WO2016065709A1 - 利用乙酸生产丁二酸的代谢工程大肠杆菌菌株构建方法和应用 - Google Patents

利用乙酸生产丁二酸的代谢工程大肠杆菌菌株构建方法和应用 Download PDF

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WO2016065709A1
WO2016065709A1 PCT/CN2014/094215 CN2014094215W WO2016065709A1 WO 2016065709 A1 WO2016065709 A1 WO 2016065709A1 CN 2014094215 W CN2014094215 W CN 2014094215W WO 2016065709 A1 WO2016065709 A1 WO 2016065709A1
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acetic acid
succinic acid
deletion
acid
escherichia coli
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李志敏
李运杰
吴辉
叶勤
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East China University of Science and Technology
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    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid

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  • the invention belongs to the field of bioengineering technology, and more particularly relates to constructing a recombinant Escherichia coli strain for producing succinic acid by using acetic acid, and synthesizing succinic acid by fermentation of the metabolic engineering Escherichia coli by using acetic acid as a main raw material.
  • Succinic acid also known as succinic acid
  • succinic acid is a four-carbon dicarboxylic acid widely used in the agricultural, food and pharmaceutical industries.
  • Succinic acid is a precursor to many important industrial chemicals, including adipic acid, 1,4-butanediol, tetrahydrofuran, N-methylpyrrolidone, 2-pyrrolidone, succinate and ⁇ -butyrolactone. It can also be used to synthesize biodegradable polymer polybutylene succinate (PBS) and polyamide polymers.
  • PBS biodegradable polymer polybutylene succinate
  • PBS polybutylene succinate
  • commercial succinic acid has been chemically synthesized from liquefied petroleum gas or mineral oil and converted into bio-fermentation production (Catal.
  • the succinic acid producing strains are mainly Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes producing succinic acid, Mannheimia succiniciproducens, recombinant Escherichia coli, Saccharomyce scerevisiae Metab Eng, 2010, 12(6): 518-525), Corynebacterium glutamicum (PLoS One, 2013, 8(4): e60659), and the like. Among them, due to the clear genetic background of E.
  • the gene operation is simple, and the fermentation substrate is mostly renewable resources, such as glucose, xylose, glycerin, wheat straw, corn stover and other lignocellulose, cassava, Jerusalem artichoke and other starchy substances, and more With industrial value.
  • E. coli HL27658k constructed by San et al. (Biotechnol Bioeng, 2005, 90(6): 775-779), blocking the TCA cycle, excising the byproduct formation pathway, releasing the glyoxylate branch repression, and achieving glucose aerobic transformation.
  • the accumulation of succinic acid was 58.3 g/L, and the yield was 0.94 ⁇ 0.07 mol/mol glucose, and the production intensity was 1.08 ⁇ 0.06 g/(Lh).
  • Acetic acid is mainly prepared by carbonylation of methanol, namely methanol and carbon monoxide. It is a large and inexpensive carbon source and one of the main by-products formed in the acid hydrolysis of lignocellulosic materials.
  • Acetic acid is the end product of anaerobic metabolism of many microorganisms and incomplete oxidation products of aerobic metabolism. Escherichia coli can aerobicly metabolize acetic acid for growth, and when acetic acid is the only carbon source for aerobic growth, glyoxylate branch and glucone The key enzyme activity of the pathway is significantly improved, which facilitates the subsequent anaerobic conversion of glucose to succinic acid (Applied and Environmental Microbiology, 2007, 73(24): 7837-7843.).
  • the invention transforms Escherichia coli from the aspects of enhancing substrate uptake, blocking downstream catabolism and diluting metabolic flux, and enabling it to directly produce succinic acid by using acetic acid as a carbon source.
  • a first object of the present invention is to provide a method for constructing a metabolically engineered E. coli strain for producing succinic acid using acetic acid.
  • a second object of the present invention is to provide a metabolically engineered E. coli strain.
  • a third object of the present invention is to provide a metabolic engineering E. coli strain for use in the production of succinic acid.
  • a fourth object of the present invention is to provide a method for producing succinic acid using a metabolically engineered Escherichia coli strain.
  • the present invention discloses the following technical solution: a method for constructing a metabolically engineered Escherichia coli strain using acetic acid to produce succinic acid, characterized in that the metabolically engineered Escherichia coli is fermented with acetic acid to produce succinic acid.
  • the pathway of transformation is to delete the succinate dehydrogenase complex gene to block the TCA cycle, and/or to delete key genes of the succinyl-CoA production pathway to block the succinic acid utilization pathway, and/or overexpress the acetate uptake gene.
  • the modification route is as follows: at least
  • the transformation pathway is: deletion of sdhAB, deletion of iclR and overexpression of gltA.
  • the transformation pathway is: deletion of sdhAB, deletion of iclR, deletion of maeB, overexpression of gltA.
  • the transformation pathway is: deletion of sdhAB, deletion of iclR, deletion of maeB, overexpression of aceA and overexpression of gltA.
  • the transformation pathway is: deletion of sdhAB, deletion of iclR, deletion of maeB, overexpression of acs and overexpression of gltA.
  • the transformation pathway is: deletion of sdhAB, deletion of iclR, deletion of maeB, overexpression of aceA, overexpression of acs and overexpression of gltA.
  • the present invention discloses the following technical solution: a metabolically engineered Escherichia coli strain obtained by the above construction method.
  • the present invention discloses the following technical solution: the use of the metabolically engineered Escherichia coli strain obtained by the above construction method for producing succinic acid.
  • the present invention discloses the following technical solution: a method for producing succinic acid by using the above-mentioned metabolic engineering Escherichia coli strain, characterized in that a common medium for Escherichia coli using acetic acid as a main carbon source is used. Fermentation is metabolically engineered with E. coli to obtain succinic acid.
  • the invention constructs a metabolic engineering Escherichia coli which utilizes acetic acid to produce succinic acid efficiently, that is, according to the existing letter
  • the E. coli pathway was analyzed, and the E. coli gene was transformed by genetic engineering, and its metabolism was analyzed to obtain a metabolic engineering Escherichia coli capable of producing succinic acid using acetic acid as a carbon source under aerobic conditions.
  • the modified metabolic engineering strain uses acetic acid as a raw material to produce succinic acid.
