CN101381696B - 构建基因工程菌增强1，3-丙二醇生产菌株抗逆性的方法 - Google Patents
构建基因工程菌增强1，3-丙二醇生产菌株抗逆性的方法 Download PDFInfo
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Abstract
一种构建基因工程菌增强1，3-丙二醇生产菌株抗逆性的方法，属于生物化工技术领域。在产生PDO的野生菌中，引入β-酮基乙酰辅酶A硫解酶、NADPH依赖型乙酰乙酰辅酶A还原酶和聚羟基脂肪酸合酶基因，使菌株在生产PDO的同时，可积累高浓度PHB，显著提高菌体对甘油和3-羟基丙醛的耐受性；本发明还可用于PHB和PDO的联产。优点在于，所构建基因工程菌显著提高了菌体对高甘油浓度和中间产物3-羟基丙醛的抗逆性，降低了工业生产中的发酵异常现象的发生；另一方面，可在生产PDO的同时积累高浓度的PHB，PHB也可作为一种产物分离，提高了产品的附加值和原料利用率，降低了生产成本。
Description
技术领域
本发明属于生物化工技术领域，特别涉及一种构建基因工程菌增强1，3-丙二醇生产菌株抗逆性的方法。
背景技术
1，3-丙二醇(简称PDO)是一种重要的化工原料，可作为有机溶剂应用于油墨、印染、涂料、润滑剂、抗冻剂等行业。PDO最主要的用途是作为聚酯和聚氨酯合成的单体，特别是与对苯二甲酸聚合生成的聚对苯二甲酸丙二酯(PTT)，显示了比以1，2-丙二醇、丁二醇、乙二醇为单体合成的聚合物更优良的性能。目前全球每年消费数千万吨聚对苯二甲酸乙二酯(PET)，而PTT的化学稳定性、生物可降解性等与PET相当，但耐污染性、韧性和回弹性及抗紫外性能等更优越。此外PTT纤维还具有耐磨、吸水性低、低静电等优点，可在地毯领域与尼龙竞争。它还可用于具有优良性能的无纺布、工程塑料、服装、家庭装饰、垫衬料、织物等方面。PTT被评为美国98年六大石化新产品之一，被认为将是PET的升级产品。
PTT的优越性能及市场潜力早在50年前就被人们所认识，只因原料PDO生产技术难度大、成本高而导致PTT很难大规模工业化生产，迄今为止，只有Dupont和Shell两家跨国公司采用传统的化学合成路线，以环氧乙烷或丙烯为原料生产仅供它们合成PTT自用的PDO。化学合成法的缺点是副产物多，选择性差，操作条件需高温高压，设备投资巨大，原料为不可再生资源，且环氧乙烷和另一路线的中间产物丙烯醛分别是易燃易爆或剧毒的危险品。由于发酵法生产PDO选择性高，操作条件温和，因此近年来受到特别的重视。
生物合成法生产PDO是利用微生物歧化甘油产生。自然界中可将甘油转化为PDO的微生物主要是厌氧或兼性厌氧菌，其中克雷伯氏肺炎杆菌(Klebsiella pneumoniae)、丁酸梭状芽胞杆菌(Clostridium butyricum)及弗氏柠檬酸菌(Citrobacter freundii)具有较高的PDO转化率，而且对甘油和产物PDO具有较高的耐受力，因此具有较高的开发价值与应用前景。
在发酵法生产1，3-丙二醇的过程中，甘油作为唯一碳源和能源沿着氧化和还原途径发生歧化反应，氧化途径中产物与糖类发酵产物一致，产生供细胞生长所必需的ATP，在某些产物形成的同时释放还原力NADH2；还原途径则消耗氧化途径中多余的还原力，生成1，3-丙二醇[Zeng.AP，Biebl H，Schlieker H et al.Pathway analysis of glycerolfermentation by Klebsiella pneumoniae：regulation of reducing equivalent balance and product formation.Enzyme Microbial Technol.1993，15：770-779.]。还原途径包括两步反应：第一步，由依赖于辅酶B12的甘油脱水酶(GDHt)催化甘油脱水生成3-羟基丙醛；第二步，由1，3-丙二醇氧化还原酶(1，3-丙二醇R)催化3-羟基丙醛还原生成1，3-丙二醇[Zeng AP，Biebl H，Schlieker H et al.Pathway analysis of glycerol fermentation by Klebsiella pneumoniae：regulation ofreducing equivalent balance and product formation.Enzyme Microbial Technol.1993，15：770-779.Zeng AP，Menzel K，Deckwer WD et al.Kinetic，Dynamic，and Pathway Studies of Glycerol Metabolismby Klebsiella Pneumoniae in Anaerobic Continuous Culture：II Analysis of metabolic rates and pathwaysunder oscillation and steady-state conditions.Biotechnol Bioeng.1996，52：561-571.Cameron DC，Altaras NE，Hoffman ML et al.Metabolic engineering of propanediol pathways.BiotechnolProg.1998，14：116-125.
