CN111849782A - 一种藻菌共培养发酵体系及用其生产化学品的方法 - Google Patents
一种藻菌共培养发酵体系及用其生产化学品的方法 Download PDFInfo
- Publication number
- CN111849782A CN111849782A CN202010494336.3A CN202010494336A CN111849782A CN 111849782 A CN111849782 A CN 111849782A CN 202010494336 A CN202010494336 A CN 202010494336A CN 111849782 A CN111849782 A CN 111849782A
- Authority
- CN
- China
- Prior art keywords
- culture
- fermentation
- escherichia coli
- phycomycete
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
-
- 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
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- 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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- 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
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01088—Hydroxymethylglutaryl-CoA reductase (1.1.1.88)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01009—Acetyl-CoA C-acetyltransferase (2.3.1.9)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
- C12Y203/0301—Hydroxymethylglutaryl-CoA synthase (2.3.3.10)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01036—Mevalonate kinase (2.7.1.36)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
- C12Y207/04002—Phosphomevalonate kinase (2.7.4.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01033—Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
- C12Y402/03027—Isoprene synthase (4.2.3.27)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/03—Intramolecular oxidoreductases (5.3) transposing C=C bonds (5.3.3)
- C12Y503/03002—Isopentenyl-diphosphate DELTA-isomerase (5.3.3.2)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
Abstract
一种藻菌共培养发酵体系及用其生产化学品的方法。本发明属于发酵生产领域。本发明为解决现有分批或分批补料发酵生产工艺复杂,周期短,难以实现连续稳定生产的问题。该体系由重组菌与光合自养微生物经发酵培养基共培养而成;所述重组菌为基因工程大肠杆菌。方法:先将重组菌和光合自养微生物分别接种到LB和BG11培养基中,培养至对数生长中后期,取菌液离心,重悬于无菌水中;然后接种到光照反应器中的发酵培养基中进行培养,培养过程中于葡萄糖耗尽后进行补加,持续培养至发酵结束。本发明提供的方法合成周期由100h延长到400h,异戊二烯产量是纯培养的8倍。