  • the method of the invention comprises the following steps: (1) using molecular biological means to strengthen the acetic acid uptake pathway and the glyoxylate cycle, and using the vectors pTrc99a and pBAD33 to express the glyoxylate cycle key enzyme genes gltA, aceA, acnB and acetic acid uptake gene acs, respectively. And ackA; (2) using red gene recombination technology to obtain genes such as sdhAB, iclR, arcA, ldhA, sfcA, maeB, sucABCD, poxB, adhE, fadR and fumC single deletion or combination deletion of host E. coli; combination (1) and (2) Construction of succinic acid to produce Escherichia coli.
  • the invention adopts wild-type Escherichia coli or other modified strains as starting strains (the wild type strain is used as a starting strain in the embodiment), and the succinyl dehydrogenase complex gene sdhAB is deleted on the one hand to block the TCA cycle.
  • succinyl dehydrogenase complex gene sdhAB is deleted on the one hand to block the TCA cycle.
  • succinic acid After the acetic acid enters the cell, it is converted into acetyl-CoA, which is metabolized by both the TCA cycle and the glyoxylate cycle. Therefore, it is possible to further select the regulatory genes iclR, arcA, and or overexpress aceA, gltA, acnB of the glyoxylate cycle and the TCA cycle.
  • sfcA and maeB reduces the decarboxylation of malic acid and maintains the supply of oxaloacetate.
  • PoxB is deleted to reduce the inefficient loop caused by the decarboxylation of pyruvic acid to produce acetic acid.
  • the byproducts of lactic acid and ethanol are deleted.
  • Another aspect of the invention is to enhance the flux of the acetic acid utilization pathway. Acetic acid was utilized by both acetyl-CoA synthetase and ack-pta, and the effects of expression of acs and ackA on the metabolism of acetic acid by E. coli were studied.
  • the invention has the advantages that: the invention modifies Escherichia coli by genetic engineering means by analyzing metabolic pathways and regulation, and the obtained strain can produce succinic acid in a medium using acetic acid as a carbon source, and there is no By-products are produced.
  • the invention is further illustrated below in conjunction with specific embodiments.
  • the experimental methods used in the following examples are conventional methods unless otherwise specified.
  • the materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It is to be understood that the examples are not intended to limit the scope of the invention.
  • the experimental methods in the following examples, which do not specify the specific conditions are usually in accordance with conventional conditions, such as the Guide to Molecular Cloning, prepared by J. Joseph Sambrook et al. The conditions described in the experiments were carried out according to the conditions recommended by the manufacturer.
  • the present invention analyzes through metabolic pathways, and in order to enhance the utilization of acetic acid, it is necessary to modify the acetic acid uptake pathway.
  • the acs and ackA were overexpressed using pBAD33 as a vector, and ligase sites were used for ligation.
  • acetic acid is primarily metabolized by the glyoxylate cycle
  • key genes overexpressing the glyoxylate cycle and the TCA cycle i.e., overexpressing gltA, aceA, and acnB, were ligated using pTrc99a as a vector.
  • the efficient accumulation of succinic acid requires blocking the TCA cycle, ie knocking out the genes sdhAB and fumC. In addition, it is also necessary to block the aerobic degradation pathway of succinic acid, that is, the succinyl-CoA production pathway, so that sucABCD is knocked out.
  • the gene deletion recombinant Escherichia coli was constructed by Red gene recombination (Folia Microbiologica, 2011, 56(3): 253-263). Using a primer with a 50 bp homology arm of the gene of interest, a kan resistance gene fragment flanked by a FRT site was amplified using pKD4 or a single deletion strain of the corresponding gene as a template. The helper plasmid pKD46 was transferred into the host strain, and the exo, bet, and gam genes which assisted homologous recombination were induced by L-arabinose at 30 ° C, and the plasmid was lost at 42 ° C for a period of time.
  • Electrotransformation promotes homologous recombination of the gene, replacing the gene of interest with the kan resistance gene.
  • the plasmid pCP20 which expresses the flip-flop recombinase (FLP) at high temperature, was transfected to eliminate the homologous recombination resistance gene on the chromosome.
  • the plasmid was a temperature-sensitive ampicillin and chloramphenicol resistant plasmid, and the FLP enzyme was induced at 42 °C. The production of the plasmid is lost.
  • the genes sdhAB, iclR, arcA, ldhA, sfcA, maeB, sucABCD, poxB, adhE, fumC, and fadR were knocked out by the red recombination technique.
  • the specific operations are as follows:
  • the primer 1 (F-sdhAB) having the sequence of SEQ ID NO: 1 and the primer 2 (R-sdhAB) having the sequence of SEQ ID NO: 2 were used, and the PCR was cloned with pKD4 as a template of about 1700 bp. DNA fragment.
  • Calcification in the host strain was introduced into plasmid pKD46, and recombinants were screened with ampicillin.
  • the recombinant strain introduced with pKD46 was cultured at 30 ° C until the OD 600 was about 0.3, and L-arabinose was added for 1 hour, and then electrotransformation was prepared using 10% glycerol.
  • Electroporation was performed using bacterial mode 1 (1.8 KV, 5 ms). Transformants that undergo homologous recombination are screened using kanamycin. Subsequently, colony PCR was performed using primer 3 (F-sdhAB-check) having the sequence of SEQ ID NO: 3 and primer 4 (R-sdhAB-check) having the sequence of SEQ ID NO: 4, knocking out successful transformants and wild bacteria
  • F-sdhAB-check primer 3
  • R-sdhAB-check primer 4
  • the size of the target fragment obtained by PCR is different, and it can be initially determined whether the recombination is successful.
  • the PCR product was sent to the sequencing.
  • the wild-type PCR gene band was identical to the sdhAB gene sequence, and the middle part of the recombinant PCR gene band was identical to the kan gene sequence, which proved that the gene was successfully knocked out.
  • Colony PCR was carried out using primer 3 (F-sdhAB-check) having the sequence of SEQ ID NO: 3 and primer 4 (R-sdhAB-check) having the sequence of SEQ ID NO: 4, and transformants and wild strains with successful resistance elimination were used.