Biebl H，Menzel K，Zeng AP et al.Microbial production of 1，3-propanediol.Appl Microbiol Biotechnol.1999，52：289-297.
Abbad-Andaloussi S，Guedon E，Spiesser E et al.Glycerol dehydratase activity：the limiting step for1，3-propanediol production by Clostridium butyricum.Lett Appl Microbiol.1996，22：311-314.]。
目前，调控PDO合成的关键酶及其基因得到了广泛的研究。在PDO的代谢路径上，3-羟基丙醛是一种重要的代谢中间产物，如图1所示，其来源于甘油的酶促脱水，随后在PDO氧化还原酶催化下还原为PDO[Ahrens K，Menzel K，Zeng AP et al.Kinetic，Dynamic，and Pathway Studies of Glycerol Metabolism by Klebsiella pneumoniae in AnaerobicContinuous Culture：III Enzymes and Fluxes of Glycerol Dissimilation and 1，3-Propanediol Formation.Biotechnol Bioeng.1998，59：544-552.]
在PDO的生产过程中发现，当起始甘油浓度大于50g/l时，发酵会出现异常中止。流加发酵中甘油浓度控制不当也会引起3-羟基丙醛积累，造成菌体死亡，发酵异常终止。因此，目前对3-羟基丙醛的研究也引起了国内外的广泛关注。
聚β-羟基脂肪酸，英文名称Polyhydroxyalkanoates，简称PHAs，泛指由单体β-羟基脂肪酸之间的羟基和羧基脱水酯化而得到的一种生物高分子材料。目前已经发现PHA聚酯有至少125种不同的单体结构，并且新的单体被不断地发现出来。另外，根据单体结构或含量的不同，PHA的性能可从坚硬到柔软到弹性变化。PHA有许多潜在的应用前景，国内外都对其进行大量的基础和应用开发研究。
PHA不仅具有与化工合成高分子材料相近的物化性能，还具有后者所没有的生物可降解性、压电性、光学活性、气体阻隔性等优良性质，因而有望在绿色包装材料、容器、电器元件外壳等方面取代或者部分取代化工合成材料。另外，PHA具有生物相容性，且其最终降解产物3-羟基脂肪酸对人体没有副作用，因而有望应用于组织工程领域，如心脏阀门、心血管修补材料等等。通过对PHA进行一些表面修饰后，其生物相容性等性能还可以进一步得到提高，在组织工程及医疗领域的应用范围有望进一步扩大。
目前，已经实现工业化生产的PHA只有PHB(聚β-羟基丁酸酯)(图2)、以及PHB和3-羟基戊酸的共聚酯(PHBV)的共聚物PHBV。分别由奥地利林茨化学公司(Chemie Linz AG)和英国帝国化学工业公司(ICI，现在称为Zeneca)在八十年代实现。从1998年以来，清华大学微生物实验室与广东江门生物技术开发中心合作，在国内外首次开发成功了羟基丁酸与羟基己酸的共聚物PHBHHx的工业化生产技术，为这种新型材料的应用开发打下了物质基础[Chen G Q，Zhang G，Park S J，et al.Industrial scaleproduction of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).Appl Microbiol Biot.2001，57(1-2)：50-55.Qiu Y Z，Han J，Chen G Q.Metabolic engineering of Aeromonas hydrophilafor the enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).ApplMicrobiol Biot.2006，69(5)：537-542.Ouyang S P，Han J，Qiu Y Z，et al.Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)production in recombinant Aeromonashydrophila 4AK4 harboring phbA，phbB and vgb genes.Macromol Symp.2005，224：21-34Qiu Y Z，Ouyang S P，Shen Z Y， et al.Metabolic engineering for the production ofcopolyesters consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate by Aeromonashydrophila.Macromol Biosci.2004，4(3)：255-261]。