CN106148373A - 一种基因工程菌及其在生产1,5‑戊二胺中的用途 - Google Patents
一种基因工程菌及其在生产1,5‑戊二胺中的用途 Download PDFInfo
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- CN106148373A CN106148373A CN201610365537.7A CN201610365537A CN106148373A CN 106148373 A CN106148373 A CN 106148373A CN 201610365537 A CN201610365537 A CN 201610365537A CN 106148373 A CN106148373 A CN 106148373A
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- pentanediamine
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Abstract
本发明涉及微生物技术领域，具体涉及一种增加大肠杆菌胞内磷酸吡哆醛（PLP）合成的基因工程菌及应用工程菌生产1,5‑戊二胺的方法。本申请利用基因工程技术，在大肠杆菌中异源表达经核糖‑5‑磷酸途径合成磷酸吡哆醛（PLP）的关键基因，从而构建一株1,5‑戊二胺高效转化生产菌株。所述基因工程菌可在不外源添加PLP条件下，4 h内转化生产1,5‑戊二胺含量达到250 g/l，远优于现有技术报道。
Description
技术领域
本发明涉及生物催化技术领域，特别涉及一种增加大肠杆菌胞内磷酸吡哆醛（PLP）合成的基因工程菌及其在生产1,5-戊二胺中的用途。
背景技术
1,5-戊二胺（简称戊二胺），即尸胺，是生物体内广泛存在的具有生物活性的含氮碱，为蛋白质腐败时赖氨酸在脱羧酶作用下发生脱羧反应时生成的产物。在农业上, 1,5-戊二胺可用于调控植物衰老过程、促进雌雄蕊的发育,改善植物果实发育,提高果实产量；医学上，亦可以作为一种有效治疗痢疾的药物成份；工业上是一种极为重要的化工原料，与己二酸、琥珀酸和癸二酸等二元酸聚合可分别形成新型材料聚酰胺5.6、聚酰胺5.4和聚酰胺5.10。
目前合成戊二胺的方法有化学合成法、酶转化法、微生物发酵生产法等。化学合成法条件苛刻、污染环境；酶转化法过程复杂、成本较高；微生物直接发酵生产法虽相对生产原料成本较低，但直接发酵生产的戊二胺含量较低，且发酵液成分复杂，副产物多，产物的分离纯化比较困难。
全细胞催化是指利用完整的生物有机体作为催化剂进行化学转化，其本质是利用细胞内的酶进行催化，是介于发酵法和提取酶催化法之间的一种生物催化技术。相比发酵法，全细胞催化克服了发酵法生产周期长、代谢产物复杂、底物转化率低、产物分离提取困难及能耗高等缺点。相比纯酶催化反应，全细胞中各酶系维持原有状态和特定位置，酶稳定性更好，半衰期更长，适应性更强，更易实现能量和辅酶的原位再生；细胞内完整的多酶体系可以实现酶的级联反应，催化效率高。全细胞催化的另一特点是不需考虑催化产物对细胞的毒害作用，非常适合类似1,5-戊二胺等对细胞有毒性的物质的生产。
专利CN 103725724 A中利用固定化的蜂房哈夫尼菌（Hafnia alvei ）AS1.1009，通过流加200 g/l的L-赖氨酸母液，转化生产得到1,5-戊二胺60.54 g/l。专利US 7189543中将E.coli W3110(ATCC 39936)的赖氨酸脱羧酶基因（cadA）克隆到质粒pUC18上，而后转化到E.coli JM109中构建了一株基因工程菌株，并利用该菌株采用全细胞催化技术生产1,5-戊二胺，其在反应液中浓度达到69 g/l，是目前报道最高产量。