  • the size of the target fragment obtained by PCR is significantly different, and it can be determined whether the resistance gene eliminates the successful recombination.
  • the knockout was sequentially performed according to the above method, and the primers used are shown in Table 3.
  • the strains obtained after gene knockout are shown in Table 1.
  • a single gene deletion strain of the phase was searched from the CGSC (http://cgsc.biology.yale.edu/index.php) strain library to obtain strain JW2447-5. Then, using primer 13 (F-maeB-check) of SEQ ID NO: 13 and primer 14 (F-maeB-check) of SEQ ID NO: 14, using JW2447-5 whole genome as a template, PCR cloning card Natamycin resistance gene. Other competent sdhAB knockout methods for competing preparation, electroporation and strain screening methods.
  • the knockout of the genes ldhA, adhE, fumC, fadR, sfcA, sucABCD, poxB was the same as that of the gene maeB, and the primers used are shown in Table 3.
  • the strains obtained after gene knockout are shown in Table 1.
  • Example 2 Overexpression of glyoxylate cycle key enzyme genes gltA, aceA and acnB.
  • the Escherichia coli-derived citrate synthase gene gltA was overexpressed using plasmid pTrc99a.
  • primer 35 pTrc99a-gltA-F (BamH1)
  • primer 36 pTrc99a-gltA-R-Hind3 having the sequence of SEQ ID NO: 30 were used, and Escherichia coli MG1655 was used as a template for PCR.
  • the gltA gene with the restriction site was cloned.
  • the pTrc99a and PCR-recovered target fragments were then digested with BamHI and HindIII, and the digested products were recovered and ligated using T4 ligase at 16 °C.
  • the calcium is then transformed into the strain of interest.
  • Successful recombinant strains were constructed using ampicillin resistance screening. Protein electrophoresis determines whether the protein is expressed.
  • the plasmid expressing the correct strain was sampled and sent for sequencing and recorded as pTrc99a-gltA.
  • Overexpression of aceA and acnB also used plasmid pTrc99a.
  • the method is the same as above, and is recorded as pTrc99a-aceA and pTrc99a-acnB, respectively.
  • gltA When gltA was expressed in tandem with aceA and acnB, respectively, the cleavage sites were ligated, and the obtained plasmids were designated as pTrc99a-aceA-gltA and pTrc99a-acnB-gltA, respectively.
  • the genes poxB, adhE, and ldhA were further knocked out, and strains MG042, MG043, and MG044 were obtained. Since the gene arcA significantly affected the yield of succinic acid to acetic acid, the strain MG04 was constructed by knocking out arcA in strain MG032.
  • each of the constructed plasmids was transformed with each of the constructed plasmids to obtain a recombinant strain overexpressing each gene.
  • the fermentation performance of each strain was verified by shake flask fermentation.
  • the performance of the recombinant strain was evaluated by culturing with LB medium supplemented with 10 g/l sodium acetate in a 250 ml shake flask.
  • the culture temperature was 37 ° C and the rotation speed was 200 rpm.
  • the amount of liquid is 50 ml.
  • the strain with the recombinant plasmid was induced by adding 1 mM IPTG after 2 hours of culture (OD 600 of about 0.4-0.6).
  • the cells were sampled and centrifuged at 12,000 rpm for 10 minutes to separate the cells and the supernatant. After the supernatant of the fermentation broth was appropriately diluted, it was filtered through a 0.22 um microporous membrane, and the organic acid in the supernatant of the fermentation broth was monitored by a Shimadzu high performance liquid chromatograph.
  • the column is a BioRadAminex HPX-87 ion column (300mm*7.8mm) with a UV detector and a refractive index detector.
  • the mobile phase was 5 mM H 2 SO 4 , the flow rate was 0.6 ml/min, and the column temperature was 65 °C.
  • the strain MG02 consumed 0.76 g/l of acetic acid to produce 0.01 g/l of succinic acid, and the yield was 0.01 mol/mol of acetic acid.
  • the strain MG02/pTrc99a-gltA consumed 3.31 g/l of acetic acid to produce 0.85 g/l of succinic acid, and the yield was 0.131 mol/mol of acetic acid.
  • the seed inoculated in the glycerol tube was inoculated with a tube containing 3 ml of LB overnight, and then transferred to an Erlenmeyer flask containing 50 ml of LB medium, inoculated in an amount of 2%, and the plasmid-containing strain was cultured for about 2 hours to add 1 mM.
  • IPTG induction or induction in M9 medium containing 10 g/l glucose, inoculum size 4%, induction by 5 mM IPTG after 5 hours of culture. After 5 hours of induction, the cells were collected by centrifugation at 8000 rpm for 10 min. The basic salt medium is washed once.
  • the bacteria were suspended in M9 medium without nitrogen source (ie, ammonium chloride was not added to the medium), and the carbon source was 5-10 g/l of sodium acetate. Add appropriate antibiotics and IPTG as needed in the medium.
  • the culture conditions were both 37 ° C and 220 rpm.
  • the composition of M9 medium is (per liter): Na 2 HPO 4 ⁇ 12H 2 O 15.12 g, KH 2 PO 4 3 g, NaCl 0.5 g, MgSO 4 ⁇ 7H 2 O 0.5 g, CaCl 2 0.011 g, ammonium chloride 1 g, 1% vitamin B 1 0.2 mL and trace element (TE) mixture 0.2 mL.
  • the composition of the trace element (TE) mixture is (per liter): Na 2 MoO 4 ⁇ 2H 2 O 2.0 g, FeSO 4 ⁇ 7H 2 O 80 g, MnSO 4 ⁇ H 2 O 10 g, ZnSO 4 ⁇ 7H 2 O 2.0 g, CoCl 2 4.0 g, CuCl 2 ⁇ 2H 2 O 1.0 g, H 3 BO 4 0.5 g, AlCl 3 ⁇ 6H 2 O 10 g.
  • the strain MG01 obtained by single knockout of the gene sdhAB increased the yield of succinic acid to 0.16 g/l, and the yield increased to 0.047 mol/mol of acetic acid.
  • the double-deletion strain MG02 obtained by knocking out the genes sdhAB and iclR had a slight increase in the yield of succinic acid, which was 0.19 g/l, and the yield did not change significantly.
