CN102575272B - 使用有产油能力的微生物生产脂肪酸烷基酯的方法 - Google Patents
使用有产油能力的微生物生产脂肪酸烷基酯的方法 Download PDFInfo
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Abstract
本发明涉及一种使用有产油能力的微生物生产脂肪酸烷基酯的方法,尤其涉及一种生产脂肪酸烷基酯的方法,该方法包括培养有产油能力的微生物,从而在微生物内积累大量油,诱导微生物内产生的油自溶以生产游离脂肪酸,并且将游离脂肪酸转变成烷基酯。根据本发明方法,可以通过代谢工程方法将在微生物中积累的油,例如甘油三酯(典型的通过微生物产生的油),高效转化为脂肪酸烷基酯。因此,本发明方法可用于脂肪酸烷基酯(最近发现可作为有效的生物柴油)的工业生产。
Description
技术领域
本发明涉及一种使用有产油能力的微生物生产脂肪酸烷基酯的方法,尤其涉及一种生产脂肪酸烷基酯的方法,所述方法包括培养有产油能力的微生物,从而在微生物内积累大量油,诱导微生物内产生的油自溶以生产游离脂肪酸,并且将该游离脂肪酸转变成烷基酯。
背景技术
近来,由于高油价以及环境方面的考虑,微生物生产生物燃料受到了极大关注。此外,生物柴油已经取代轻油或者出现了生物柴油和轻油的混合物作为替代燃料应用于柴油发动机,因此生物柴油的市场迅速扩大。2008年,欧盟(EU)生产了660万吨生物柴油,市场份额为55亿欧元(Biodiesel Market,Frost&Sullivan)。另外,2006年美国生产了30亿加仑生物柴油(Biodiesel Market,GlobalIndustry Analysts Inc,2006.5)。
生物柴油的优势在于其具有高燃烧率,因而有毒气体排放量低,较之轻油热值约低10%,但燃点更高,表明其在存储和运输期间更为稳定。生物柴油主要是通过处理动物和植物的脂肪成分来生产,从而使其具有类似于轻油的性质,或者通过植物油脂(米糠、烹饪废油、大豆油、菜籽油等等)与乙醇反应而制得。然而,在这种情况下的缺点是难以大量生产生物柴油。因此,如果能通过使用微生物大量生产生物柴油作为轻油的代用燃料,将减少原油的进口及温室气体的排放,使得环境改善。
同时,油是一种能量载体,当微生物富有碳源而缺乏其它生长因子(氮、磷、氧、硫、等等)时,油可在微生物细胞内合成并积累。当微生物的生长环境改变,而向微生物提供其它生长因子时,所积累的油将被降解并且用作能源。众所周知,由于产油微生物的种类、所提供的化学原料的种类、培养条件等的不同,油可由超过100种单体组成。
近来,已经开发了向植物(如甘蔗)脂肪酸中添加醇来生产脂肪酸烷基酯的技术,所生产的脂肪酸烷基酯目前正被用作生物柴油。此外,欧洲专利EP127104A、EP184740A和美国专利4,164,506中也公开了酯化游离脂肪酸的方法。根据这些专利的公开内容,酯化反应通过加热脂肪酸和甘油三酯以及甲醇的混合物来进行。另外,欧洲专利申请EP708813A公开了一种提高油脂生产脂肪酸烷基酯的产量的方法,其中将游离脂肪酸与酯交换得到的甘油分离,然后进行酯化。
然而,很难用这种方法获得大量脂肪酸或者游离脂肪酸。另外,由于植物的生长期很长而且生产植物脂肪酸的代谢工程方法有些困难,因而很难增加目前常用的植物脂肪酸的积累和产生。
因此,本发明人通过大量努力,提出了一种通过代谢工程途径高效且高产地生产被用作生物柴油的脂肪酸烷基酯的新方法,并且结果发现使用代谢工程方法,通过使产油微生物最大化地生产油,然后诱导微生物中的油自溶生产游离脂肪酸,然后游离脂肪酸转变为烷基酯,可高效生产脂肪酸烷基酯,从而完成本发明。
发明内容
本发明的目的是提供一种通过代谢工程途径高效且高产地大量生产可用作生物柴油的脂肪酸烷基酯的新方法。
为了达到上述目标,本发明提供一种生产脂肪酸烷基酯的方法,该方法包括以下步骤:
(a)培养有产油能力的微生物以生产油;
(b)诱导微生物内产生的油自溶以生产游离脂肪酸;以及