Description
技术领域
本发明属于发酵生产领域;具体涉及一种藻菌共培养发酵体系及用其生产化学品的方法。
背景技术
为了实现化工行业的绿色可持续发展,许多燃料和平台化合物已通过发酵法工业化生产。随着生物技术的进一步发展,发酵产品将更多地替代石油基化学品。然而,目前微生物发酵大多采用分批或补料分批的发酵方式,工艺复杂,耗水量大,生产成本高,固定资产投资大。我国发酵工业每年约排放80亿吨废水,占工业总排放量的10%。因此,如果能够开发出连续稳定高效运行的生产方式,对发酵工业具有重要的意义。
光合自养和异养微生物的共培养体系能够在连续发酵模式的建立中起到关键作用。藻菌共培养的研究表明,生态位的互补性和功能的冗余性使共培养系统在面对生物或非生物干扰时具有较高的效率和稳定性。Hays等描述了模式蓝藻聚球藻PCC 7942可以与枯草芽孢杆菌、大肠杆菌或酿酒酵母在较长时间内保持稳定的共培养状态。通过共培养Scenedesmus sp.D202,好氧菌种BaciLLus sp.D320和Rhodobacter sphaeroides、固氮菌Methanobacteria sp.D422和SpiruLina sp.D11,实现了连续的共生生长,并维持了3.5年。Cong等人工构建了热带假丝酵母与Scenedesmus obLiquus共培养体系。与纯培养体系相比,Scenedesmus obLiquus的生物量和光合活性分别增加了30.3%和61%。假单胞菌相关的GM41菌株与集胞藻PCC6803共培养使得蓝藻生物量增加8倍。Zhang等研究表明与纯培养相比,产油酵母RhodotoruLa gLutinis与微藻ChLoreLLa vuLgaris共培养可使生物量增加17.3%,油脂产量增加70.9%。Shu等通过共培养小球藻和酿酒酵母来增加CO2固定和产油。因此,光合自养和异养微生物的共培养体系有助于高效、稳定的发酵。
大肠杆菌具有遗传背景清楚,基因操作方便,生长速度快等优势,是用于生物合成和发酵工业的重要菌株。虽然蓝藻、小球藻等光合微生物在自然环境中可以与异样的细菌、真菌共生,但目前还没有蓝藻、小球藻等与大肠杆菌的天然共生关系的报道。在人工共培养体系方面,Hays等描述了以分泌蔗糖的聚球藻PCC 7942为柔性平台,构建了与包括大肠杆菌等异养微生物的共培养体系。然而,这种新型的共培养体系是一种完全光驱动的系统,细胞密度低,产物合成效率低,这限制了其在发酵工业的应用。
化学品生产以异戊二烯为例说明,异戊二烯是合成橡胶的理想原料,95%的异戊二烯产品用于合成橡胶的生产。此外,异戊二烯还可用于生产高质量的航空燃料等。稳定可再生的异戊二烯资源对国家安全和经济发展至关重要。随着石化资源的日益枯竭和全球环境问题的日益加剧,生物异戊二烯将逐渐替代石油基异戊二烯。利用枯草芽孢杆菌、大肠杆菌、聚球藻和酿酒酵母等的纯培养进行异戊二烯的生物合成已取得了一定的进展。Genencor已经取得了突破,开发出了一种基于大肠杆菌的生产系统,能够生产60g/L的异戊二烯。然而,目前还没有关于共培养发酵生产异戊二烯的报道,因此,如何建立连续稳定生产的发酵体系是生物基化学品生产领域需要解决的关键问题之一。
发明内容
本发明为解决现有分批或分批补料发酵生产工艺复杂,周期短,难以实现连续稳定生产的问题,而提供了一种藻菌共培养发酵体系及用其生产化学品的方法。
本发明的一种藻菌共培养发酵体系由重组菌与光合自养微生物经发酵培养基共培养而成;所述重组菌为基因工程大肠杆菌。
进一步限定,所述基因工程大肠杆菌为游离态大肠杆菌或经包埋的固定化大肠杆菌。
进一步限定,所述经包埋的固定化大肠杆菌是经海藻酸钠或卡拉胶包埋的。
进一步限定,所述的基因工程大肠杆菌的宿主为BL21(DE3)、MG1655、JM109、JM109(DE3)、DH5α、K12、C41、C41(DE3)或XL1-bLue。
进一步限定,所述的光合自养微生物为聚球藻PCC 7942、聚球藻UTEX 2973、聚球藻PCC7002、集胞藻PCC6803、小球藻、螺旋藻、栅藻、黄丝藻、杜氏盐藻或新月藻。