  • the three-deletion strain MG032 which is a strain obtained by further knocking out the gene maeB on the basis of MG02, consumes 1.98 g/l of acetic acid to produce 0.75 g/l of succinic acid, and the yield is increased by 30 times, and the yield reaches 0.194 mol/mol of acetic acid.
  • the growth of the five-deletion strain MG05 was significantly inhibited, the yield of succinic acid was 0.42 g/l, and the yield was significantly increased to 0.421 mol/mol acetic acid, which was close to the theoretical yield of 0.5 mol/mol.
  • the citrate synthase gene gltA was expressed, and the strain MG02pTrc99a-gltA (see Table 6) was obtained, which consumed 1.9 g/l of acetic acid to produce 0.80 g/l of succinic acid, which was produced relative to the wild MG1655.
  • the increase was 33.3 times and the yield was further increased to 0.214 mol/mol acetic acid.
  • the combination MG032pTrc99a-gltA was optimized, 2.7 g/l acetic acid was consumed, and 1.50 g/l succinic acid was produced, and the yield was 0.283 mol/mol acetic acid.
  • the combination MG032pTrc99a-aceA-gltA was optimized and consumed 3.5 g/l of acetic acid to produce 2.2 g/l succinic acid with a yield of 0.320 mol/mol acetic acid.
  • the cells can be cultured using a basic salt medium using glucose as a carbon source.
  • the fermentation results are shown in Table 7.
  • the strain MG02pTrc99a-gltA consumed 2.34 g/l of acetic acid, yielding 0.84 g/l of succinic acid, and the yield was 0.184 mol/mol.
  • the strain MG02pTrc99a-gltA consumed 1.53 g/l of acetic acid, yielding 0.97 g/l of succinic acid, and the yield was 0.322 mol/mol.
  • the obtained recombinant Escherichia coli can produce succinic acid using acetic acid as a carbon source.
  • Example 5 Fermentation of a succinic acid producing strain in a fed batch fermentation tank
  • the fermentation performance of the above-described Succinic acid-producing bacteria was evaluated by means of fed-batch culture in a 3 L fermentor.
  • the fermentation medium was: 10 g/l peptone, 5 g/l yeast extract, 10 g/l sodium chloride, 10 g/l sodium acetate.
  • a small test tube containing 3 ml of LB medium was used, and cultured at 37 ° C for about 9 hours, and the resulting culture was used as a primary seed. Then, the primary seed was transferred to a flask containing 30 ml of LB, and the inoculum amount was 2%. After 2 hours of culture, 1 mM IPTG was added for 7 hours to induce secondary seed. The secondary seed is introduced into the 3L fermentor to start fermentation. The fermentation temperature was 37 ° C, the inoculum amount was 2%, and stirring and aeration were adjusted to maintain the dissolved oxygen higher than the 30% saturated oxygen concentration. The pH was adjusted to 7.0 using 4M sodium hydroxide and 2M sulfuric acid.
  • the strain MG032pTrc99a-gltA was fermented for 56 hours, and the cells were grown to an OD600 of 10.2 to produce 5 g/l of succinic acid.