许多PHA合成菌能够在有机物以及重金属离子污染区生存，而在活性污泥中也发现了大量的PHA合成菌，这提示我们PHA可能与细菌的抗逆能力有一定的关系。对PHA的合成菌及其PhaC或者PhaZ缺失型突变株的对比研究也证实了PHA的合成或代谢与菌株抗逆性存在相关性，并获得了一些生化以及蛋白水平上的证据。人们通过对具有PHB合成能力的巨大芽孢杆菌(Bacillus megaterium)和罗尔斯通氏菌(Ralstonia eutropha)及其相应的PHB合成缺陷型突变株研究后发现，原始菌在不同的培养条件下均表现出更好的生存能力[Lopez NI，Floccari M E，Steinbuchel A，et al.Effect of Poly(3-Hydroxybutyrate)(Phb)Content on theStarvation-Survival of Bacteria in Natural-Waters.FEMS Microbiol Ecol.1995，16(2)：95-101]。PHA降解酶PhaZ对PHA合成菌的抗逆性也有重要的影响，研究发现，原始菌对饥饿、渗透压、热激、紫外照射等不利因素的抗逆程度比PhaZ缺失型突变株明显较高，并具有更高的生长速率[Kadouri D，Jurkevitch E，Okon Y.Poly beta-hydroxybutyrate depolymerase(PhaZ)inAzospirillum brasilense and characterization of a phaZ mutant.Arch Microbiol.2003，180(5)：309-18]。因此，可以通过向工业生产菌引入PHA代谢相关基因，从而改变菌株的代谢流，以改进菌株的生产性能。
目前对对野生PDO生产菌株的基因工程改造主要集中在以下几个方面：
(1)通过基因工程方法强化表达还原途径中限速酶(如甘油脱水酶，PDO氧化还原酶)
[Sun JB.，Heuvel J.，Soucaille P.，Qu Y.，and Zeng A.P.Comparative GenomicAnalysis of dha Regulon and Related Genes for Anaerobic Glycerol Metabolism in Bacteria.Biotechnol.Prog.2003 19：263-272][黄日波等，产气荚膜梭菌甘油脱水酶基因及其PDO的生产方法中国专利申请号：200610019452.X]
(2)敲除无益副产物编码基因，阻断副产物代谢途径；
[张延平，刘铭，曹竹安.醛脱氢酶基因敲除的K.pneumoniae重组菌的构建.中国生物工程杂志，2005，25(12)：34～38][杨光.1，3-丙二醇产生菌肺炎克氏杆菌的分子育种[D].北京：中国农业大学，2003]。
(3)在PDO生产菌中构建辅酶再生系统；
[黄志华，张延平，曹竹安等.甲酸脱氢酶在Klebsiella pneumoniae中的表达和功能分析.微生物学报，2007，47(1)：64～68][16黄志华，张延平，曹竹安.在Klebsiellapneumoniae醛脱氢酶失活菌中构建NADH2再生系统.中国生物工程杂志，2006，26(12)：75～80]
(4)在E.coli中构建利用葡萄糖合成PDO的基因工程菌
[Bulthuis B A，Gatenby A A，Haynie S L，et al.Method for the Productionof Glycerol by Recombinant Organisms[P].United States Patent：6 358 716，2002-05-19.Diaz-Torres M，Dunn-Coleman N S，Chase M W，et al.Method for theRecombinant Production of 1，3-Propanediol[P].United States Patent：6 136 576，2000-10-24.Emptage M，Haynie S L，Laffend L A，et al.Process for the BiologicalProduction of 1，3-Propanediol with High Titer.United States Patent：6 514 733，2003-08-21.]