进一步限定,所述发酵培养基由0.1g/L~2g/L的NaNO3、0.01g/L~0.5g/L的CaCL2、0.01g/L~2g/L的NaCO3、0.1mg/L~5mg/L的Na2EDTA·2H2O、0.1g/L~5g/L的葡萄糖、0.1g/L~2g/L的MgSO4、0.1g/L~6g/L的KH2PO4·3H2O、0.1g/L~2g/L的甜菜碱、0.1g/L~2g/L的硫酸铵、0.1g/L~2g/L的柠檬酸、0.05g/L~1g/L的柠檬酸铁铵、0.1mL/L~0.5mL/L的微量元素储液和抗生素组成。
进一步限定,所述微量元素储液的配置方法为:将0.37g的(NH4)6Mo7O24·4H2O、0.29g的ZnSO4·7H2O、2.47g的H3BO4、0.25g的CuSO4·5H2O和1.58g的MnCl2·4H2O用蒸馏水定容至100mL,然后过滤除菌,得到微量元素储液。
进一步限定,所述抗生素为0~100mg/L氨苄青霉素、0~50mg/L卡那霉素、0~34mg/L氯霉素、0~20mg/L庆大霉素、0~50mg/L壮观霉素、0~50mg/L链霉素和0~50mg/L四环素中的一种或几种的混合物。
本发明的利用一种藻菌共培养发酵体系生产化学品的方法按以下步骤进行:
一、将光合自养微生物接种到BG11培养基中,接种量为培养基体积的1%~20%,培养至对数生长中后期,取菌液离心收集光合自养微生物藻体,重悬于无菌水中至终浓度2g/L~10g/L;
二、将重组菌接种到LB培养基中,接种量为培养基体积的1%~20%,培养至对数生长中后期,取菌液离心收集菌体,重悬于无菌水中至终浓度2g/L~10g/L;
三、将步骤一的无菌液和步骤二的无菌液按比例混合,然后将混合液接种到光照反应器中的发酵培养基中,于温度为20~35℃,pH值为5~12,搅拌转速为50rpm~500rpm,通气比为0.5vvm~2vvm的条件下,采用不同的光照强度进行培养,培养至OD600为1~90左右,加入诱导剂IPTG至终浓度为50μM~500μM诱导表达,诱导后继续培养,培养过程中于葡萄糖耗尽后进行补加,保持残糖浓度在1g/L以下,持续培养至发酵结束。
进一步限定,步骤一中所述重组菌为基因工程大肠杆菌。
进一步限定,步骤三中所述步骤一的无菌液和步骤二的无菌液的质量比为1:(0.1~10)。
进一步限定,步骤三中所述混合液的接种量为发酵培养基体积的1%~20%。
进一步限定,步骤三中所述光照强度为30μmoL m-2s-1~1200μmoLm-2s-1。
本发明与现有技术相比具有的显著效果如下:
1)本发明提供的采用藻菌共培养生产异戊二烯的方法,解决了常规的发酵生产工艺复杂,周期短,难以实现连续稳定生产的问题,合成周期由100h延长到400h,异戊二烯产量是纯培养的8倍。
2)光合自养和异样微生物的共培养体系能够在连续发酵模式中起到关键作用,解决现有发酵模式的瓶颈问题。
3)大肠杆菌遗传背景清楚,基因操作方便,生长速度快,能够以葡萄糖为碳源,在无机盐培养基中实现高密度发酵,本发明建立的藻类与大肠杆菌的共培养高密度发酵体系,效率更高。
附图说明
图1为具体实施方式二和三所述共培养过程中生物量曲线图;
图2为具体实施方式二和三所述共培养过程中异戊二烯产量曲线图;
图3为具体实施方式二和三所述共培养过程中异戊二烯产率曲线图。
具体实施方式
具体实施方式一:本实施方式的一种藻菌共培养发酵体系由重组菌与光合自养微生物经发酵培养基共培养而成;所述重组菌为基因工程大肠杆菌,所述基因工程大肠杆菌为携带异戊二烯合成途径质粒的重组菌,所述基因工程大肠杆菌的宿主为大肠杆菌BL21(DE3),所述基因工程大肠杆菌为游离态大肠杆菌;
所述重组菌携带异戊二烯合成途径的质粒(见Yang,pLosone,2012),具体表达方式为将乙酰辅酶A乙酰基转移酶/HMG辅酶A还原酶基因mvaE、HMG辅酶A合成酶基因mvaS和异戊二烯合成酶基因ispS连接到质粒载体pACYCDuet-1上,将磷酸甲羟戊酸激酶基因erg8、甲羟戊酸激酶基因erg12、甲羟戊酸焦磷酸脱羧酶基因erg19和IPP异构酶基因idi1连接到载体pTrchis2B;
所述的光合自养微生物为聚球藻PCC 7942;
所述发酵培养基由0.75g/L的NaNO3、0.014g/L的CaCL2、0.01g/L的NaCO3、0.5mg/L的Na2EDTA·2H2O、5g/L的葡萄糖、0.21g/L的MgSO4、4.9g/L的KH2PO4·3H2O、0.1g/L的甜菜碱、1g/L的硫酸铵、1g/L的柠檬酸、0.15g/L的柠檬酸铁铵、0.2mL/L的微量元素储液和3.4μg/mL氯霉素,10μg/mL氨苄青霉素组成。
