毕赤酵母和高山被孢霉脂肪酸脱氢酶基因功能及表达调控的研究
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摘要
多数不饱和脂肪酸，尤其是多不饱和脂肪酸在人类疾病的预防和治疗方面具有重要作用。许多微生物能够积累多不饱和脂肪酸，但是关于其在微生物中的作用的报道相对较少。一些研究证明，在微生物体内，多不饱和脂肪酸也能够行使某些特异功能。但是，对于其在细胞生长发育过程中，具体参与了哪些细胞过程，发挥了哪些细胞功能,目前尚不清楚。因此，有必要进行系统研究。
随着人们对于多不饱和脂肪酸需求量的日益增加，急需为多不饱和脂肪酸的生产寻找新的可替代来源。利用产油真菌等微生物发酵生产以及利用转基因方法生产有价值的多不饱和脂肪酸具有广阔的应用前景。要利用微生物发酵技术和转基因技术实现多不饱和脂肪酸的大量和优质生产，首先需要了解该微生物多不饱和脂肪酸合成的信号系统和调控机制。然而，到目前为止，对于多不饱和脂肪酸合成的关键基因—脂肪酸脱氢酶基因表达调控的分子机制的了解还相当少。因此，从分子水平探索脱氢酶基因的表达调控机制显得尤为重要。
巴斯德毕赤酵母是表达外源蛋白的理想宿主。与酿酒酵母相比，其体内具有相对完整的多不饱和脂肪酸代谢系统，是研究脂肪酸代谢理想的真核模式菌株。产油丝状真菌高山被孢霉是α-亚麻酸、二高-γ-亚麻酸、花生四烯酸和二十碳五烯酸的主要生产菌株。本文选择高山被孢霉ATCC16266作为研究对象，其α-亚麻酸产量可达总脂肪酸的20%以上，是α-亚麻酸生产的优良菌株。
为了阐明各种不饱和脂肪酸在微生物生长发育过程中的作用，我们利用同源重组的方法构建毕赤酵母脱氢酶基因缺失株。Fad9A基因或Fad9B基因的单缺失并未影响菌株的正常生长；当两者同时缺失时，引起了菌株的死亡。外源添加油酸恢复了菌株的生长。△Fad12突变株的生长速度明显变慢，而△Fad15突变株生长速度基本没有变化。以上结果显示：与亚油酸和α-亚麻酸相比，油酸对于菌株的生长发育更为重要；亚油酸的缺失也会影响菌株的生长速度，而α-亚麻酸的缺失似乎对菌株的生长影响不大。这些结果也在分子水平上阐明了毕赤酵母不饱和脂肪酸的合成途径，明确了每一步骤中的催化酶类及其编码基因：Fad9A基因和Fad9B基因可能属于同源基因，两者的编码蛋白是同工酶，均能行使△~9-脱氢酶的功能，催化硬脂酸生成油酸；Fad12基因编码产生了△12-脱氢酶，负责将油酸转化成亚油酸；Fad15基因编码产生了△15-脱氢酶，能够在亚油酸的基础上进一步催化脱氢生成α-亚麻酸。
同时，我们证明了油酸与毕赤酵母的低温耐受性和乙醇耐受性成正相关关系，而亚油酸和α-亚麻酸可能与毕赤酵母低温耐受性和乙醇耐受性之间没有明显的关系。此外，油酸、亚油酸、α-亚麻酸与毕赤酵母的甲醇耐受性之间没有关系。
通过实时定量PCR技术检测了在低温和外源添加脂肪酸条件下毕赤酵母脱氢酶基因mRNA表达水平的动态变化。结果表明，低温对于脱氢酶基因转录的激活作用和外源不饱和脂肪酸对于脱氢酶基因转录的抑制作用都是快速和短暂的，而饱和脂肪酸硬脂酸的添加对于几种脱氢酶基因的表达影响不大。
通过构建Fad15基因启动子与lacZ基因的融合载体并测定β-半乳糖苷酶活性的方法来确定在低温和外源脂肪酸刺激下Fad15基因启动子转录活性的变化情况。结果表明，低温能够在短时间内增加PFAD15启动子的活性，而且这种激活作用随着时间的延长而持续增强。PFAD15启动子活性在添加饱和脂肪酸后变化不大；而添加不饱和脂肪酸后，PFAD15启动子活性明显下降，不饱和程度越高，抑制作用越强。而且，不饱和脂肪酸的浓度越高、作用时间越长，抑制作用越强。这一结果与Fad15基因在转录水平上的变化规律并不一致，这说明毕赤酵母脱氢酶基因的表达可能受到细胞内脂肪酸组成变化的反馈调节作用。通过气相色谱法比较了低温和外源脂肪酸刺激后脂肪酸组成的变化情况。结果表明，在低温刺激下，油酸在总脂肪酸中的相对比例表现为持续增加；而亚油酸和α-亚麻酸的相对含量随着时间变化并没有明显的变化规律。同时，在这两种条件下，脱氢酶基因转录水平的变化与对应脂肪酸产物的变化没有相关性。这说明低温和外源不饱和脂肪酸除了在转录水平上调控基因表达发生变化之外，可能主要是在转录后水平上介导了胞内脂肪酸组成的变化。