(5)在甘油产生菌中构建合成PDO的基因工程菌
[Cameron DC，Altaras NE，Hoffman ML et.al.Metabolic Engineering ofPropanediol Pathways.Biotechnol.Prog.1998，14：116-125]在酿酒酵母(Saccharomyces cerevisia)]
目前还未有引入PHB基因以提高PDO生产菌株抗三羟基丙醛的报道。
发明内容
本发明的目的是提供一种特别涉及构建基因工程菌增强1，3-丙二醇生产菌株抗逆性的方法。在PDO产生菌中，引入β-酮基乙酰辅酶A硫解酶(β-ketothiolase，PhbA)、NADPH依赖型乙酰乙酰辅酶A还原酶(NADPH-dependent acetoacetyl-CoA，PhbB)和聚羟基脂肪酸合酶基因(Polyhydroxyalkanoates synthase，PhbC)，使菌株在生产PDO的同时，可积累高浓度PHB，显著提高菌体对甘油和3-羟基丙醛的耐受性。本发明还可用于发酵联产PHB和PDO；见(图3)。
其具体内容如下：
1.基因工程菌的构建
(1)以携带有β-酮基乙酰辅酶A硫解酶(phbA)、NADPH依赖型乙酰乙酰辅酶A还原酶(phbB)和聚羟基脂肪酸合酶(phbC)基因的质粒pBHR68为模板，通过PCR(聚合酶联反应)的方法将其操纵子(phbCAB)完整克隆下来。
(2)把PCR产物β-酮基乙酰辅酶A硫解酶、NADPH依赖型乙酰乙酰辅酶A还原酶和聚羟基脂肪酸合酶基因片段纯化后与克隆载体(如：pMD18-T-vector)连接。
(3)筛选阳性克隆载体并分别用EcoRI和BamHI酶切，回收，连接表达载体(如表达载体pDK6)，构建重组表达载体pDK-CAB(图4)。
(4)将构建好的重组表达载体pDK-CAB转化到感受态大肠杆菌中(如SM10)，在卡那霉素抗性平板上筛选阳性克隆。
(5)从阳性克隆里提取重组表达载体pDK-CAB，并通过电转化或化学转化法将其转化到克雷伯氏菌属、柠檬酸菌属、肠杆菌属等可产生PDO的野生型菌株感受态细胞里，鉴定并分离出阳性克隆，即为目的菌株。
2.发酵
将所构建的基因工程菌在固体培养基上培养16～24h，接入种子培养基30～37℃有氧培养16～24h，以1％～5％的接种量接入以甘油为发酵底物的发酵培养基。发酵温度30～37℃。发酵过程中流加甘油使发酵液中甘油浓度控制在30～60g/L，pH值控制在5.0～8.0，40～60h后停止流加至发酵结束。发酵过程中向罐中通入0.4-1.0vvm的空气，搅拌转速150-250rpm。
本发明所述的用于构建基因工程菌的野生菌株包括克雷伯氏菌属、柠檬酸梭菌属、肠杆菌属、沙雷氏菌属等能产生PDO的菌株。所构建的基因工程菌发酵底物为甘油、甘油发酵液、生物柴油副产物粗甘油或肥皂行业的副产物粗甘油。
步骤2中发酵是采用有氧流加发酵的方式，并在发酵过程中流加甘油使发酵液中甘油浓度控制在30～60g/L。
本发明的有益效果：
所构建基因工程菌显著提高了菌体对高甘油浓度和中间产物3-羟基丙醛的抗逆性，降低了工业生产中的发酵异常现象的发生；另一方面，可在生产PDO的同时积累高浓度的PHB，PHB也可作为一种产物分离，提高了产品的附加值和原料利用率，降低了生产成本。本发明还可用于PDO和PHB的联产。
附图说明
图1为3-羟基丙醛的产生及转化示意图。
图2为台湾沃特斯氏菌Wautersia eutropha H16中的PHB合成途径。其中：PhbA，β-酮基硫解酶(β-ketothiolase)；PhbB，NADPH依赖的乙酰乙酰辅酶A还原酶(NADPH-dependent acetoacetyl-CoA reductase；PhbC，PHA合酶(PHA synthase)。
图3为甘油发酵联产PDO和PHB示意图。其中：Glycerol：甘油，3-Hydroxypropionaldehyde：3-羟基丙醛，1，3-Propanediol：1，3-丙二醇，Acetyl-CoA：乙酰辅酶A，Acetoacetyl-CoA：乙酰乙酰辅酶A，3-Hydroxybutyryl-CoA：3-羟基丁酸辅酶A，PHB：聚羟基丁酸酯。