(c)向所生产的游离脂肪酸中添加醇,并且使醇与游离脂肪酸反应以生产脂肪酸烷基酯。
本发明还提供了一种使用有产油能力且包含脂酶编码基因的微生物来生产脂肪酸甲酯的方法,该方法包括以下步骤:
(a)培养有产油能力且包含脂酶编码基因的微生物以生产油;
(b)诱导微生物内产生的油自溶以生产游离脂肪酸;以及
(c)向所生产的游离脂肪酸中添加甲醇,并且使甲醇与游离脂肪酸反应以生产脂肪酸甲酯。
本发明的其他特征和实施例在下文的详细说明和附加权利要求中显而易见。
附图说明
图1示出用不透明红球菌(Rhodococcus opacus)PD630从典型油(甘油三酯TAG)高效生产脂肪酸甲酯的方法。
图2示出重组载体rpROUC18(a)和rpROUC18_KM(b)的遗传图谱。
图3示出包含sdp1和MSMEG_0220基因的重组载体pRUCSdpMag的遗传图谱。
图4示出基于表1所示的信息得到的质粒的类型和信息。
图5示出游离脂肪酸的气相色谱分析结果。
图6和7示出脂肪酸甲酯的气相色谱分析结果。
具体实施方式
除非另行定义,否则在此使用的所有技术和科学术语的含义与本领域技术人员所通常理解的相同。在此使用的命名法和实验方法是本领域众所周知并且经常使用的。
发明详述中的主要术语的定义如下。
如在此使用,术语″油″是指一种能量载体,当微生物富有碳源而缺乏其它生长因子(氮、磷、氧、硫、等等)时,其可在微生物细胞内合成并积累。它是一种游离脂肪酸前体,可水解为游离脂肪酸和甘油。
如在此使用,术语″脂肪酸″指饱和或者不饱和的单羧酸类型的链。这些脂肪酸根据碳链长度和饱和度进行分类,油(即,脂肪)水解而产生的脂肪酸被称为″游离脂肪酸″。
一方面,本发明涉及一种使用有产油能力的微生物生产脂肪酸烷基酯的方法,尤其涉及一种生产脂肪酸烷基酯的方法,该方法包括以下步骤:
(a)培养有产油能力的微生物以生产油;
(b)诱导微生物内产生的油自溶以生产游离脂肪酸;并且
(c)向所生产的游离脂肪酸中添加醇,并且使醇与游离脂肪酸反应以生产脂肪酸烷基酯。
在本发明中,油可以是微生物生产的任何油,其实例包括但不限于,甘油三酯(TAG)、二酰甘油(DAG)、单酰甘油(MAG)、磷脂、甾醇类脂、鞘脂类、糖脂、异戊烯醇类脂以及聚酮化合物。
在此,通过分解油而产生的游离脂肪酸可以是饱和或者不饱和脂肪酸,其中不饱和脂肪酸指具有一个或多个碳链内双键的脂肪酸,其实例包括油酸、亚油酸、亚麻酸、棕榈油酸、蓖麻油酸、11-十八碳烯酸、顺9-二十碳烯酸、花生四烯酸、EPA(5,8,11,14,17-二十碳五烯酸)、芥子酸、DHA(4,7,10,13,16,19-二十二碳六烯酸)等。另外,饱和脂肪酸指没有碳链内双键的脂肪酸,其实例包括丁酸、己酸、辛酸、癸酸、月桂酸、豆蔻酸、棕榈酸、硬脂酸、花生酸、正廿二烷酸、二十四(烷)酸等。用于本发明的脂肪酸可被选自下述基团的取代基取代,包括但不限于芳环基、环氧基、氰基基团和卤素基团。
在本发明中,作为游离脂肪酸的前体的油是由有产油能力的微生物生产的。有产油能力的微生物包括气单胞菌属(Aeromonas sp.)、无色杆菌(Achromobacter sp.)、德氏食酸菌(Acidovorax delafieldii)、敏捷食酸菌(Acidovax facilis)、不动细菌属(Acinetobacter sp.)、放线菌属(Actinomycessp.)、气单胞菌属(Aeromonas sp.)、产碱杆菌属(Alcaligenes sp.)、交替单胞菌属(Alteromonas sp.、破囊壶菌属(Althornia sp.)、Aplanochytrium sp.、曲霉菌属(Aspergillus sp.)、可变杆菌属(Amoebobacter sp.)、隐球蓝细菌属(Aphanocapsa sp.)、隐杆蓝细菌属(Aphanothece sp.)、自养水螺菌(Aquaspirillum autotrophicum)、茎瘤固氮根瘤菌(Azorhizobium caulinodans)、固氮螺菌属(Azospirillum sp.)