已知两种存在于大肠杆菌中赖氨酸脱羧酶(CadA和LdcC)，都需要磷酸吡哆醛（PLP）作为辅酶。经实验验证（详见实施例2），在全细胞转化生产戊二胺过程中加入赖氨酸脱羧酶的辅酶磷酸吡哆醛，对维持生产菌株的1,5-戊二胺转化能力有显著作用。同时也表明，菌株胞内自身合成的PLP无法满足赖氨酸脱羧酶高效转化L-赖氨酸生成1,5-戊二胺的需求。其部分原因是大肠杆菌合成PLP主要依赖1-脱氧-5-磷酸木酮糖途径，该途径需7种酶参与，合成效率较低；相比较，生物体中存在另一种依赖核糖-5-磷酸的PLP合成途径，该途径只需两个酶参与即可利用细胞中较充足存在的核糖-5-磷酸、甘油醛-3-磷酸和谷氨酰胺一步合成PLP，且该途径广泛分布于包括真菌、植物及部分原核生物细胞中，参与PLP合成的酶为YaaD（又称PdxS、Pdx1或SnzP）和 YaaE （又称PdxT、 Pdx2, 或SnoP）。此外，菌体胞内的PLP含量是受严格调控的，其含量过高或过低均会对菌体造成伤害。（见Pease A J, Roa BR, Luo W, Winkler M E. Positive Growth Rate-Dependent Regulation of the pdxA,ksgA, and pdxB Genes of Escherichia coli K-12 [J]. Journal of bacteriology,2002, 184(5): 1359-69.）因此，要增加菌体胞内PLP的含量，不仅要改造菌体胞内PLP的合成途径，还需要优化菌体培养方法，从而实现在不影响菌体生长的前提下，提高菌体积累PLP的能力，进而构建出高效的1,5-戊二胺生产菌株。
发明内容
本发明所要解决的技术问题是提供一种增加大肠杆菌胞内磷酸吡哆醛（PLP）合成的基因工程菌及该菌在生产1,5-戊二胺中的用途。
为解决的上述技术问题，本发明公开了一种重组表达载体，其含有磷酸吡哆醛（PLP）合成基因。
本发明所述核糖-5-磷酸途径依赖的磷酸吡哆醛（PLP）合成基因来源没有特别限制，但是优选使用来源于（例如）芽孢杆菌属（Bacillus spp.）等原核生物。
进一步的，所述的表达yaaD的氨基酸序列为SEQ ID NO：2所示的氨基酸序列或其突变体。
进一步的，所述的表达yaaE氨基酸序列为SEQ ID NO：3所示的氨基酸序列或其突变体。
另一方面，本发明还公开了一种基因工程菌，其含有上述所述的重组表达载体。
进一步的，所述的基因工程菌，还包括质粒pETDuet-pelB-CadB-CadA。
本发明中所述产戊二胺的基因工程菌所使用的载体系列包括：pETDuet系列质粒、pACYCDuet系列、pRSFDuet系列、pCDFDuet系列、pET系列、pGEX系列、pMAL系列、、pTXB1系列、pTYB系列。
本发明中所述产戊二胺的基因工程菌，其特征在于所述的能高效表达外源基因的宿主菌为下列之一：BL21系列、Rosetta系列、Origami系列、Tuner系列。
另一方面，本申请公开了所述基因工程菌在生产1,5-戊二胺中的用途。
优选的，基因工程菌的培养基以甘油与乳糖作为碳源，二者比例为1:1~7:3，总糖量为2%；以氯化铵或硫酸铵为氮源，培养基C/N比为3:2~3:4。
另外，
本发明中，种子培养基为用于培养大肠杆菌的常规培养基，如LB培养基。
本发明中，发酵培养基，可以使用含有可同化的碳源、氮源及无机盐类等的营养培养基。作为碳源，可以使用例如葡萄糖、甘油、果糖、蔗糖、麦芽糖、淀粉水解产物等，优选甘油。作为氮源，可以使用氨、氯化铵、硫酸铵、碳酸铵、醋酸铵等各种无机和有机铵盐类，尿素，其它含氮化合物，以及肉类提取物、酵母提取物、玉米浆、大豆水解产物等含氮有机物，优选氯化铵和硫酸铵。作为无机盐，可以使用磷酸二氢钾、磷酸氢二钾、硫酸镁、碳酸钙、氯化钠、硫酸铵、钼酸铵等。此外，根据需要，可以添加生物素、硫胺素、维生素B6等微量营养源。
所述的底物L-赖氨酸优选为L-赖氨酸盐酸盐，也可以为其他赖氨酸盐，例如赖氨酸·己二酸盐、赖氨酸·癸二酸盐、赖氨酸·琥珀酸酸盐等。
对于转化过程pH的调节，优选使用盐酸或二羧酸。pH调节到5~8，优选控制在pH5.6。
本发明中，表达yaaD和yaaE的核酸序列来源广泛，真菌、植物及部分原核生物细胞中的YaaD（又称PdxS、Pdx1或SnzP）和 YaaE （又称PdxT、 Pdx2, 或SnoP）核酸序列序列均可。