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Abstract

一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法和应用，代谢工程改造的大肠杆菌使用乙酸为原料发酵生产丁二酸，其改造途径为阻断TCA循环，并和/或阻断丁二酸利用途径，并和/或增强乙酸摄取和草酰乙酸供给，强化乙醛酸循环，和/或缺失副产物生成途径，和/或减少从丙酮酸脱羧生产乙酸引起的无效循环，和/或疏导乙酰CoA节点代谢流。通过对代谢途径和调控的分析，利用基因工程手段对大肠杆菌进行了改造，获得的菌株在以乙酸为碳源的培养基中，能够产生丁二酸，且没有副产物产生。

Description

利用乙酸生产丁二酸的代谢工程大肠杆菌菌株构建方法和应用 技术领域
本发明属于生物工程技术领域，更具体地讲，涉及构建利用乙酸生产丁二酸的重组大肠杆菌菌株，以及通过乙酸为主要原料利用该代谢工程大肠杆菌发酵合成丁二酸。
背景技术
丁二酸，又名琥珀酸，是一种四碳二羧酸，广泛应用于农业、食品及医药行业。丁二酸是许多重要工业化学品的前体，包括已二酸，1,4-丁二醇，四氢呋喃，N-甲基吡咯烷酮，2-吡咯烷酮，丁二酸盐及γ-丁内酯。还可用于合成可生物降解的聚合物聚丁二酸丁二醇酯(PBS)及聚酰胺聚合物
Figure PCTCN2014094215-appb-000001
目前，商业用丁二酸已经由液化石油气或矿物油经化学合成生产，转变为生物发酵生产(Catal.Today(2014),http://dx.doi.org/10.1016/j.cattod.2014.05.035)，利用可再生资源通过生物发酵法生产丁二酸比石油炼制更具经济和社会效益，因此受到广泛关注(Enzyme and microbial technology,2006,39(3):352-361)。
丁二酸生产菌株主要为产丁二酸厌氧螺菌Anaerobiospirillum succiniciproducens，产丁二酸放线杆菌Actinobacillus succinogenes，曼海姆产丁二酸菌Mannheimia succiniciproducens，重组大肠杆菌Escherichia coli，酿酒酵母Saccharomyce scerevisiae(Metab Eng,2010,12(6):518-525)，谷氨酸棒杆菌Corynebacterium glutamicum(PLoS One,2013,8(4):e60659)等。其中大肠杆菌由于基因遗传背景清楚，基因操作简单，同时发酵底物多为可再生资源，如葡萄糖，木糖，甘油，麦草、玉米秸秆等木质纤维素，木薯、菊芋等淀粉类物质，而更具有工业价值。
利用重组大肠杆菌发酵丁二酸的生产，大量工作针对葡萄糖的代谢途径改造，包括在好氧和厌氧条件下葡萄糖到丁二酸的代谢流引导。San等(Biotechnol Bioeng,2005,90(6):775-779)构建的大肠杆菌HL27658k(pKK313)，阻断TCA循环，切除副产物形成途径，解除乙醛酸支路阻遏，实现葡萄糖好氧转化积累丁二酸58.3g/L，得率为0.94±0.07mol/mol葡萄糖，生产强度为1.08±0.06g/(L.h)。Zhao等(Journal of Industrial Microbiology&Biotechnology,2013,40(12):1461-1475) 构建的大肠杆菌E2-Δsdh-ppc-sucAB，阻断TCA循环和过表达磷酸烯醇式丙酮酸羧化酶、琥珀酰辅酶A合成酶的改造下，在甘油基本盐培养基中，丁二酸的最大体积生产速率和平均体积生产速率分别为19.2和6.55mM h-1。
乙酸主要由甲醇的羰基化制备，即甲醇和一氧化碳制备，是一种大量而廉价的碳源，同时也是木质纤维素材料酸水解中形成的主要副产物之一。乙酸是许多微生物厌氧代谢的终产物和有氧代谢的不完全氧化产物，大肠杆菌可以好氧代谢乙酸进行生长，并且以乙酸为唯一碳源好氧生长时，乙醛酸支路和糖异生途径的关键酶活性会得到显著提高，有利于随后进行葡萄糖到丁二酸的厌氧转化(Applied and Environmental Microbiology,2007,73(24):7837-7843.)。但是目前为止尚未有采用乙酸作为碳源直接生物合成丁二酸的研究报道。本发明从增强底物摄取、阻断下游分解代谢和疏导代谢流等多方面对大肠杆菌进行改造，使其能够直接利用乙酸为碳源生产丁二酸。
发明内容
本发明的第一个目的在于提供一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法。
本发明的第二个目的在于提供一种代谢工程大肠杆菌菌株。
本发明的第三个目的在于提供一种代谢工程大肠杆菌菌株在生产丁二酸中的应用。
本发明的第四个目的在于提供一种利用代谢工程大肠杆菌菌株生产丁二酸的方法。
为实现以上目的，本发明公开以下技术方案：一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法，其特征在于，代谢工程改造的大肠杆菌使用乙酸为原料发酵生产丁二酸，其改造途径为缺失丁二酸脱氢酶复合物基因以阻断TCA循环，并和/或缺失琥珀酰CoA生成途径关键基因以阻断丁二酸利用途径，并和/或过表达乙酸摄取基因、和/或缺失乙醛酸循环和TCA循环的调节基因、和/或过表达乙醛酸循环和TCA循环关键酶基因以增强乙酸摄取和草酰乙酸供给，强化乙醛酸循环，和/或减少苹果酸的脱羧反应以缺失副产物生成途径，和/或减少从丙酮酸脱羧生产乙酸引起的无效循环，和/或缺失乳酸和乙 醇生产途径中关键基因以疏导乙酰CoA节点代谢流。
作为一个优选方案，其改造途径为：至少包括
(1)缺失sdhAB
以及以下途径中的一种或几种：
(2)缺失sucABCD和/或fumC；
(3)过表达acs和/或ackA；
(4)缺失iclR、fadR和/或arcA；
(5)过表达gltA和/或aceA和/或acnB；
(6)缺失sfcA和/或maeB；
(7)缺失poxB；
(8)缺失adhE和/或ldhA。
作为一个优选方案，其改造途径为：缺失sdhAB，缺失iclR和过表达gltA。
作为一个优选方案，其改造途径为：缺失sdhAB，缺失iclR，缺失maeB，过表达gltA。
作为一个优选方案，其改造途径为：缺失sdhAB，缺失iclR，缺失maeB，过表达aceA和过表达gltA。
作为一个优选方案，其改造途径为：缺失sdhAB，缺失iclR，缺失maeB，过表达acs和过表达gltA。
作为一个优选方案，其改造途径为：缺失sdhAB，缺失iclR，缺失maeB，过表达aceA，过表达acs和过表达gltA。
为实现本发明第二个目的，本发明公开以下技术方案：利用上述构建方法得到的代谢工程大肠杆菌菌株。