通过同源重组介导敲除的方法构建了毕赤酵母Spt23基因缺失突变株，结果发现Spt23基因缺失严重影响了菌株的生长速度，这说明Spt23p蛋白可能在菌株生长发育过程中起到了重要作用。进一步分析表明Spt23基因缺失突变株生长变缓的原因可能是由于油酸含量的降低所致。实时定量PCR的结果说明：稳定期△Spt23菌株中Fad9A基因和Fad9B基因的相对表达量显著下降、Fad12基因和Fad15基因的相对表达量变化不大。在低温和外源添加不饱和脂肪酸条件下，Spt23基因的存在与否对于Fad12基因和Fad15基因的表达并无影响，所以这两种基因的表达可能并不受Spt23p蛋白的调控。而Spt23基因缺失株Fad9A基因和Fad9B基因的表达变化情况与野生株完全不同，低温的激活作用和外源不饱和脂肪酸的抑制作用消失，说明Spt23p蛋白可能参与了低温和外源脂肪酸对于毕赤酵Fad9A基因和Fad9B基因表达的调控作用。
由于对非模式微生物的丝状真菌的遗传操作较为困难，所以，高山被孢霉等产油丝状真菌中多不饱和脂肪酸合成的调控机制至今还不清楚。我们首先利用农杆菌介导T-DNA转化的方法成功构建了高山被孢霉的遗传转化体系，从而为后续的分子遗传操作奠定基础。
接下来研究了不同碳源对于高山被孢霉菌体生长、油脂产生以及脱氢酶基因表达的影响情况。结果证实了三种脱氢酶基因的表达以及多不饱和脂肪酸的合成依赖于不同的碳源类型而变化，这可能是机体在面对不同生长条件时所作出的不同的应答反应，也说明了多不饱和脂肪酸的合成同样可能也受到了不同碳源代谢网络的调控。
通过实时定量PCR技术和启动子报告基因融合载体的方法，研究低温和外源不饱和脂肪酸对于高山被孢霉三种脱氢酶基因表达随时间进程的影响。实时定量PCR的结果表明：低温对于三种脱氢酶基因的转录具有激活作用，外源不饱和脂肪酸对基因转录起抑制作用，而且这两种作用都是快速响应的，随时间延长逐渐减弱并消失。脂肪酸组成测定结果证明了基因转录水平变化与对应产物变化之间没有相关性。低温能够在短时间内诱导PFAD6启动子活性增加，并随时间延长而持续增强；外源不饱和脂肪酸对PFAD6启动子活性起抑制作用，其不饱和度和浓度越高，抑制作用越强，而且抑制作用是快速且持续的。以上结果说明低温和外源不饱和脂肪酸除了在转录水平上调控脱氢酶基因表达发生变化之外，可能主要在转录后水平上介导了胞内脂肪酸组成的变化。而且，脱氢酶基因的表达可能受到胞内脂肪酸组成变化的反馈调节作用。
本文首次在转录水平上对毕赤酵母和高山被孢霉脱氢酶基因的表达调控机制进行了探索，为深入了解产油菌株体内脱氢酶基因表达及多不饱和脂肪酸合成对外界信号的应答机制提供了有用信息，也对应用微生物发酵和转基因技术生产多不饱和脂肪酸具有指导意义。
Nearly all unsaturated fatty acids (UFAs), especially those polyunsaturated fatty acids(PUFAs) have important functions in prevention and treatment of various humandiseases. Many microorganisms capable of producing PUFAs have been isolated andcharacterized so far, but the exact molecular and biochemical role of PUFAsaccumulated in these oleaginous microbes for themselves remains unclear. Theamount of information that is available from research in this area is much lesscompared with that in mammalian cells. Toward this end, in order to betterunderstand the nature and function of UFAs in different organisms, more thoroughstudy regarding the relationship between specific UFAs and growth, development andother physiological responses in various organisms should be continued, includingbacteria, fungi, algae, plants, fish, etc.