所述微量元素储液的配置方法为:将0.37g的(NH4)6Mo7O24·4H2O、0.29g的ZnSO4·7H2O、2.47g的H3BO4、0.25g的CuSO4·5H2O和1.58g的MnCl2·4H2O用蒸馏水定容至100mL,然后过滤除菌,得到微量元素储液。
具体实施方式二:用具体实施方式一所述的一种藻菌共培养发酵体系生产异戊二烯的方法按以下步骤进行:
一、将聚球藻PCC 7942接种到BG11培养基中,接种量为培养基体积的10%,28℃,200rpm摇床培养至对数生长中后期,取菌液离心收集光合自养微生物藻体,重悬于无菌水中至终浓度2g/L;
二、将重组菌接种到LB培养基中,接种量为培养基体积的1%,37℃,200rpm摇床培养至对数生长中后期,取菌液离心收集菌体,重悬于无菌水中至终浓度2g/L;
所述重组菌为基因工程大肠杆菌,所述基因工程大肠杆菌为携带异戊二烯合成途径质粒的重组菌,所述基因工程大肠杆菌的宿主为大肠杆菌BL21(DE3),所述基因工程大肠杆菌为游离态大肠杆菌;所述重组菌携带异戊二烯合成途径的质粒(见Yang,pLosone,2012),具体表达方式为将乙酰辅酶A乙酰基转移酶/HMG辅酶A还原酶基因mvaE、HMG辅酶A合成酶基因mvaS和异戊二烯合成酶基因ispS连接到质粒载体pACYCDuet-1上,将磷酸甲羟戊酸激酶基因erg8、甲羟戊酸激酶基因erg12、甲羟戊酸焦磷酸脱羧酶基因erg19和IPP异构酶基因idi1连接到载体pTrchis2B;
三、将步骤一的无菌液和步骤二的无菌液按比例1:1混合,然后将混合液接种到光照反应器中的发酵培养基中,发酵培养基体积为150mL,接种量为发酵培养基体积的8%,于温度为30℃,pH值为7,搅拌转速为100rpm,通气比为1vvm的条件下,于光照强度为30μmoLm- 2s-1进行培养,培养至OD600为5,加入诱导剂IPTG至终浓度为100μM诱导表达,诱导后继续培养,培养过程中于葡萄糖耗尽后进行补加,保持残糖浓度在1g/L以下,持续培养至发酵结束。
具体实施方式三:本实施方式与具体实施方式二不同的是:步骤三中所述步骤一的无菌液和步骤二的无菌液的质量比1:4。其他步骤及参数与具体实施方式二相同。
检测试验
(一)对具体实施方式二和三过程中的聚球藻PCC 7942和大肠杆菌BL21(DE3)含量进行检测,得到如图1所示的共培养过程中生物量曲线图,从图1可以看出,与纯培养相比,藻菌共培养过程的生物量大大增加。
(一)对具体实施方式二和三过程中的异戊二烯产量和产率进行检测,得到如图2所示的共培养过程中异戊二烯产量曲线图和如图3所示的共培养过程中异戊二烯产率曲线图,从图2和图3可以看出,与纯培养相比,藻菌共培养合成周期由100h延长到400h,异戊二烯产量达到0.4g/L,是纯培养的8倍,产率保持不变。
具体实施方式四:本实施方式的一种藻菌共培养发酵体系由重组菌与光合自养微生物经发酵培养基共培养而成;所述重组菌为基因工程大肠杆菌,所述基因工程大肠杆菌为携带异戊二烯合成途径质粒的重组菌,所述基因工程大肠杆菌的宿主为大肠杆菌C41(DE3),所述基因工程大肠杆菌为采用海藻酸钠包埋的固定化大肠杆菌;
所述重组菌携带异戊二烯合成途径的质粒(见Yang,pLosone,2012),具体表达方式为将乙酰辅酶A乙酰基转移酶/HMG辅酶A还原酶基因mvaE、HMG辅酶A合成酶基因mvaS和异戊二烯合成酶基因ispS连接到质粒载体pACYCDuet-1上,将磷酸甲羟戊酸激酶基因erg8、甲羟戊酸激酶基因erg12、甲羟戊酸焦磷酸脱羧酶基因erg19和IPP异构酶基因idi1连接到载体pTrchis2B;
所述的光合自养微生物为小球藻;
所述发酵培养基由2g/L的NaNO3、0.5g/L的CaCL2、0.1g/L的NaCO3、0.1mg/L的Na2EDTA·2H2O、0.1g/L的葡萄糖、0.1g/L的MgSO4、0.1g/L的KH2PO4·3H2O、2g/L的甜菜碱、0.1g/L的硫酸铵、0.1g/L的柠檬酸、0.05g/L的柠檬酸铁铵、0.5mL/L的微量元素储液和17μg/mL氯霉素、50μg/mL氨苄青霉素组成。
所述微量元素储液的配置方法为:将0.37g的(NH4)6Mo7O24·4H2O、0.29g的ZnSO4·7H2O、2.47g的H3BO4、0.25g的CuSO4·5H2O和1.58g的MnCl2·4H2O用蒸馏水定容至100mL,然后过滤除菌,得到微量元素储液。
具体实施方式五:用具体实施方式四所述的一种藻菌共培养发酵体系生产异戊二烯的方法按以下步骤进行:
一、将小球藻接种到500mL的BG11培养基中,接种量为培养基体积的1%,30℃,200rpm摇床培养至对数生长中后期,取菌液离心收集光合自养微生物藻体,重悬于10mL无菌水中;
二、将重组菌接种到500mL的LB培养基中,接种量为培养基体积的1%,30℃,200rpm摇床培养至对数生长中后期,加入诱导剂IPTG至终浓度为500μM诱导表达3h,取菌液离心收集菌体,重悬于5mL无菌水中,用5mL的4%海藻酸钠溶液混合,滴入2%的CaCL2溶液中,获得固定化的重组菌,所述重组菌为基因工程大肠杆菌,所述基因工程大肠杆菌为携带异戊二烯合成途径质粒的重组菌,所述基因工程大肠杆菌的宿主为大肠杆菌C41(DE3),所述重组菌携带异戊二烯合成途径的质粒(见Yang,pLosone,2012),具体表达方式为将乙酰辅酶A乙酰基转移酶/HMG辅酶A还原酶基因mvaE、HMG辅酶A合成酶基因mvaS和异戊二烯合成酶基因ispS连接到质粒载体pACYCDuet-1上,将磷酸甲羟戊酸激酶基因erg8、甲羟戊酸激酶基因erg12、甲羟戊酸焦磷酸脱羧酶基因erg19和IPP异构酶基因idi1连接到载体pTrchis2B;
三、将步骤一的无菌液和步骤二的固定化细胞无菌液混合,然后将混合液接种到光照反应器中的发酵培养基中,发酵培养基体积的150mL,于温度为20℃,pH值为12,搅拌转速为50rpm,通气比为0.5vvm的条件下,于光照强度为400μmoLm-2s-1进行培养,培养过程中于葡萄糖耗尽后进行补加,保持残糖浓度在1g/L以下,持续培养至发酵结束。
本实施方式中异戊二烯产量达到2g/L。
具体实施方式六:本实施方式的一种藻菌共培养发酵体系由重组菌与光合自养微生物经发酵培养基共培养而成;所述重组菌为基因工程大肠杆菌,所述基因工程大肠杆菌为携带脂肪酸合成途径质粒的重组菌,所述基因工程大肠杆菌的宿主为大肠杆菌JM109(DE3),所述基因工程大肠杆菌为游离态大肠杆菌;
所述重组菌携带脂肪酸合成途径的质粒(见Liu,microbiaL ceLL factories,2012),具体表达方式为将去除信号肽的硫酯酶基因’tesA连接到质粒载体pACYCDuet-1上;
所述的光合自养微生物为集胞藻PCC6803;
所述发酵培养基由0.1g/L的NaNO3、0.25g/L的CaCL2、2g/L的NaCO3、5mg/L的Na2EDTA·2H2O、2.5g/L的葡萄糖、2g/L的MgSO4、6g/L的KH2PO4·3H2O、1g/L的甜菜碱、2g/L的硫酸铵、2g/L的柠檬酸、1g/L的柠檬酸铁铵、0.1mL/L的微量元素储液和1.7μg/mL氯霉素组成。
所述微量元素储液的配置方法为:将0.37g的(NH4)6Mo7O24·4H2O、0.29g的ZnSO4·7H2O、2.47g的H3BO4、0.25g的CuSO4·5H2O和1.58g的MnCl2·4H2O用蒸馏水定容至100mL,然后过滤除菌,得到微量元素储液。
具体实施方式七:用具体实施方式六所述的一种藻菌共培养发酵体系生产脂肪酸的方法按以下步骤进行:
一、将集胞藻PCC6803接种到BG11培养基中,接种量为培养基体积的20%,28℃,200rpm摇床培养至对数生长中后期,取菌液离心收集光合自养微生物藻体,重悬于无菌水中至终浓度10g/L;
二、将重组菌接种到LB培养基中,接种量为培养基体积的1%,37℃,200rpm摇床培养至对数生长中后期,取菌液离心收集菌体,重悬于无菌水中至终浓度5g/L;
所述重组菌为基因工程大肠杆菌,所述基因工程大肠杆菌为携带脂肪酸合成途径质粒的重组菌,所述基因工程大肠杆菌的宿主为大肠杆菌JM109(DE3),所述基因工程大肠杆菌为游离态大肠杆菌;
所述重组菌携带脂肪酸合成途径的质粒(见Liu,microbiaL ceLL factories,2012),具体表达方式为将去除信号肽的硫酯酶基因’tesA连接到质粒载体pACYCDuet-1上;
三、将步骤一的无菌液和步骤二的无菌液按比例1:10混合,然后将混合液接种到光照反应器中的发酵培养基中,发酵培养基体积为150mL,接种量为发酵培养基体积的20%,于温度为35℃,pH值为5,搅拌转速为500rpm,通气比为2vvm的条件下,于光照强度为1200μmoLm-2s-1进行培养,培养至OD600为90,加入诱导剂IPTG至终浓度为50μM诱导表达,诱导后继续培养,培养过程中于葡萄糖耗尽后进行补加,保持残糖浓度在1g/L以下,持续培养至发酵结束。