、固氮菌属(Azospirillum sp.)、杆菌属(Bacillussp.)、贝日阿托菌属(Beggiatoa sp.)、贝氏固氮菌属(Beijerinckia sp.)、贝内克菌属(Beneckea sp.)、布拉霉属(Blakeslea sp.)、百日咳鲍特菌(Bordetellapertussis)、大豆慢生根瘤菌(Bradyrhizobium japonicum)、阔显核菌(Caryophanon latum)、柄杆菌属(Caulobacter sp.)、绿胶蓝细菌(Chlorogloeasp.)、紫色硫光合细菌(Chromatium sp.)、色杆菌属(Chromobacterium sp.)、梭状芽胞杆菌(Clostridium sp.)、铵氧化细菌(Comamonas sp.)、棒状杆菌属(Corynebacterium sp.)、隐甲藻(Crypthecodinium sp.)、蓝藻细菌属(Cyanobacteria sp.)、德克斯氏菌属(Derxia sp.)、脱硫线菌属(Desulfonemasp.)、脱硫叠球菌(Desulfosarcina variabilis)、食皂脱硫弧菌(Desulfovibriosapovorans)、外硫红螺菌属(Ectothiorhodospira sp.)、Elina菌属、虫瘟霉属(Entomophthora sp.)、氧化铁杆菌(Ferrobacillus ferroxidans)、黄杆菌属(Flavobacterium sp.)、流感嗜血杆菌属(Haemophilus influenzae)、盐杆菌属(Halobacterium sp.)、噬盐菌(Haloferax mediterranei)、网胰藻(Hydroclathratusclathratus)、敏捷氢细菌(Hydrogenomonas facilis)、氢噬胞菌属(Hydrogenophaga sp.)、生丝微菌属(Hyphomicrobium sp.)、德氏泥杆菌(Ilyobacter delafieldii)、Japonochytrium菌属、Labrys monachus、Lamprocystisroseopersicina、Lampropedia hyalina、军团杆菌属(Legionella sp.)、Leptothrixdiscophorus、甲基杆菌属(Methylobacterium sp.)、甲烷氧化菌属(Methylosinussp.)、细球菌属(Micrococcus sp.)、被孢霉属(Mortierella sp.)、分支杆菌属(Mycobacterium sp.)、硝化杆菌属(Nitrobacter sp.)、诺卡菌属(Nocardiasp.)、Paracoccus dentrificans、灰颤藻(Oscillatoria limosa)、圆弧青霉(Penicillium cyclopium)、发光细菌属(Photobacterium sp.)、Physarumploycephalum、须霉属(Phycomyces sp.)、假单胞菌属(Pseudomonas sp.)、丝状真菌属(Pythium sp.)、雷尔氏菌属(Ralstonia sp.)、根瘤菌属(Rhizobiumsp.)、Rhodobacillus菌属、红细菌属(Rhodobacter sp.)、红球菌属(Rhodococcus sp.)、红环菌属(Rhodocyclus sp.)、万尼氏红微菌(Rhodomicrobium vannielii)、红假单胞菌属(Rhodopseudomonas sp.)、红螺菌属(Rhodospirillum sp.)、裂殖壶菌属(Schizochytrium sp.)、少动鞘氨醇单胞菌(Sphingomonas paucimobilis)、螺菌属(Spirillum sp.)、螺旋藻属(Spirulinasp.)