CN110885777A - 一种利用BmoR生物传感器筛选异丁醇高产菌株的方法 - Google Patents
一种利用BmoR生物传感器筛选异丁醇高产菌株的方法 Download PDFInfo
- Publication number
- CN110885777A CN110885777A CN201910765520.4A CN201910765520A CN110885777A CN 110885777 A CN110885777 A CN 110885777A CN 201910765520 A CN201910765520 A CN 201910765520A CN 110885777 A CN110885777 A CN 110885777A
- Authority
- CN
- China
- Prior art keywords
- isobutanol
- screening
- bmor
- yield
- biosensor
- 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.)
- Pending
Links
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 title claims abstract description 104
- 238000012216 screening Methods 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 28
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 230000035772 mutation Effects 0.000 claims abstract description 11
- QHKABHOOEWYVLI-UHFFFAOYSA-N 3-methyl-2-oxobutanoic acid Chemical compound CC(C)C(=O)C(O)=O QHKABHOOEWYVLI-UHFFFAOYSA-N 0.000 claims abstract description 9
- 101100162204 Aspergillus parasiticus (strain ATCC 56775 / NRRL 5862 / SRRC 143 / SU-1) aflH gene Proteins 0.000 claims abstract description 7
- 101150024743 adhA gene Proteins 0.000 claims abstract description 7
- 108700008625 Reporter Genes Proteins 0.000 claims abstract description 3
- 239000013612 plasmid Substances 0.000 claims description 15
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 238000002703 mutagenesis Methods 0.000 claims description 6
- 231100000350 mutagenesis Toxicity 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 5
- 238000005457 optimization Methods 0.000 claims description 4
- 108020004705 Codon Proteins 0.000 claims description 3
- 230000010076 replication Effects 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 15
- 230000004151 fermentation Effects 0.000 description 14
- 238000000855 fermentation Methods 0.000 description 14
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 12
- 239000005090 green fluorescent protein Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000004817 gas chromatography Methods 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 5
- 230000037353 metabolic pathway Effects 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 150000001298 alcohols Chemical class 0.000 description 3
- 239000002551 biofuel Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000012269 metabolic engineering Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 2
- 101150078509 ADH2 gene Proteins 0.000 description 2
- 101150026777 ADH5 gene Proteins 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101100098786 Bacillus subtilis (strain 168) tapA gene Proteins 0.000 description 2
