WO2017080111A1 - 一株生产戊二胺的基因工程菌及其制备戊二胺的方法 - Google Patents

一株生产戊二胺的基因工程菌及其制备戊二胺的方法 Download PDF

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WO2017080111A1
WO2017080111A1 PCT/CN2016/071793 CN2016071793W WO2017080111A1 WO 2017080111 A1 WO2017080111 A1 WO 2017080111A1 CN 2016071793 W CN2016071793 W CN 2016071793W WO 2017080111 A1 WO2017080111 A1 WO 2017080111A1
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genetically engineered
fermentation
pentamethylenediamine
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田康明
路福平
王正祥
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天津科技大学
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  • the invention belongs to the technical field of microorganisms, and particularly relates to a genetic engineering bacteria capable of efficiently producing pentanediamine and an application thereof.
  • 1,5-pentanediamine also known as cadaverine, 1,5-diaminopentane, pentamethylenediamine or cadaveric toxin
  • cadaverine 1,5-diaminopentane, pentamethylenediamine or cadaveric toxin
  • aliphatic biogenic amine including spermine, putrescine, sub One of spermine, pentamethylenediamine, and the like.
  • Ludwig Brieger a physician in Berlin, Germany, first discovered the amine in a corrupted body and named it cadaverine.
  • pentamethylenediamine is an extension reaction of the lysine synthesis pathway, which is produced by decarboxylation of lysine under the action of lysine decarboxylase (EC 4.1.1.18) (as shown in Figure 1).
  • Aminoamines are found in spoilage and, like the decarboxylation product of ornithine, putrescine, are one of the odors produced by the decay of biological corpses.
  • This diamine is not only related to the role of spoilage, but also a small amount of pentamethylenediamine in the living metabolism, which is a biologically active nitrogenous base widely present in living organisms.
  • Pentylamine has many important physiological functions, such as pentanediamine, which is the main component of the "iron affinity system” for regulating iron ion concentration in microbial cells and some strictly anaerobic Gram-negative peptidoglycans; Amines also play an important role in shutting down microporous channels and protecting E. coli from oxygen toxicity; secretion of endogenous pentamethylenediamine and accumulation of intracellular high concentrations of pentamethylenediamine can lead to decreased outer membrane permeability, inhibition The role of certain antibiotics such as cephalosporins.
  • Pentadecylamine has a wide range of applications in agriculture, medicine and industry. In agriculture, exogenous application of pentamidine can improve fruit setting and promote fruit development and increase fruit yield; in medicine, it can also be used as an effective treatment for dysentery; in industry, pentamethylenediamine and dibasic acid The polymerization reaction can be carried out to synthesize high-quality polymer materials.
  • pentamidine is a novel and potentially competitive production route, while the prior art is more concerned with the expression of lysine decarboxylase and the construction of recombinant bacteria.
  • the Chinese patent "Method for producing 1,5-pentanediamine” which is filed by Toray Industries Co., Ltd., Japan, discloses a 1,5-pentanediamine fermented by microorganisms.
  • the microorganism used is a coryneform bacterium having an LDC gene in a chromosome, and the coryneform bacterium can maintain a lysine decarboxylase activity of 20 mU/mg or more in the culture process.
  • a method for producing cadaverine by the publication of CN 101389765 A by BASF Europe, Germany discloses a method for producing cadaverine by constructing a recombinant microorganism and cultivating the microorganism.
  • the prior art does not carry out technologically innovative research on how to more efficiently prepare catalytic cells for the synthesis of pentamethylenediamine.
  • the invention realizes the large-scale preparation process of pentamethylenediamine by establishing a catalytic cell required for the synthesis of pentanediamine and an efficient preparation method thereof.
  • the present invention provides a genetically engineered bacteria capable of efficiently producing pentamethylenediamine and an efficient preparation process for preparing pentamethylenediamine therefrom.
  • One of the technical solutions provided by the present invention is to provide a genetically engineered bacteria capable of efficiently producing pentamidine, wherein the lysine decarboxylase promoter ldcCp is replaced with an environmental/nutrition factor-controlled promoter. Therefore, the production strain does not synthesize the target enzyme in a large amount during the growth process, that is, the trace synthesis of the target enzyme does not affect the cell growth, and the target enzyme and its cofactor are rapidly overexpressed by changing the environmental/nutritive factors after the growth is completed.
  • the genetically engineered bacteria may be Escherichia coli K12, DH5 ⁇ , W3110, BL21, MG1655, etc.;
  • the starting strain is Escherichia coli B0013-070;
  • the environmental/nutrition factor-controlled promoter may be a controlled promoter such as pH, temperature, dissolved oxygen, or a controlled promoter such as lactose, xylose or arabinose;
  • control promoter is a temperature-regulated promoter p R -p L promoter
  • nucleotide sequence of the p R -p L promoter is as shown in SEQ ID NO: 1 in the Sequence Listing;
  • the genetically engineered bacteria express the signal peptide while replacing the promoter, so that the recombinant strain can express the lysine decarboxylase in a large amount in the periplasmic space;
  • the signal peptide is encoded by the gene pelBs, and the nucleotide sequence is as shown in SEQ ID NO: 2 in the Sequence Listing;
  • the genetically engineered bacteria provided by the present invention is Escherichia coli 42#, and the strain has been deposited on the General Microbiology Center of the China Microbial Culture Collection Management Committee on December 24, 2014 (Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101), deposit number CGMCC No.10240.