、葡萄球菌属(Staphylococcus sp.)、斯特拉菌属(Stella sp.)、链霉菌属(Streptomyces sp.)、沃氏互养单胞菌(Syntrophomonas wolfei)、嗜热蓝藻细菌(Thermophilic cyanobacteria)、栖热菌(Thermus thermophilus)、噬硫杆菌A2(Thiobacillus A2)、噬硫杆菌属(Thiobacillus sp.)、荚硫菌属(Thiocapsasp.)、海洋破囊壶菌属(Thraustochytrium sp.)、硫囊菌(Thiocystis violacea)、鳗弧菌(Vibrio parahaemolyticus)、自养黄色杆菌(Xanthobacter autotrophicus)、噬麦芽黄单胞菌(Xanthomonas maltophilia)、菌胶团(Zoogloea sp.)以及转化有有产油能力的酶编码基因的微生物。另外,显然任何能产油的微生物都可用于本发明的方法。
在本发明中,步骤(a)中的培养可包括用于微生物生长的第一步培养和用于产油的第二步培养,其中,用于产油的培养优选在含有限制氮源的培养基中进行以提高油的产量。
微生物生产的油是在微生物里自溶,且步骤(b)中的自溶可由脂酶进行。脂酶的实例包括甘油三酯脂酶(EC:3.1.1.34、3.1.1.13)、单酰甘油脂酶(EC:3.1.1.23)以及溶血磷脂酶(EC:3.1.1.5)。
优选地,编码脂酶的基因可被导入有产油能力的微生物中或者或在该微生物中进行扩增。更优选,可将通过与底物反应而被激活的脂酶基因导入微生物中。在本发明的一个实施例中,为了使微生物中的油自溶,使用导入有SEQ ID NOS:5和8的脂酶基因的微生物菌株。在另一个实施例中,使用单独导入有SEQ IDNOS:17、18和19的甘油三酯脂酶基因,或者单独导入SEQ ID NO:20的单酰甘油脂酶,或者导入它们的组合的微生物菌株。
在本发明中,步骤(c)中添加的醇可以是伯醇、仲醇或者叔醇。优选地,可使用具有1到8个碳原子的醇或者两种或更多种具有1到8个碳原子的醇的混合物。更优选,可使用甲醇。
另外,步骤(c)的反应可在80-120℃进行1-24小时。此外,步骤(c)的反应可在有机溶剂,优选氯仿存在的情况下进行。
在本发明的一个实施例中,使用不透明红球菌(Rhodococcus opacus)PD630作为有产油能力的微生物,而且执行由用于微生物细胞生长的第一步培养和用于产油的第二步培养的两步培养方法。在用于产油的第二步微生物培养中,采用限制氮源的培养基以诱导产油。对于所产油的自溶,将可被乙酰胺激活的脂酶基因导入微生物菌株中,并且使用通过向微生物中加入乙酰胺而被激活的脂酶基因以在体内产生约0.27g/L游离脂肪酸。
另外,将所获得的游离脂肪酸溶液冻干以去除水分,然后向其中加入氯仿和含有H2SO4的甲醇,并在100℃反应12个小时。然后向其中加入水,分离有机溶剂层,从而获得游离脂肪酸甲酯。所生产的游离脂肪酸甲酯的浓度是0.2g/L,表明游离脂肪酸已经转变为游离脂肪酸甲酯(图1)。这证明使用本发明的方法可更容易、环保且高效地生产脂肪酸甲酯,表明本发明方法对于生产生物柴油(轻油或类似物的代用品)非常有用。
另一方面,本发明涉及一种使用有产油能力且包含脂酶编码基因的微生物生产脂肪酸甲酯的方法,该方法包括以下步骤:
(a)培养有产油能力且包含脂酶编码基因的微生物以生产油;
(b)通过脂酶诱导微生物内产生的油自溶以生产游离脂肪酸;以及
(c)向所生产的游离脂肪酸中添加甲醇,并且使甲醇与游离脂肪酸反应以生产脂肪酸甲酯。
在本发明的另一个实施例中,使用单独导入有甘油三酯脂酶基因或单独导入有单酰甘油脂酶基因或导入它们的组合的微生物菌株,然后可见,与导入甘油三酯脂酶基因或单酰甘油脂酶基因相比,当甘油三酯脂酶基因与单酰甘油脂酶基因被一起导入微生物菌株时,相同量的葡萄糖可产生更多的游离脂肪酸,因而,当甘油三酯脂酶基因与单酰甘油脂酶基因被一起导入时,脂肪酸甲酯的生产更为高效。因此,本发明优选将甘油三酯脂酶基因与单酰甘油脂酶基因一起导入微生物菌株中。