- 101100321116 Escherichia coli (strain K12) yqhD gene Proteins 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010000700 Acetolactate synthase Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 101100460671 Aspergillus flavus (strain ATCC 200026 / FGSC A1120 / IAM 13836 / NRRL 3357 / JCM 12722 / SRRC 167) norA gene Proteins 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 101100269269 Drosophila mayaguana Adh gene Proteins 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 101100054943 Mus musculus Adh4 gene Proteins 0.000 description 1
- 101100125907 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) ilvC1 gene Proteins 0.000 description 1
- 241001670068 Thauera butanivorans Species 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000012824 chemical production Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- ADHFGLVXSIGCIG-UHFFFAOYSA-N diazanium sulfate hydrochloride Chemical compound [NH4+].[NH4+].Cl.[O-]S([O-])(=O)=O ADHFGLVXSIGCIG-UHFFFAOYSA-N 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000002735 gasoline substitute Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 101150090497 ilvC gene Proteins 0.000 description 1
- 101150043028 ilvD gene Proteins 0.000 description 1
- 101150105723 ilvD1 gene Proteins 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 238000000424 optical density measurement Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Optics & Photonics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种利用BmoR生物传感器筛选异丁醇高产菌株的方法,包括以下步骤:步骤1、构建BmoR生物传感器:构建以gfp为报告基因的BmoR生物传感器;步骤2、构建高产菌株的筛选体系:共表达BmoR生物传感器与2‑酮异戊酸合成异丁醇的基因kivd和adhA,通过检测荧光强度来筛选高产菌株;步骤3、筛选高产菌株:对出发菌株进行突变文库的构建,利用步骤2构建的筛选系统筛选出异丁醇高产菌株并进一步优化提高产量。利用本发明的方法筛选出的异丁醇高产菌株其产量可以达到野生型的2倍。
Description
技术领域
本发明涉及生物工程技术领域,特别涉及一种利用BmoR生物传感器筛选异丁醇高产菌株的方法。
背景技术
随着全球的能源和环境问题的逐步恶化,绿色环保的可再生生物燃料已经日益受到重视。与传统生物燃料乙醇相比,高级醇作为汽油替代品具有高能量密度和低吸湿性等优势,因而有望成为新一代生物燃料。随着代谢工程与合成生物学的发展,通过设计基因线路,代谢途径来改造微生物宿主使其合成人类所需产物,已经逐渐取代了低效率和高污染的化工生产。高级醇的生物合成近几年来受到广泛关注,已经在很多微生物宿主中通过不同的策略实现。异丁醇作为支链高级醇,相较于直链醇(正丁醇)具有更高的辛烷值,从而会产生更高的热效应,因而异丁醇的生物合成受到了更加广泛的关注和应用。异丁醇的合成前体来源于缬氨酸合成途径的2-酮异戊酸(KIV),2008年James Liao教授课题组通过挖掘来自枯草芽孢杆菌(Bacillus subtilis)中乙酰乳酸合酶AlsS,来自乳酸菌(Lactococcuslactis)的脱羧酶KivD和醇脱氢酶Adh2,并过表达大肠杆菌自身基因ilvC和ilvD,构建整条从葡萄糖到异丁醇的生物合成途径到宿主JCL260中。并对宿主进行耐受性改造,最终得到了异丁醇耐受菌株SA481,其异丁醇的产量在112h达到22g/L,达到理论产率的86%。2010年该课题组的Shota Atsumi等人通过比较三种来源于不同物种的异丁醇合成途径最后一步的基因yqhD(大肠杆菌),adh2(酿酒酵母)和adhA(乳酸菌),最终发现在大肠基因组表达yqhD和高拷贝质粒共表达adhA时产量最高,超过8g/L。在此基础上2011年通过发酵罐实验,在1-L发酵罐中通过不断补加葡萄糖进行连续发酵,最终产量达到50.8±1.1g/L。尽管目前异丁醇已经实现了较高的产量,但是仍然无法满足工业生产的需求。