  • the transcription of the target enzyme encoding gene ldcC is strongly inhibited at a lower temperature, such as 25 to 36 ° C; and at a higher temperature, such as 37 to 50 ° C, the target enzyme encoding gene ldcC The transcription is strongly initiated.
  • the second technical solution provided by the invention is: an efficient process for preparing pentamethylenediamine, wherein the preparation process is to produce pentanediamine by fermentation of the genetically engineered bacteria described in the first scheme, in the initial stage of fermentation.
  • the culture temperature is controlled at 25-36 °C, and the rapid growth of the cells is carried out; in the remaining fermentation stage, the temperature is controlled at 37-50 °C, the enzyme production is induced for 1 ⁇ 5h, and the conversion is 2-8h, and the pentanediamine level is produced.
  • the medium used in the fermentation process is a fully synthetic medium
  • the key enzyme efficient preparation process and the rapid conversion process established by the invention are not limited to the preparation of pentamethylenediamine, and include other chemicals having a similar reaction process, such as pyruvic acid, alanine, lactic acid, ⁇ -ketopentane. Diacids, succinic acid, itaconic acid, and a variety of functional sugars, and the like.
  • the genetically engineered bacteria provided by the invention have obvious ability to efficiently convert lysine to glutaramine, and the strain is cultured at 25-50 ° C for 6-12 hours, and the enzyme is induced for 1 to 5 hours, and the transformation is 2-8 hours. , from the cultivation of cells to the completion of the conversion of pentamethylenediamine 9-25h, the production of pentamidine to reach 10.6-11.6% (w / v) or more, the yield reached 106-116.8g / L;
  • the process of expressing the enzyme used in the present invention to express the enzyme is completed in the cell membrane, and the finally expressed enzyme is interposed between the inner membrane of the cell and the cell wall, and is not released into the fermentation system, thereby enabling the repeated use of the catalytic cell.
  • the cells expressing the catalyst can simultaneously synthesize the cofactor, and the intact cell can be used in the process of converting lysine into pentamethylenediamine.
  • the substrate-to-product conversion is carried out directly, and the cells simultaneously serve as a site for providing a catalyst and protecting the activity of the catalyst, wherein the presence of the cofactor provides a guarantee for the high activity of the catalyst.
  • the genetically engineered bacteria of the pentamethyleneamine biotransformation of the present invention adopts a fully synthetic medium in the process of culturing the cells, and the culture solution is clarified, which is advantageous for the separation and extraction of the subsequent products; the raw materials used in the conversion process of the present invention may be The treated lysine fermentation feed liquid is extracted; the efficiency of the conversion process of the present invention is not affected by the residue of the lysine fermentation feed liquid; the pentamethylene salt formed after the completion of the conversion process of the present invention is easy to be extracted and purified.
  • the pentaamine biotransformation genetic engineering bacteria of the invention has a high level of glutaramine accumulation in the process of cell culture, induction of enzyme production and high-efficiency transformation, which provides convenience for subsequent extraction and purification.
  • the high-efficiency conversion process of pentamethylenediamine of the invention the cell growth temperature for catalysis is rapidly grown by using glucose at 25-36 ° C 6 ⁇ 12h, the cells were formed; after 1 to 5 hours of rapid induction of enzyme production, lysine was rapidly converted to pentanediamine at 37-50 °C.
  • the production process of the pentamethylenediamine only needs to change the fermentation temperature control parameter, and the glutaramine can be efficiently produced by using lysine as a raw material.
  • Figure 2 shows the physical map of the recombinant plasmid pT-ldcC
  • Figure 3 shows the results of functional identification of the lysine decarboxylase promoter
  • Figure 4 shows the results of functional identification of lysine decarboxylase signal peptide
  • Figure 5 is a flow chart for efficient preparation of pentamethylenediamine
  • Figure 7 is a small test level of pentanediamine conversion process
  • Figure 8 shows the conversion process of pentamethylenediamine under scale production.
  • Example 1 Escherichia coli lysine decarboxylase temperature-regulated promoter and signal peptide replacement
  • the 915 bp and 2.7 kb fragments were released by EcoRI digestion of the recombinant plasmid pLDC-UP; the DNA of the recombinant plasmid pLDC-up was used as a template, and the primers ldc-invF (SEQ ID NO: 5) and ldc-invR (SEQ ID NO: 6) were used.
  • the PCR product is the promoter portion of the upstream part of the ldcC gene, that is, the LDC-up size is -3.57 kb.