同时,本发明的下述例子只是阐明具体的培养基和培养方法。使用糖解液如乳清或者CSL(玉米浆)以及其它培养基,以及使用各种培养方法如流加培养或者连续培养,如文献(Lee et al.,Bioprocess Biosyst.Eng.,26:63,2003;Lee et al.,Appl.Microbiol.Biotechnol.,58:663,2002;Lee et al.,Biotechnol.Lett.,25:111,2003;Lee et al.,Appl.Microbiol.Biotechnol.,54:23,2000;Lee et al.,Biotechnol.Bioeng.,72:41,2001)所报道的方法对本领域技术人员而言是显而易见的。
实施例
下文,将参照实施例进一步详细说明本发明。对本领域技术人员而言显而易见的是,所述实施例仅作阐述之用,且不应解释为限制本发明的范围。
特别地,虽然下列实施例仅是阐明以不透明红球菌(Rhodococcus opacus)PD630作为宿主微生物的方法,但根据其所揭示的内容,本领域技术人员显而易见其它任何有产油能力的微生物或任何经转化而具有产油能力的微生物都可应用于本方法。
另外,虽然下列实施例阐明甲醇作为醇用于酯化游离脂肪酸的方法中,但本领域技术人员显而易见其它任何醇都可以用来酯化游离脂肪酸,从而产生各种各样的脂肪酸烷基酯。
实施例1:导入有诱导油自溶的基因的重组不透明红球菌(Rhodococcusopacus)PD630的制备(1)
1-1、pRUCSdp质粒的构建
由pUC18质粒(Phamacia,Biotech,Uppsala,Sweden)制备遗传图谱如图2所示的重组载体rpROUC18(SEQ ID NO:1)和重组载体rpROUC18_KM(SEQ ID NO:2),然后将基因片段以下述方式导入其中。
首先,以Arabidopsis thaliana col.基因组DNA为模板,SEQ ID NO:3和4为引物,进行PCR,由此构建甘油三酯脂酶编码基因片段sdp1。
SEQ ID NO:3:5`-TATAGGCGCCATGGATATAAGTAATGAGGC-3`
SEQ ID NO:4:5`-TGTCCTGCAGCTAAGCATCTATAACACTAC-3`
然后,用限制性内切酶NarI和PstI处理所制备的sdp1片段(SEQ ID NO:5),再用T4DNA连接酶连接到rpROUC 18质粒(Phamacia,Biotech,Uppsala,Sweden)上,从而构建重组质粒pRUCSdp。
1-2、pRUCSdpMag质粒的构建
以Mycobacterium smegmatis(KCTC 9108)基因组DNA为模板,SEQ ID NO:6和7为引物,进行PCR,从而构建单酰甘油脂酶编码基因片段MSMEG_0220。
SEQ ID NO:6:5`-TATATCTAGAACAACGGGGAGGACAACCGAATGGTGAGCAGCACCCGCAGTGAACAC-3`
SEQ ID NO:7:5`-TATATCTAGATCACAGATGACTCACGATCCATGAG-3`
然后,用限制性内切酶(XbaI)处理MSMEG_0220片段(SEQ ID NO:8),再用T4DNA连接酶连接到pRUCSdp质粒上,从而构建如图3所示的重组质粒pRUCSdpMag。然后,将制备的重组质粒pRUCSdpMag导入不透明红球菌PD630DSM 44193株(Deutsche Sammlung von Mikroorganismen und Zellkulturen(DSMZ),Germany)中,从而构建导入有可被乙酰胺激活的脂酶基因的重组菌株。
实施例2:导入有诱导油自溶的基因的重组不透明红球菌PD630的制备(2)
2-1、质粒的构建