Claims (10)
1.一种藻菌共培养发酵体系,其特征在于,该体系由重组菌与光合自养微生物经发酵培养基共培养而成;所述重组菌为基因工程大肠杆菌。
2.根据权利要求1所述的一种藻菌共培养发酵体系,其特征在于,所述基因工程大肠杆菌为游离态大肠杆菌或经包埋的固定化大肠杆菌,所述经包埋的固定化大肠杆菌是经海藻酸钠或卡拉胶包埋的。
3.根据权利要求1所述的一种藻菌共培养发酵体系,其特征在于,所述的基因工程大肠杆菌的宿主为BL21(DE3)、MG1655、JM109、JM109(DE3)、DH5α、K12、C41、C41(DE3)或XL1-blue。
4.根据权利要求1所述的一种藻菌共培养发酵体系,其特征在于,所述的光合自养微生物为聚球藻PCC 7942、聚球藻UTEX 2973、聚球藻PCC7002、集胞藻PCC6803、小球藻、螺旋藻、栅藻、黄丝藻、杜氏盐藻或新月藻。
5.根据权利要求1所述的一种藻菌共培养发酵体系,其特征在于,所述发酵培养基由0.1g/L~2g/L的NaNO3、0.01g/L~0.5g/L的CaCl2、0.01g/L~2g/L的NaCO3、0.1mg/L~5mg/L的Na2EDTA·2H2O、0.1g/L~5g/L的葡萄糖、0.1g/L~2g/L的MgSO4、0.1g/L~6g/L的KH2PO4·3H2O、0.1g/L~2g/L的甜菜碱、0.1g/L~2g/L的硫酸铵、0.1g/L~2g/L的柠檬酸、0.05g/L~1g/L的柠檬酸铁铵、0.1ml/L~0.5ml/L的微量元素储液和抗生素组成。
6.根据权利要求5所述的一种藻菌共培养发酵体系,其特征在于,所述抗生素为0~100mg/L氨苄青霉素、0~50mg/L卡那霉素、0~34mg/L氯霉素、0~20mg/L庆大霉素、0~50mg/L壮观霉素、0~50mg/L链霉素和0~50mg/L四环素中的一种或几种的混合物。
7.根据权利要求1所述的一种藻菌共培养发酵体系,其特征在于,所述微量元素储液的配置方法为:将0.37g的(NH4)6Mo7O24·4H2O、0.29g的ZnSO4·7H2O、2.47g的H3BO4、0.25g的CuSO4·5H2O和1.58g的MnCl2·4H2O用蒸馏水定容至100mL,然后过滤除菌,得到微量元素储液。
8.利用如权利要求1~7任意一项权利要求所述的一种藻菌共培养发酵体系生产化学品的方法,其特征在于,该方法按以下步骤进行:
一、将光合自养微生物接种到BG11培养基中,接种量为培养基体积的1%~20%,培养至对数生长中后期,取菌液离心收集光合自养微生物藻体,重悬于无菌水中至终浓度2g/L~10g/L;
二、将重组菌接种到LB培养基中,接种量为培养基体积的1%~20%,培养至对数生长中后期,取菌液离心收集菌体,重悬于无菌水中至终浓度2g/L~10g/L;
三、将步骤一的无菌液和步骤二的无菌液混合,然后将混合液接种到光照反应器中的发酵培养基中,于温度为20~35℃,pH值为5~12,搅拌转速为50rpm~500rpm,通气比为0.5vvm~2vvm的条件下,采用不同的光照强度进行培养,培养至OD600为1~90左右,加入诱导剂IPTG至终浓度为50μM~500μM诱导表达,诱导后继续培养,培养过程中于葡萄糖耗尽后进行补加,保持残糖浓度在1g/L以下,持续培养至发酵结束。
9.根据权利要求8所述的一种藻菌共培养发酵体系生产化学品的方法,其特征在于,步骤三中所述步骤一的无菌液和步骤二的无菌液的质量比为1:(0.1~10)。
10.根据权利要求8所述的一种藻菌共培养发酵体系生产化学品的方法,其特征在于,步骤三中所述混合液的接种量为发酵培养基体积的1%~20%。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010494336.3A CN111849782B (zh) | 2020-06-03 | 2020-06-03 | 一种藻菌共培养发酵体系及用其生产化学品的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010494336.3A CN111849782B (zh) | 2020-06-03 | 2020-06-03 | 一种藻菌共培养发酵体系及用其生产化学品的方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111849782A true CN111849782A (zh) | 2020-10-30 |