  • PCR amplification of the pL promoter of pPL451 (Gene, 1996, 176:49-53) (primers pL-F (SEQ ID NO: 7) and pL-R (SEQ ID NO: 8)), PCR product size ⁇ 1.37 kb .
  • the PCR product was digested with BamHI and SpeI (BcuI)
  • pET-20b was ligated with BglII and XbaI
  • the temperature-controlled promoter fragment pL and the signal peptide sequence pelBs were ligated to obtain plasmid pPL-pelBs, which was 5.0 kb in size; SmaI and BamHI were digested.
  • the PCR product LDC-up was digested with BamHI, digested with pMa-pelBs by SmaI+BamHI, and the pL-pelBs (1.48 kb) fragment was recovered by ligation; the plasmid pLDC::pL-pelBs was obtained by ligation into E. coli, and 2.7 kb was digested with EcoRI.
  • the +2.36 kb fragment was cloned into the gentamicin resistance gene difGm cassette at the SmaI site to obtain the plasmid pLDC::pL-pelBs-Gm.
  • the 2.7 kb+3.36 kb fragment was digested with EcoRI; the 3.36 kb fragment of pLDC::pL-pelBs-Gm/EcoRI was recovered by gel (31.66 kb fragment was prepared by PCR using primers ldc-up1 and ldc-up2, DpnI digestion, purification After electroporation), electroporation was carried out in the B0013-070 strain already containing pKD46. A recombinant strain 41# strain in which the ldcC gene promoter was replaced with pL was obtained.
  • SmaI and BamHI were double-digested with pPL-pelBs, and the pL-pelBs fragment was isolated by gel, and reversed with primers Ec-RlC3 (SEQ ID NO: 9) and Ec-RlC4 (SEQ ID NO: 10) using pET20b-ldcC as a template.
  • PCR amplification and restriction enzyme digestion such as BglII digestion to obtain ldcC gene product ligation, to obtain recombinant plasmid pT-cI ts 857-p R -p L - pelBs-ldcC abbreviation, pT-ldcC, its physical map As shown in Fig.
  • the recombinant plasmid comprises a temperature-controlled promoter, a signal peptide and an ldcC intact gene, and has a function of temperature-controlled secretion of ldcC.
  • the recombinant plasmid pT-ldcC was electrotransformed into the 41# strain to obtain a recombinant strain 42# (pT-ldcC) having a patent deposit number of CGMCC No. 10240.
  • the patent strain 42# (pT-ldcC) and the starting strain B0013-070 were cultured at 25-36 ° C and 37-50 ° C for 2-10 h, and the medium was (g/L): yeast extract 15, peptone 0.5, anhydrous MgSO 4 0.25, glucose 5.
  • yeast extract 15 yeast extract 15, peptone 0.5, anhydrous MgSO 4 0.25, glucose 5.
  • LDC cell disruption lysine decarboxylase
  • the strain 42# produced only a very low amount of LDC activity at 30 °C.
  • the LDC activity of the strain 42# (pT-ldcC) was 20 times that of the starting strain 42#, which satisfies the need for rapid formation of pentanediamine.
  • the LDC cultured at 42 °C of the strain 42# (pT-ldcC) was 100% than the enzyme activity value, whereas the LDC enzyme activity was significantly decreased when the strain was grown at 30 °C. It is indicated that the p R -p L promoter of the strain 42# (pT-ldcC) is controlled by the change in temperature to effectively control the expression of the ldcC gene.
  • the patent strain 42#(pT-ldcC) and the starting strain B0013-070 are cultured at 25-36 ° C and 37-50 ° C for 2-10 h, and the medium is (g/L): yeast paste 0-20, peptone 0 ⁇ 20, anhydrous MgSO 4 0 ⁇ 10, glucose 5. And the expression process of lysine decarboxylase is enhanced by adding lactose, IPTG and the like.
  • the fermentation broth was centrifuged at 6000 rpm for 8 min to take the supernatant and directly measure the enzyme activity as the enzyme activity in the fermentation broth; the centrifuged cells were resuspended in the medium to the initial volume and the enzyme activity was determined as the enzyme activity in the periplasmic space; The measured enzyme activity was used as the periplasmic space and the total intracellular enzyme activity. Typical measurement results are shown in Figure 4.
  • the strain LDC than the enzyme activity value of the cell disruption solution after culture and induction of the enzyme 42# (pT-ldcC) was 100%.
  • the enzyme activity in the fermentation broth is extremely low, and the cell periplasmic space enzyme activity is close to the enzyme activity of the cell disruption solution. It can be seen that the signal peptide effectively expresses lysine decarboxylase to the periplasmic space of the cell.
  • Example 47L Fermentation tank induces enzyme production and conversion of lysine to pentamethylenediamine
  • the strain 42# (pT-ldcC) was subjected to decarboxylation of lysine to form pentamethylenediamine in a 7 L fermentor to examine the effect of temperature-regulated LDC expression under controlled production conditions.