In recent years, there is an urgent need to search for alternative sources ofPUFAs with a rapid increasing demand for them. Among them, the accumulation ofsome valuable PUFAs using traditional fermentation techniques by cultivation ofoleaginous fungi and using genetic engineering strains by introducing specific genesinto a suitable host has wide application prospects. In order to achieve large-scale andhigh-quality production of PUFAs adopting these two approaches, it is very importantto understand the signaling pathways and the genetic regulatory mechanisms ofPUFAs biosynthesis. However, up to now, due to the regulatory mechanisms ofPUFAs biosynthesis and the control of desaturase gene expression in many organismsare not clearly understood. So it seemed very essential to assess the regulatorymechanism of the expression of desaturase genes at the molecular level.
The methylotrophic yeast Pichia pastoris has been proved to be an excellentsystem for high-level expression of heterologous proteins because of manyadvantages and unlike the yeast Saccharomyces cerevisiae, P. pastoris possesses arelatively complete system of PUFAs biosynthesis. For the reasons above, P. pastorisis a better choice for constructing a transgenic strain for the production of specificfatty acids and a good eukaryotic model organism for studies on the regulation mechanism of fatty acid biosynthesis. Mortierella alpina is viewed as a representativeof important oleaginous fungi, which has been utilized for the industrial production ofα-linolenic acid (ALA), dihomo-γ-linolenic acid (DGLA), arachidonic acid (ARA)and eicosapentaenoic acid (EPA). M. alpina ATCC16266used in this study is asuperior strain capable of producing ALA which accounted for more than20%of thetotal fatty acids.
In an effort to investigate the potential significance of UFAs for growth anddevelopment in microbes, we constructed△Fad9A,△Fad9B,△Fad12,△Fad15null mutants and△Fad9A;△Fad9B double null mutants of P. pastoris byhomologous recombination in this study. There was no difference among△Fad9Amutant,△Fad9B mutant and wild type in the fatty acid composition and growth rates.However, simultaneous deletion of Fad9A and Fad9B was lethal and exogenoussupplementation of oleic acid (OA) to the medium could restore growth of thismutant.△Fad12mutant grew much slower than wild type and△Fad15mutant grewat almost the same rate as wild type. From these data, we can conclude that OA isnecessary and plays a greater role than LA and ALA during growth and developmentof P. pastoris; the absence of LA decreased the growth rate obviously but the absenceof ALA did not lead to obvious physiological changes. These results also clarified thebiosynthetic pathway of PUFA in P. pastoris on the molecular level: in the first step,possibly both Fad9A and Fad9B encode△9-fatty acid desaturase (FAD9) thatconverts saturated fatty acids (SFA) to monounsaturated fatty acid (MUFA), primarilyfrom stearic acid (SA) into OA, and then OA is converted to LA catalyzed by△12-fatty acid desaturase (FAD12), which is encoded by Fad12gene, and△15-fattyacid desaturase (FAD15) encoded by Fad15catalyzed the final step in the synthesisof ALA from LA.
Further analysis showed that OA, but not LA or ALA was probably involved inthe response process of cold tolerance and ethanol tolerance in P. pastoris. In addition,we showed that tolerance of P. pastoris to high concentration of methanol wasindependent of these three UFAs.
Time-course studies of gene expression by real-time PCR showed that mRNAlevels of four desaturase genes were rapidly and transiently enhanced by low temperature and suppressed by exogenous OA. SA showed no obvious effect onmRNA levels of four desaturase genes.
