槐糖脂产生菌拟威克酵母的诱变育种、遗传改造及槐糖脂体内和体外抗肿瘤机制研究
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摘要
表面活性剂是一类最重要的精细化工产品,广泛应用于家庭、工业和农业。人们生活工作的各个方面几乎都离不开表面活性剂。随着人类社会的进步与发展以及人们环保意识的增强,生物表而活性剂替代化学合成的表面活性剂将成为必然趋势。槐糖脂是一类由酵母产生的糖脂类生物表面活性剂,由于其具有高产量、生物可降解性、低毒性和良好的环境兼容性等许多优点,可广泛应用于石油、环境保护、化妆品、洗涤剂、药学及纳米科技等工业领域,尤其是在医药领域的应用得到了研究者的广泛关注。因此,槐糖脂被认为是一种最有发展和应用潜力的、可能替代化学表面活性剂的可再生的生物表面活性剂,对其开发及应用将会带来极大的经济效益和生态效益。
本论文的主要研究内容及结果如下：
1.低能离子束注入诱变选育高产槐糖脂菌株
目前,对产槐糖脂菌株的诱变育种研究较少。本论文对实验室现有的产槐糖脂菌株W. domercqiae Y2A采用低能N+注入的方式进行了诱变育种。
选取N+作为离子束注入的离子源,发现经过不同剂量N+注入后,菌株Y2A的存活率曲线是典型的“马鞍型”曲线,即随着注入剂量增加,存活率呈现出先降低后升高再降低的趋势。选取的诱变剂量是3×1015ions/cm2,经过注入诱变选育后,18个突变株菌的总槐糖脂产量较出发株提高了20%以上,18个突变株的内酯型槐糖脂产量较出发株提高了100%以上；5个突变株的酸型槐糖脂产量较出发株提高了30%以上。其中,菌株N3-18在摇瓶水平发酵时总槐糖脂产量可达104g/L,较出发株提高了约84.71%,酸型槐糖脂产量达77g/L,较出发株提高了1.48倍；在5-L发酵罐水平,其总槐糖脂产量可达135g/L,较出发株产量提高一倍左右。
出发株和5株高产菌株所产的总槐糖脂组成的HPLC分析表明,各突变株所产槐糖脂组成同出发株相同,但是,突变株的多数槐糖脂分子的峰面积较原菌株有较大提高,某些槐糖脂组分在总槐糖脂中所占的比例同野生株有较大变化,得到了内酯型或者酸型槐糖脂比例增加的突变株,而这些特定的高产菌株可以更好的应用于不同的工业领域。
2.新型槐糖脂的生产、分离纯化及结构鉴定
槐糖脂的结构会影响其理化活性和生物学活性。天然合成的槐糖脂脂肪酸部分通常是16C或18C,本工作旨在获得脂肪酸部分碳链>18C或者为C18：3的新型槐糖脂。
分别以菜籽油、亚麻油和鱼油为疏水性碳源发酵槐糖脂,得到了三种组分差异较大的槐糖脂混合物。利用制备型HPLC制备得到了这三种槐糖脂粗品中的主要组分,并用MS、MS/MS和GC-MS分析了这几种槐糖脂纯品的结构。以亚麻油为底物时,首次在W. domercqiae中得到了脂肪酸部分为C18：3的双乙酰化内酯型槐糖脂,并且,该物质的抗肿瘤活性未有报道。以富含长链脂肪酸的鱼油为底物时得到了碳链长度大于18C的新型槐糖脂,包括：脂肪酸部分为C20：5的,不含、含有一个或两个乙酰基的酸型槐糖脂；脂肪酸部分为C22：6的双乙酰化酸型槐糖脂：脂肪酸部分为C22：3的无乙酰基的内酯型槐糖脂：脂肪酸部分为C20：0的无乙酰基的内酯型槐糖脂。这些新型槐糖脂可能会拓宽槐糖脂的应用领域。
3.槐糖脂脂肪酸部分结构对其抗宫颈癌活性影响、体外诱导细胞凋亡的途径和体内抗宫颈癌活性的研究
虽然之前的研究已经证明了槐糖脂纯品的乙酰化程序、脂肪酸部分的不饱和度及是否存在分子内内酯化等都会影响其抗肿瘤活性,脂肪酸的碳链长度和多不饱和度(双键>2)对槐糖脂抗肿瘤活性的影响还没有被系统研究过。另外,槐糖脂引起细胞调亡的信号通路也不清楚。目前,槐糖脂的抗肿瘤活性多限于体外实验,体内抗肿瘤活性报道非常少。
选取6种双乙酰化内酯型槐糖脂分子,其脂肪酸部分分别为C18：0,C18：1,C18：2,C18：3,C16：0和C16：1,对这些槐糖脂分子对HeLa和CaSki(?)胞的细胞毒性进行了研究。结果农明,槐糖脂的细胞毒性大小不仅受脂肪酸不饱和度影响,同时也受脂肪酸链长影响。研究脂肪酸链长对槐糖脂体外抗宫颈癌活性的影响发现：碳链长度越长,槐糖脂的细胞毒性越大。不同槐糖脂分子的细胞毒性应该是脂肪酸不饱和度、碳链长度和槐糖部分乙酰化共同作用的结果。
对抗宫颈癌活性最好的脂肪酸部分为C18：1的双乙酰化内酯型槐糖脂组分的体外抗宫颈癌机制进行了深入研究,利用相差显微镜、电子显微镜及TUNEL等证明了该组分可以诱导HeLa细胞发生凋亡,流式细胞术检测细胞周期分布发现,该槐糖脂作用后出现了明显的二倍体亚峰,且多数细胞被阻滞在G0期,少部分细胞被阻滞在G2期。利用荧光定量PCR分析发现槐糖脂可以诱导HeLa细胞内质网应激的特异性蛋白CHOP和Bip蛋白的表达,而caspase-12和caspase-3也同样被激活,说明槐糖脂可以通过ER应激通路来诱导HeLa细胞发生细胞凋亡。而对线粒体膜电位变化及胞质中细胞色素C的含量检测发现,槐糖脂作用前后二者变化不大,说明线粒体通路可能没有参与槐糖脂诱导的细胞凋亡。
建立了小鼠宫颈癌U14细胞的移植瘤模型,以研究槐糖脂的体内抗宫颈癌活性,发现内酯型槐糖脂粗品对人宫颈癌裸鼠移植瘤有较好的抑制作用,低、中、高剂量的槐糖脂给药组抑瘤率分别可以达到15.73%、26.40%和34.83%。并且,槐糖脂在体内也可以诱导肿瘤组织发生细胞凋亡。
4.主要酯酶／脂肪酶和单加氧酶在槐糖脂合成中的功能的初步研究
目前,还没有发现催化酸型槐糖脂分子形成内酯的内酯化酶。通常,催化合成内酯的酶类可能为硫酯酶或者酯酶／脂肪酶及特定的单加氧酶。本工作利用W.domercqiae基因组注释及该菌株在硫酸铵和酵母粉两种条件下基因表达水平的差异对可能完成催化内酯化反应的酯酶／脂肪酶及单加氧酶进行了筛选,研究其在槐糖脂合成中的作用。