用下表1所示的引物、反应条件和基因模板,将甘油三酯脂酶和单酰甘油脂酶导入实施例1-1的质粒rpROUC18和rpROUC18_KM中,从而构建如图4所述的多种质粒。另外,还将甘油三酯脂酶和单酰甘油脂酶同时导入所述质粒中。下表1阐明了限制性酶的种类和基因的来源,且可导入不同种类的基因。
如实施例1所述,在rpROUC18质粒中使用乙酰胺诱导的启动子使所导入的基因在想要的时间工作,乙酰胺的使用浓度为0.5%(w/w)。
ARA_f引物(SEQ ID NO:9)
5′-TATATTCCATGGGGAGGACAACATATAAGTAATGAGGCTAGT-3′
ARAT-r引物(SEQ ID NO:10)
5′-CCGCCTGCAGCTAAGCATCTATAACACTAC-3′
ATAG7_f引物(SEQ ID NO:11)
5′-TATTGACGTCGACAACGGGGAGGACAACCGAATGGAACGCGGATCC
ACTTG-3′
ATAG7-r引物(SEQ ID NO:12)
5′-CTTGTACTAAGTCCCGGGTTAGTGGACGACCTCGAAGC-3′
Mlip2_f引物(SEQ ID NO:13)
5′-TATTGGCGCCGACAACGGGGAGGACAACCGAATGGTGAGCAGCACC
CGCAGTGAA-3′
Mlip2_r引物(SEQ ID NO:14)
5′-CCACGATGGACACGTTGTACTAAGTCTGCAGTCACAGATGACTCACG
ATCC-3′
PAO_f引物(SEQ ID NO:15)
5′-TATAGACGTCATGAAGAAGAAGTCTCTGCTCCCC-3′
PAO_r引物(SEQ ID NO:16)
[表1]
图4中,被导入到rpROUC18KM_Ara质粒和rpROUC18KM_Ara_MAG质粒中的拟南芥的TAG脂酶基因片段具有SEQ ID NO:17的核苷酸序列,被导入到rpROUC18KM_Af7G质粒和rpROUC18KM_Af7G_MAG质粒中的烟曲霉的TAG7G脂酶基因片段具有SEQ ID NO:18的核苷酸序列。此外,被导入到rpROUC18KM_PAO质粒和rpROUC18KM_PAO_MAG质粒中的铜绿假单胞菌的TAG脂酶基因片段具有SEQ ID NO:19的核苷酸序列,被导入到rpROUC18KM_MAG质粒、rpROUC18KM_Ara_MAG质粒、rpROUC18KM_Af7G_MAG质粒和rpROUC18KM_PAO_MAG质粒中的耻垢分枝杆菌的MAG脂酶基因片段具有SEQ ID NO:20的核苷酸序列。
实施例3:用重组不透明红球菌PD630生产脂肪酸甲酯
3-1、用实施例1的重组不透明红球菌PD630生产游离脂肪酸(1)
为了培养实施例1的导入有被乙酰胺激活的脂酶基因的重组不透明红球菌PD630菌株,在限制氮源的培养基中执行两步法培养以生产油。
首先,在第一步培养中,将实施例1的重组菌株在含有100ml NB(营养肉汤)的250毫升烧瓶中,于30℃,250rpm培养24小时。
将肉汤培养液于6000rpm离心10分钟收集微生物细胞,再用MSM培养基(第二步培养时所使用的)清洗以去除NB成分。然后,将细胞溶液于6000rpm离心10分钟收集微生物细胞,再重悬于100ml MSM培养基。MSM培养基(pH7.0)组成如下:每升蒸馏水含有0.8g KH2PO4、5.58g Na2HPO4、0.1g(NH4)2SO4、0.12g MgSO47H2O、0.5mg FeSO45H2O、1.54mg MnSO45H2O、2.86mg H3BO3、0.039mg CuSO45H2O、0.041mg CoCl26H2O、0.021mg ZnCl2、0.025mgNa2MoO42H2O以及11.6mg CaCl22H2O。
向重悬有微生物细胞的100ml MSM培养基中加入20g/l葡萄糖作为碳源,然后于30℃,250rpm培养微生物细胞24小时。然后用显微镜实时监测微生物菌株内的油积累。然后将0.5%(w/v)乙酰胺加到微生物细胞中,于30℃培养48小时,激活脂酶以生产游离脂肪酸。