Using promoter-reporter constructs (PFAD15, containing promoter region of-1000bp/+27bp from the ORF of Fad15/β-galactosidase gene), we demonstrated thatthe PFAD15promoter activity was induced by low temperature in a time-dependentmanner and reduced in a continuous, dose-and time-dependent manner by addition ofunsaturated fatty acids to the media and ALA containing three double bonds appearedto have a more effective inhibition than LA and OA, whereas neither palmitic acid(PA) nor SA had significant effect on reporter activity. The responses of promoteractivities to low temperature and exogenous fatty acids appeared to be different withFad15gene expression profile changes which were basically rapid and transientduring the test period. This observation suggested that there may be an unknownend-product (changes in fatty acid compositions) feedback regulation in thetranscription of desaturase genes to maintain cellular UFAs’ homeostasis in P.pastoris. In order to characterize the relationship between desaturase gene expressionand fatty acid production, we measured the relative abundance of the correspondingfatty acid products using GC at the product level. There was no obvious changingtrend in the content of corresponding desaturation products OA+LA+ALA, LA+ALAand ALA after low-temperature shift. Surprisingly, a substantial increase of OAamount compared with control was observed. Likewise, changes in desaturase geneexpression did not correspond with the changes observed in fatty acid compositionunder these two conditions. These results indicated that the correlation betweenchanges in mRNA transcript abundance and fatty acid products profiles was variedand there may be post-transcriptional control and other modes of regulation of UFAssynthesis in P. pastoris under these two conditions.
To test whether the Spt23p protein of P. pastoris has functions in the regulationof its desaturase genes, we constructed the Spt23disruptant by homologousrecombination.△Spt23strain grew much more slowly than wild-type cells undernormal growth conditions which showed that Spt23p protein probably playedimportant roles in growth and development of P. pastoris. Further analysis showedthat a decrease in the relative abundance of OA was observed which may be the reason for the lower growth rates of P. pastoris. Results of real-time PCR showed thatthe mRNA levels of Fad9A and Fad9B in the stationary phase cells of△Spt23mutant were markedly reduced compared with wild-type. By contrast, the mRNAlevels of Fad12and Fad15did not change significantly. Time-course expressionstudy showed that there were no clear differences in the gene expression profiles ofFad12and Fad15in response to low temperature and exogenous OA betweenwild-type strain and△Spt23strain. While there were no clear changes in theexpression for Fad9A and Fad9B of△Spt23strain in response to low temperatureand exogenous OA during the test period which was not consistent with the resultsobtained in wild type strain. These data indicated that Spt23p are probably necessaryfor the control over the transcription of Fad9A and Fad9B internally and involved inthe regulation of Fad9A and Fad9B genes transcription in response to lowtemperature and exogenous UFA, but not Fad12and Fad15.
Until now, the molecular and genetic manipulation for non-model filamentousfungi has not been sufficiently developed and is normally difficult to perform. So, theregulation mechanism of PUFA biosynthesis and the control of fatty acid desaturasegene expression in filamentous fungi, such as M. alpina are not clearly understood. Inthis study, we established the Agrobacterium tumefaciens-mediated genetictransformation system of M. alpina ATCC16266, which was the efficient method, aswell as the basis for subsequent molecular genetic manipulation.
And then, the effect of different carbon sources on steady-state fatty aciddesaturase gene expression, biomass accumulation and fatty acid biosynthesis wasinvestigated in M. alpina. The results showed that expression levels of differentdesaturases and production of PUFAs respond differently to different types of carbonsources, and even the same type of desaturase in different organisms may havedifferent response manners. These findings demonstrated that the organisms mayadjust the physiological properties and functions of cellular membranes and someorganells through this response mechanism when facing different growth conditionsand also the production of PUFAs was possibly regulated by the differentcarbon-source metabolism and its metabolites.
We performed time-course studies of fatty acid desaturase gene expression by real-time PCR and fatty acid desaturase gene promoter activity usingpromoter-reporter constructs. Relative expression results in real-time PCR showedthat the mRNA levels of three fatty acid desaturase genes (Fad6, Fad12and Fad3)were rapidly and transiently enhanced after1h of shifting to low temperature, incontrast, high concentration of exogenous OA suppressed the transcription of thesegenes and the transcriptional response appears to be rapid and transient. Also, therewas no absolute correlation between mRNA abundance and production ofcorresponding fatty acids. The PFAD6promoter activity was induced by lowtemperature in a time-dependent manner and reduced in a dose-and time-dependentmanner by addition of unsaturated fatty acids to the media, and ALA containing threedouble bonds appeared to have a more effective inhibition than LA and OA. Theseresults indicated that there may be post-transcriptional control and other modes ofregulation of UFAs synthesis in M. alpina when facing different stimuli such as lowtemperature and exogenous unsaturated fatty acids besides the regulation in thetranscription of fatty acid desaturase genes at the initial stage. Also, there may be anunknown end-product (changes in fatty acid compositions) feedback regulation in thetranscription of fatty acid desaturase genes to maintain cellular UFAs’ homeostasis inM. alpina.