为实现本发明第三个目的，本发明公开以下技术方案：利用上述构建方法得到的代谢工程大肠杆菌菌株在生产丁二酸中的应用。
为实现本发明第四个目的，本发明公开以下技术方案：一种利用上述代谢工程大肠杆菌菌株生产丁二酸的方法，其特征在于，利用以乙酸为主要碳源的大肠杆菌常用培养基，发酵代谢工程大肠杆菌，得到丁二酸。
本发明构建高效利用乙酸生产丁二酸的代谢工程大肠杆菌，即根据已有信 息对大肠杆菌途径进行分析，利用基因工程手段对大肠杆菌基因进行改造，并对其代谢情况进行分析，获得好氧条件下能够以乙酸作为碳源生产丁二酸的代谢工程大肠杆菌，并利用改造后的代谢工程菌株以乙酸为原料生产丁二酸。
本发明的方法：(1)采用分子生物学手段，对乙酸摄取途径和乙醛酸循环进行加强，分别使用载体pTrc99a和pBAD33表达乙醛酸循环关键酶基因gltA、aceA、acnB和乙酸摄取基因acs和ackA；(2)利用red基因重组技术获得基因sdhAB、iclR、arcA、ldhA、sfcA、maeB、sucABCD、poxB、adhE、fadR和fumC等单缺失或组合缺失的宿主大肠杆菌；组合(1)和(2)构建丁二酸生产大肠杆菌。
本发明是以野生型大肠杆菌或已有其他改造的菌株为出发菌株(实施例用的是野生型菌株为出发菌株)，一方面缺失丁二酸脱氢酶复合物基因sdhAB，阻断TCA循环以达到积累丁二酸的目的。乙酸进入细胞后被转化为乙酰辅酶A，乙酰辅酶A通过TCA循环和乙醛酸循环两种途径被代谢。故而可以选择进一步缺失乙醛酸循环和TCA循环的调节基因iclR、arcA，和或过表达aceA、gltA、acnB。缺失sfcA和maeB可以减少苹果酸的脱羧反应，维持草酰乙酸的供给。缺失poxB来减少从丙酮酸脱羧生产乙酸引起的无效循环。同时，缺失乳酸和乙醇的副产物产生途径。本发明的另一方面是增强乙酸利用途径的通量。乙酸通过乙酰辅酶A合成酶和ack-pta两种途径被利用，研究过表达acs和ackA对大肠杆菌代谢乙酸的影响。
本发明的优点在于：本发明通过对代谢途径和调控的分析，利用基因工程手段对大肠杆菌进行了改造，获得的菌株在以乙酸为碳源的培养基中，能够产生丁二酸，且没有副产物产生。
具体实施方式
下面结合具体实施例，进一步阐述本发明。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。应理解，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，例如J.萨姆布鲁克(Joseph Sambrook)等编写的《分子克隆实验指南》 中所述的条件，或按照制造厂商所推荐的条件进行实验。
本发明通过代谢途径分析，为了加强乙酸的利用，需要对乙酸摄取途径进行改造。采用pBAD33作为载体过表达acs及ackA，使用酶切位点进行连接。
由于乙酸主要通过乙醛酸循环进行代谢，需要加强流向乙醛酸循环的代谢通量。一方面，需要敲除乙醛酸循环及TCA循环的调节基因，解除调节基因对代谢流的抑制，即敲除iclR、fadR、arcA。另一方面，过表达乙醛酸循环及TCA循环的关键基因，即过表达gltA、aceA和acnB，采用pTrc99a作为载体，使用酶切位点进行连接。
琥珀酸的高效积累，需要阻断TCA循环，即敲除基因sdhAB、fumC。另外，还需阻断琥珀酸的有氧降解途径，即琥珀酰CoA生成途径，故敲除sucABCD。
为减少苹果酸的脱羧反应，维持草酰乙酸的供给，缺失sfcA和maeB。
避免由丙酮酸脱羧生成乙酸，形成无效循环，缺失poxB。
敲除可能的代谢副产物途径，使代谢流流向目标产物，同时避免代谢副产物的生成，节约下游分离成本，故而敲除乳酸和乙醇产生途径中的关键基因ldhA和adhE。
上述1-8项中每一项均作为改造的方向，但每一项中不是全部进行改造。多种组合仅是为了提高生产效率和产量。
采用Red基因重组的方法构建基因缺失重组大肠杆菌(Folia Microbiologica,2011,56(3):253-263)。使用带有目的基因50bp同源臂的引物，以pKD4或相应基因的单缺失菌为模板扩增两侧带有FRT位点的kan抗性基因片段。宿主菌中转入辅助质粒pKD46，30℃下以L-阿拉伯糖诱导表达协助同源重组的exo、bet、gam基因，42℃下培养一段时间质粒丢失。电转化促使基因发生同源重组，将目的基因替换为kan抗性基因。最后转入高温诱导表达翻转酶重组酶(FLP)的质粒pCP20，消除染色体上同源重组的抗性基因，该质粒是温度敏感型氨苄青霉素和氯霉素抗性质粒，42℃时诱导FLP酶的产生，同时质粒丢失。
实施例1.敲除基因以获得丁二酸生产菌株
采用red重组技术敲除基因sdhAB、iclR、arcA、ldhA、sfcA、maeB、sucABCD、poxB、adhE、fumC、fadR。具体操作如下：
对于基因sdhAB的敲除，首先用序列为SEQ ID NO:1的引物1(F-sdhAB)和序列为SEQ ID NO:2的引物2(R-sdhAB)，以pKD4为模板PCR克隆约1700bp的DNA片段。宿主菌中钙转化导入质粒pKD46，以氨苄青霉素筛选重组子。导入pKD46的重组菌30℃培养至OD600约为0.3时，加入L-阿拉伯糖诱导1小时，然后使用10％的甘油制备电转感受态。电转化采用细菌模式1(1.8KV，5ms)进行。使用卡那霉素筛选发生同源重组的转化子。随后使用序列为SEQ ID NO:3的引物3(F-sdhAB-check)和序列为SEQ ID NO:4的引物4(R-sdhAB-check)进行菌落PCR，敲除成功的转化子与野生菌PCR得到的目的片段大小有差异，能够初步判定是否重组成功。PCR产物送测序，野生菌PCR基因条带与sdhAB基因序列相同，而重组菌PCR基因条带中间部分与kan基因序列相同，则证明基因确实敲除成功。
sdhAB敲除成功的重组菌，37℃培养5-6小时后，转入42℃培养过夜，分离单菌落，然后验证抗性。只有卡那霉素抗性无氨苄青霉素抗性的为pKD46已消除的菌落。然后在该菌中转入质粒pCP20，30℃培养一段时间后，转入42℃培养过夜，分离单菌落，然后验证抗性。挑取只在无抗性平板上生长，而卡那霉素抗性平板和氨苄青霉素抗性平板上均不生长的菌落进行鉴定。使用序列为SEQ ID NO:3的引物3(F-sdhAB-check)和序列为SEQ ID NO:4的引物4(R-sdhAB-check)进行菌落PCR，抗性消除成功的转化子与野生菌PCR得到的目的片段大小有明显差异，能够判定抗性基因是否消除重组成功。
对于iclR、arcA基因，按照上述方法依次敲除，所用引物见表3。基因敲除后获得的菌株见表1。