本发明所述的产戊二胺基因工程菌的构建、表达及应用的效果具体体现在：
提供了一种有效提高胞内PLP含量的基因构建策略，实现胞内PLP的高效合成，并保障菌体的正常生长。
（1）提供了一种利用胞内PLP合成积累的培养策略，并实现与L-赖氨酸脱羧酶及赖氨酸-尸胺反向转运蛋白协调表达策略，从而实现在不另外添加PLP的情况下，高效转化生产戊二胺。
（2）所得基因工程菌菌体活性高，实现胞内PLP含量达到1144 nmol/gDCW，单位菌体的1,5-戊二胺生产强度达到25 g/gDCW/h，1,5-戊二胺含量达到210 g/l，远优于现有技术报道。
附图说明
图1为质粒pRSF-yaaDE构建示意图。
图2为磷酸吡哆醛（PLP）的添加对基因工程菌转化生产1,5-戊二胺的影响。
图3为基因工程菌胞内磷酸吡哆醛（PLP）含量及1,5-戊二胺产率。
图4为基因工程菌培养条件优化。
图5、图6、图7为补料分批全细胞转化生产1,5-戊二胺。
图8为基因工程菌全细胞转化产物MS分析结果（单丹酰尸胺）。
图9为基因工程菌全细胞转化产物MS分析结果（双丹酰尸胺）。
具体实施方式
以下结合具体实施例，进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1 产1,5-戊二胺菌株的构建
根据枯草芽孢杆菌Bacillus subtilis (GenBank: AL009126.3)
的磷酸吡哆醛合成酶基因序列设计引物：
YaaDE-NcoI-F：CATGccATGGCTCAAACAGGTACTGAACG；
YaaDE-SalI-R:ACGCGTCGACTTATACAAGTGCCTTTTGCTTATATTCCTCAACC。
PCR得到的yaaDE基因克隆至质粒pRSF质粒上，构建得到质粒pRSF-yaaDE (图1)。yaaDE核苷酸序列如SEQ ID NO：1，将质粒pETDuet-pelB-CadB-CadA（见本发明人的公开专利申请文献：发明名称为一种表达重组载体及其应用，申请公布号CN104498519A）和pRSF-yaaDE用常规方法均导入大肠杆菌BL21 (DE3)，菌株命名为BL-BADE，并以甘油菌或冻干菌种形式保存。
实施例2 外源添加磷酸吡哆醛（PLP）对1,5-戊二胺合成的影响
挑取BL- DAB（见本发明人的公开专利申请文献：发明名称为一种表达重组载体及其应用，申请公布号CN104498519A）单菌落于5ml含100 ug/ml 氨苄青霉素的LB液体培养基中，37℃、200 rpm过夜活化菌种。活化的菌种按1%接种量转接到50 ml 含相应抗生素（100 ug/ml 氨苄青霉素）的液体发酵培养基中， 37℃、200 rpm 培养至OD600约为0.5。加入IPTG至终浓度0.5 mM，30 ℃，200 rpm诱导培养12h后4000 x g 离心收集菌体细胞，用0.9%的氯化钠溶液洗涤两遍后用转化反应液重悬到细胞浓度为4 g(DCW)/l，转化反应液含L-赖氨酸盐酸盐至终浓度125 g/l （相当于L-赖氨酸浓度为100 g/l）。重悬菌液分为两组，一组添加终浓度为0.1 mM PLP；另一组为对照组（即不添加PLP组）。37 ℃，250 rpm转化，每隔三小时取样检测上清中L-赖氨酸及1,5-戊二胺含量。
L-赖氨酸含量使用SBA-40E型生物传感仪进行检测。1,5-戊二胺含量用丹磺酰氯衍生化后，反相高效液相色谱检测。衍生步骤如下：移取100 ul离心后的转化液到5 ml离心管中，依次加入2 M NaOH溶液200 μl, 饱和NaHCO3溶液300 μl ，10 g/1的1,7-二氨基庚烷溶液（内标）100 μl及10 g/l 的丹磺酰氯溶液1 ml。混匀后置于40 ℃水浴中避光反应45min，而后加入25%的氨水100 μl，混匀后室温避光静置30 min。反应结束后，用乙腈定容至5ml。HPLC分析使用agilent 1290 infinity system，色谱柱为Prevail C18反向柱(250 mm× 4.6 mm × 5 μm)。HPLC条件为：流动相A：100% 乙腈，流动相B：0.1 M 乙酸铵溶液，采用梯度洗脱，条件如下：初始：50% A；19 min：90% A；20-30 min：50% A；流速：1.0 ml/min；柱温：40±1 °C；进样量：10 μl。检测使用荧光检测器 (FLD G1321B)，激发波长：320 nm；发射波长：523 nm。