We assessed mechanisms of transcriptional regulation of fatty acid desaturasegene expression in P. pastoris and M. alpina for the first time. We wished to make itpossible to obtain a better understanding of the mechanisms and got some theoreticalknowledge to offer some guidance to the industrial production of PUFAs bytransgenic technology and microbial fermentation technology.
随着人们对于多不饱和脂肪酸需求量的日益增加，急需为多不饱和脂肪酸的生产寻找新的可替代来源。利用产油真菌等微生物发酵生产以及利用转基因方法生产有价值的多不饱和脂肪酸具有广阔的应用前景。要利用微生物发酵技术和转基因技术实现多不饱和脂肪酸的大量和优质生产，首先需要了解该微生物多不饱和脂肪酸合成的信号系统和调控机制。然而，到目前为止，对于多不饱和脂肪酸合成的关键基因—脂肪酸脱氢酶基因表达调控的分子机制的了解还相当少。因此，从分子水平探索脱氢酶基因的表达调控机制显得尤为重要。
巴斯德毕赤酵母是表达外源蛋白的理想宿主。与酿酒酵母相比，其体内具有相对完整的多不饱和脂肪酸代谢系统，是研究脂肪酸代谢理想的真核模式菌株。产油丝状真菌高山被孢霉是α-亚麻酸、二高-γ-亚麻酸、花生四烯酸和二十碳五烯酸的主要生产菌株。本文选择高山被孢霉ATCC16266作为研究对象，其α-亚麻酸产量可达总脂肪酸的20%以上，是α-亚麻酸生产的优良菌株。
为了阐明各种不饱和脂肪酸在微生物生长发育过程中的作用，我们利用同源重组的方法构建毕赤酵母脱氢酶基因缺失株。Fad9A基因或Fad9B基因的单缺失并未影响菌株的正常生长；当两者同时缺失时，引起了菌株的死亡。外源添加油酸恢复了菌株的生长。△Fad12突变株的生长速度明显变慢，而△Fad15突变株生长速度基本没有变化。以上结果显示：与亚油酸和α-亚麻酸相比，油酸对于菌株的生长发育更为重要；亚油酸的缺失也会影响菌株的生长速度，而α-亚麻酸的缺失似乎对菌株的生长影响不大。这些结果也在分子水平上阐明了毕赤酵母不饱和脂肪酸的合成途径，明确了每一步骤中的催化酶类及其编码基因：Fad9A基因和Fad9B基因可能属于同源基因，两者的编码蛋白是同工酶，均能行使△~9-脱氢酶的功能，催化硬脂酸生成油酸；Fad12基因编码产生了△12-脱氢酶，负责将油酸转化成亚油酸；Fad15基因编码产生了△15-脱氢酶，能够在亚油酸的基础上进一步催化脱氢生成α-亚麻酸。
同时，我们证明了油酸与毕赤酵母的低温耐受性和乙醇耐受性成正相关关系，而亚油酸和α-亚麻酸可能与毕赤酵母低温耐受性和乙醇耐受性之间没有明显的关系。此外，油酸、亚油酸、α-亚麻酸与毕赤酵母的甲醇耐受性之间没有关系。
通过实时定量PCR技术检测了在低温和外源添加脂肪酸条件下毕赤酵母脱氢酶基因mRNA表达水平的动态变化。结果表明，低温对于脱氢酶基因转录的激活作用和外源不饱和脂肪酸对于脱氢酶基因转录的抑制作用都是快速和短暂的，而饱和脂肪酸硬脂酸的添加对于几种脱氢酶基因的表达影响不大。
通过构建Fad15基因启动子与lacZ基因的融合载体并测定β-半乳糖苷酶活性的方法来确定在低温和外源脂肪酸刺激下Fad15基因启动子转录活性的变化情况。结果表明，低温能够在短时间内增加PFAD15启动子的活性，而且这种激活作用随着时间的延长而持续增强。PFAD15启动子活性在添加饱和脂肪酸后变化不大；而添加不饱和脂肪酸后，PFAD15启动子活性明显下降，不饱和程度越高，抑制作用越强。而且，不饱和脂肪酸的浓度越高、作用时间越长，抑制作用越强。这一结果与Fad15基因在转录水平上的变化规律并不一致，这说明毕赤酵母脱氢酶基因的表达可能受到细胞内脂肪酸组成变化的反馈调节作用。通过气相色谱法比较了低温和外源脂肪酸刺激后脂肪酸组成的变化情况。结果表明，在低温刺激下，油酸在总脂肪酸中的相对比例表现为持续增加；而亚油酸和α-亚麻酸的相对含量随着时间变化并没有明显的变化规律。同时，在这两种条件下，脱氢酶基因转录水平的变化与对应脂肪酸产物的变化没有相关性。这说明低温和外源不饱和脂肪酸除了在转录水平上调控基因表达发生变化之外，可能主要是在转录后水平上介导了胞内脂肪酸组成的变化。
