgca 540
accgtgctgg tccccggcgg cgcgacgctg cccgcaaagg atggagaaga caacggcaag 600
gtgcaaacca ttggcgcacg cttcgccaat ggcgaattat ctctacagga cgcacgccgt 660
gcgggctgta aagcctgtgc ctcttccggc ggcggctgtc aatttttggg cactgccggg 720
acatctcagg tggtggccga aggattggga ctggcaatcc cacattcagc cctggcccct 780
tccggtgagc ctgtgtggcg ggagatcgcc agagcttccg cgcgagctgc gctgaacctg 840
agtcaaaaag gcatcaccac ccgggaaatt ctcaccgata aagcgataga gaatgcgatg 900
acggtccatg ccgcgttcgg tggttcaaca aacctgctgt tacacatccc ggcaattgct 960
caccaggcag gttgccatat cccgaccgtt gatgactgga tccgcatcaa caagcgcgtg 1020
ccccgactgg tgagcgtact gcctaatggc ccggtttatc atccaacggt caatgccttt 1080
atggcaggtg gtgtgccgga agtcatgttg catctgcgca gcctcggatt gttgcatgaa 1140
gacgttatga cggttaccgg cagcacgctg aaagaaaacc tcgactggtg ggagcactcc 1200
gaacggcgtc agcggttcaa gcaactcctg ctcgatcagg aacaaatcaa cgctgacgaa 1260
gtgatcatgt ctccgcagca agcaaaagcg cgcggattaa cctcaactat caccttcccg 1320
gtgggcaata ttgcgccaga aggttcggtg atcaaatcca ccgccattga cccctcgatg 1380
attgatgagc aaggtatcta ttaccataaa ggtgtggcga aggtttatct gtccgagaaa 1440
agtgcgattt acgatatcaa acatgacaag atcaaggcgg gcgatattct ggtcattatt 1500
ggcgttggac cttcaggtac agggatggaa gaaacctacc aggttaccag tgccctgaag 1560
catctgtcat acggtaagca tgtttcgtta atcaccgatg cacgtttctc gggcgtttct 1620
actggcgcgt gcatcggcca tgtggggcca gaagcgctgg ccggaggccc catcggtaaa 1680
ttacgcaccg gggatttaat tgaaattaaa attgattgtc gcgagcttca cggcgaagtc 1740
aatttcctcg gaacccgtag cgatgaacaa ttaccttcac aggaggaggc aactgcaata 1800
ttaaatgcca gacccagcca tcaggattta cttcccgatc ctgaattgcc agatgatacc 1860
cggctatggg caatgcttca ggccgtgagt ggtgggacat ggaccggttg tatttatgat 1920
gtaaacaaaa ttggcgcggc tttgcgcgat tttatgaata aaaactga 1968
<210> 4
<211> 1011
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgaaggctg cagttgttac gaaggatcat catgttgacg ttacgtataa aacactgcgc 60
tcactgaaac atggcgaagc cctgctgaaa atggagtgtt gtggtgtatg tcataccgat 120
cttcatgtta agaatggcga ttttggtgac aaaaccggcg taattctggg ccatgaaggc 180
atcggtgtgg tggcagaagt gggtccaggt gtcacctcat taaaaccagg cgatcgtgcc 240
agcgtggcgt ggttctacga aggatgcggt cattgcgaat actgtaacag tggtaacgaa 300
acgctctgcc gttcagttaa aaatgccgga tacagcgttg atggcgggat ggcggaagag 360
tgcatcgtgg tcgccgatta cgcggtaaaa gtgccagatg gtctggactc ggcggcggcc 420