培养结束后,将肉汤培养液于6000rpm离心10分钟收集微生物细胞,将所收集的细胞用蒸馏水清洗一次,然后在干燥器中于100℃干燥24小时。
将干燥后的细胞用配有毛细管柱的Agilent 6890N系列气相色谱***(Chiraldex G-TA of Astec,USA)进行分析,从而测量细胞内合成的游离脂肪酸的含量。结果表明两步法烧瓶培养的游离脂肪酸的产量浓度为0.27g/l。
3-2、用实施例2的重组不透明红球菌PD630菌株生产游离脂肪酸(2)
为了培养实施例2的导入有被乙酰胺激活的脂酶基因的重组不透明红球菌PD630菌株,在限制氮源的培养基中执行两步法培养以生产油。
首先,在第一步培养中,将实施例2的重组菌株各在含有200ml TSB(胰蛋白胨大豆肉汤)的250毫升烧瓶中,于30℃,200rpm培养16小时。
将肉汤培养液于3000rpm离心30分钟,收集微生物细胞,再用MSM培养基清洗以去除TSB成分。然后,将细胞溶液于3000rpm离心30分钟,收集微生物细胞,再重悬于200ml MSM培养基。MSM培养基(pH 7.0)组成如下:每升蒸馏水含有0.8g KH2PO4、5.58g Na2HPO4、0.1g(NH4)2SO4、0.12g MgSO47H2O、1.0mg FeSO45H2O、3.08mg MnSO45H2O、5.72mg H3BO3、0.078mg CuSO45H2O、0.082mg CoCl26H2O、0.042mg ZnCl2、0.050mg Na2MoO42H2O以及23.2mgCaCl22H2O。
向重悬有微生物细胞的200ml MSM培养基中加入20g/l葡萄糖作为碳源,然后于30℃,200rpm培养48小时。然后用显微镜实时监测微生物菌株内的油积累。然后将0.5%(w/v)乙酰胺添加到微生物细胞中,于30℃培养24小时,激活脂酶以生产游离脂肪酸。
培养结束后,将肉汤培养液于3000rpm离心30分钟,收集微生物细胞。上清于-45℃,10mm Torr冷冻干燥48小时。所收集的细胞用蒸馏水清洗一次,然后在干燥器中于80℃干燥24小时。从得到的材料中各取0.1g,按产商说明书用微生物鉴定***(Microbial ID,Inc.,Network,Del.,USA)进行处理以制备气相色谱样品。
所制备的每份样品用配有毛细管柱的Agilent 6890N系列气相色谱***(Chiraldex G-TA of Astec,USA)进行分析,从而测量细胞内合成的游离脂肪酸的含量。
上述两步烧瓶培养法的结果表明上清中游离脂肪酸的含量显著高于细胞中游离脂肪酸的含量,表明游离脂肪酸是细胞外分泌的。图5显示出在冷冻干燥的上清中游离脂肪酸的测量结果。由图5可见,游离脂肪酸是以各种长度的游离脂肪酸混合物形式产生的。另外可见,与单独导入甘油三酯脂酶相比,当甘油三酯脂酶与单酰甘油脂酶被一起导入时,相同量的葡萄糖可产生更多的游离脂肪酸。
3-3、游离脂肪酸向脂肪酸甲酯的转变
向在实施例3-1所获得的干燥菌种中加入2ml氯仿和1ml含3%(v/v)H2SO4的甲醇。混合物于100℃反应12个小时。
反应结束后,将混合物冷却至室温,加入1ml蒸馏水,强烈搅拌5分钟,使混合物分离成有机溶剂(氯仿)层和水(水溶液)层。将得到的材料于10,000rpm离心10分钟,仅收集有机溶剂层,并用配有毛细管柱的Agilent 6890N系列气相色谱***(Chiraldex G-TA of Astec,USA)进行分析,从而测量有机溶剂层中脂肪酸甲酯的浓度。
结果如图6所示,发现所生产的C13脂肪酸甲酯的浓度为0.2g/L。这表明游离脂肪酸被转变为脂肪酸甲酯。
另外,将上述同样的甲醇加入实施例3-2所获得的上清中进行反应,然后测量反应液中所产生的脂肪酸甲酯的浓度。