首先,建立了W.domercqiae的遗传操作系统：确定潮霉素为抗性筛选标记,甘油三磷酸脱氢酶的启动子Pgpd可作为外源基因表达的启动子,电转化的方法可以高效完成外源基因的导入。
通过基因组及表达谱分析,选取了酵母粉条件下较硫酸铵条件下表达显著上调的8个酯酶／脂肪酶基因及4个单加氧酶,利用double-joint PCR的方法成功构建了基因的敲除盒,对这12个基因进行了成功敲除,并对其在槐糖脂合成过程中的功能进行了初步分析。lipB基因可以影响多种槐糖脂组分的合成,ΔlipB敲除株槐糖脂产量较野生株大幅下降,当以油酸为单一碳源时,ΔlipB敲除株无法检测到槐糖脂产生。通过胞外蛋白分析及荧光定量PCR检测槐糖脂合成过程中关键酶的表达情况,推测敲除株产量降低的原因包括两个方面：菌株内与槐糖脂合成相关的关键酶的表达量下调；胞外糖苷水解酶含量增加,可能通过水解槐糖脂的糖苷键而使槐糖脂产量下降。△moA(?)敲除株所产酸型槐糖脂C18:2DASL在总槐糖脂中比例增加而内酯型槐糖脂C18：2DLSL所占比例则有较大下降,推测(?)moA(?)基因的功能应该是同脂肪酸部分为C18：2的酸型槐糖脂的内酯化相关。noD基因影响了C18:2NASL的乙酰化。在以葡萄糖和豆油为底物时,同野生株相比,ΔlipD、ΔlipE、 ΔlipF和ΔlipG敲除株发酵所得槐糖脂产量均有所降低,槐糖脂中乙酰化程度较高的组分含量降低,而乙酰化程度相对较低的组分含量增加,推测这种现象的出现是由于脂肪酶/酯酶的缺失使菌株将油脂水解为脂肪酸的能力下降,从而间接影响槐糖脂的合成,同时,脂肪酶可能也有催化槐糖脂乙酰化的功能,它的缺失导致槐糖脂乙酰化程度降低；而当以葡萄糖和油酸为底物时,由于油酸为单一脂肪酸,这几个敲除株槐糖脂产量及组成无显著变化。lipH基因同脂肪酸部分为C18：2的酸型槐糖脂的内酯化有一定关系,并且对菌株将C18：1整合入槐糖脂的过程也有一定影响。除moA和lipH基因同脂肪酸部分为C18：2的酸型槐糖脂的内酯化相关外,其它几个基因的敲除多对槐糖脂内酯化无显著影响,推测菌体内的内酯化酶应有多种。
5.鼠李糖基转移酶在拟威克酵母中的异源表达
为获得具有更好的生物学活性或表面活性的槐糖脂分子,对槐糖脂的结构修饰通常是通过化学法或酶催化法进行,而本论文则成功构建了鼠李糖基转移酶A和B基因(rhlA和rhlB)在W. domercqiae中的表达载体,对W. domercqiae Y2A进行遗传改造,筛选得到了相应的重组菌株Y2A-2,使其具有表达鼠李糖基转移酶1的能力。利用HPLC-MS的方法对重组菌株发酵的糖脂结构进行鉴定,发现两个组分(保留时间分别为16.74和17.47)的分子量分别为554(C26H50O12)和536(C26H48O11),前者可能为含1分子葡萄糖、1分子鼠李糖,脂肪酸部分为C14：0的无乙酰基的酸型糖脂,后者可能是含有两个鼠杍糖的、脂肪酸部分为C14：1的糖脂,为我们所要获得的目的糖脂；利用制备型HPLC,分离得到了含有一个鼠李糖的、脂肪酸部分为C18：3的酸型糖脂和既含有葡萄糖也含有鼠李糖、脂肪酸部分为C14：1的酸型糖脂。此外,同野生株相比,重组菌株产酸型槐糖脂的能力提高,而产内酯型槐糖脂能力下降。
Surfactants are one of the most important industrial bulk chemicals which have been widely applied in industrial, household and agricultural sectors. However, with the development of society and growing environmental awareness, biosurfactants have attracted increasing attention and will play more important roles than chemically synthetic surfactants in our daily life. Sophorolipids (SLs) are important glycolipid biosurfactants synthesized by a selected number of non-pathogenic yeast species. They have great potential to be widely applied in many industrial sectors, including petroleum, environment, cosmetics, detergent industries, also including nanotechnology and especially pharmaceutical industry due to their high yields, biodegradability, low toxicity, good emulsification ability, tolerance to temperature, and good biological activities. They are one of the most promising biosurfactants which can bring great social, economic and ecological benefits.