  • the strain 42# (pT-ldcC) is aerobic cultured at 25-36 ° C until the OD 600 value is about 15-40, the fermenter temperature is set to 37-50 ° C, the aerobic culture is continued for 0-120 min, and then the aeration is performed.
  • the amount is set to 0 to 0.2 vvm for oxygen-limited fermentation, the fermentation temperature in the oxygen-limited stage is 37 to 50 ° C, and the amount of lysine added is 166 to 176 g/L.
  • the fermentation medium is (g/L): diammonium phosphate 0-25, potassium dihydrogen phosphate 0-5, disodium hydrogen phosphate, 0-25, sodium chloride 0-5, MgSO 4 0-0.5, FeSO 40 to 1, FeCl 3 0 to 1, CoCl 2 0 to 1, CuCl 2 0 to 1, CoCl 2 0 to 1, Na 2 MoO 4 0 to 1, H 3 BO 3 0 to 1, MnCl 2 0 to 1 , thiamine 0 to 1, IPTG 0 to 5, lactose 0 to 10, glucose 0 to 50, pH 6.0 to 7.5.
  • the flow chart of efficient preparation of pentamethylenediamine is shown in Fig. 5.
  • the HPLC detection results of pentamethylenediamine are shown in Fig. 6.
  • the lysine, pentamethylenediamine, lysine decarboxylase activity and cell concentration during fermentation were as shown in Fig. 7.
  • Fermentation results of strain 42# (pT-ldcC) fermenter showed that glucose was successfully used in the aerobic phase for the accumulation of bacterial cells, and the conversion phase rapidly converted lysine to pentamethylenediamine.
  • the yield of pentamethylenediamine is as high as 106.5 to 116.8 g/L.
  • the lysine conversion rate reached 91% to 97% of the theoretical conversion rate.
  • Example 4 The fermentation process in Example 4 was scaled up to a scale of 10 tons. Fermentation tanks and flow tanks were selected to meet the continuous addition of glucose and lysine, and the pre-run fermenter preparation was completed according to the normal operation in the factory.
  • One main fermenter one glucose feed tank, one lysine feed tank and one seed tank. Configure 70% glucose, heat and dissolve, and sterilize for use.
  • the concentrated lysine extraction solution is sterilized and stirred for use.
  • the culture medium is set, sterilized, and then inoculated, fermentation is started, 25 to 36 ° C, and the mixture is stirred at 180 to 340 L/h for 0 to 600 r/min. After 12 hours, it entered the induction enzyme production stage, and the fermentation temperature was increased to 37-50 °C.
  • the glucose content was measured every 2 hours, 1 to 5% of the initial sugar was consumed, the ventilation was stopped, and the stirring speed was reduced to 0 to 180 r/min.
  • the ceramic membrane concentrates the above high-activity cells, and adds 8 tons of fermentation broth with a final concentration of 17% lysine, and the transformation temperature is 37-50 °C. After 3 h, the lysine was consumed to below -0.6 g/L, and the conversion process was terminated, followed by product extraction and crystal preparation.
  • the ceramic membrane is filtered to recover the cells, and re-feeding is carried out to carry out the conversion process of the next batch. Complete 5 batch conversion processes.
  • the lysine, pentamethylenediamine, lysine decarboxylase activity and cell concentration during fermentation are shown in Fig. 8.
  • the present invention dynamically regulates the expression of a gene encoding a lysine decarboxylase on a chromosome of a starting strain by genetic engineering technology, thereby realizing the efficient production of pentanediamine from a catalytic lysine by a recombinant strain.
  • Simple preparation process
  • the technology of the present invention can be used for other industrially important microbial metabolites after simple modification, but is not limited to various organic acids such as L-lactic acid, acetic acid, pyruvic acid, succinic acid, malic acid, etc.; , alanine, lysine, methionine, glutamic acid, arginine and other amino acids; thiamine, vitamin B 12 and other microorganisms; or, ethanol, propanol and other short-chain alcohol strain construction, The establishment and application of fermentation production and new process technologies.