图4为重组质粒pDK-CAB。
具体实施方式
本发明的目的是提供一种特别涉及构建基因工程菌增强1，3-丙二醇生产菌株抗逆性的方法。在PDO产生菌中，引入β-酮基乙酰辅酶A硫解酶(β-ketothiolase，PhbA)、NADPH依赖型乙酰乙酰辅酶A还原酶(NADPH-dependent acetoacetyl-CoA，PhbB)和聚羟基脂肪酸合酶基因(Polyhydroxyalkanoates synthase，PhbC)，使菌株在生产PDO的同时，可积累高浓度PHB，显著提高菌体对甘油和3-羟基丙醛的耐受性。本发明还可用于PHB和PDO的联产(图3)。
下面再举具体实施例对本发明予以进一步说明。
实例1：
(1)基因工程菌的构建
(i)野生菌株：克雷伯氏菌AC01
(ii)以携带有β-酮基乙酰辅酶A硫解酶(phbA)、NADPH依赖型乙酰乙酰辅酶A还原酶(phbB)和聚羟基脂肪酸合酶(phbC)基因的质粒pBHR68为模板，通过PCR(聚合酶联反应)的方法将其操纵子(phbCAB)完整克隆下来。PCR扩增条件如下：96℃预变性8min；96℃变性1min，62.5℃退火1min，72℃延伸4.5min，30个循环；72℃延伸10min。
phbL：5’-CCCGAATTC(EcoRI)CTGACGGCAGAGAGACAATC-3’
phbR：5’-TATGGATCC(BamHI)TGCCGACTGGTTGAACCAG-3’
(iii)把PCR产物β-酮基乙酰辅酶A硫解酶、NADPH依赖型乙酰乙酰辅酶A还原酶和聚羟基脂肪酸合酶基因片段纯化后与克隆载体pMD18-T-vector连接，转化到感受态大肠杆菌中，在卡那霉素抗性平板上筛选单菌落。PCR产物基因序列如下：
GGATCCCTGCCGACTGGTTGAACCAGGCCGGCAGGTCAGCCCATATGCAGGCC
GCCGTTGAGCGAGAAGTCGGCGCCGGTCGAGAAACCGGACTCCTCCGACGACA
ACCAGGCGCAGATCGAGGCGATCTCTTCCGGCAGGCCCAGGCGCTTGACCGGG
ATCGTCGCGACGATCTTGTCGAGCACGTCCTGGCGGATCGCCTTGACCATGTCG
GTGGCGATATAGCCCGGAGAGACCGTGTTGACGGTCACGCCCTTGGTCGCCAC
TTCCTGCGCCAGTGCCATGGTGAAGCCATGCAGGCCGGCCTTGGCGGTGGAGT
AGTTGGTCTGGCCGAACTGGCCCTTCTGCCCGTTCACCGACGAGATGTTGACGA
TGCGGCCCCAGCCACGGTCGGCCATGCCGTCGATCACCTGCTTGGTGACGTTGA
ACAGCGAGGTCAGGTTGGTGTCGATCACCGCATCCCAGTCGGCGCGGGTCATC
TTGCGGAACACCACGTCGCGGGTGATACCGGCGTTGTTGATCAGCACATCAAC
CTCGCCGACCTCGGACTTGACCTTGTCGAATGCGGTCTTGGTCGAGTCCCAGTC
AGCCACATTGCCTTCCGAGGCAATGAAATCGAAGCCCAGGGCCTTCTGCTGCTC
CAGCCACTTTTCGCGGCGCGGCGAGTTGGGGCCGCAACCGGCCACCACACGAA
AGCCATCCTTGGCCAGCCGCTGGCAAATGGCGGTTCCGATACCACCCATGCCGC
CGGTCACATACGCAATGCGCTGAGTCATGTCCACTCCTTGATTGGCTTCGTTAT
CGTCGCCGGGTCCGCGCCAACCGCGCGCGGCCCCGGAAAACCCCTTCCTTATTT
GCGCTCGACTGCCAGCGCCACGCCCATGCCGCCGCCGATGCACAGCGAGGCCA
GGCCCTTCTTCGCGTCACGGCGCTTCATCTCGTGCAGCAGCGTCACCAGGATAC