The main research contents and results are as follows:
1. Mutation breeding of high sophorolipid-producing strains by low-energy nitrogen ion implantation
Wickerhamiella domercqiae Y2A (screened and preserved by our laboratory) is one strain which can produce SLs. To meet the increasing demands of SLs as biosurfactant and bioactive compounds, it is necessary to obtain more high sophorolipid-producing strains. The wild strain was treated by low energy N+ion beam implantation with energy of15keV and doses ranging from1.5×1015to5.25×1015ions/cm2. The results showed that the survival curve of the strain took a "saddle" shape, and in the "saddle" region, the better mutagenic effect can be obtained. According to the survival curve of stain Y2A, the mutation dose was selected as3×1015ions/cm2. About114strains were selected out from YEPD agar plates after nitrogen ion implantation. The total sophorolipid production from18strains increased by more than20%compared with that of the wild strain. The lactonic SLs yields from 18strains increased by100%more than that of the wild strain.5strains that produced acidic SLs30%more than that of the wild strain were obtained. Especially, the sophorolipid production of strain N3-18reached104g/L at200rpm,30℃in shaking flask, which increased84.71%than that of the wild strain. The production of SLs was elevated to135g/L in5L fermentor.
HPLC analysis showed that the composition of every sophorolipid mixture from different strains was similar except that the contents of most components from mutants were higher than that from wild strain. Two mutants N1-32and N3-18, produced more acidic sophorolipid components; three lactonic sophorolipid molecules with good anticancer activities were greatly enhanced in several mutants, especially mono-acetylated lactonic SL with a C18:1fatty acid, were enhanced by153%and211%in strain N1-32and N3-18. Low-energy nitrogen ion beam implantation was efficient for obtaining a variety of high and specific sophorolipid-producing mutants to be applied in food, cosmetic, environmental and pharmaceutical sectors etc.
2. Production, purification and structure elucidation of novel sophorolipid molecules
Different SLs have different biological and physicochemical activities. Compared with SLs with shorter chain hydroxy fatty acid, SLs with longer chain hydroxy fatty acid might have better properties. Due to the differences in the properties of SLs, it is necessary to obtain more novel SLs. To obtain novel sophorolipid molecules differing in fatty acid part, three different hydrophobic carbon sources, rapeseed oil and two uncommonly-used oils (linseed oil and fish oil) were used for the production of SLs. The compositions of SLs produced by W. domercqiae var. sophorolipid grown on different hydrophobic carbon sources were very different. Sophorolipid molecules were purified by preparative HPLC and their structures were identified by MS, MS/MS and GC-MS. The di-acetylatcd lactonic sophorolipid with a C18:3fatty acid whose anticancer activity was not reported before was first obtained from W. domercqiae on linseed oil. Several unconventional acidic sophorolipid molecules with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DMA) including non-, mono-and di-acetylated acidic SLs with an EPA, di-acetylated acidic sophorolipid with a DHA, two unconventional lactonic sophorolipid molecules, non-acetylated lactonic sophorolipid with a docosatrienoic acid and non-acetylated lactonic sophorolipid with an eicosanoic acid were obtained using fish oil as hydrophobic carbon source.
3. The study of the relationship between fatty acid part of sophorolipid and their cytotoxicity to human cervical cancer cells, the signaling pathway of apoptosis induced by sophorolipid and the antitumor activity of SLs in vivo
It was found that differences of sophorolipid structures in acetylation degree of sophorose, unsaturation degree of hydroxy fatty acid, and lactonization or not could all affect the cytotoxicities of SLs. However, the influence of carbon chain length and polyunsaturation (the number of double bonds>2) of fatty acid moiety on the anticancer activity of SLs has not been detailedly studied, the apoptotic signaling pathway needs to be further revealed. Besides, the in vivo anti-tumor activity of SLs has not been reported before.