agcagcatta cctgtgcggg agtcaccacc tacaaagccg ttaagctgtc aaaaattcgt 480
ccagggcagt ggattgctat ctacggtctt ggcggtctgg gtaacctcgc cctgcaatac 540
gcgaagaatg tctttaacgc caaagtgatc gccattgatg tcaatgatga gcagttaaaa 600
ctggcaaccg aaatgggcgc agatttagcg attaactcac acaccgaaga cgccgccaaa 660
attgtgcagg agaaaactgg tggcgctcac gctgcggtgg taacagcggt agctaaagct 720
gcgtttaact cggcagttga tgctgtccgt gcaggcggtc gtgttgtggc tgtcggtcta 780
ccgccggagt ctatgagcct ggatatccca cgtcttgtgc tggatggtat tgaagtggtc 840
ggttcgctgg tcggcacgcg ccaggattta actgaagcct tccagtttgc cgccgaaggt 900
aaagtggtgc cgaaagtcgc cctgcgtccg ttagcggaca tcaacaccat ctttactgag 960
atggaagaag gcaaaatccg tggccgcatg gtgattgatt tccgtcacta a 1011
Claims (7)
1.重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,其特征在于,构建表达木糖脱氢酶xylb、木糖酸脱水酶kdcA、酮酸脱羧酶yjhG和醛还原酶adhp的重组大肠杆菌,制备重组大肠杆菌的种子液,并接种至含有纤维素、纤维素酶、酵母粉、蛋白胨、氯化钠、木糖的柠檬酸钠缓冲液中发酵生产1,2,4-丁三醇。
2.根据权利要求1所述的重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,其特征在于,所述木糖脱氢酶基因xylb的核苷酸序列如SEQ ID NO.1所示,木糖酸脱水酶kdcA的核苷酸序列如SEQ ID NO.2所示,酮酸脱羧酶基因yjhG的核苷酸序列如SEQ ID NO.3所示,醛还原酶adhp基因的核苷酸序列如SEQ ID NO.4所示。
3.根据权利要求1所述的重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,其特征在于,将重组大肠杆菌制得的种子液按4%的体积比的接种量接种。
4.根据权利要求1所述的重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,其特征在于,所述发酵为37℃下培养至OD600=2时加入IPTG至终浓度为1 mmol/L,加入IPTG后在33℃培养66h至发酵结束。
5.根据权利要求1所述的重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,其特征在于,重组大肠杆菌的构建方法包括如下步骤:
(1)构建重组质粒pTRC99a-xylb-kdcA和重组质粒PCWJ-yjhG-adhp;
(2)再将重组质粒转化宿主菌,所述的宿主菌为大肠Trans 1 T1,得到产D-1,2,4丁三醇的重组大肠杆菌。
6.根据权利要求1所述的重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,其特征在于,所述柠檬酸钠缓冲液中各组分的含量为:5g/L酵母粉,10g/L蛋白胨,10g/L纤维素,15FPU纤维素酶、10g/L氯化钠、10g/L木糖;溶剂为0.5 mol/L柠檬酸钠缓冲液。
7.根据权利要求6所述的重组大肠杆菌基于纤维素一步发酵产1,2,4-丁三醇的方法,其特征在于,0.5 mol/L柠檬酸钠缓冲液配制方法为分别配制0.5 mol/L柠檬酸溶液和0.5mol/L柠檬酸钠溶液,再用0.5 mol/L柠檬酸溶液加入0.5 mol/L柠檬酸钠溶液调节pH至6,即可。
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CN111172143A (zh) * | 2020-01-10 | 2020-05-19 | 南京工业大学 | D-木糖酸脱水酶及其应用 |
CN111593014A (zh) * | 2020-06-24 | 2020-08-28 | 江南大学 | 联产1,3-丙二醇和d-1,2,4-丁三醇的方法 |
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CN111172143A (zh) * | 2020-01-10 | 2020-05-19 | 南京工业大学 | D-木糖酸脱水酶及其应用 |
CN111593014A (zh) * | 2020-06-24 | 2020-08-28 | 江南大学 | 联产1,3-丙二醇和d-1,2,4-丁三醇的方法 |
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