Lipid characterization of an arachidonic acid-rich oil producing fungus Mortierella alpina☆

Wenjia Wu *,Jiacheng Yan 2,*,Xiaojun Ji**,Xin Zhang ,Jingsheng Shang Lina Sun Lujing Ren He Huang

1 State Key Laboratory of Materials-Oriented Chemical Engineering,College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211816,China

2 Faculty of Science,Hong Kong Baptist University,Hong Kong 999077,China

3 College of Sciences,Nanjing Tech University,Nanjing 211816,China

Keywords:Arachidonic acid Lipid characterization Triacylglycerol Mortierella alpina

ABSTRACT Mortierella alpina has been considered as the most effective producer of arachidonic acid(ARA)-rich oil.It was found that several methods could improve the percentage of ARA in total lipids successfully,as they activated the desaturation system on the endoplasmic reticulum.Additionally,in M.alpina the ARA exists in several forms,such as triacylglycerol(TAG),and diacylglycerol(DAG).These forms are caused by different acyltransferases and they determine the nutrient value of the microbial oil.However,few works revealed detailed fatty acid distribution among lipid classes,which to some extent impeded the accurate regulation in ARA accumulation.Herein,this paper gives information on the accumulation process of main lipid classes and the changes of fatty acid composition in these lipids during ARA accumulation period in M.alpina.The result demonstrates that TAG was the dominant component of the total lipids,and it is the main form for ARA storage.The ARA enrichment stage occurred during 168–192 h when the amount of total lipids maintained steady.Further analysis indicated that the newly formed ARA-TAG might come from the incorporation and modification of saturated and monounsaturated fatty acids in other lipid classes.This work could be helpful for furtheroptimization of ARA-rich TAG production.

1.Introduction

Arachidonic acid(ARA,5,8,11,14-cis-eicosatetraenoic acid),one of the ω-6 class polyunsaturated fatty acids(PUFAs),plays an important role in many physiological processes[1].Furthermore,it is an essential fatty acid for growth and brain development of infants.Because of its unique biological properties,it has been widely applied into infant formula.Meanwhile,it was also extensively used in medicine,cosmetics,and other fields[2,3].

Mortierella alpina has been extensively investigated for its high productivity of ARA-rich oil,and has been applied for the industrial scale production[4,5].Various methods have been taken to improve the ARA-rich oil production efficiency,including the strain breeding[6,7]and some fermentation strategies[8,9].These methods were proved effective since they increased the activities of enzymes involved in ARA biosynthesis.Most of these enzymes exist in the pathway of fatty acid biosynthesis,supplying NADPH and enhancing the desaturation process of fatty acids.Compared with the control,regulating these enzymes obviously improved the percentage of ARA in total oil.

However,microbial oil needs to be refined before being used,because it's the mixture of several types of lipids.When fatty acid-CoA was formed,it could be incorporated into different lipid classes(such as triacylglycerol(TAG),diacylglycerol(DAG))(Fig.1).When fermentation is completed,we obtained the compound of these lipids.Furthermore,these different forms have respective physiological effects[10,11].Therefore,besides the ARA biosynthesis,the distribution of ARA in different lipid forms is also very important.Few reports revealed the relationship between storage of fatty acids and individual lipid class[12–14].To some degree,this restricted some further understanding of key factors involved in accumulation of specific fatty acids.

Acetyl-CoA was the common precursor for all lipid biosynthesis.When fatty acid-CoA was formed,it could be incorporated into different lipid classes.Sterol esters and triacylglycerol are end-product of this way,and structure lipids were transformed form DAG.Free fatty acid was released from TAG and sterol esters to serve energy and for the new biomass formation.

Fig.1.Biosynthetic pathway of major lipid classes in M.alpina.

In this work,the lipid accumulation process of M.alpina was studied.Detailed fatty acid composition of individual lipid class during the ARA storage phase was also analyzed.Investigating these,we hope to get better understanding of the lipid metabolic pathway in M.alpina and provide an accurate guidance for the industrialization.

2.Materials and Methods

2.1.Microorganism and culture condition

M.alpina R807(CCTCC M 2012118),preserved in the China Centre for Type Culture Collection,was used in this study[7].Itwas maintained in PDA slant,and transferred every 3 months.Fermentation medium contained(g·L-1):glucose 80,yeast extract 10,KH2PO44,NaNO33,and MgSO4·7H2O 1.Fermentation experiments were performed in 500-ml baffled flasks containing 100 ml medium,incubated in a rotary shaker at 125 r·min-1and 25 °C for 8 days.