Six sophorolipid molecules including di-acetylated lactonic SLs with C16:1, C16:0, C18:3C18:1, C18:2or C18:0, were all employed and the relationship between the structures of sophorolipid molecules and their cytotoxicities to HeLa and Caski cells were investigated in detail. Cytotoxicity of sophorolipid molecules with different unsaturation degree was also influenced by carbon chain length of SLs. The influence of carbon chain length of fatty acid in SLs was also investigated and the results showed that the longer the carbon chain length of sophorolipid, the stronger its cytotoxicity to HeLa and Caski cells.
To investigate if SLs could induce the apoptosis or differentiation of human cervical cancer, cell morphological changes, TUNEL, cell cycle distribution, induction expression of CHOP and Bip, caspase-12and caspase-3activities were measured after they were treated with di-acetylated lactonic sophorolipid with a C18:1fatty acid. The cells developed many features of apoptosis such as cell shrinkage, chromatin condensation inside the nuclear membrane and margination to be a block or crescent shape, nuclear fragmentation, and appearance of apoptotic bodies. Cell cycle was blocked at Go phase and partly at G2phase by sophorolipid. The induction expression of CHOP/GADD153and Bip/GRP78in endoplasmic reticulum (ER) signaling pathway, and the activation of apoptotic caspase-12and caspase-3demonstrated that the apoptosis induced by SLs might be triggered through ER signaling pathway. However, Cyt. C was not released from mitochondria and no loss of MMP was detected in HeLa cells treated with sophorolipid, which revealed that mitochondria was not involved in the apoptosis induced by sophorolipid.
The anti-tumor activity of lactonic SLs in human cervical tumor xenografts in BALB/c mice were first investigated.5,50,500mg/kg lactonic SLs showed15.73%,26.40%and34.83%of inhibition without significant non-tumor toxicity in tumor-bearing mice. TUNEL results showed that lactonic SLs could induce apoptosis of tumors in vivo.
4. The primary study of the roles of main esterase/lipase and monooxygenases in sophorolipid biosynthesis
No lactone esterase has been identified in W. domercqiae or in other SL producing species. Normally, two types of enzyme which might catalyze the lactonization reaction were thioesterase or esterase/lipase and specific monooxygenases. The present work analyzed the genome of W. domercqiae and differences on gene expression level of W. domercqiae with two different nitrogen sources, yeast extract and ammonium sulphate, to screen the possible esterase/lipase and monooxygenase enzyme that catalyze lactonization reaction. Their roles in SL synthesis were primarily investigated.
The genetic engineering system of W. domercqiae was established:hygromycin B was chosen as the selection marker; electroporation method was an efficient transformation method; the promoter for glycerol3'-phosphate dehydrogenase Pgpd could be used as the promoter of the exogenous expression.
Eight esterase/lipase and four monooxygenases were selected, because their expression levels were up-regulated significantly when yeast extract was used as the nitrogen source. Gene deletion cassette was successfully constructed by double-joint PCR method and then the deletion strains were obtained. The roles of the12genes in SL biosynthesis were preliminarily analyzed. lipB can greatly affect the yields and the compositions of SLs. Compared with the wild strain, the yields of SLs produced by ΔlipB strain dropped significantly and no SLs could be detected when oleic acid was used as the sole carbon source. There might be two reasons for the reduction of SL yields:the down-regulated expression of enzymes involved in SL biosynthesis including cytochrome P450monooxygenase and UDP-glucosyltransferase; increase of extracellular glycoside hydrolase, which might hydrolyze the glycosidic bond of SLs. The proportion of di-acetylated acidic SL with C18:2fatty acid enhanced while that of di-acetylated lactonic SL with C18:2fatty acid decreased in the SLs produced by ΔmoA strain, which might demonstrate that moA might be related to the lactonization of the SLs with a C18:2fatty acid. moD gene influenced the acetylation degree of acidic SL with C18:2fatty acid. Compared with the wild strain, the SL yields of AlipD to AlipG strains were decreased when using glucose and soya-been oil as carbon source, which might be due to the decreased ability of the deletion strains to hydrolyze the glycerides into fatty acids. Besides, the contents of SLs with a higher acetylation degree produced by the four deletion strains were also decreased, suggesting that lipases might catalyze the acetylation reaction of SLs. The ability of AlipH strain to incorporate C18:1into SLs was reduced and the proportion of di-acetylated lactonic SL with C18:2fatty acid was reduced. The deletion of other genes had no significant influence on SL biosynthesis.
5. Heterologous expression of rhamnosyltransferase in W. domercqiae
Commonly, in order to obtain SLs with better biological activity or surface activity, structural modification of SLs was performed by chemical or enzymatic method in vitro. Rhamnosyltransferase expression vector in W. domercqiae was successfully constructed and one recombinant strain Y2A-2was obtained which could heterologous express the rhamnosyltransferase Rh1A and Rh1B.