GGCAGCCCGACGCGCCGATCGGGTGGCCGATGGCGATGGCGCCGCCGTTCACA
TTGACCTTGGAGGTGTCCCAGCCCATCTGCTGGTGCACCGCCAGCGCCTGCGCG
GCAAAGGCCTCGTTGATCTCCATCAGGTCCAGGTCTTGCGGGGTCCACTCGGCG
CGCGACAGGGCGCGCTTGGAGGCCGGCACCGGGCCCATGCCCATCACCTTGGG
ATCGACACCGGCGTTGGCATAGCTCTTGATCGTGGCCAGCGGGGTCAGGCCCA
GTTCCTTGGCCTTGGCCGCCGACATCACCACCACCGCGGCGGCGCCGTCGTTCA
GGCCCGAGGCGTTGGCCGCGGTCACCGTGCCGGCCTTGTCGAAGGCGGGCTTG
AGGCCGGACATGCTGTCCAGCGTGGCGCCCTGGCGCACGAACTCGTCGGTCTT
GAAGGCCACCGGGTCGCCCTTGCGCTGCGGGATCAGCACCGGGACGATCTCTT
CGTCAAACTTGCCGGCCTTCTGCGCGGCTTCGGCCTTGTTCTGCGAGCCGACGG
CGAACTCATCCTGCGCCTCGCGTGTGATGCCGTATTCCTTGGCCACGTTCTCGG
CGGTGATGCCCATGTGGTACTGGTTGTACACGTCCCACAGGCCGTCGACGATCA
TGGTGTCGACCAGCTTGGCATCGCCCATGCGGAAACCATCGCGCGAGCCCGGC
AGCACGTGCGGGGCGGCGCTCATGTTTTCCTGGCCGCCGGCCACCACGATCTCG
GCGTCGCCCGCCATGATCGCGTTGGCGGCCAGCATCACGGCCTTCAGGCCCGA
GCCGCACACCTTGTTGATGGTCATGGCCGGCACCATCGCCGGCAGGCCGGCCTT
GATCGCGGCCTGGCGTGCGGGGTTCTGGCCCGAACCGGCGGTCAGCACCTGGC
CCATGATGACTTCGCTCACCTGCTCCGGCTTGACGCCGGCGCGCTCCAGCGCGG
CCTTGATGACCACGGCACCCAGTTCCGGTGCCGGGATCTTGGCCAGCGAGCCG
CCAAACTTGCCGACCGCGGTGCGGGCGGCGGATACGATGACAACGTCAGTCAT
TGTGTAGTCCTTTCAATGGAAACGGGAGGGAACCTGCAGGCCTGCCGGCGCCG
TGCATGACGCACGCCGGCACTCATGCAAGCGTCATGCCTTGGCTTTGACGTATC
GCCCAGGCGCGGGTTCGATTGCGCGATAGCGCGCATTGCCATAGTTGGCGGGC
GCGGCGCGTTTCGCGCCGGCCTGCCCGGCCAGCCATGCGGTCCAGTCCGGCCA
CCAGCTGCCGTGATGCTCGATGGCGCCGGCCAGCCATTGCTGCGGCGACTCCG
GCAGCGCATCGTTAGTCCAGTGGCTGCGCTTGTTCTTGGCCGGCGGGTTGATCA
CACCGGCGATATGGCCCGACGCACCCAGCACGAAGCGCAGCTTGTTCGCCAGC
AGCGCGGTCGAGGCATAGGCCGCGGTCCACGGCACGATATGGTCTTCGCGCGA
GCCGTAGATATAGGTCGGCACGTCGATGCTGGCCAGGTCCACCGGCACGCCGC
ACACGGTCAGCTTGCCCGGTACCTTGAGCTCGTTCTGCAGGTAGGTGTGGCGCA
GGTACCAGCAGTACCACGGCCCCGGCAGGTTGGTGGCGTCGCCGTTCCAGAAC
AGCAGGTCGAACGGCACCGGCGTGTTGCCCTTCAGGTAGTTGTCGACCACGTA