2.2.Analytical methods

For the determination of dry cell mass,the fungal cells from the fermentation broth were harvested by filtration and washed twice with distilled water,and stoved at 55°C to obtain a constant mass.

For measuring the glucose concentration,a biosensor with glucose oxidase electrode(SBA-40C,Institute of Biology,Shandong Academy of Sciences,Shandong,China)was used.

For the lipid content determination,the dried mycelia were ground into a fine powder,and then extracted three times with chloroform/methanol(1:2,volume ratio)[15].After extraction,the solvent was removed by evaporation to obtain the microbial lipid.

For lipid separation and analysis,thin-layer chromatography(TLC)was used to determine the compositions of lipid fractions,and developed with hexane/diethylether/acetic acid(70:30:1,volume ratio)on silica gel plates.After development,the plate was dried and then sprayed with iodine vapor.The bands corresponding to individual lipid classes were scraped off and directly transmethylated for GC analysis.

For the determination of fatty acids,gas chromatography was performed by using capillary column DB-23(60 m × 0.25 mm × 0.25 μm)and a FID detector.Tridecane acid methyl ester was used as internal standard.Samples(1 μl)were analyzed with the column held initially at 100 °C and then increased to 196 °C with a gradient of 25 °C·min-1.Finally the temperature was increased to 220°C with a gradient of 2 °C·min-1and hold for 6 min[16].

For sterol detection,the extracted lipids were firstly saponified,the reactant was then extracted with petroleum ether,and finally washed to get neutral pH.After rotary evaporation the nonsaponifiable matter was collected.Sterols contained in the nonsaponifiable fraction were analyzed by GC–MS.The chromatographic column for the analysis was a DB-5 capillary column(30 m × 0.25 mm × 0.25 μm).Helium was used as the carrier gas with a flow rate of 1.0 ml·min-1.Samples(1 μl)were injected under split less mode at 250 °C,and analyzed with the column held initially at 70°C for 1.5 min,and then increased to 200 °C with a gradient of 10 °C·min-1.Finally the temperature was increased to 280°C and hold for 20 min.The MS device was operated in scan mode,starting after 7 min with a mass range of 50–500 AMU.The mass source(Electron Impact mode,EI:70 eV)temperature was 230 °C,and analyzed temperature was 250 °C.

3.Results and Discussion

3.1.Lipid classes accumulation process in M.alpina

The fermentation process of M.alpina was shown in Fig.2.Cells were obtained after 8 days of cultivation.The accumulation of lipids was consistent with cell growth.Further analysis of lipids was conducted to illustrate detailed information of lipid accumulation.

Fig.2.Fermentation process of M.alpina.

As shown in Fig.3,TAG was the major component of lipids(81.0%of total lipids,and 13.7 g·L-1at 192 h).During the whole process of fermentation,TAG maintained at a high percentage.The phenomenon also occurred in other oleaginous microorganisms[17,18,19].In M.alpina the accumulation process of TAG could be divided into two stages,judged by its distinct accumulation rate.In the early stage(24–96 h),it accumulated quickly,but the rate slowed down later(96–192 h).

Fig.3.Accumulation process of different lipid classes in M.alpina.Triacylglycerol(TAG),diacylglycerol(DAG),monoacylglycerol(MAG),free fatty acid(FFA).

Analysis of fatty acid composition in total lipids(Fig.4)revealed that during the later period the percentage of saturated and monounsaturated fatty acid decreased with time,while PUFAs increased.As a result,ARA increasingly became the dominant fatty acid at 192 h.Other study demonstrated that when sufficient carbon resource existed in the medium,the pathway of de novo fatty acid biosynthesis was active,leading to high proportion of saturated and monounsaturated fatty acids in total lipids[20].Therefore,this high supply rate of these fatty acid-CoAs resulted in quick accumulation of TAG during 24–96 h.However,the biosynthesis of ARA takes much more time owing to the desaturation and elongation process[21].As ARA became the prevailing fatty acids in the laterstage,the supply of fatty acid-CoAs could not be as quick as it was.Correspondingly,TAG was accumulated in a relatively low speed during 96–192 h.

Fig.4.Fatty acid composition in total lipids.Palmitic acid(C 16:0),stearic acid(C 18:0),oleic acid(C 18:1),γ-linoleic acid(C 18:2,ω-6),γ-linolenic acid(C 18:3,ω-6),arachidonic acid(C 20:4,ω-6).