随着代谢工程与合成生物学的发展,通过改造宿主,挖掘高性能的酶以及代谢途径调控是提高目标化合物产量的常用手段。改造宿主是代谢工程中必不可少的步骤,通常通过物理的紫外线诱变(UV),化学的亚硝基胍(NTG)诱变以及近几年兴起的常温常压等离子体诱变(ARTP)诱变方法等方法构建宿主突变文库,但是如何在这些广泛的文库中确定最好的变体是一个缓慢和繁琐的工程。传统筛选依赖于气相色谱(GC),高效液相色谱(HPLC),超高效液相色谱(UPLC),气相色谱-质谱(GC-MS)以及液相色谱-质谱(LC-MS)等检测方法,都可以精确地定性和定量代谢产物,但是限于成本和时间的消耗,这样的技术不能以高通量的方式进行。生物传感器生物传感器(Biosensor)作为一种合成生物学中新兴的工具,是一种基于自身基因线路编码的组件。通过设计和构建生物传感器,可以使其动态地响应信号物质浓度的变化从而输出便于检测的蛋白信号,因此生物传感器是一种快速和高通量评估候选途径变体的有前景的工具。已经广泛应用于高产菌株筛选或富集,代谢途径中相关酶的高通量筛选,以及代谢途径调控等方面。
BmoR是假单胞杆菌(Pseudomonas butanovora)的正烷烃代谢途径中的转录因子,调控烷烃单加氧酶的σ54依赖性启动子Pbmo,其信号分子时包括正丁醇和异丁醇在内的多种高级醇。在之前的研究中,Jay D.Keasling等人已经将BmoR成功应用于大肠杆菌中筛选正丁醇高产菌株,但是还没有通过BmoR生物传感器进行异丁醇高产菌株的筛选。
发明内容
本发明要解决的技术问题是提供一种利用BmoR生物传感器筛选异丁醇高产菌株的方法,以解决现有技术中异丁醇产量无法满足工业生产的需求。
为了解决上述技术问题,本发明的技术方案为:
一种利用BmoR生物传感器筛选异丁醇高产菌株的方法,包括以下步骤:
步骤1、构建BmoR生物传感器:构建以gfp为报告基因的BmoR生物传感器;
步骤2、构建高产菌株的筛选体系:共表达BmoR生物传感器与2-酮异戊酸合成异丁醇的基因kivd和adhA,通过检测荧光强度来筛选异丁醇高产菌株;
步骤3、筛选高产菌株:对出发菌株进行突变文库的构建,利用步骤2构建的筛选系统筛选出异丁醇高产菌株并进一步优化提高产量。
进一步的,所述步骤1具体包括:
将bmor基因经过大肠杆菌中密码子优化后通过PCR合成Pbmo,Pbmo合成之后连接gfp基因,构建含有不同复制起点的生物传感器质粒,所述生物传感器质粒即Bmor生物传感器。
进一步的,所述步骤3具体包括:
通过ARTP诱变的方法对出发菌株进行突变文库的构建,并对突变库中的突变体进行荧光筛选得到异丁醇高产菌株。
采用上述技术方案,利用本发明的方法筛选出的异丁醇高产菌株其产量可以达到野生型的2倍。通过进一步对高产菌株进行优化,可以将其异丁醇产量提高至14g/L。
附图说明
图1为本发明的方法中图BmoR生物传感器的外源添加验证和体内验证流程图;
图2为本发明的方法中图BmoR生物传感器的筛选原理图;
图3为本发明的方法中基于BmoR生物传感器从出发菌株的突变库筛选的流程;
图4为本发明的方法中异丁醇高产菌株的筛选和验证图。
具体实施方式
下面结合附图对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
一种利用BmoR生物传感器筛选异丁醇高产菌株的方法,包括以下步骤:
(1)质粒的pYH1和pYH10的构建:在NCBI网站上查找到假单胞菌中bmo操纵子的完整序列(AY093933.3),从其中选出bmor基因和启动子Pbmo的序列。
首先将bmor基因的CDS序列在大肠杆菌中进行密码子优化,优化后的bmor基因序列与Pbmo的序列分别在Gene Design网站的Building Block Design(length overlap)模块设计合成bmor基因的引物yh1-66与合成Pbmo的引物yh67-80,通过两步PCR的方法合成bmor基因与Pbmo(534bp)。以实验室保存的质粒以pSB1C3质粒为模板,yh81,82为引物扩增gfp基因(836bp)并胶回收。以pEL54为模板,yh83,84为引物扩增colE1+ampicillin(2089bp),Dpn Ⅰ消化模板质粒后胶回收。以yh95,96为引物,通过OE-PCR将Pbmo与gfp基因进行构建Pbmo+gfp片段(1353bp)胶回收。最终通过Gibson连接将colE1+ampicillin,bmor,Pbmo+gfp进行连接(分别加1,1和0.5μL)并化转大肠杆菌YH。设计bmor与Pbmo连接处的引物yh91,yh92,挑23个单克隆进行菌落PCR验证。接种条带大小正确(797bp)的单克隆提质粒,送金唯智公司测序,引物为yh99,100,101,102和yh103,测通bmor,Pbmo+gfp序列,最终成功构建质粒pYH1。
构建质粒pYH10的方法与上述方法基本相同,在此不再赘述。
(2)外源添加实验验证:在实验室菌株YHS中转化质粒pYH1,构建菌株YH1。通过异丁醇,正丁醇和3-甲基-1-丁醇浓度梯度的外源添加实验,测定不同浓度条件下的响应值GFP/OD600,做浓度-响应曲线,结果如图1(a);进一步在出发菌株YHS中同时转化生物传感器质粒pYH10和表达kivd和adhA的质粒pYH14,构建菌株YHS5。进行发酵实验,测定不同时间异丁醇产量与荧光强度,结果如图1(b),具体包括以下步骤:
挑平板上的YH1单克隆,接种到5mL LB(100μg/mL ampicillin)液体培养基中,37℃,220rpm培养8小时作为种子液。之后的外源滴加实验在灭菌的2mL96深孔板中进行,每个孔先用移液枪添加950μL新鲜的LB(100μg/mL Ampicillin)培养基中,然后在并在培养基中分别加入异丁醇,正丁醇和3-甲基-1-丁醇(3-MB)使它们终浓度分别为0,0.1,1,10,20,40,50和100mM,每种醇的每个浓度做三个重复组,然后取50μL种子液接种到每个孔中(3个孔只接1mL LB作为背景对照),封好封口膜之后,将深孔板放于30℃,250rpm培养16小时。吸取200μL菌液或发酵液到黑色96孔板中和透明96孔板中,分别用酶标仪(BioTek Cytation3)测量荧光强度和OD600。绿色荧光蛋白GFP的检测使用470nm激发波长和510nm发射波长,增益值为50,每个孔读三次求平均值,菌体密度是在600nm波长下进行测定。得到的每个孔的GFP和OD600值先减去背景对照的值,在此基础上计算每个孔的GFP/OD600作为相对荧光强度值。每种醇分别以GFP/OD600为纵坐标,浓度为横坐标,用OriginPro 8.5软件作图,并用Hill1方程进行拟合,测定BmoR对不同醇的Km,最大响应强度等参数。