CN111849782B CN111849782B (zh) | 2022-06-03 |
Family
ID=72985644
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010494336.3A Active CN111849782B (zh) | 2020-06-03 | 2020-06-03 | 一种藻菌共培养发酵体系及用其生产化学品的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111849782B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107287123A (zh) * | 2017-07-27 | 2017-10-24 | 扬州大学 | 一种通过固定化微生物共培养利用蔗糖异养培养微藻的方法 |
CN111088166A (zh) * | 2019-12-26 | 2020-05-01 | 南昌大学 | 一种藻菌共培养体系促进微生物诱导碳酸钙沉淀的方法 |
-
2020
- 2020-06-03 CN CN202010494336.3A patent/CN111849782B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107287123A (zh) * | 2017-07-27 | 2017-10-24 | 扬州大学 | 一种通过固定化微生物共培养利用蔗糖异养培养微藻的方法 |
CN111088166A (zh) * | 2019-12-26 | 2020-05-01 | 南昌大学 | 一种藻菌共培养体系促进微生物诱导碳酸钙沉淀的方法 |
Non-Patent Citations (2)
Title |
---|
STEPHANIE G. HAYS等: "Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction", 《JOURNAL OF BIOLOGICAL ENGINEERING》 * |
沈佳佳: "利用磁性纳米颗粒共价固定化重组大肠杆菌", 《中国优秀博硕士学位论文全文数据库(硕士) 基础科学辑》 * |
Also Published As
Publication number | Publication date |
---|---|
CN111849782B (zh) | 2022-06-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ren et al. | A new lipid-rich microalga Scenedesmus sp. strain R-16 isolated using Nile red staining: effects of carbon and nitrogen sources and initial pH on the biomass and lipid production | |
RU2459871C2 (ru) | Способ ферментативного получения 2-гидрокси-2-метилкарбоновых кислот | |
Yang et al. | Repeated batch production of 1, 3-propanediol from biodiesel derived waste glycerol by Klebsiella pneumoniae | |
KR102502571B1 (ko) | Dha의 생산을 위해 클로라이드가 없고 소듐이 없는 배양 배지 내에서 아우란티오키트리움 속의 미세조류를 배양하는 방법 | |
Palamae et al. | Production of renewable biohydrogen by Rhodobacter sphaeroides S10: a comparison of photobioreactors | |
JP2016503651A (ja) | ニトリルヒドラターゼを産生するロドコッカス・アエセリボランス(Rhodococcusaetherivorans)VKMAc−2610D菌株、その培養方法およびアクリルアミドを生成するための方法 | |