结果如图2所示，不添加PLP条件下，单位质量菌体的1,5-戊二胺产率从3 h的3.91g/gDCW/h下降到9 h的2.30 g/gDCW/h，降低了41%。而当添加0.1 mM PLP时，单位质量菌体的1,5-戊二胺产率仅下降5%。结果表明，外源PLP的添加对维持菌体活力有显著效果。
实施例3 基因工程菌转化生产1,5-戊二胺
挑取单菌落BL-BADE于5ml含100 ug/ml 氨苄青霉素的LB液体培养基中，37℃、200 rpm过夜活化菌种。活化的菌种按1%接种量转接到50 ml 含相应抗生素（100 ug/ml 氨苄青霉素，170 μg/ml氯霉素）的液体发酵培养基中， 37℃、200 rpm 培养至OD600约为0.5。加入IPTG至终浓度0.5 mM，30 ℃，200 rpm诱导培养12h后4000 x g 离心收集菌体细胞，取10mg（干重）菌体细胞检测胞内PLP含量。剩余菌体用0.9%的氯化钠溶液洗涤两遍后用转化反应液重悬到细胞浓度为2 g(DCW)/l，转化反应液含L-赖氨酸盐酸盐至终浓度12.5 g/l （相当于L-赖氨酸浓度为10 g/l），37 ℃，250 rpm转化30分钟后，6000 x g离心后，检测上清中L-赖氨酸及1,5-戊二胺含量。
L-赖氨酸和1,5-戊二胺含量检测方法见实施例2。
胞内PLP含量检测采用反相高效液相色谱法，条件为：流动相A（pH 3.0）：0.1 M 磷酸二氢钾, 0.1 M 高氯酸钠和0.5 g/L 亚硫酸钠;流动相B（pH 3.0）：0.1 M 磷酸二氢钾,0.1 M 高氯酸钠，0.5 g/L 亚硫酸钠和20% 乙腈。 采用梯度洗脱，条件如下：0-4 min :100% A；4.1 to 13 min：27% B；流速：1.0 ml/min；柱温：25 ± 1°C；进样量：20 μl。检测使用荧光检测器 (FLD G1321B)，激发波长：300 nm；发射波长：400 nm。
结果如图3所示，经30分钟转化后，基因工程菌BL-BADE的胞内PLP含量达到533nmol/gDCW，1,5-戊二胺产率为0.21 g /gDCW /h, 分别是对照菌株BL-DAB的1.5倍和1.6倍；是初始菌株BL21 (DE3)的5.3倍和4.7倍。
实施例4 基因工程菌培养条件优化
挑取BL-BADE单菌落于5ml含100 ug/ml 氨苄青霉素和170 μg/ml氯霉素的LB液体培养基中，37℃、200 rpm过夜活化菌种。活化的菌种按1%接种量转接到50 ml 含100 ug/ml 氨苄青霉素和170 ug/ml氯霉素的液体发酵培养基中， 37℃、200 rpm 培养12 h后转接优化培养基中。优化培养基以甘油与乳糖作为碳源，二者比例为1:1~7:3，总糖量为2%；以氯化铵为氮源，培养基C/N比为3:2~3:4；对比试验以LB培养基使用IPTG诱导作为对照。37 ℃，200rpm诱导培养6 h后4,000 × g离心10 min收集菌体并进行全细胞转化试验及检测胞内PLP含量。
全细胞转化条件同实施例3。转化液中L-赖氨酸、1,5-戊二胺和胞内PLP含量检测方法见实施例2和实施例3。
结果如图4所示，利用优化培养基得到的菌株BL-BADE胞内PLP含量达到1051nmol/gDCW，菌株1,5-戊二胺产率达到0.7 g /gDCW/h，与LB培养基添加IPTG诱导相比，分别提高了1.97倍和3.5倍。且生物量没有显著差异。
实施例5 补料分批全细胞转化生产1,5-戊二胺
