Study On Function And Regulation Of Fatty Acid Desaturase Genes In Pichia Pastoris And Mortierella Alpina

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.