HPLC-MS was used to identify the structures of the glycolipids produced by Y2A-2. There were two novel components (retention time at16.74and17.47min, respectively) with molecular weight of554(C26H50O12) and536(C26H48O11), the possible structure of the former was acidic C14:0glycolipid containing non-acetylated glucose and rhamnose, and the structure of the latter was acidic C14:1glycolipid containing two rhamnose. Preparative HPLC was also applied to obtain pure glycolipid and the structure of pure glycolipid was elucidated by MS method. Two novel glycolipids, acidic C18:3glycolipid containing one rhamnose and acidic C14:1glycolipid containing both glucose and rhamnose were obtained. However, because of their low contents, it was difficult for them to be prepared in large scale. Therefore, their accurate structures were not further identified by NMR and other methods. Compared with the wild strain, the recombinant strain produced more acidic SLs and less laconic SLs.
本论文的主要研究内容及结果如下：
1.低能离子束注入诱变选育高产槐糖脂菌株
目前,对产槐糖脂菌株的诱变育种研究较少。本论文对实验室现有的产槐糖脂菌株W. domercqiae Y2A采用低能N+注入的方式进行了诱变育种。
选取N+作为离子束注入的离子源,发现经过不同剂量N+注入后,菌株Y2A的存活率曲线是典型的“马鞍型”曲线,即随着注入剂量增加,存活率呈现出先降低后升高再降低的趋势。选取的诱变剂量是3×1015ions/cm2,经过注入诱变选育后,18个突变株菌的总槐糖脂产量较出发株提高了20%以上,18个突变株的内酯型槐糖脂产量较出发株提高了100%以上；5个突变株的酸型槐糖脂产量较出发株提高了30%以上。其中,菌株N3-18在摇瓶水平发酵时总槐糖脂产量可达104g/L,较出发株提高了约84.71%,酸型槐糖脂产量达77g/L,较出发株提高了1.48倍；在5-L发酵罐水平,其总槐糖脂产量可达135g/L,较出发株产量提高一倍左右。
出发株和5株高产菌株所产的总槐糖脂组成的HPLC分析表明,各突变株所产槐糖脂组成同出发株相同,但是,突变株的多数槐糖脂分子的峰面积较原菌株有较大提高,某些槐糖脂组分在总槐糖脂中所占的比例同野生株有较大变化,得到了内酯型或者酸型槐糖脂比例增加的突变株,而这些特定的高产菌株可以更好的应用于不同的工业领域。
2.新型槐糖脂的生产、分离纯化及结构鉴定
槐糖脂的结构会影响其理化活性和生物学活性。天然合成的槐糖脂脂肪酸部分通常是16C或18C,本工作旨在获得脂肪酸部分碳链>18C或者为C18：3的新型槐糖脂。
分别以菜籽油、亚麻油和鱼油为疏水性碳源发酵槐糖脂,得到了三种组分差异较大的槐糖脂混合物。利用制备型HPLC制备得到了这三种槐糖脂粗品中的主要组分,并用MS、MS/MS和GC-MS分析了这几种槐糖脂纯品的结构。以亚麻油为底物时,首次在W. domercqiae中得到了脂肪酸部分为C18：3的双乙酰化内酯型槐糖脂,并且,该物质的抗肿瘤活性未有报道。以富含长链脂肪酸的鱼油为底物时得到了碳链长度大于18C的新型槐糖脂,包括：脂肪酸部分为C20：5的,不含、含有一个或两个乙酰基的酸型槐糖脂；脂肪酸部分为C22：6的双乙酰化酸型槐糖脂：脂肪酸部分为C22：3的无乙酰基的内酯型槐糖脂：脂肪酸部分为C20：0的无乙酰基的内酯型槐糖脂。这些新型槐糖脂可能会拓宽槐糖脂的应用领域。
3.槐糖脂脂肪酸部分结构对其抗宫颈癌活性影响、体外诱导细胞凋亡的途径和体内抗宫颈癌活性的研究
虽然之前的研究已经证明了槐糖脂纯品的乙酰化程序、脂肪酸部分的不饱和度及是否存在分子内内酯化等都会影响其抗肿瘤活性,脂肪酸的碳链长度和多不饱和度(双键>2)对槐糖脂抗肿瘤活性的影响还没有被系统研究过。另外,槐糖脂引起细胞调亡的信号通路也不清楚。目前,槐糖脂的抗肿瘤活性多限于体外实验,体内抗肿瘤活性报道非常少。
选取6种双乙酰化内酯型槐糖脂分子,其脂肪酸部分分别为C18：0,C18：1,C18：2,C18：3,C16：0和C16：1,对这些槐糖脂分子对HeLa和CaSki(?)胞的细胞毒性进行了研究。结果农明,槐糖脂的细胞毒性大小不仅受脂肪酸不饱和度影响,同时也受脂肪酸链长影响。研究脂肪酸链长对槐糖脂体外抗宫颈癌活性的影响发现：碳链长度越长,槐糖脂的细胞毒性越大。不同槐糖脂分子的细胞毒性应该是脂肪酸不饱和度、碳链长度和槐糖部分乙酰化共同作用的结果。
对抗宫颈癌活性最好的脂肪酸部分为C18：1的双乙酰化内酯型槐糖脂组分的体外抗宫颈癌机制进行了深入研究,利用相差显微镜、电子显微镜及TUNEL等证明了该组分可以诱导HeLa细胞发生凋亡,流式细胞术检测细胞周期分布发现,该槐糖脂作用后出现了明显的二倍体亚峰,且多数细胞被阻滞在G0期,少部分细胞被阻滞在G2期。利用荧光定量PCR分析发现槐糖脂可以诱导HeLa细胞内质网应激的特异性蛋白CHOP和Bip蛋白的表达,而caspase-12和caspase-3也同样被激活,说明槐糖脂可以通过ER应激通路来诱导HeLa细胞发生细胞凋亡。而对线粒体膜电位变化及胞质中细胞色素C的含量检测发现,槐糖脂作用前后二者变化不大,说明线粒体通路可能没有参与槐糖脂诱导的细胞凋亡。