结果如图7所示,游离脂肪酸转变为脂肪酸甲酯。特别地,与单独导入甘油三酯脂酶(TAG脂酶)或单酰甘油脂酶(MAG脂酶)相比,当甘油三酯脂酶(TAG脂酶)与单酰甘油脂酶(MAG脂酶)被一起导入时,可产生显著更大量的脂肪酸甲酯。如上所述,根据本发明使用微生物生产脂肪酸烷基酯的方法生产效率高,可立即用于生物燃料的生产。此外,显然本发明的脂肪酸甲酯生产效率比本领域公知的现有方法要高得多。
换句话说,这证明用本发明的方法可更容易、环保且高效地生产脂肪酸甲酯,表明本发明方法对于生产生物柴油(轻油或类似物的代用品)非常有用。
工业实用性
如上所述,根据本发明方法,可以通过代谢工程方法将在微生物中积累的油,例如甘油三酯(典型的通过微生物生产的油),高效转变为脂肪酸烷基酯。因此,本发明方法可用于脂肪酸烷基酯(最近发现可作为有效的生物柴油)的工业生产。
虽然本发明参照具体的特征作了详细描述,但本领域技术人员显然可知该描述只是优选实施例,并非限制本发明的范围。因而,本发明的实质范围将由随附的权利要求书和其等同形式限定。
Claims (12)
1.一种使用有产油能力的不透明红球菌(Rhodococcus opacus)生产脂肪酸烷基酯的方法,所述不透明红球菌包括编码甘油三酯脂酶的基因和编码单酰甘油脂酶的基因,该方法包括以下步骤:
(a)培养所述不透明红球菌,由此生产油;
(b)诱导产生的油在所述不透明红球菌内自溶,由此生产游离脂肪酸;并且
(c)向所生产的游离脂肪酸中添加醇,并且使醇与游离脂肪酸反应,由此生产脂肪酸烷基酯。
2.权利要求1的生产脂肪酸烷基酯的方法,其中所述油选自甘油三酯(TAG)、二酰甘油(DAG)、单酰甘油(MAG)、磷脂、甾醇类脂、鞘脂、糖脂、异戊烯醇类脂和聚酮化合物。
3.权利要求1的生产脂肪酸烷基酯的方法,其中步骤(a)在包含有限氮源的培养基中进行。
4.权利要求1的生产脂肪酸烷基酯的方法,其中所述游离脂肪酸是饱和或者不饱和脂肪酸。
5.权利要求4的生产脂肪酸烷基酯的方法,其中所述游离脂肪酸选自油酸、亚油酸、亚麻酸、棕榈油酸、蓖麻油酸、11-十八碳烯酸、二十碳烯酸、花生四烯酸、EPA(5,8、11、14、17二十碳五烯酸)、芥子酸、DHA(4,7,10,13,16,19-docosahexaenoic酸)、丁酸、已酸、辛酸酸、癸酸、月桂酸、豆蔻酸、软脂酸、硬脂酸、花生酸、正廿二烷酸和二十四(烷)酸。
6.权利要求1的生产脂肪酸烷基酯的方法,其中所述游离脂肪酸被选自芳环基、环氧基、氰基基团和卤素基团之一的取代基取代。
7.权利要求1的生产脂肪酸烷基酯的方法,其中步骤(b)通过脂酶进行。
8.权利要求7的生产脂肪酸烷基酯的方法,其中所述脂酶是甘油三酯脂酶、单酰甘油脂酶和溶血磷脂脂酶中的任意一种或多种。
9.权利要求1的生产脂肪酸烷基酯的方法,编码脂酶的基因具有选自SEQID NO:5、SEQ ID NO:8、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQID NO:20中的任意一种序列。
10.权利要求1的生产脂肪酸烷基酯的方法,其中步骤(c)在80-120℃进行1-24个小时。
11.一种使用有产油能力的不透明红球菌生产脂肪酸甲酯的方法,所述不透明红球菌包含编码甘油三酯脂酶的基因和编码单酰甘油脂酶的基因,该方法包括以下步骤:
(a)培养所述不透明红球菌,由此生产油;
(b)通过脂酶诱导产生的油在所述不透明红球菌内自溶,由此生产游离脂肪酸;以及
(c)向所生产的游离脂肪酸中添加甲醇,并且使甲醇与游离脂肪酸反应,由此生产脂肪酸甲酯。
12.权利要求11的生产脂肪酸甲酯的方法,其中编码甘油三酯脂酶的基因具有选自SEQ ID NO:5、SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19中的任意一种序列,而且编码单酰甘油脂酶的基因具有SEQ ID NO:8或SEQ ID NO:20中的任意一种序列。
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