图1(a)为YH1外源添加异丁醇,正丁醇和3-甲基-1-丁醇响应曲线,图1(b)为异丁醇生产菌株发酵过程中的产量曲线和荧光曲线,其结果证明了荧光强度的高低与异丁醇产量能够一一对应,因此该BmoR生物传感器可以用于高产菌株的筛选。
(3)高产菌株的筛选体系的构建得到异丁醇:筛选原理如图2:培养基中葡萄糖进入大肠杆菌,通过糖酵解途径生成丙酮酸,进一步在内源酶的催化下生成2-酮异戊酸。当异源表达从2-酮异戊酸合成异丁醇的酶KivD和AdhA时,会催化2-酮异戊酸合成异丁醇。
异丁醇分子会与六聚体BmoR蛋白结合使其与Eσ54闭合复合体发生异构化反应,起始启动子Pbmo的转录,从而表达绿色荧光蛋白。这样一来,绿色荧光强度的高低代表着宿主生产异丁醇的多少,因此我们可以通过检测荧光强度来筛选高产菌株。
(4)突变库的构建方法:如图3所示,本文中使用无锡源清天木生物科技有限公司ARTP诱变育种仪(ARTP-ПS)对出发菌株YHS进行诱变构建突变文库,首先将平板划线的单菌落YHS接种到5mL LB培养基中,37℃,220rpm摇床培养4小时,使其达到对数生长期。先准备6个灭菌1.5mLEP管中各加入500μL灭菌的LB,然后用移液枪各吸取10μL菌液浸泡在6个灭菌不锈钢载片上,在ARTP下暴露60秒,诱变后的载片会被放置于1.5mL EP管中。拿出EP管,涡旋振荡1分钟使载片上的菌液分布到培养基中,吸出菌液加入到含有3mL新鲜LB培养基的试管中,37℃复苏6小时后,将菌液保存于15%甘油中,放到-80℃冰箱备用。
(5)突变库的筛选流程:在构建了宿主突变库之后,通过转化中拷贝生物传感器系统pYH10,与表达kivd,adhA的质粒pYH14,得到的平板上单克隆均来源于不同突变的宿主。挑平板上的单克隆,接种到5mLLB(100μg/mL ampicillin和25μg/mL chloramphenicol全称)液体培养基中,37℃,220rpm培养8小时作为种子液。首先对每管种子液进行保菌。之后的外源滴加实验在灭菌的2mL96深孔板中进行,每个孔先用移液枪添加1mL新鲜的M9(20g/L葡萄糖,0.1mM IPTG,100μg/mL ampicillin和25μg/mL chloramphenicol)培养基中,然后分别接入50μL新鲜的种子液,每管种子液做三个重复组,封好封口膜之后,将深孔板放于30℃,250rpm发酵24小时。吸取200μL菌液或发酵液到黑色96孔板中和透明96孔板中,分别用酶标仪(BioTek Cytation3)测量荧光强度和OD600。绿色荧光蛋白GFP的检测使用470nm激发波长和510nm发射波长,增益值为50,每个孔读三次求平均值,菌体密度是在600nm波长下进行测定,在此基础上计算每个孔的GFP/OD600作为相对荧光强度值。
(6)发酵实验检测产量:为了验证生物传感器是否能真正筛选到异丁醇高产菌株,以及对发酵条件的进一步优化,需要对出发菌株以及筛选到的突变体10号进行发酵验证检测产物。具体的过程包括以下几个步骤。种子液培养:挑取新鲜转化的平板或者平板划线的单菌落接到LB液体培养基(加相应抗生素)中,37℃,220rpm过夜培养,作为种子液,每株菌挑取3个单菌落接种子液,作为重复组。接种摇瓶:使用250mL螺旋盖锥形瓶进行发酵,首先在灭菌的摇瓶加入20mL配制好的灭菌M9培养基,然后加入相应的抗生素和2μL IPTG母液(终浓度0.1mM,诱导启动子PLlacO1表达),最后接种200μL新鲜的种子液进去,30℃摇床,250rpm进行发酵,在12,24,36,48和60小时各自取样1mL。样品处理:首先各自吸取200μL菌液用酶标仪进行GFP和OD600的测定,剩下的菌液12000rpm离心10分钟,吸取197.5μL上清加到气相小瓶的套管里面,再加入配制好的2.5μL 10%正戊醇(终浓度为1g/L)作为气相色谱的检测的内标,拧好气相小瓶的盖子,充分震荡均匀。异丁醇的检测:异丁醇标品的配制:先吸取195μL蒸馏水加到气相小瓶的套管里面,再加入配制好的2.5μL 10%异丁醇和2.5μL10%正戊醇作为内标,将套管放入气相小瓶,拧好气相小瓶的盖子,充分震荡均匀。气相色谱检测程序:使用常州磐诺仪器公司的A91气相色谱仪(GC),DB-FFAP毛细管柱(30m×0.32mm×0.25μm;安捷伦科技)进行异丁醇定量测定,GC炉温最初在80℃保持3分钟,以115℃每分钟的梯度升高至230℃后,保持1分钟。载气为氮气,保持进样器和检测器温度分别为250℃和280℃。进样器进样量为0.2μL,分流比为30:1。使用正戊醇作为内标,标品的峰图和结果如图4。以异丁醇为参照,用软件批处理功能通过内标法计算每个样品的异丁醇含量。
通过对200个单克隆进行荧光筛选,筛选到突变体10号突变体达到野生型的1.4倍(图4a),进一步发酵验证产量在48h小时达到野生型的2倍(图4b)。在此基础上,通过在突变体10号中转化质粒pYH15,表达丙酮酸到2-酮异戊酸的酶AlsS,IlvC和IlvD,构建异丁醇高产菌。
发酵培养基M9的配方:6g/L NaH2PO4,3g/L KH2PO4,0.5g/L NaCl,1g/L NH4Cl,1mMMgSO4,0.1mM CaCl2,10mg/L VB1和40g/L葡萄糖。M9盐溶液:称量6.0g NaH2PO4,3.0gKH2PO4,0.5g NaCl,1.0g NH4Cl和相应量的酵母粉(部分实验不需要添加)用蒸馏水溶解定容于900mL,121℃下灭菌20分钟。冷却至室温后,分别取100mL灭菌的40%葡萄糖溶液,1mL1M MgSO4溶液,1mL 0.1M CaCl2溶液和1mL 10mg/mL vB1溶液盐酸硫铵溶液加入灭菌的900mL M9盐溶液中混匀即可。
以上结合附图对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (3)
1.一种利用BmoR生物传感器筛选异丁醇高产菌株的方法,其特征在于:包括以下步骤:
步骤1、构建BmoR生物传感器:构建以gfp为报告基因的BmoR生物传感器;
步骤2、构建高产菌株的筛选体系:共表达BmoR生物传感器与2-酮异戊酸合成异丁醇的基因kivd和adhA,通过检测荧光强度来筛选异丁醇高产菌株;
步骤3、筛选高产菌株:对出发菌株进行突变文库的构建,利用步骤2构建的筛选系统筛选出异丁醇高产菌株并进一步优化提高产量。
2.根据权利要求1所述的方法,其特征在于:所述步骤1具体包括:
将bmor基因经过大肠杆菌中密码子优化后通过PCR合成Pbmo,Pbmo合成之后连接gfp基因,构建含有不同复制起点的生物传感器质粒,所述生物传感器质粒即BmoR生物传感器。
3.根据权利要求2所述的方法,其特征在于:所述步骤3具体包括:
通过ARTP诱变的方法对出发菌株进行突变文库的构建,并对突变库中的突变体进行荧光筛选得到异丁醇高产菌株。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910765520.4A CN110885777A (zh) | 2019-08-19 | 2019-08-19 | 一种利用BmoR生物传感器筛选异丁醇高产菌株的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910765520.4A CN110885777A (zh) | 2019-08-19 | 2019-08-19 | 一种利用BmoR生物传感器筛选异丁醇高产菌株的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110885777A true CN110885777A (zh) | 2020-03-17 |
Family
ID=69745861
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910765520.4A Pending CN110885777A (zh) | 2019-08-19 | 2019-08-19 | 一种利用BmoR生物传感器筛选异丁醇高产菌株的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110885777A (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115160415A (zh) * | 2021-04-02 | 2022-10-11 | 北京理工大学 | 对正丁醇特异性响应的BmoR蛋白突变体及其应用 |
CN115181168A (zh) * | 2021-04-02 | 2022-10-14 | 北京理工大学 | 对乙醇不敏感对正丁醇特异性响应的BmoR蛋白突变体 |
CN115181167A (zh) * | 2021-04-02 | 2022-10-14 | 北京理工大学 | 一种基于BmoR突变体的乙醇生产菌株的筛选方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110065105A1 (en) * | 2009-04-24 | 2011-03-17 | The Regents Of The University Of California | Novel transcription factor-based biosensor |
-
2019
- 2019-08-19 CN CN201910765520.4A patent/CN110885777A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110065105A1 (en) * | 2009-04-24 | 2011-03-17 | The Regents Of The University Of California | Novel transcription factor-based biosensor |
Non-Patent Citations (1)
Title |
---|
HUAN YU等: "Establishment of BmoR-based biosensor to screen isobutanol overproducer", 《MICROB CELL FACT》 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115160415A (zh) * | 2021-04-02 | 2022-10-11 | 北京理工大学 | 对正丁醇特异性响应的BmoR蛋白突变体及其应用 |
CN115181168A (zh) * | 2021-04-02 | 2022-10-14 | 北京理工大学 | 对乙醇不敏感对正丁醇特异性响应的BmoR蛋白突变体 |
CN115181167A (zh) * | 2021-04-02 | 2022-10-14 | 北京理工大学 | 一种基于BmoR突变体的乙醇生产菌株的筛选方法 |
CN115181167B (zh) * | 2021-04-02 | 2023-08-08 | 北京理工大学 | 一种基于BmoR突变体的乙醇生产菌株的筛选方法 |
CN115160415B (zh) * | 2021-04-02 | 2024-02-09 | 北京理工大学 | 对正丁醇特异性响应的BmoR蛋白突变体及其应用 |
CN115181168B (zh) * | 2021-04-02 | 2024-02-09 | 北京理工大学 | 对乙醇不敏感对正丁醇特异性响应的BmoR蛋白突变体 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Dietrich et al. | Transcription factor-based screens and synthetic selections for microbial small-molecule biosynthesis | |