通过同源重组介导敲除的方法构建了毕赤酵母Spt23基因缺失突变株，结果发现Spt23基因缺失严重影响了菌株的生长速度，这说明Spt23p蛋白可能在菌株生长发育过程中起到了重要作用。进一步分析表明Spt23基因缺失突变株生长变缓的原因可能是由于油酸含量的降低所致。实时定量PCR的结果说明：稳定期△Spt23菌株中Fad9A基因和Fad9B基因的相对表达量显著下降、Fad12基因和Fad15基因的相对表达量变化不大。在低温和外源添加不饱和脂肪酸条件下，Spt23基因的存在与否对于Fad12基因和Fad15基因的表达并无影响，所以这两种基因的表达可能并不受Spt23p蛋白的调控。而Spt23基因缺失株Fad9A基因和Fad9B基因的表达变化情况与野生株完全不同，低温的激活作用和外源不饱和脂肪酸的抑制作用消失，说明Spt23p蛋白可能参与了低温和外源脂肪酸对于毕赤酵Fad9A基因和Fad9B基因表达的调控作用。
由于对非模式微生物的丝状真菌的遗传操作较为困难，所以，高山被孢霉等产油丝状真菌中多不饱和脂肪酸合成的调控机制至今还不清楚。我们首先利用农杆菌介导T-DNA转化的方法成功构建了高山被孢霉的遗传转化体系，从而为后续的分子遗传操作奠定基础。
接下来研究了不同碳源对于高山被孢霉菌体生长、油脂产生以及脱氢酶基因表达的影响情况。结果证实了三种脱氢酶基因的表达以及多不饱和脂肪酸的合成依赖于不同的碳源类型而变化，这可能是机体在面对不同生长条件时所作出的不同的应答反应，也说明了多不饱和脂肪酸的合成同样可能也受到了不同碳源代谢网络的调控。
通过实时定量PCR技术和启动子报告基因融合载体的方法，研究低温和外源不饱和脂肪酸对于高山被孢霉三种脱氢酶基因表达随时间进程的影响。实时定量PCR的结果表明：低温对于三种脱氢酶基因的转录具有激活作用，外源不饱和脂肪酸对基因转录起抑制作用，而且这两种作用都是快速响应的，随时间延长逐渐减弱并消失。脂肪酸组成测定结果证明了基因转录水平变化与对应产物变化之间没有相关性。低温能够在短时间内诱导PFAD6启动子活性增加，并随时间延长而持续增强；外源不饱和脂肪酸对PFAD6启动子活性起抑制作用，其不饱和度和浓度越高，抑制作用越强，而且抑制作用是快速且持续的。以上结果说明低温和外源不饱和脂肪酸除了在转录水平上调控脱氢酶基因表达发生变化之外，可能主要在转录后水平上介导了胞内脂肪酸组成的变化。而且，脱氢酶基因的表达可能受到胞内脂肪酸组成变化的反馈调节作用。
本文首次在转录水平上对毕赤酵母和高山被孢霉脱氢酶基因的表达调控机制进行了探索，为深入了解产油菌株体内脱氢酶基因表达及多不饱和脂肪酸合成对外界信号的应答机制提供了有用信息，也对应用微生物发酵和转基因技术生产多不饱和脂肪酸具有指导意义。
Nearly all unsaturated fatty acids (UFAs), especially those polyunsaturated fatty acids(PUFAs) have important functions in prevention and treatment of various humandiseases. Many microorganisms capable of producing PUFAs have been isolated andcharacterized so far, but the exact molecular and biochemical role of PUFAsaccumulated in these oleaginous microbes for themselves remains unclear. Theamount of information that is available from research in this area is much lesscompared with that in mammalian cells. Toward this end, in order to betterunderstand the nature and function of UFAs in different organisms, more thoroughstudy regarding the relationship between specific UFAs and growth, development andother physiological responses in various organisms should be continued, includingbacteria, fungi, algae, plants, fish, etc.