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Abstract

提供了一种生产戊二胺的基因工程菌株，其中该基因工程菌株的赖氨酸脱羧酶启动子ldcCp被替换为环境/营养因素控制型启动子的同时表达信号肽，可在细胞周质空间大量表达赖氨酸脱羧酶，且该酶的活力可通过环境温度进行调节。还提供了一种利用该基因工程菌株生产戊二胺的方法，包括发酵培养菌体，变温高效产酶和目标产物快速转化三个阶段。

Description

一株生产戊二胺的基因工程菌及其制备戊二胺的方法 技术领域：
本发明属于微生物技术领域，特别涉及一株能高效生产戊二胺的基因工程菌及其应用。
背景技术：
1,5-戊二胺，又名尸胺(cadaverine)、1,5-二氨基戊烷、五亚甲基二胺或尸毒素，是脂肪族生物胺类(包括精胺、腐胺、亚精胺和戊二胺等)中的一种。1885年，德国柏林的医师Ludwig Brieger在腐败的尸体中首次发现该胺类，并以此得名尸胺。在细胞内，戊二胺是赖氨酸合成途径的延伸反应，是赖氨酸在赖氨酸脱羧酶(E.C.4.1.1.18)的作用下脱羧产生的(如附图1)，作为一种肉毒胺存在于腐败物中，与鸟氨酸的脱羧产物腐胺一样都是生物尸体腐败产生的气味中的一种成分。这种二胺不仅与腐败作用有关，生物活体在生命代谢中也会产生少量的戊二胺，是生物体内广泛存在的具有生物活性的含氮碱。
戊二胺具有许多重要的生理功能，如戊二胺是微生物细胞内调节铁离子浓度的“铁亲和系统”和一些严格厌氧的革兰氏阴性菌肽聚糖的主要组成成分；戊二胺在关闭微孔蛋白通道和保护大肠杆菌免受氧的毒害方面也起着重要的作用；内源性戊二胺的分泌以及胞内高浓度戊二胺积累可导致外膜渗透性降低，抑制某些抗生素如头孢霉素类抗生素的作用。
戊二胺在农业、医学和工业上均具有广泛的应用。在农业上，外源施加戊二胺可以改善坐果和促进果实发育，提高果实的产量；在医学上，它也可作为一种有效治疗痢疾的药物；在工业上，戊二胺与二元酸进行聚合反应可合成优质高分子材料。
微生物法生产戊二胺，是一条新颖且具有潜在竞争力的生产途径，而现有技术更多的关注在赖氨酸脱羧酶的表达和重组菌的构建等。
如：由日本的东丽株式会社申请的专利号为公开号为CN 102844440 A的中国专利《1,5-戊二胺的制造方法》公开了一种利用微生物发酵的1,5-戊二胺的制造方法，所使用的微生物是染色体中具有LDC基因的棒状杆菌，可使棒状杆菌在培养过程中持续保持20mU/mg蛋白以上的赖氨酸脱羧酶活性。
德国的巴斯夫欧洲公司申请的公开号为CN 101389765 A的专利《生产尸胺的方法》公开了一种通过构建重组微生物并培养所述微生物来生产尸胺的方法
德国的赢创德固赛有限责任公司申请的公开号为CN 101240258A的专利则公布了戊二胺制备相关专利关注的是发酵条件优化和重组菌构建过程的保护。
可见，现有技术并没有在如何更高效的制备用于戊二胺合成的催化细胞方面进行技术创新性研究。本发明则通过建立戊二胺合成所需催化细胞及其高效制备方法，从而实现戊二胺的大规模制备过程。
发明内容：
为了解决上述技术问题，本发明提供一种可高效生产戊二胺的基因工程菌以及利用其制备戊二胺的高效制备工艺。
本发明所提供的技术方案之一是，提供一种可高效生产戊二胺的基因工程菌，所述基因工程菌其赖氨酸脱羧酶启动子ldcCp被替换为环境/营养因素控制型启动子，使得生产菌株在生长过程中不大量合成目标酶，即目标酶的痕量合成不影响细胞生长，而在生长完成后通过改变环境/营养因素快速过量表达目标酶及其辅因子。
进一步的，所述基因工程菌其出发菌株，可以为大肠杆菌K12、DH5α、W3110、BL21、MG1655等；
更进一步的，所述出发菌株为大肠杆菌B0013-070；
进一步的，所述环境/营养因素控制型启动子可以是pH、温度、溶氧等控制型启动子，也可以是多种营养因素如乳糖、木糖、阿拉伯糖等控制型启动子；
更进一步的，所述控制型启动子为温度调控性启动子pR-pL启动子；
所述pR-pL启动子的核苷酸序列如序列表中SEQ ID NO:1所示；
进一步的，所述基因工程菌在替换启动子的同时表达信号肽，使得重组菌株可以在细胞周质空间大量表达赖氨酸脱羧酶；
所述信号肽由基因pelBs编码，核苷酸序列如序列表中SEQ ID NO:2所示；
进一步的，本发明所提供的基因工程菌为大肠埃希氏菌(Escherichia coli)42#,所述菌株已于2014年12月24日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号，邮编100101)，保藏编号CGMCC No.10240。