建立了小鼠宫颈癌U14细胞的移植瘤模型,以研究槐糖脂的体内抗宫颈癌活性,发现内酯型槐糖脂粗品对人宫颈癌裸鼠移植瘤有较好的抑制作用,低、中、高剂量的槐糖脂给药组抑瘤率分别可以达到15.73%、26.40%和34.83%。并且,槐糖脂在体内也可以诱导肿瘤组织发生细胞凋亡。
4.主要酯酶／脂肪酶和单加氧酶在槐糖脂合成中的功能的初步研究
目前,还没有发现催化酸型槐糖脂分子形成内酯的内酯化酶。通常,催化合成内酯的酶类可能为硫酯酶或者酯酶／脂肪酶及特定的单加氧酶。本工作利用W.domercqiae基因组注释及该菌株在硫酸铵和酵母粉两种条件下基因表达水平的差异对可能完成催化内酯化反应的酯酶／脂肪酶及单加氧酶进行了筛选,研究其在槐糖脂合成中的作用。
首先,建立了W.domercqiae的遗传操作系统：确定潮霉素为抗性筛选标记,甘油三磷酸脱氢酶的启动子Pgpd可作为外源基因表达的启动子,电转化的方法可以高效完成外源基因的导入。
通过基因组及表达谱分析,选取了酵母粉条件下较硫酸铵条件下表达显著上调的8个酯酶／脂肪酶基因及4个单加氧酶,利用double-joint PCR的方法成功构建了基因的敲除盒,对这12个基因进行了成功敲除,并对其在槐糖脂合成过程中的功能进行了初步分析。lipB基因可以影响多种槐糖脂组分的合成,ΔlipB敲除株槐糖脂产量较野生株大幅下降,当以油酸为单一碳源时,ΔlipB敲除株无法检测到槐糖脂产生。通过胞外蛋白分析及荧光定量PCR检测槐糖脂合成过程中关键酶的表达情况,推测敲除株产量降低的原因包括两个方面：菌株内与槐糖脂合成相关的关键酶的表达量下调；胞外糖苷水解酶含量增加,可能通过水解槐糖脂的糖苷键而使槐糖脂产量下降。△moA(?)敲除株所产酸型槐糖脂C18:2DASL在总槐糖脂中比例增加而内酯型槐糖脂C18：2DLSL所占比例则有较大下降,推测(?)moA(?)基因的功能应该是同脂肪酸部分为C18：2的酸型槐糖脂的内酯化相关。noD基因影响了C18:2NASL的乙酰化。在以葡萄糖和豆油为底物时,同野生株相比,ΔlipD、ΔlipE、 ΔlipF和ΔlipG敲除株发酵所得槐糖脂产量均有所降低,槐糖脂中乙酰化程度较高的组分含量降低,而乙酰化程度相对较低的组分含量增加,推测这种现象的出现是由于脂肪酶/酯酶的缺失使菌株将油脂水解为脂肪酸的能力下降,从而间接影响槐糖脂的合成,同时,脂肪酶可能也有催化槐糖脂乙酰化的功能,它的缺失导致槐糖脂乙酰化程度降低；而当以葡萄糖和油酸为底物时,由于油酸为单一脂肪酸,这几个敲除株槐糖脂产量及组成无显著变化。lipH基因同脂肪酸部分为C18：2的酸型槐糖脂的内酯化有一定关系,并且对菌株将C18：1整合入槐糖脂的过程也有一定影响。除moA和lipH基因同脂肪酸部分为C18：2的酸型槐糖脂的内酯化相关外,其它几个基因的敲除多对槐糖脂内酯化无显著影响,推测菌体内的内酯化酶应有多种。
5.鼠李糖基转移酶在拟威克酵母中的异源表达
为获得具有更好的生物学活性或表面活性的槐糖脂分子,对槐糖脂的结构修饰通常是通过化学法或酶催化法进行,而本论文则成功构建了鼠李糖基转移酶A和B基因(rhlA和rhlB)在W. domercqiae中的表达载体,对W. domercqiae Y2A进行遗传改造,筛选得到了相应的重组菌株Y2A-2,使其具有表达鼠李糖基转移酶1的能力。利用HPLC-MS的方法对重组菌株发酵的糖脂结构进行鉴定,发现两个组分(保留时间分别为16.74和17.47)的分子量分别为554(C26H50O12)和536(C26H48O11),前者可能为含1分子葡萄糖、1分子鼠李糖,脂肪酸部分为C14：0的无乙酰基的酸型糖脂,后者可能是含有两个鼠杍糖的、脂肪酸部分为C14：1的糖脂,为我们所要获得的目的糖脂；利用制备型HPLC,分离得到了含有一个鼠李糖的、脂肪酸部分为C18：3的酸型糖脂和既含有葡萄糖也含有鼠李糖、脂肪酸部分为C14：1的酸型糖脂。此外,同野生株相比,重组菌株产酸型槐糖脂的能力提高,而产内酯型槐糖脂能力下降。
Surfactants are one of the most important industrial bulk chemicals which have been widely applied in industrial, household and agricultural sectors. However, with the development of society and growing environmental awareness, biosurfactants have attracted increasing attention and will play more important roles than chemically synthetic surfactants in our daily life. Sophorolipids (SLs) are important glycolipid biosurfactants synthesized by a selected number of non-pathogenic yeast species. They have great potential to be widely applied in many industrial sectors, including petroleum, environment, cosmetics, detergent industries, also including nanotechnology and especially pharmaceutical industry due to their high yields, biodegradability, low toxicity, good emulsification ability, tolerance to temperature, and good biological activities. They are one of the most promising biosurfactants which can bring great social, economic and ecological benefits.