In recent years, there is an urgent need to search for alternative sources ofPUFAs with a rapid increasing demand for them. Among them, the accumulation ofsome valuable PUFAs using traditional fermentation techniques by cultivation ofoleaginous fungi and using genetic engineering strains by introducing specific genesinto a suitable host has wide application prospects. In order to achieve large-scale andhigh-quality production of PUFAs adopting these two approaches, it is very importantto understand the signaling pathways and the genetic regulatory mechanisms ofPUFAs biosynthesis. However, up to now, due to the regulatory mechanisms ofPUFAs biosynthesis and the control of desaturase gene expression in many organismsare not clearly understood. So it seemed very essential to assess the regulatorymechanism of the expression of desaturase genes at the molecular level.
The methylotrophic yeast Pichia pastoris has been proved to be an excellentsystem for high-level expression of heterologous proteins because of manyadvantages and unlike the yeast Saccharomyces cerevisiae, P. pastoris possesses arelatively complete system of PUFAs biosynthesis. For the reasons above, P. pastorisis a better choice for constructing a transgenic strain for the production of specificfatty acids and a good eukaryotic model organism for studies on the regulation mechanism of fatty acid biosynthesis. Mortierella alpina is viewed as a representativeof important oleaginous fungi, which has been utilized for the industrial production ofα-linolenic acid (ALA), dihomo-γ-linolenic acid (DGLA), arachidonic acid (ARA)and eicosapentaenoic acid (EPA). M. alpina ATCC16266used in this study is asuperior strain capable of producing ALA which accounted for more than20%of thetotal fatty acids.
In an effort to investigate the potential significance of UFAs for growth anddevelopment in microbes, we constructed△Fad9A,△Fad9B,△Fad12,△Fad15null mutants and△Fad9A;△Fad9B double null mutants of P. pastoris byhomologous recombination in this study. There was no difference among△Fad9Amutant,△Fad9B mutant and wild type in the fatty acid composition and growth rates.However, simultaneous deletion of Fad9A and Fad9B was lethal and exogenoussupplementation of oleic acid (OA) to the medium could restore growth of thismutant.△Fad12mutant grew much slower than wild type and△Fad15mutant grewat almost the same rate as wild type. From these data, we can conclude that OA isnecessary and plays a greater role than LA and ALA during growth and developmentof P. pastoris; the absence of LA decreased the growth rate obviously but the absenceof ALA did not lead to obvious physiological changes. These results also clarified thebiosynthetic pathway of PUFA in P. pastoris on the molecular level: in the first step,possibly both Fad9A and Fad9B encode△9-fatty acid desaturase (FAD9) thatconverts saturated fatty acids (SFA) to monounsaturated fatty acid (MUFA), primarilyfrom stearic acid (SA) into OA, and then OA is converted to LA catalyzed by△12-fatty acid desaturase (FAD12), which is encoded by Fad12gene, and△15-fattyacid desaturase (FAD15) encoded by Fad15catalyzed the final step in the synthesisof ALA from LA.
Further analysis showed that OA, but not LA or ALA was probably involved inthe response process of cold tolerance and ethanol tolerance in P. pastoris. In addition,we showed that tolerance of P. pastoris to high concentration of methanol wasindependent of these three UFAs.
Time-course studies of gene expression by real-time PCR showed that mRNAlevels of four desaturase genes were rapidly and transiently enhanced by low temperature and suppressed by exogenous OA. SA showed no obvious effect onmRNA levels of four desaturase genes.
Using promoter-reporter constructs (PFAD15, containing promoter region of-1000bp/+27bp from the ORF of Fad15/β-galactosidase gene), we demonstrated thatthe PFAD15promoter activity was induced by low temperature in a time-dependentmanner and reduced in a continuous, dose-and time-dependent manner by addition ofunsaturated fatty acids to the media and ALA containing three double bonds appearedto have a more effective inhibition than LA and OA, whereas neither palmitic acid(PA) nor SA had significant effect on reporter activity. The responses of promoteractivities to low temperature and exogenous fatty acids appeared to be different withFad15gene expression profile changes which were basically rapid and transientduring the test period. This observation suggested that there may be an unknownend-product (changes in fatty acid compositions) feedback regulation in thetranscription of desaturase genes to maintain cellular UFAs’ homeostasis in P.pastoris. In order to characterize the relationship between desaturase gene expressionand fatty acid production, we measured the relative abundance of the correspondingfatty acid products using GC at the product level. There was no obvious changingtrend in the content of corresponding desaturation products OA+LA+ALA, LA+ALAand ALA after low-temperature shift. Surprisingly, a substantial increase of OAamount compared with control was observed. Likewise, changes in desaturase geneexpression did not correspond with the changes observed in fatty acid compositionunder these two conditions. These results indicated that the correlation betweenchanges in mRNA transcript abundance and fatty acid products profiles was variedand there may be post-transcriptional control and other modes of regulation of UFAssynthesis in P. pastoris under these two conditions.