对于基因maeB的敲除，从CGSC(http://cgsc.biology.yale.edu/index.php)菌种库中查找相的单基因缺失菌，得到菌株JW2447-5。然后使用序列为SEQ ID NO:13的引物13(F-maeB-check)和序列为SEQ ID NO:14的引物14(F-maeB-check)，以JW2447-5全基因组为模板，PCR克隆卡那霉素抗性基因。其他感受态制备、电转化及菌株筛选方法同基因sdhAB敲除方法。
基因ldhA、adhE、fumC、fadR、sfcA、sucABCD、poxB的敲除，与基因maeB的敲除方法相同，所用引物见表3。基因敲除后获得的菌株见表1。
实施例2.过表达乙醛酸循环关键酶基因gltA、aceA和acnB。
大肠杆菌来源的柠檬酸合成酶基因gltA使用质粒pTrc99a过量表达。首先使用序列为SEQ ID NO:29的引物35(pTrc99a-gltA-F(BamH1))和序列为SEQ ID NO:30的引物36(pTrc99a-gltA-R-Hind3)，以大肠杆菌MG1655为模板PCR克隆带有酶切位点的gltA基因。然后使用BamHⅠ和HindⅢ对pTrc99a和PCR回收的目的片段双酶切，酶切产物回收后，使用T4连接酶16℃连接。然后钙转化进入目的菌株中。使用氨苄青霉素抗性筛选构建成功的重组菌。蛋白电泳确定蛋白是否表达。表达正确的菌株抽质粒，送测序，记为pTrc99a-gltA。
aceA及acnB的过量表达同样使用质粒pTrc99a。方法同上，分别记为pTrc99a-aceA、pTrc99a-acnB。
gltA分别与aceA、acnB串联表达时，以酶切位点进行连接，得到的质粒分别记为pTrc99a-aceA-gltA、pTrc99a-acnB-gltA。
过表达acs及ackA采用载体pBAD33，该质粒能与pTrc99a同时存在于大肠杆菌中。重组质粒的构建方法同上。构建成功的质粒见表2。
表1菌种
Figure PCTCN2014094215-appb-000002
Figure PCTCN2014094215-appb-000003
表2质粒
Figure PCTCN2014094215-appb-000004
表3引物列表
Figure PCTCN2014094215-appb-000005
Figure PCTCN2014094215-appb-000006
Figure PCTCN2014094215-appb-000007
实施例3.产丁二酸菌株复合培养基摇瓶发酵
以大肠杆菌MG1655为出发菌株，敲除基因sdhAB，阻断TCA循环，得到产琥珀酸基础菌株MG01。
在单缺失菌MG01的基础上，分别敲除基因iclR、fadR、arcA和fumC,获得菌株MG02、MG022、MG023、MG024。经比较发现，MG02效果最优，故接下来以MG02为出发菌株。
在双缺失菌MG02的基础上，验证阻断补给途径和激活TCA循环对发酵产琥珀酸的影响，故又分别敲除基因arcA、maeB、sfcA,获得菌株MG03、MG032、MG033。发酵结果表明，MG032效果最优，故以MG032为基础进行后续改造。
为了解缺失副产物生成途径对琥珀酸发酵的影响，又进一步敲除了基因poxB、adhE、ldhA，获得菌株MG042、MG043、MG044。由于基因arcA显著影响琥珀酸对乙酸的得率，故在菌株MG032中敲除了arcA，构建了菌株MG04。
在MG042的基础上敲除基因arcA,得到菌株MG05，敲除基因adhE和ldhA 得到菌株MG06。
上述菌株构建完成后，保存于甘油中(25％v/v)。并应用构建完成的各种质粒转化各缺失菌株，获得过表达各基因的重组菌。摇瓶发酵验证各菌株的发酵性能。
使用添加10g/l乙酸钠的LB培养基于250ml摇瓶中培养以评价重组菌的性能。培养温度37℃，转速200rpm。装液量50ml。根据需要加入卡那霉素或者氨苄青霉素。带有重组质粒的菌株，培养2小时(OD600约为0.4-0.6)后，加入1mM IPTG诱导。
培养结束后取样，12000rpm离心10分钟分离菌体和上清。发酵液上清适当稀释后，经0.22um微孔膜过滤，采用日本岛津高效液相色谱仪监测发酵液上清中的有机酸。色谱柱为BioRadAminex HPX-87离子色谱柱(300mm*7.8mm)，配有UV检测器和示差折光检测器。流动相为5mM的H2SO4，流速0.6ml/min，柱温65℃。
生物量的测定采用分光光度计法测定600nm下的吸光值。
该培养条件下野生菌MG1655消耗7.11g/l乙酸，没有琥珀酸产生。
该培养条件下菌株MG1655pTrc99a-gltA，没有琥珀酸产生。
该培养条件下菌株MG02消耗0.76g/l乙酸，生成0.01g/l丁二酸，得率为0.01mol/mol乙酸。
而该培养条件下菌株MG02/pTrc99a-gltA消耗3.31g/l乙酸，生成0.85g/l丁二酸，得率为0.131mol/mol乙酸。
表4不同菌种复合培养基摇瓶发酵结果
Figure PCTCN2014094215-appb-000008
ND：未检测到产物。
实施例4.乙酸生物转化生产丁二酸
为了提高琥珀酸的生产速率，采用生物转化法摇瓶培养验证各菌株发酵性 能。具体操作如下：
保存在甘油管中的种子接种与装有3ml LB的试管中培养过夜，然后转接到装有50ml LB培养基的锥形瓶中，接种量2％，含有质粒的菌株培养约2小时加入1mM IPTG诱导(或转接于含10g/l葡萄糖的M9培养基中，接种量4％，培养5小时后加入1mM IPTG诱导)。诱导5小时后，8000rpm离心10min收集菌体。基本盐培养基洗涤一次。菌体重悬于不含氮源的M9培养基(即培养基中不添加氯化铵)中，碳源为5-10g/l的乙酸钠。培养基中根据需要加入适当的抗生素和IPTG。培养条件均为37℃，220rpm。
M9培养基组成为(每升)：Na2HPO4·12H2O 15.12g，KH2PO43g，NaCl 0.5g，MgSO4·7H2O 0.5g，CaCl20.011g，氯化铵1g，1％维生素B10.2mL以及微量元素(TE)混合液0.2mL。微量元素(TE)混合液组成为(每升)：Na2MoO4·2H2O 2.0g，FeSO4·7H2O 80g，MnSO4·H2O 10g，ZnSO4·7H2O 2.0g，CoCl24.0g，CuCl2·2H2O1.0g，H3BO40.5g，AlCl3·6H2O 10g。
有机酸的测定方法及生物量的测定同实施例3。
摇瓶发酵结果如表5、6所示。
由表5可见，以LB培养菌体进行转化实验时，野生型大肠杆菌MG1655只产生0.024g/l琥珀酸，得率仅为0.003mol/mol乙酸。
经过改造，单敲除基因sdhAB得到的菌株MG01，琥珀酸产量上升至0.16g/l，得率上升至0.047mol/mol乙酸。敲除基因sdhAB和iclR得到的双缺失菌株MG02，琥珀酸的产量有轻微上升，为0.19g/l，得率没有明显改变。