更进一步地，本发明菌种在较低的温度下，如25～36℃，目标酶编码基因ldcC的转录被强烈抑制；而在较高温度下，如37～50℃，目标酶编码基因ldcC的转录被强烈启动。
本发明所提供的技术方案之二：是一种制备戊二胺的高效工艺，所述制备工艺是利用技术方案一所述的基因工程菌为生产菌株发酵生产戊二胺，在发酵初期的6～12h内，培养温度控制在25～36℃，进行菌体的快速生长；在余下的发酵阶段温度控制在37～50℃，诱导产酶1～5h，转化2～8h，产戊二胺水平达到10.6～11.6％(w/v)或以上；其工艺特征是：25～50℃的条件下，分别经过发酵培养菌体，变温高效产酶和目标产物快速转化三个阶段，产戊二胺水平达到106-116.8g/L，赖氨酸转化率达到了理论转化率的91％～97％；
进一步的，发酵过程中使用的培养基为全合成培养基；
更进一步地，本发明建立的关键酶高效制备工艺和快速转化工艺不限于戊二胺的制备，还包括具有类似反应过程的其他化学品，如丙酮酸、丙氨酸、乳酸、α-酮戊二酸、丁二酸、衣康酸以及多种功能糖等等。
有益效果：
1、本发明提供的基因工程菌具有明显的高效转化赖氨酸合成戊二胺的能力，菌种在25～50℃的条件下培养6～12h，诱导产酶1～5h，转化2～8h，从培养细胞到戊二胺转化完成工耗时9-25h,产戊二胺水平达到10.6～11.6％(w/v)或以上，产量达到106-116.8g/L；
2、本发明中使用的表达宿主菌其表达酶的过程在细胞膜完成，最终表达的酶介于细胞内膜与细胞壁之间，而不释放的发酵体系中，进而可以实现催化细胞的重复使用。
3、由于本发明所制备的基因工程就其出发菌株为大肠杆菌，因此表达催化剂的细胞同时可以完成辅因子的合成，在用于赖氨酸转化为戊二胺的过程中可以使用完整的细胞直接进行底物到产物的转化，细胞同时起到了提供催化剂作用的场所和保护催化剂活性的作用，其中辅因子的存在为催化剂的高活性提供了保证。
4、本发明的戊二胺生物转化基因工程菌，在菌体培养过程中，采用全合成培养基，培养液澄清，有利于后续产品分离提取；本发明转化过程使用的原料可以是未经后提取处理的赖氨酸发酵料液；本发明转化过程的效率不受赖氨酸发酵料液残余物的影响；本发明涉及的转化过程完成后形成的戊二胺盐易于后提取和纯化。5、本发明的戊二胺生物转化基因工程菌，在菌体培养、诱导产酶和高效转化过程中，戊二胺积累达较高水平，为后续提取纯化提供了便捷。
6、本发明的戊二胺高效转化过程：用于催化的菌体细胞生长温度在25～36℃下利用葡萄糖快速生长 6～12h，形成菌体；快速诱导产酶1～5h后，在37～50℃下快速转化赖氨酸合成戊二胺。即：运用本发明的重组菌及其戊二胺制备工艺，戊二胺的生产过程仅需改变发酵温度控制参数，即可实现以赖氨酸为原料高效生成戊二胺。
附图说明
图1赖氨酸脱羧反应；
图2重组质粒pT-ldcC的物理图谱；
图3赖氨酸脱羧酶启动子功能鉴定结果；
图4赖氨酸脱羧酶信号肽功能鉴定结果；
图5戊二胺高效制备流程图；
图6戊二胺HPLC检测图谱；
图7小试水平戊二胺转化进程；
图8规模化生产下戊二胺转化进程。
具体实施方式：
实施例1：大肠杆菌染色体赖氨酸脱羧酶温度调控型启动子和信号肽的替换
1，ldcC基因部分序列的克隆
以大肠杆菌B0013-070染色体DNA为模板，用引物ldc-up1(SEQ ID NO:3)和ldc-up2(SEQ ID NO:4)进行PCR扩增ldcC 5'端及其上游序列(ldcC-up)，两端通过引物加入EcoRI位点。PCR产物大小～920bp；将PCR产物克隆入pMD-18T-simple，获得重组质粒pLDC-UP；
ldc-up1：
Figure PCTCN2016071793-appb-000001
；EcoRI
ldc-up2：
Figure PCTCN2016071793-appb-000002
；EcoRI
2，ldcC基因重组同源臂的获得
EcoRI酶切重组质粒pLDC-UP释放915bp和2.7kb片段；以重组质粒pLDC-up的DNA为模板，用引物ldc-invF(SEQ ID NO:5)和ldc-invR(SEQ ID NO:6)进行反向PCR扩增，PCR产物为ldcC基因上游部分序列含启动子部分，即LDC-up大小～3.57kb。
ldc-invF：
Figure PCTCN2016071793-appb-000003
；BamHI
ldc-invR：
Figure PCTCN2016071793-appb-000004
3，温控型启动子和分泌型信号肽的获得
PCR扩增pPL451(Gene,1996,176:49～53)的pL启动子(引物pL-F(SEQ ID NO:7)和pL-R(SEQ ID NO:8))，PCR产物大小～1.37kb。PCR产物用BamHI和SpeI(BcuI)酶切、pET-20b用BglII和XbaI、连接转化温控型启动子片段pL和信号肽序列pelBs获得质粒pPL-pelBs,大小5.0kb；SmaI和BamHI双酶切pPL-pelBs，胶分离pL-pelBs片段，大小1.48kb。