The main research contents and results are as follows:
1. Mutation breeding of high sophorolipid-producing strains by low-energy nitrogen ion implantation
Wickerhamiella domercqiae Y2A (screened and preserved by our laboratory) is one strain which can produce SLs. To meet the increasing demands of SLs as biosurfactant and bioactive compounds, it is necessary to obtain more high sophorolipid-producing strains. The wild strain was treated by low energy N+ion beam implantation with energy of15keV and doses ranging from1.5×1015to5.25×1015ions/cm2. The results showed that the survival curve of the strain took a "saddle" shape, and in the "saddle" region, the better mutagenic effect can be obtained. According to the survival curve of stain Y2A, the mutation dose was selected as3×1015ions/cm2. About114strains were selected out from YEPD agar plates after nitrogen ion implantation. The total sophorolipid production from18strains increased by more than20%compared with that of the wild strain. The lactonic SLs yields from 18strains increased by100%more than that of the wild strain.5strains that produced acidic SLs30%more than that of the wild strain were obtained. Especially, the sophorolipid production of strain N3-18reached104g/L at200rpm,30℃in shaking flask, which increased84.71%than that of the wild strain. The production of SLs was elevated to135g/L in5L fermentor.
HPLC analysis showed that the composition of every sophorolipid mixture from different strains was similar except that the contents of most components from mutants were higher than that from wild strain. Two mutants N1-32and N3-18, produced more acidic sophorolipid components; three lactonic sophorolipid molecules with good anticancer activities were greatly enhanced in several mutants, especially mono-acetylated lactonic SL with a C18:1fatty acid, were enhanced by153%and211%in strain N1-32and N3-18. Low-energy nitrogen ion beam implantation was efficient for obtaining a variety of high and specific sophorolipid-producing mutants to be applied in food, cosmetic, environmental and pharmaceutical sectors etc.
2. Production, purification and structure elucidation of novel sophorolipid molecules
Different SLs have different biological and physicochemical activities. Compared with SLs with shorter chain hydroxy fatty acid, SLs with longer chain hydroxy fatty acid might have better properties. Due to the differences in the properties of SLs, it is necessary to obtain more novel SLs. To obtain novel sophorolipid molecules differing in fatty acid part, three different hydrophobic carbon sources, rapeseed oil and two uncommonly-used oils (linseed oil and fish oil) were used for the production of SLs. The compositions of SLs produced by W. domercqiae var. sophorolipid grown on different hydrophobic carbon sources were very different. Sophorolipid molecules were purified by preparative HPLC and their structures were identified by MS, MS/MS and GC-MS. The di-acetylatcd lactonic sophorolipid with a C18:3fatty acid whose anticancer activity was not reported before was first obtained from W. domercqiae on linseed oil. Several unconventional acidic sophorolipid molecules with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DMA) including non-, mono-and di-acetylated acidic SLs with an EPA, di-acetylated acidic sophorolipid with a DHA, two unconventional lactonic sophorolipid molecules, non-acetylated lactonic sophorolipid with a docosatrienoic acid and non-acetylated lactonic sophorolipid with an eicosanoic acid were obtained using fish oil as hydrophobic carbon source.
3. The study of the relationship between fatty acid part of sophorolipid and their cytotoxicity to human cervical cancer cells, the signaling pathway of apoptosis induced by sophorolipid and the antitumor activity of SLs in vivo
It was found that differences of sophorolipid structures in acetylation degree of sophorose, unsaturation degree of hydroxy fatty acid, and lactonization or not could all affect the cytotoxicities of SLs. However, the influence of carbon chain length and polyunsaturation (the number of double bonds>2) of fatty acid moiety on the anticancer activity of SLs has not been detailedly studied, the apoptotic signaling pathway needs to be further revealed. Besides, the in vivo anti-tumor activity of SLs has not been reported before.
Six sophorolipid molecules including di-acetylated lactonic SLs with C16:1, C16:0, C18:3C18:1, C18:2or C18:0, were all employed and the relationship between the structures of sophorolipid molecules and their cytotoxicities to HeLa and Caski cells were investigated in detail. Cytotoxicity of sophorolipid molecules with different unsaturation degree was also influenced by carbon chain length of SLs. The influence of carbon chain length of fatty acid in SLs was also investigated and the results showed that the longer the carbon chain length of sophorolipid, the stronger its cytotoxicity to HeLa and Caski cells.