GTTCCACACCAGGTCGTTCGGGCGCAAGAACGAGAAGGTATTGGCCAGCTCAA
GGCCGCGCAGCAGCGCGCACGGCGCGCCGGCGCCGCCGCCCAGCGTGGCCTCG
CGCAACTGCACATGGCCCTCGTCGACAAAGACGTCGAGGATGCCCGTGTCGGC
AAAGTCCAGCAGCGTGGTCAGCAGCGTGACGCTGGCGGCCGGGTGCTCGCCGC
GCGCGGCCAGCACCGCCAGCGCGGTCGAGACAATGGTGCCGCCCACGCAGAAG
CCGAGCACGTTGATCTTGTCCTGGCCGCTGATGTCGCGCGCGACTTCGATGGCG
CGGATGGCCGCGTGCTCGATGTAGTCGTCCCAGGTGCTGCCGGCCATGCTGGCG
TCCGGATTGCGCCACGACACCAGAAACACCGTATGTCCCTGCTCCACCACATGG
CGCACCAGCGAGCTCTCCGGCTGCAGGTCCAGGATGTAGTACTTGTTGATGCAC
GGCGGCACCATCAGCAGCGGGCGCGCGTGCACCTTGTCGGTCAGCGGCTTGTA
CTGCAACAGCTGGAAGTACTCGTTCTCGAAGACCACGGCGCCTTCGGTCACCGC
GACATTGCGGCCGACCTCAAACGCGCTCTCGTCGGTCTGCGAGATCTTGCCGCG
TGTCAGGTCTTCCATCATGTTGCGCACGCCGGCACGCAGCGATTCGCCGCCCGA
CTCGATCAGCAGGCGCTGCGCCTCGGGATTGGTGGCAAGGAAGTTGGCGGGCG
ACATCGCATCGACCCATTGCGAGATCGCGAAGCGGATGCGCTGGCGGGTCTTG
GCATCGGCCTCGACGGCATCGGCCAGCTCGGTCAAGGCGCGCGCATTGAGCAG
GTAGAACGCGGCAGCGAAGCGATATGGGAGGTTGGTGCGCCATGCGTCGCCGG
CGAAGCGCCGGTCGTGCAGCGGACCGGTGGCCTCGGCCTTGCCCTCGGCCATG
GCCTGCCACAGCGCTGAGAAGTCCTTCATGTAGCGCTGCTGGATATCACCCAGC
TGCGCCGGCGCGATCTTGACGCCTGCCAGCGCATCCAGGCCCGGAATGCCGGA
CGCGGCCGCGTGGCCGTTGCCTTCAGTGCCCTGCCACTGGCGGGACCATTCCAG
CCATGTGGCTGGATCGAATGGCCCCGGCGTGACCTTGAATGGTTGGGACTTGCC
TTCCTGCGTGGAAGCTGCCGCGCCTTTGCCGGTCGCCATGATTTGATTGTCTCTC
TGCCGTCAGAATTC
(iv)将阳性克隆载体和表达载体pDK6分别用EcoRI和BamHI酶切，连接，构建重组载体pDK-CAB。
(v)将构建好的重组表达载体pDK-CAB转化到感受态大肠杆菌SM10中，在卡那霉素抗性平板上筛选阳性克隆。
(vi)从阳性克隆里提取重组表达载体pDK-CAB，并通过电转化法将其转化到野生型克雷伯氏菌AC01的感受态细胞里，鉴定并分离出阳性克隆，即为目的菌株AC01-PHB。
(2)发酵
(i)菌种：所构建的目的菌株AC01-PHB
(ii)培养基：
表1 培养基组成
*铁溶液的配制：每升水中加入FeSO4·H2O5.0g，37％的浓盐酸4ml。
(iii)发酵方式：将所构建的基因工程菌在固体培养基上培养16h，将菌种接入含有30g/L甘油的种子培养基中(250ml三角瓶，装液量100ml)，培养温度30℃，摇床转速150rpm，好氧培养16h。以1％的接种量接入含初始甘油为30g/l的发酵培养基。发酵采用5L发酵罐，发酵温度30℃。pH值控制在7.0，通气量0.5vvm空气，转速250rpm，发酵过程中流加甘油使发酵液中甘油浓度控制在30～40g/L，60h后停止流加至发酵结束。