pL-F：
Figure PCTCN2016071793-appb-000005
；BamHI/SmaI
pL-R:
Figure PCTCN2016071793-appb-000006
；SpeI
4，替换启动子和信号肽突变盒的获得
PCR产物LDC-up用BamHI酶切、SmaI+BamHI酶切pPL-pelBs，胶回收pL-pelBs(1.48kb)片段；连接转化大肠杆菌获得质粒pLDC::pL-pelBs，用EcoRI酶切释放2.7kb+2.36kb片段；在SmaI位点克隆入庆大霉素抗性基因difGm盒，获得质粒pLDC::pL-pelBs-Gm。
5，ldcC基因启动子和信号肽同源重组替换
用EcoRI酶切释放2.7kb+3.36kb片段；胶回收pLDC::pL-pelBs-Gm/EcoRI的3.36kb片段(用引物ldc-up1和ldc-up2进行PCR制备3.36kb片段，DpnI酶切，纯化后电转化)，电转化已经含有pKD46的B0013-070菌株中。获得ldcC基因启动子替换为pL的重组菌41#菌株。
6，游离质粒中ldcC基因启动子和信号肽的替换
SmaI和BamHI双酶切pPL-pelBs，胶分离pL-pelBs片段，与以pET20b-ldcC为模板用引物Ec-RlC3(SEQ ID NO:9)和Ec-RlC4(SEQ ID NO:10)进行反向PCR扩增并用限制性内切酶，如BglII进行酶切获得ldcC基因产物连接，从而获得重组质粒pT-cIts857-pR-pL-pelBs-ldcC简称，pT-ldcC，其物理图谱如图2所示，该重组质粒包含温控型启动子，信号肽和ldcC完整基因，具备温控分泌表达ldcC的功能。将重组质粒pT-ldcC电转化41#菌株，获得重组菌种42#(pT-ldcC)，该菌种专利保藏号为：CGMCC No.10240。
Ec-RlC3：
Figure PCTCN2016071793-appb-000007
Ec-RlC4：
Figure PCTCN2016071793-appb-000008
；BglII
实施例2：菌种42#(pT-ldcC)中pR-pL启动子活性的确定
专利菌株42#(pT-ldcC)和出发菌株B0013-070在25～36℃和37～50℃进行培养2～10h，其培养基为(g/L)：酵母膏15，蛋白胨0.5，无水MgSO40.25，葡萄糖5。并测定它们的细胞破碎液赖氨酸脱羧酶(LDC)比酶活，典型的结果如图3所示。其中细胞破碎液的制备过程如下，30mL发酵液于50mL离心管中，6000rpm离心8min；弃去上清，加入10mL ddH2O，漩涡震荡混匀，补加ddH2O至30mL，6000rpm离心8min；弃去上清，加入10mL PBS，漩涡震荡混匀，补加PBS至30mL，6000rpm离心8min。重复洗涤细胞1次。加入10mL PBS重悬菌体，超声破碎。(超声破碎条件：超声3s，间隙2s，温度25℃，破碎时间30min，功率40％)；4℃，8000rpm离心15min，除去细胞碎片。
菌种42#在30℃仅产生极低量的LDC活性。在42℃培养，菌种42#(pT-ldcC)的LDC活性是出发菌株42#的20倍，可以满足戊二胺快速形成的需要。菌种42#(pT-ldcC)在42℃培养的LDC比酶活值标定为100％，与此相比，该菌种在30℃生长时LDC酶活显著降低。说明通过温度的变化来控制菌种42#(pT-ldcC)的pR-pL启动子有效地控制了ldcC基因的表达。
实施例3菌种42#(pT-ldcC)细胞活性及赖氨酸脱羧酶的分泌表达过程鉴定
专利菌株42#(pT-ldcC)和出发菌株B0013-070在25～36℃和37～50℃进行培养2～10h，其培养基为(g/L)：酵母膏0～20，蛋白胨0～20，无水MgSO40～10，葡萄糖5。并通过添加乳糖，IPTG等加强赖氨酸脱羧酶的表达过程。发酵液6000rpm离心8min取上清直接测定酶活作为发酵液中的酶活；离心收集的细胞经培养基重悬为起始体积后测定的酶活作为细胞周质空间的酶活；细胞破碎液测定的酶活作为细胞周质空间和胞内总的酶活。典型测定结果如图4所示。
菌种42#(pT-ldcC)培养并诱导产酶后细胞破碎液的LDC比酶活值标定为100％。发酵液中酶活极低，细胞周质空间酶活接近细胞破碎液的酶活。可见信号肽有效的将赖氨酸脱羧酶表达至细胞的周质空间。
实施例47L发酵罐中菌体培养诱导产酶及转化赖氨酸形成戊二胺