To investigate if SLs could induce the apoptosis or differentiation of human cervical cancer, cell morphological changes, TUNEL, cell cycle distribution, induction expression of CHOP and Bip, caspase-12and caspase-3activities were measured after they were treated with di-acetylated lactonic sophorolipid with a C18:1fatty acid. The cells developed many features of apoptosis such as cell shrinkage, chromatin condensation inside the nuclear membrane and margination to be a block or crescent shape, nuclear fragmentation, and appearance of apoptotic bodies. Cell cycle was blocked at Go phase and partly at G2phase by sophorolipid. The induction expression of CHOP/GADD153and Bip/GRP78in endoplasmic reticulum (ER) signaling pathway, and the activation of apoptotic caspase-12and caspase-3demonstrated that the apoptosis induced by SLs might be triggered through ER signaling pathway. However, Cyt. C was not released from mitochondria and no loss of MMP was detected in HeLa cells treated with sophorolipid, which revealed that mitochondria was not involved in the apoptosis induced by sophorolipid.
The anti-tumor activity of lactonic SLs in human cervical tumor xenografts in BALB/c mice were first investigated.5,50,500mg/kg lactonic SLs showed15.73%,26.40%and34.83%of inhibition without significant non-tumor toxicity in tumor-bearing mice. TUNEL results showed that lactonic SLs could induce apoptosis of tumors in vivo.
4. The primary study of the roles of main esterase/lipase and monooxygenases in sophorolipid biosynthesis
No lactone esterase has been identified in W. domercqiae or in other SL producing species. Normally, two types of enzyme which might catalyze the lactonization reaction were thioesterase or esterase/lipase and specific monooxygenases. The present work analyzed the genome of W. domercqiae and differences on gene expression level of W. domercqiae with two different nitrogen sources, yeast extract and ammonium sulphate, to screen the possible esterase/lipase and monooxygenase enzyme that catalyze lactonization reaction. Their roles in SL synthesis were primarily investigated.
The genetic engineering system of W. domercqiae was established:hygromycin B was chosen as the selection marker; electroporation method was an efficient transformation method; the promoter for glycerol3'-phosphate dehydrogenase Pgpd could be used as the promoter of the exogenous expression.
Eight esterase/lipase and four monooxygenases were selected, because their expression levels were up-regulated significantly when yeast extract was used as the nitrogen source. Gene deletion cassette was successfully constructed by double-joint PCR method and then the deletion strains were obtained. The roles of the12genes in SL biosynthesis were preliminarily analyzed. lipB can greatly affect the yields and the compositions of SLs. Compared with the wild strain, the yields of SLs produced by ΔlipB strain dropped significantly and no SLs could be detected when oleic acid was used as the sole carbon source. There might be two reasons for the reduction of SL yields:the down-regulated expression of enzymes involved in SL biosynthesis including cytochrome P450monooxygenase and UDP-glucosyltransferase; increase of extracellular glycoside hydrolase, which might hydrolyze the glycosidic bond of SLs. The proportion of di-acetylated acidic SL with C18:2fatty acid enhanced while that of di-acetylated lactonic SL with C18:2fatty acid decreased in the SLs produced by ΔmoA strain, which might demonstrate that moA might be related to the lactonization of the SLs with a C18:2fatty acid. moD gene influenced the acetylation degree of acidic SL with C18:2fatty acid. Compared with the wild strain, the SL yields of AlipD to AlipG strains were decreased when using glucose and soya-been oil as carbon source, which might be due to the decreased ability of the deletion strains to hydrolyze the glycerides into fatty acids. Besides, the contents of SLs with a higher acetylation degree produced by the four deletion strains were also decreased, suggesting that lipases might catalyze the acetylation reaction of SLs. The ability of AlipH strain to incorporate C18:1into SLs was reduced and the proportion of di-acetylated lactonic SL with C18:2fatty acid was reduced. The deletion of other genes had no significant influence on SL biosynthesis.
5. Heterologous expression of rhamnosyltransferase in W. domercqiae
Commonly, in order to obtain SLs with better biological activity or surface activity, structural modification of SLs was performed by chemical or enzymatic method in vitro. Rhamnosyltransferase expression vector in W. domercqiae was successfully constructed and one recombinant strain Y2A-2was obtained which could heterologous express the rhamnosyltransferase Rh1A and Rh1B.
HPLC-MS was used to identify the structures of the glycolipids produced by Y2A-2. There were two novel components (retention time at16.74and17.47min, respectively) with molecular weight of554(C26H50O12) and536(C26H48O11), the possible structure of the former was acidic C14:0glycolipid containing non-acetylated glucose and rhamnose, and the structure of the latter was acidic C14:1glycolipid containing two rhamnose. Preparative HPLC was also applied to obtain pure glycolipid and the structure of pure glycolipid was elucidated by MS method. Two novel glycolipids, acidic C18:3glycolipid containing one rhamnose and acidic C14:1glycolipid containing both glucose and rhamnose were obtained. However, because of their low contents, it was difficult for them to be prepared in large scale. Therefore, their accurate structures were not further identified by NMR and other methods. Compared with the wild strain, the recombinant strain produced more acidic SLs and less laconic SLs.
引文
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