The amounts of sterols and sterol esters were also studied.Both of them are biosynthesized from acetyl-CoA,and stored in lipid bodies[22].Therefore,they will compete with TAG for substrate and storage space.In M.alpina,sterols and their esters were present in low quantities,just 0.27 g·L-1and 1.05 g·L-1respectively,at the end of fermentation.The same phenomenon was also observed in other oleaginous microorganism,in which the amount of sterols and their esters was much lower than TAG[23].These studies showed that in oleaginous microorganisms,TAG takes an absolute advantage in lipid biosynthetic pathway.

Monoacylglycerol(MAG)and DAG were in small amounts.Compared with DAG,MAG was in much smaller quantities,because its strong detergent property would have a disruptive effect on cell membrane.Overall,the variation tendency of the two components was in consistent with each other.In addition,as they were key metabolites in TAG metabolism,their amounts were affected by TAG.Their highest amounts occurred at 132 h,when the TAG accumulation rate was apparently slower than the early period.Possibly,the relatively high amounts of DAG and MAG were caused by the distinct accumulation rate of TAG.

The amount of free fatty acid(FFA)was trace.Its quantity increased gradually,which is quite different with other lipid classes.FFA was released from TAG and sterol esters by specific hydrolases[24].The increasing quantity suggests that TAG and sterol esters were in dynamic state that some fatty acids were released constantly.

Other lipids were just 0.44 g·L-1,at 192 h.They were structure lipids(phospholipid,glycolipid,etc.)of the cell,participating in cell function,such as signal transmission and material transport.Their amounts and composition were strictly related with the cell physiology and the environment[25].In this work,they were notseparated further but recognized as a whole reflecting the physiological condition of the cell.Their amount was consistent with biomass.Owing to their hypersensitivity to the culture environment,their amount declined immediately when the glucose has been exhausted and the cell biomass stopped increasing.

3.2.Fatty acid distribution during ARA accumulation stage

Fig.4 indicated that ARA was enriched from 168 h to 192 h,as its percentage in total lipids increased from 40.6%to 46.5%.Composition of individual lipid class during the span was analyzed to illustrate the fluctuation inside lipid metabolism.Generally,this kind of fluctuation is caused by the flow of fatty acid among all lipid fractions.Several factors contributing to this change are as follows:bioconversion or degradation of fatty acid in the lipid pool,selective incorporation of fatty acid from other pools,and the de novo biosynthesis of the fatty acid in the pool.Physical condition of the cell determined which pathway the fungus will take for the transition[13].

Table 1 showed that TAG has a higher percentage of ARA at 192 h(40.3%).Combined with Fig.2,it is obviously that the most ARA existed in this form.Similarly,other studies also showed that the major PUFA were stored in TAG[26].Since the absolute amounts of other fatty acids in TAG did not change sharply and there was no carbon source,we conclude that the main cause of ARA enrichment in TAG was the incorporation of fatty acids from other lipid pools.And the bioconversion of other fatty acids in the TAG pool played a minor role,as the amounts of palmitic acid(C 16:0)and stearic acid(C 18:0)decreased slightly.

However,the percentage of ARA in TAG fraction to the total ARA was lower at 192 h than that at 168 h,which was 86.1%and 90.1%,respectively.Composition of individual lipid class demonstrated that this phenomenon resulted from the much higher percentage of ARA in otherlipid classes.By the way,this factproved that enzymes in relation to the ARA biosynthesis still remained high activity leveleven at the end stage of fermentation.

Table 1 Fatty acid composition of different lipid classes during the transition stage in M.alpina(%)

The increased amount of ARA in FFA was about two fold than the decrease of other fatty acids,suggesting that the incorporation and modification of fatty acids in other lipid pools and the bioconversion of saturated and monounsaturated fatty acids in the FFA pool had equivalent effects.Besides FFA,the percentage of ARA in MAG also had a significant increase.In some way,this phenomenon maybe caused by the physiological status,since the two forms of ARA play an important role in cell protection and death[27–29].

Amounts of ARA in DAG just remained steady,while they had a higher percentage.In the two fractions,the amounts of other fatty acid decreased apparently(especially the saturated and monounsaturated fatty acids).The decreased amount in Others fraction equals to the increased ARA in TAG.Therefore,we supposed that these fatty acids were released in the cytoplasm,and were re-modified to be transformed into ARA–TAG.

In the fraction of sterolesters,amounts of saturated and monounsaturated fatty acids decreased.Possibly,they might be channeled to ARA-sterol ester.On the other hand,as a kind of storage lipid,these fatty acids might be used for β-oxidation for energy supply.