菌种42#(pT-ldcC)在7L发酵罐中进行赖氨酸脱羧形成戊二胺来检验温度调控的LDC表达在可控生产条件下的效果。菌种42#(pT-ldcC)在25～36℃进行好氧培养至OD600值约为15～40，将发酵罐温度设定为37～50℃继续好氧培养0～120min，再将通气量设为0～0.2vvm进行限氧发酵，限氧阶段发酵温度为37～50℃，赖氨酸添加量为166～176g/L。其发酵培养基为(g/L)：磷酸氢二铵0～25，磷酸二氢钾0～5，磷酸氢二钠，0～25，氯化钠0～5，MgSO40～0.5，FeSO40～1，FeCl30～1，CoCl20～1，CuCl20～1，CoCl20～1，Na2MoO40～1，H3BO30～1，MnCl20～1，硫胺素0～1，IPTG 0～5，乳糖0～10，葡萄糖0～50，pH 6.0～7.5。戊二胺高效制备流程图如图5所示。戊二胺HPLC检测结果如图6所示。发酵过程中赖氨酸、戊二胺、赖氨酸脱羧酶酶活和菌体浓度如图7所示。
菌种42#(pT-ldcC)发酵罐发酵结果表明在好氧阶段成功将葡萄糖用于菌体量的积累，转化阶段快速将赖氨酸转化为戊二胺。最终发酵液中，戊二胺产量高达106.5～116.8g/L。赖氨酸转化率达到了理论转化率的91％～97％。
实施例5——菌种10吨发酵罐中5批次发酵转化制备戊二胺
将实施例4中的发酵工艺放大至10吨规模。以满足葡萄糖和赖氨酸的连续补加为参考指标对发酵罐和流加罐进行选择，并按照工厂里的常规操作完成运行前发酵罐的准备工作。主发酵罐一个，葡萄糖补料罐一个，赖氨酸补料罐一个和种子罐一个。配置70％葡萄糖，加热溶解后，灭菌备用。浓缩后赖氨酸提取溶液，灭菌后，搅拌备用。配置培养基，并灭菌，然后进行接种，开始发酵，25～36℃，通风180～340L/h搅拌0～600r/min。12h后进入诱导产酶阶段，发酵温度提高为37～50℃。每隔2个小时测定葡萄糖含量，1～5％初糖耗尽，停止通风，搅拌速度降至0～180r/min。进入转化制备戊二胺阶段，陶瓷膜浓缩上述高活性细胞，并添加8吨赖氨酸终浓度为17％的发酵料液，转化温度为37～50℃。3h后，赖氨酸消耗至～0.6g/L以下后结束转化过程，进行产品的后提取和晶体制备。单批转化结束，陶瓷膜过滤回收菌体，并重新投料进行下批次的转化过程。连续完成5批转化过程。发酵过程中赖氨酸、戊二胺、赖氨酸脱羧酶酶活和菌体浓度如图8所示。
表1：五批次10吨罐连续转化生产结果
Figure PCTCN2016071793-appb-000009
综上所述，本发明通过基因工程技术对出发菌染色体上的赖氨酸脱羧酶编码基因的表达进行简易条件下的动态调控，从而实现了重组菌从催化赖氨酸高效生产戊二胺的简洁制备工艺。本发明技术经过简单修改后，同样可以用于其它工业上重要的微生物代谢产物，但不限于，如L-乳酸、乙酸、丙酮酸、丁二酸、苹果酸等多种有机酸；脯氨酸、丙氨酸、赖氨酸、蛋氨酸、谷氨酸、精氨酸等多种氨基酸；硫胺素、维生素B12等多种微生物；或，乙醇、丙醇等短链醇的菌种构建、发酵生产和新工艺技术的建立与应用。

Claims (8)

  1. 一种可高效生产戊二胺的基因工程菌，其特征在于，所述基因工程菌其赖氨酸脱羧酶启动子ldcCp被替换为环境/营养因素控制型启动子的同时表达信号肽。
  2. 如权利要求1所述的一种可高效生产戊二胺的基因工程菌，其特征在于，出发菌株为大肠杆菌K12或DH5α或W3110或BL21或MG1655。
  3. 如权利要求1所述的一种可高效生产戊二胺的基因工程菌，其特征在于，所述环境/营养因素控制型启动子是pH、温度、溶氧、乳糖、木糖、阿拉伯糖控制型启动子。
  4. 如权利要求1或3所述的一种可高效生产戊二胺的基因工程菌，其特征在于，所述环境/营养因素控制型启动子是pR-pL启动子，核苷酸序列如序列表中SEQ ID NO:1所示。
  5. 如权利要求1所述的一种可高效生产戊二胺的基因工程菌，其特征在于，所述信号肽由pelBs基因编码，核苷酸序列如序列表中SEQ ID NO:2所示。
  6. 如权利要求1所述的一种可高效生产戊二胺的基因工程菌，其特征在于，所述基因工程菌为大肠埃希氏菌(Escherichia coli)42#,保藏编号CGMCC No.10240。
  7. 一种利用权利要求1所述基因工程菌生产戊二胺的方法，其特征在于，在发酵初期的6～12h内，培养温度控制在25～36℃，进行菌体的快速生长；在余下的发酵阶段温度控制在37～50℃，诱导产酶1～5h，转化生产戊二胺2～8h。
  8. 如权利要求7所述的一种生产戊二胺的方法，其特征在于，发酵过程中使用的培养基为全合成培养基。
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