To test whether the Spt23p protein of P. pastoris has functions in the regulationof its desaturase genes, we constructed the Spt23disruptant by homologousrecombination.△Spt23strain grew much more slowly than wild-type cells undernormal growth conditions which showed that Spt23p protein probably playedimportant roles in growth and development of P. pastoris. Further analysis showedthat a decrease in the relative abundance of OA was observed which may be the reason for the lower growth rates of P. pastoris. Results of real-time PCR showed thatthe mRNA levels of Fad9A and Fad9B in the stationary phase cells of△Spt23mutant were markedly reduced compared with wild-type. By contrast, the mRNAlevels of Fad12and Fad15did not change significantly. Time-course expressionstudy showed that there were no clear differences in the gene expression profiles ofFad12and Fad15in response to low temperature and exogenous OA betweenwild-type strain and△Spt23strain. While there were no clear changes in theexpression for Fad9A and Fad9B of△Spt23strain in response to low temperatureand exogenous OA during the test period which was not consistent with the resultsobtained in wild type strain. These data indicated that Spt23p are probably necessaryfor the control over the transcription of Fad9A and Fad9B internally and involved inthe regulation of Fad9A and Fad9B genes transcription in response to lowtemperature and exogenous UFA, but not Fad12and Fad15.
Until now, the molecular and genetic manipulation for non-model filamentousfungi has not been sufficiently developed and is normally difficult to perform. So, theregulation mechanism of PUFA biosynthesis and the control of fatty acid desaturasegene expression in filamentous fungi, such as M. alpina are not clearly understood. Inthis study, we established the Agrobacterium tumefaciens-mediated genetictransformation system of M. alpina ATCC16266, which was the efficient method, aswell as the basis for subsequent molecular genetic manipulation.
And then, the effect of different carbon sources on steady-state fatty aciddesaturase gene expression, biomass accumulation and fatty acid biosynthesis wasinvestigated in M. alpina. The results showed that expression levels of differentdesaturases and production of PUFAs respond differently to different types of carbonsources, and even the same type of desaturase in different organisms may havedifferent response manners. These findings demonstrated that the organisms mayadjust the physiological properties and functions of cellular membranes and someorganells through this response mechanism when facing different growth conditionsand also the production of PUFAs was possibly regulated by the differentcarbon-source metabolism and its metabolites.
We performed time-course studies of fatty acid desaturase gene expression by real-time PCR and fatty acid desaturase gene promoter activity usingpromoter-reporter constructs. Relative expression results in real-time PCR showedthat the mRNA levels of three fatty acid desaturase genes (Fad6, Fad12and Fad3)were rapidly and transiently enhanced after1h of shifting to low temperature, incontrast, high concentration of exogenous OA suppressed the transcription of thesegenes and the transcriptional response appears to be rapid and transient. Also, therewas no absolute correlation between mRNA abundance and production ofcorresponding fatty acids. The PFAD6promoter activity was induced by lowtemperature in a time-dependent manner and reduced in a dose-and time-dependentmanner by addition of unsaturated fatty acids to the media, and ALA containing threedouble bonds appeared to have a more effective inhibition than LA and OA. Theseresults indicated that there may be post-transcriptional control and other modes ofregulation of UFAs synthesis in M. alpina when facing different stimuli such as lowtemperature and exogenous unsaturated fatty acids besides the regulation in thetranscription of fatty acid desaturase genes at the initial stage. Also, there may be anunknown end-product (changes in fatty acid compositions) feedback regulation in thetranscription of fatty acid desaturase genes to maintain cellular UFAs’ homeostasis inM. alpina.
We assessed mechanisms of transcriptional regulation of fatty acid desaturasegene expression in P. pastoris and M. alpina for the first time. We wished to make itpossible to obtain a better understanding of the mechanisms and got some theoreticalknowledge to offer some guidance to the industrial production of PUFAs bytransgenic technology and microbial fermentation technology.
引文
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