挑取单菌落于5ml含100 ug/ml 氨苄青霉素和170 μg/ml氯霉素的LB液体培养基中，37℃、200 rpm过夜活化菌种。活化的菌种按1%接种量转接到250 ml 含100 ug/ml 氨苄青霉素和170 μg/ml氯霉素的种子培养基中，37℃、200 rpm 培养8h。而后按10 %比例接种到4 L发酵培养基中，37℃，200 rpm, 1 vvm，培养12小时，离心收集菌体。种子培养基为LB培养基；发酵培养基成分为：1.2% (W/V) 蛋白胨、2.4% (W/V) 酵母提取物、0.4% (V/V) 甘油、17 mM KH2PO4、72 mM K2HPO4。
转化体系包括：菌体8 gDCW/l， L-赖氨酸量150 g/l。转化条件为37 ℃，200 rpm，转化4小时，其间补加5 M 盐酸维持pH稳定。转化过程中定期检测转化液中L-赖氨酸含量，当赖氨酸消耗至2.5 ~ 4 g /g DCW时，补加底物至150 g/l。
转化液中L-赖氨酸含量使用SBA-40E型生物传感仪进行检测。1,5-戊二胺含量用丹磺酰氯衍生化后，HPLC-MS分析。衍生化操作见实施例2。
HPLC-MS分析使用电喷雾离子质谱，色谱柱为Prevail C18反向柱(250 mm × 4.6mm × 5 μm)。HPLC条件为：流动相A：100% 乙腈，流动相B：0.1 M 乙酸铵溶液，采用梯度洗脱，条件如下：初始：50% A；19 min：90% A；20-30 min：50% A；流速：1.0 ml/min；柱温：40±1 °C；进样量：10 μl。检测使用紫外检测器，检测波长254 nm。
结果如图5所示，菌株BL-BADE在不添加PLP情况下的产率相当于(p=0.143, α=0.05) 添加0.1 mM PLP的菌株BL-DAB的产量，转化液中1,5-戊二胺含量达到210 g/l,最大1,5-戊二胺产率可达到 25 g /gDCW/h，是菌株BL-DAB在不添加PLP情况下的2.9倍；且菌株可维持该产率达1小时，第一小时1,5-戊二胺的平均产率为24 ± 1 g /gDCW/h，是菌株BL-DAB在添加0.1 mM PLP条件下的产率（20 ± 1 g /gDCW/h）的1.2倍（如图6所示）。经过4小时的转化，单位质量菌株BL-BADE可生产1,5-戊二胺76 g/L，略高于菌株BL-DAB在添加了0.1 mM PLP下的产率（71.9 g/L）（如图7所示）。
转化产物经HPLC-MS分析，单丹酰尸胺（图8）分子量为335（336-1=335），双丹酰尸胺（图9）分子量为568（569-1=568），分别于单丹酰尸胺和双丹酰尸胺的分子量吻合。
本实施例结果说明本发明所公开的1,5-戊二胺生产菌构建策略可有效协调赖氨酸的摄取、赖氨酸脱羧及戊二胺外排等过程，并可高效从头合成脱羧反应所需的辅酶磷酸吡哆醛（PLP），以满足菌体高效转化生产1,5-戊二胺的需求。所构建的菌种生产能力强，配合本发明公开的培养策略，可转化生产得到高浓度的1,5-戊二胺的溶液，远优于现有技术报道。
本发明的范围不受所述具体实施方案的限制，所述实施方案只作为阐明本发明各个方面的单个例子，本发明范围内还包括功能等同的方法和组分。实际上，除了本文所述的内容外，本领域技术人员参照上文的描述和附图可以容易地掌握对本发明的多种改进。所述改进也落入所附权利要求书的范围之内。上文提及的每篇参考文献皆全文列入本文作为参考。
序列表
<110> 南京工业大学
<120> 一种基因工程菌及其在生产1,5-戊二胺中的用途
<130> xb16052702
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<170> PatentIn version 3.3
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aaagcgcgta tcggacatat tgttgaagcg cgtgtgcttg aagctatggg tgttgactat 300