To get more details of lipid flux during the span,the composition of unsaponifiable matters(mainly including squalene and sterol lipids)was measured.Seven ingredients were detected,and desmosterol was the major one(86.4%of total unsaponifiable matters,at192 h)followed by squalene.Other components were all in trace amounts.Limited to our knowledge,we did not find all these matters in the metabolic pathway of lipid(Fig.3).Maybe they were the precursors of desmosterol.Table 2 showed that all these matters decreased except desmosterol.Maybe they were transformed into desmosterol during the transition stage.Owing to the lack of studies about sterol in oleaginous organisms,it is hard to know the reason about the changes in the proportion of these matters.Anyway,the result provided an insight into the metabolic pathway of sterol.

Table 2 Composition of unsaponifiable matters during the transition stage in M.alpina(%)

3.3.Possible mechanism for ARA rich–TAG accumulation

As FFA is toxic to cells,it could not be accumulated in large quantity.Meanwhile,other lipids are also closely linked to cell function,which results in strict limitation in their amounts.Therefore,the best way for ARA accumulation is in the form of TAG.In oleaginous microorganisms,extra fatty acid-CoA would be transformed into TAG continuously atendoplasmic reticulum.First,they gathered together forming microdroplets(the precursors of lipid bodies)inside the endoplasmic reticulum.When reaching a certain size,these micro-droplets would bud off the endoplasmic reticulum,and became lipid bodies[23].Once these lipid bodies formed,more TAG would be incorporated in.So these lipid bodies could grow up rapidly.Benefited from high efficiency and nontoxic property,TAG would be the suitable form for fatty acid accumulation.

Further,to achieve ARA-rich TAG accumulation,the supply of ARACoA should be enhanced.However,there are still some doubts persisting in the processes of the biosynthesis of ARA-CoA and the incorporation of it into TAG.We suggest that two pathways might exist in the processes respectively.The conventional route for ARA-rich TAG biosynthesis demonstrates that ARA-CoA is directly derived from the modification of saturated fatty acid-CoA.Then the ARA-CoA is distributed to TAG by diacylgycerol acyltransferase,forming ARA-rich TAG.In this process,ARA could not have a dominant percentage in TAG fraction,because other easier formed fatty acid-CoAs have the priority of speed and space to enter into TAG.Thus,there might be another pathway for the accumulation of ARA.Higher percentage of ARA was often obtained by strict measures,such as low temperature[30],aging[31]and so on.It should be noticed that these measures were all taken after the cell got a high biomass and lipid content.Then ARA is concentrated rapidly under the adverse conditions.Maybe these measures triggered some mechanisms of the ARA formation and accumulation.Together with our analysis above,it is very likely that the ARA-CoA in TAG came from the further modified saturated fatty acid-CoAs in the cell structure lipid pools.Under normal conditions when sufficient glucose existing in the culture,abundant saturated and monounsaturated fatty acid-CoAs will be biosynthesized,resulting rapid accumulation of microbial oils.When the culture environment becomes extreme,such as starvation and low temperature,the membrane structure lipids(especially the phospholipid)tend to incorporate PUFAs to maintain the membrane fluidity[32].During this period,the extra PUFA would be incorporated into TAG.Therefore,suitable culture condition is necessary for the ARA-rich TAG production.

In addition,the dynamic of TAG also might be beneficial for ARA-rich TAG accumulation.Since most of ARA was stored in TAG inside the lipid bodies,enzymes on the surface of lipid bodies(mainly the diacylglycerol acyltransferase and lipase)would influence of the percentage of ARA in TAG.It has been reported that the genetically modified diacylglycerol acyltransferase could successfully improve the TAG content and change the fatty acid composition[33,34].As well,some studies found that TAG which contains specific fatty acid was hydrolyzed slower than other TAG molecular species by lipase,causing the specific fatty acid occupying a relatively high percentage in TAG[13].The selectivity of the two enzymes could be used for the enhancing ARA in TAG.

In conclusion,the characterization of lipid composition in M.alpina was studied in this work.The data demonstrated that TAG was preferentially accumulated and contained the most ARA.Further,to get the best efficiency for ARA storage in TAG,the accumulation rate should be controlled.We suggest that the significant step in ARA accumulation lies in the transformation process rather than the precursors supplement.Thus,conditions should be created for this transition potentiality.This opinion offers another choice for efficient ARA-rich oil fermentation.