而三缺失菌株MG032，即在MG02基础上进一步敲除基因maeB获得的菌株，消耗1.98g/l乙酸，生成0.75g/l琥珀酸，产量提高30倍，得率达到0.194mol/mol乙酸。
在MG032的基础上，进一步敲除基因poxB得到菌株MG042，相比野生菌MG1655，琥珀酸的产量和得率均有明显上升，分别为0.67g/l和0.169mol/mol，但是并没有优于菌株MG032。
五缺失菌株MG05的生长受到明显的抑制，琥珀酸的产量为0.42g/l，而得率明显上升，为0.421mol/mol乙酸，接近理论得率0.5mol/mol。
在菌株MG02的基础上，表达柠檬酸合成酶基因gltA，获得菌株MG02pTrc99a-gltA(见表6)，该菌株消耗1.9g/l乙酸，生成0.80g/l琥珀酸，相对于野生菌MG1655，产量提高了33.3倍，得率进一步提高到0.214mol/mol乙酸。
优化组合MG032pTrc99a-gltA，消耗2.7g/l乙酸，生成1.50g/l琥珀酸，得率为0.283mol/mol乙酸。
优化组合MG032pTrc99a-aceA-gltA，消耗3.5g/l乙酸，生成2.2g/l琥珀酸，得率为0.320mol/mol乙酸。
优化组合MG032pTrc99a-aceA-gltA pBAD-acs，消耗3.6g/l乙酸，生成2.6g/l琥珀酸，得率为0.367mol/mol乙酸。
其他菌株发酵结果见表5、6。
除了使用LB为培养基培养菌体外，还可使用以葡萄糖为碳源的基本盐培养基培养菌体。发酵结果如表7所示。
此培养条件下，菌株MG02pTrc99a-gltA消耗乙酸2.34g/l，产生琥珀酸0.84g/l，得率为0.184mol/mol。菌株MG02pTrc99a-gltA消耗乙酸1.53g/l，产生琥珀酸0.97g/l，得率为0.322mol/mol。
综上所述，获得的重组大肠杆菌能够以乙酸为碳源生产丁二酸。
表5以LB培养菌体进行转化实验摇瓶结果1
Figure PCTCN2014094215-appb-000009
表6以LB培养菌体进行转化实验摇瓶结果2
Figure PCTCN2014094215-appb-000010
表7以葡萄糖培养菌体进行转化实验摇瓶结果
Figure PCTCN2014094215-appb-000011
实施例5.产丁二酸菌株在补料分批发酵罐中的发酵
使用补料分批培养的方式，在3L发酵罐中培养以评价上述产丁二酸菌的发酵性能。
发酵培养基为：10g/l蛋白胨、5g/l酵母浸提物、10g/l氯化钠、10g/l乙酸钠。
菌株预培养时，使用装有3ml LB培养基的小试管，37℃培养9小时左右，所得培养物作为一级种子。然后将一级种子转接于装有30ml LB的三角瓶中，接种量2％，培养2小时左右加入1mM IPTG诱导7小时作为二级种子。二级种子接入3L发酵罐中开始发酵。发酵温度37℃，接种量2％，调节搅拌和通气以保持溶解氧高于30％饱和氧浓度。使用4M氢氧化钠及2M硫酸调节pH为7.0。发酵初始，根据需要添加适当抗生素。培养物培养至OD600为2左右时，加入1mM IPTG诱导。发酵过程中补加乙酸，控制残乙酸浓度不高于5g/l。
菌株MG032pTrc99a-gltA发酵56小时，菌体生长至OD600为10.2，产生琥珀酸5g/l。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法，其特征在于，代谢工程改造的大肠杆菌使用乙酸为原料发酵生产丁二酸，其改造途径为缺失丁二酸脱氢酶复合物基因以阻断TCA循环，并和/或缺失琥珀酰CoA生成途径关键基因以阻断丁二酸利用途径，并和/或过表达乙酸摄取基因、和/或缺失乙醛酸循环和TCA循环的调节基因、和/或过表达乙醛酸循环和TCA循环关键酶基因以增强乙酸摄取和草酰乙酸供给，强化乙醛酸循环，和/或减少苹果酸的脱羧反应以缺失副产物生成途径，和/或减少从丙酮酸脱羧生产乙酸引起的无效循环，和/或缺失乳酸和乙醇生产途径中关键基因以疏导乙酰CoA节点代谢流。
  2. 根据权利要求1所述的一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法，其特征在于，其改造途径为：至少包括
    (1)缺失sdhAB
    以及以下途径中的一种或几种：
    (2)缺失sucABCD和/或fumC；
    (3)过表达acs和/或ackA；
    (4)缺失iclR、fadR和/或arcA；
    (5)过表达gltA和/或aceA和/或acnB；
    (6)缺失sfcA和/或maeB；
    (7)缺失poxB；
    (8)缺失adhE和/或ldhA。
  3. 根据权利要求2所述的一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法，其特征在于，其改造途径为：
    缺失sdhAB，缺失iclR和过表达gltA。
  4. 根据权利要求2所述的一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法，其特征在于，其改造途径为：
    缺失sdhAB，缺失iclR，缺失maeB和过表达gltA。
  5. 根据权利要求2所述的一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法，其特征在于，其改造途径为：
    缺失sdhAB，缺失iclR，缺失maeB，过表达aceA和过表达gltA。
  6. 根据权利要求2所述的一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法，其特征在于，其改造途径为：
    缺失sdhAB，缺失iclR，缺失maeB，过表达acs和过表达gltA。
  7. 根据权利要求2所述的一种利用乙酸生产丁二酸的代谢工程大肠杆菌菌株的构建方法，其特征在于，其改造途径为：
    缺失sdhAB，缺失iclR，缺失maeB，过表达aceA，过表达acs和过表达gltA。
  8. 利用权利要求1或2所述的构建方法得到的代谢工程大肠杆菌菌株。
  9. 利用权利要求1或2所述的构建方法得到的代谢工程大肠杆菌菌株在生产丁二酸中的应用。
  10. 一种利用权利要求8所述代谢工程大肠杆菌菌株生产丁二酸的方法，其特征在于，利用以乙酸为主要碳源的大肠杆菌常用培养基，发酵权利要求8所述代谢工程大肠杆菌，得到丁二酸。
PCT/CN2014/094215 2014-10-30 2014-12-18 利用乙酸生产丁二酸的代谢工程大肠杆菌菌株构建方法和应用 Ceased WO2016065709A1 (zh)

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