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tcagacaacc ctgctaaatt tgcgaaagca attgtggaag caacaactca ctttactgat 780
tacaaattaa tcgctgagtt gtcaaaagag cttggtactg caatgaaagg gattgaaatc 840
tcaaacttac ttccagaaca gcgtatgcaa gaacgcggct ggtaagaaca taggagcgct 900
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Claims (8)
1.一种重组表达载体，其特征在于，该表达载体含有磷酸吡哆醛（PLP）合成基因。
2.如权利要求1所属的重组表达载体，其特征在于，所述的磷酸吡哆醛（PLP）合成基因为表达yaaD的核酸序列及表达yaaE的核酸序列。
3.如权利要求2所述的重组表达载体，其特征在于，所述的表达yaaD的氨基酸序列为SEQ ID NO：2所示的氨基酸序列或其突变体。
4.如权利要求2所述的重组表达载体，其特征在于，所述的表达yaaE氨基酸序列为SEQID NO：3所示的氨基酸序列或其突变体。
5.一种基因工程菌，其特征在于所述宿主菌含有权利要求1-4所述重组表达载体。
6.权利要求5所述的基因工程菌，其特征在于还包括质粒pETDuet-pelB-CadB-CadA。
7.权利要求6所述基因工程菌在生产1,5-戊二胺中的用途。
8.如权利要求7所述的基因工程菌在生产1,5-戊二胺中的用途，其特征在于，所述基因工程菌的培养基以甘油与乳糖作为碳源，二者比例为1:1～7:3，总糖量为2%；以氯化铵或硫酸铵为氮源，培养基C/N比为3:2～3:4。
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| WO2024000368A1 (zh) * | 2022-06-30 | 2024-01-04 | 江南大学 | 一种重组大肠杆菌及其构建方法与合成1,5-戊二胺的方法 |
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| CN106967660A (zh) * | 2017-03-28 | 2017-07-21 | 浙江省环境保护科学设计研究院 | 一种生产复苏促进因子的基因工程菌及其应用 |
| CN108795835A (zh) * | 2018-06-21 | 2018-11-13 | 浙江大学 | 一种基因工程菌及其在制备l-草铵膦中的应用 |
| CN108795835B (zh) * | 2018-06-21 | 2020-08-04 | 浙江大学 | 一种基因工程菌及其在制备l-草铵膦中的应用 |
| CN109082448A (zh) * | 2018-08-20 | 2018-12-25 | 南京工业大学 | 一种大肠杆菌及其在发酵生产1,5-戊二胺中的应用 |
| CN111117940A (zh) * | 2019-12-04 | 2020-05-08 | 天津大学 | 一种高产戊二胺的大肠杆菌工程菌与方法 |
| CN111117940B (zh) * | 2019-12-04 | 2022-06-28 | 天津大学 | 一种高产戊二胺的大肠杆菌工程菌与方法 |
| CN113817762A (zh) * | 2021-09-23 | 2021-12-21 | 南京工业大学 | 一种产戊二胺的重组大肠杆菌及其应用 |
| CN113817762B (zh) * | 2021-09-23 | 2023-06-20 | 南京工业大学 | 一种产戊二胺的重组大肠杆菌及其应用 |
| WO2024000368A1 (zh) * | 2022-06-30 | 2024-01-04 | 江南大学 | 一种重组大肠杆菌及其构建方法与合成1,5-戊二胺的方法 |
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