(iv)发酵结果：
72小时后，PHB含量达到24.70％(g/g cell)，PDO产量58.68g/l，乳酸产量35.93g/l。菌体发酵抗3-HPA和高甘油浓度能力增强，发酵过程中3-HPA浓度可达9.51mmol/L，发酵并无异常终止。
实例2：
(1)基因工程菌的构建
(i)野生菌株：克雷伯氏菌HR521
(ii)同实施例一
(iii)同实施例一
(iv)同实施例一
(v)同实施例一
(vi)从阳性克隆里提取重组表达载体pDK-CAB，并通过电转化法将其转化到野生型克雷伯氏菌HR521的感受态细胞里，鉴定并分离出阳性克隆，即为目的菌株HR521-PHB。
(2)发酵
(i)菌种：所构建的目的菌株HR521-PHB
(ii)培养基：同实施例一
(iii)发酵方式：将所构建的基因工程菌在固体培养基上培养24h，将菌种接入含有30g/L甘油的种子培养基中(250ml三角瓶，装液量100ml)，培养温度37℃，摇床转速150rpm，好氧培养18h。以5％的接种量接入含初始甘油为30g/l的发酵培养基。发酵采用5L发酵罐，发酵温度37℃。pH值控制在6.0，通气量0.2vvm空气，转速250rpm，发酵过程中流加甘油使发酵液中甘油浓度控制在40～60g/L，60h后停止流加至发酵结束。
(iv)发酵结果：
72小时后，OD650nm达到9；PHB含量达到34.39％(g/g cell)，PDO产量达到67.05g/l，乳酸产量达到37.72g/l。菌体发酵抗3-HPA和高甘油浓度能力增强，开始流加后甘油浓度一直保持在50g/L以上，发酵过程中3-HPA浓度可达9.72mmol/L，。发酵并不异常终止，且可同时得到较高浓度的PDO和PHB。
Claims (4)
1.一种构建基因工程菌增强1，3-丙二醇生产菌株抗逆性的方法，其特征在于，在产生PDO的野生菌中，引入β-酮基乙酰辅酶A硫解酶、NADPH依赖型乙酰乙酰辅酶A还原酶和聚羟基脂肪酸合酶基因，使菌株在生产PDO的同时，积累高浓度PHB，提高菌体对甘油和3-羟基丙醛的耐受性；或用于发酵联产PHB和PDO；
所述的基因工程菌的构建步骤包括：
(1)以携带有β-酮基乙酰辅酶A硫解酶、NADPH依赖型乙酰乙酰辅酶A还原酶和聚羟基脂肪酸合酶基因的质粒pBHR68为模板，通过PCR聚合酶联反应的方法将其操纵子phbCAB完整克隆下来；
(2)把PCR产物β-酮基乙酰辅酶A硫解酶、NADPH依赖型乙酰乙酰辅酶A还原酶和聚羟基脂肪酸合酶基因片段纯化后与克隆载体连接；
(3)筛选阳性克隆载体并分别用EcoRI和BamHI酶切，回收，连接表达载体，构建重组表达载体pDK-CAB；
(4)将构建好的重组表达载体pDK-CAB转化到感受态大肠杆菌中，在卡那霉素抗性平板上筛选阳性克隆；
(5)从阳性克隆里提取重组表达载体pDK-CAB，并通过电转化或化学转化法将其转化到克雷伯氏菌感受态细胞里，鉴定并分离出阳性克隆，为目的菌株；
所构建的基因工程菌用于提高菌体对甘油和3-羟基丙醛的耐受性或发酵联产PDO和PHB；其工艺为：
将所构建的基因工程菌在固体培养基上培养16～24h，接入种子培养基30～37℃有氧培养16～24h，以1％～5％的体积接种量接入以甘油为发酵底物的发酵培养基；发酵温度30～37℃，发酵过程中流加甘油使发酵液中甘油浓度控制在30～60g/L，pH值控制在5.0～8.0，40～60h后停止流加至发酵结束；发酵过程中向罐中通入0.4-1.0vvm的空气，搅拌转速150-250rpm；
所述的用于构建基因工程菌的野生型菌株为克雷伯氏菌；
所构建的基因工程菌发酵底物为甘油。
2.按照权利要求1所述方法，其特征在于：所构建的基因工程菌发酵底物为甘油发酵液、生物柴油副产物粗甘油或肥皂行业的副产物粗甘油。
3.按照权利要求1所述方法，其特征在于：所述的克隆载体为：pMD18-T-vector。
4.按照权利要求1所述方法，其特征在于：所述的大肠杆菌为SM10。
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