Qiu et al. | Combining genetically-encoded biosensors with high throughput strain screening to maximize erythritol production in Yarrowia lipolytica | |
Burgard et al. | Development of a commercial scale process for production of 1, 4-butanediol from sugar | |
Liang et al. | Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply | |
Seok et al. | Directed evolution of the 3-hydroxypropionic acid production pathway by engineering aldehyde dehydrogenase using a synthetic selection device | |
Yan et al. | Enantioselective synthesis of pure (R, R)-2, 3-butanediol in Escherichia coli with stereospecific secondary alcohol dehydrogenases | |
Yang et al. | Expanded synthetic small regulatory RNA expression platforms for rapid and multiplex gene expression knockdown | |
CN110885777A (zh) | 一种利用BmoR生物传感器筛选异丁醇高产菌株的方法 | |
Chen et al. | Protein design and engineering of a de novo pathway for microbial production of 1, 3‐propanediol from glucose | |
Yu et al. | Establishment of BmoR-based biosensor to screen isobutanol overproducer | |
Guo et al. | De novo phenol bioproduction from glucose using biosensor‐assisted microbial coculture engineering | |
Li et al. | A synthetic anhydrotetracycline-controllable gene expression system in Ralstonia eutropha H16 | |
Wang et al. | Developing E. coli-E. coli co-cultures to overcome barriers of heterologous tryptamine biosynthesis | |
Han et al. | Improvement of L-valine production by atmospheric and room temperature plasma mutagenesis and high-throughput screening in Corynebacterium glutamicum | |
Zhang et al. | Production of α‐ketobutyrate using engineered Escherichia coli via temperature shift | |
Novak et al. | Microbial upgrading of acetate into 2, 3-butanediol and acetoin by E. coli W | |
EP3008178A1 (en) | Microbial production of 3-hydroxypropionic acid | |
Matson et al. | Adaptive laboratory evolution for improved tolerance of isobutyl acetate in Escherichia coli | |
Liu et al. | Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production | |
Yang et al. | Improving the performance of solventogenic clostridia by reinforcing the biotin synthetic pathway | |
CN110615832A (zh) | 一种高效筛选异丁醇高产菌株的BmoR突变体 | |
Zhang et al. | Strengthening the (R)-pantoate pathway to produce D-pantothenic acid based on systematic metabolic analysis | |
Wang et al. | 2, 3-Dihydroxyisovalerate production by Klebsiella pneumoniae | |
Ku et al. | Metabolic engineering of Escherichia coli for efficient biosynthesis of butyl acetate | |
Tang et al. | Multidimensional engineering of Escherichia coli for efficient synthesis of L-tryptophan |
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 | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200317 |