Elkahlout et al. | Long-term biological hydrogen production by agar immobilized Rhodobacter capsulatus in a sequential batch photobioreactor | |
Scherhag et al. | Removal of sugars in wastewater from food production through heterotrophic growth of Galdieria sulphuraria | |
Burgstaller et al. | The influence of different carbon sources on growth and single cell oil production in oleaginous yeasts Apiotrichum brassicae and Pichia kudriavzevii | |
Liu et al. | Characteristics for production of hydrogen and bioflocculant by Bacillus sp. XF-56 from marine intertidal sludge | |
CN111849782B (zh) | 一种藻菌共培养发酵体系及用其生产化学品的方法 | |
Wang et al. | Research on separation, identification, and kinetic characterization of mixed photosynthetic and anaerobic culture (MPAC) for hydrogen production | |
CN1724637A (zh) | 通过pH值反馈控制补碳培养微藻的方法 | |
Santos et al. | Zymomonas mobilis immobilized on loofa sponge and sugarcane bagasse for levan and ethanol production using repeated batch fermentation | |
WO2020050113A1 (ja) | 発酵プロセス利用による有用物質の製造方法 | |
CN1321174C (zh) | 工业化生物制氢菌种连续流培养及生物制氢系统强化方法 | |
CN108179112B (zh) | 蛋白核小球藻联合菌类产氢的方法 | |
Li et al. | Spatially-ordered layer-by-layer biofilms of a two-species microbial consortium promote hydrogen production | |
Leesing et al. | Producing of microalgal lipid by isolated microalgae under photoautotrophic and heterotrophic cultivations | |
Guo et al. | Effects of culture condition and nutrition on the co-production of microbial oil and exopolysaccharide by Sporidiobolus pararoseus JD-2 | |
CN114317623B (zh) | 基于脂肪酶大肠杆菌表面展示菌株的丁酸丁酯合成方法 | |
Voit et al. | Production of a microbial lipase by Staphylococcus carnosus (pLipMut2) in a bubble column reactor and a centrifugal field bioreactor | |
JP2009148212A (ja) | マンニトールの発酵製造方法及びその実施に用いる微生物 | |
CN109868294A (zh) | 一种连续发酵生产长链二元酸的方法 | |
CN117701486B (zh) | 一种生产pha的重组菌及其构建方法与应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |