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Mar 12, 2009 - Lu-Jing Ren Æ He Huang Æ Ai-Hua Xiao Æ Min Lian Æ. Li-Jing Jin Æ ... 4 g/L malic acid at the rapid lipid accumulation stage. Total lipid ...
Bioprocess Biosyst Eng (2009) 32:837–843 DOI 10.1007/s00449-009-0310-4

ORIGINAL PAPER

Enhanced docosahexaenoic acid production by reinforcing acetyl-CoA and NADPH supply in Schizochytrium sp. HX-308 Lu-Jing Ren Æ He Huang Æ Ai-Hua Xiao Æ Min Lian Æ Li-Jing Jin Æ Xiao-Jun Ji

Received: 27 November 2008 / Accepted: 22 February 2009 / Published online: 12 March 2009 Ó Springer-Verlag 2009

Abstract Docosahexaenoic acid (DHA) production in Schizochytrium sp. HX-308 was evaluated by detecting enzymatic activities of ATP:citrate lyase (EC 4.1.3.8), malic enzyme (EC 1.1.1.40) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) at different fermentation stages. According to the analysis, a regulation strategy was proposed which reinforced acetyl-CoA and NADPH supply at a specific fermentation stage. DHA content of total fatty acids was increased from 35 to 60% by the addition of 4 g/L malic acid at the rapid lipid accumulation stage. Total lipid content also showed an apparent increase of 35% and reached 19 g/L when 40 mL ethanol/L was added at the late lipid accumulation stage. Keywords DHA  Acetyl-CoA  NADPH  Schizochytrium sp.

Introduction Docosahexaenoic acid (DHA) (C22:6), a member of x-3 polyunsaturated fatty acids (PUFAs), is believed to be an important structural component of neural and retinal tissues [1]. In addition, DHA plays significant roles in enhancing

L.-J. Ren  H. Huang (&)  M. Lian  L.-J. Jin  X.-J. Ji College of Life Science and Pharmacy, Nanjing University of Technology, No. 5 Xinmofan Road, Gulou District, 210009 Nanjing, China e-mail: [email protected] H. Huang  A.-H. Xiao Jiangsu Provincial Innovation Center for Industrial Biotechnology (JPICIB), No. 30 Puzhunan Road, Pukou District, 211816 Nanjing, China

human health and preventing human diseases such as atherosclerosis, rheumatoid arrhythmia, psoriasis, diabetes, and cancers [2]. The production of DHA by marine microorganisms has become the subject of intensive research and increasing commercial attention because of its high physiological importance [3]. Schizochytrium sp. are heterotrophic marine traustochytrids that produce about 35% of their total fatty acids as DHA [4, 5]. They are considered to be a highly promising source for DHA production [6, 7]. During the past 20 years, studies dealing with DHA microbial production by this organism focused on the effects of cultivation conditions, cultivation styles, medium compositions, including carbon, nitrogen sources, inorganic salt and mineral addition, on DHA productivity [8–10]. However, the mechanism of lipid accumulation is still not well investigated. In order to achieve efficient lipid accumulation in microorganisms, two conditions should be satisfied simultaneously [11, 12]. One is a continuous supply of acetyl-CoA directly in the cytosol of the cell as a necessary precursor for fatty acid synthetase (FAS), and the other is a sufficient supply of NADPH as the essential reductant used in fatty acid biosynthesis. According to the metabolic pathway of DHA biosynthesis by Schizochytrium sp., the formation of acetylCoA in oleaginous microorganism is facilitated by the presence of ATP:citrate lyase (ACL). The production of NADPH for fatty acid biosynthesis is dependent on malic enzyme (ME) [13] and glucose-6-phosphate dehydrogenase (G6PDH) (Fig. 1). The present investigation focused on exploring the relationship between the lipid accumulation and the supply of acetyl-CoA and NADPH during fermentation so as to develop some strategies to enhance DHA production. The variation of acetyl-CoA and NADPH formation of Schizochytrium sp. was analyzed by detecting the changes

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Bioprocess Biosyst Eng (2009) 32:837–843 Glucose NADP

NADPH

Gluconate-6-phosphate

Glucose-6-phosphate G6PDH

HMP

EMP NADPH

Pyruvate

Pyruvate

Mitochondria

NADP PDH

ME "transhydrogenase" cycle

Malate

Acetyl-CoA

Oxaloacetate

MDH NAD

solution was filter-sterilized (0.22 lm) and contained (mg/L): thiamine 50, biotin 1 and cyanocobalamin 10. In the fermentation culture, with initial glucose concentration 120 g/L and 5% (v/v) inoculum size, Schizochytrium sp. HX-308 cells were grown at 25 °C, 160 rpm.

Oxaloacetate NADH

Determination of cell dry weight

CS ATP+CoASH

Citrate

Citrate

TCA Cycle

ACL

Acetyl-CoA FAS

Isocitrate

ICDH

2-Oxo-glutarate

The cells were collected by centrifugation, washed with distilled water, placed on filter paper, and then dried at 65 °C until the drying did not reduce the weight.

Fatty acid

Lipid extraction PDH: pyruvatedehydrogenase MDH:malate dehydrogenase CS:citrate synthase ACL:ATP:citrate lyase ICDH:isocitrate dehydrogenase FAS:Fatty acid synthase ME:malic enzyme "transhydrogenase" cycle : NADH + NADP+ +ATP→NAD+ + NADPH + Pi

Fig. 1 Supply of acetyl-CoA and NADPH for fatty acid synthesis

of three key enzymatic activities at different stages including early lipid accumulation stage, fast lipid accumulation stage and late lipid accumulation stage. To avoid the deficient supply of acetyl-CoA and NADPH and improve DHA yield, malic acid, the activator of malic enzyme [14], was introduced to the fermentation system to enhance NADPH supply. Ethanol and sodium acetate, the two interesting alternative carbon sources for DHA production [15–17], which could directly be converted into acetyl-CoA by acetyl-CoA synthetase in eukaryotes, were introduced to the experiment to enhance acetyl-CoA supply [16].

Materials and methods Microorganism Schizochytrium sp. HX-308 was used in the present study. This strain was isolated from seawater and was preserved in 20% (v/v) glycerol at -80 °C. Culture conditions The main culture medium contained 40 g/L glucose and 0.4 g/L yeast extract, which were dissolved in artificial sea water. This medium also contained trace elements in a prepared solution (2 mL/L) and vitamin solution (2 mL/L). All medium components were separately heat sterilized (121 °C). The trace element solution contained the following amounts (g/L): Na2EDTA 6, FeSO4 0.29, MnCl2 4H2O 0.86, ZnSO4 0.8, CoCl2 6H2O 0.01, Na2MoO4 2H2O 0.01, NiSO4 6H2O 0.06 and CuSO4 5H2O 0.6. Vitamin

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40 mL fermentation broth was added to the high-pressure homogenizer to crush the algal cells and then lipids were extracted with three times volume of n-hexane/ethanol (2:1, v/v) mixture solution. Hexane was removed using a rotary vacuum evaporator. Fatty acid analysis Fatty acid methyl esters (FAMEs) were prepared by the modified method [18, 19] as follows: 1 mL 2 M KOHmethanol was added to a tube containing 0.3 g dried algal cell. The tubes were heated in a water bath at 60 °C for 2 h, and then cooled down to room temperature, at which point a mixture of 2 mL methanol and 1.5 mL 45% BF3ether was added. The tubes were then heated in a water bath at 60 °C for 10 min again, and then 3 mL hexane was added when the tubes cooled down to room temperature. The liquid in the tubes were mixed through a vertex for 1 min, and then settled for separation of two phases after adding 1 mL saturated sodium chloride solution. The upper phase containing FAMEs was transferred to a clean centrifuge tube, diluted 5 times with hexane and dried with anhydrous Na2SO4. This FAMEs samples were then analyzed by GC-MS system (Thermo/Finnigan Trace GCMS, USA). The GC was equipped with a capillary column (DB-5MS, 30 m by 0.22 mm). Helium was used as the carrier gas. The injector was maintained at 250 °C, with an inject volume of 0.4 uL. The column was raised from 80 to 210 °C at 20 °C/min. and then increased to 280 at 10 °C/min. Fatty acids were identified by comparison to external standards (Sigma, USA) and MS structure database. Preparation of cell extracts The microorganism was harvested by centrifugation, washed with ice-cold distilled water and then by a washing buffer [400 mM Tris–HCl buffer, pH 7.4, containing 20%(w/v) glycerol, 1 mM dithioerythreitol (DTT)].

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Finally, the microorganism is suspended in clean washing buffer with 0.5 mM phenyl-methane-sulfonyl fluoride (PMSF). After being disrupted by ultrasonic disrupter for 15 min, the material was centrifuged (120,000 rpm for 15 min at 4 °C) and the supernatant was used immediately for the determination of enzyme activity. Protein was determined using the Bradford method with bovine serum albumin (BSA) as a standard [20]. Total protein content of crude enzyme was calculated by an empirically determined calibration equation (protein content = 0.18333 9 OD595nm - 0.00338). Enzyme assay ME and G6PDH activities were determined spectrophotometrically by monitoring the rate of NADPH formation at 28 and 25 °C, respectively [21, 22]. ACL activity was detected according to the method of Takeda [23]. A unit of enzyme activity (U) was defined as the formation of the quantitative amount of NADPH/min, which was equivalent to an increase in OD340 of 0.001/min. Specific activity (U/mg protein) was defined as the units of activity/mg of protein.

Result and discussion Batch cultivation The time course of cell growth, glucose exhaustion and lipid production of Schizochytrium sp. HX-308 during the batch cultivation is shown in Fig. 2. It is apparent that glucose was used mainly for cell growth during fermentation. Biomass and lipid content gradually increased with time after inoculation. Three different stages were noticed

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in the following statement. Schizochytrium sp. HX-308 showed lower growth rate with a little lipid accumulated before 16 h with the new cultivation environment. This stage was called early lipid accumulation stage. From 16 to 40 h, cell growth was fast and biomass reached the maximum of 58.5 g/L at 40 h. Meanwhile, total lipid content reached 10.7 g/L. Depletion of the nitrogen source causing excess carbon to divert to lipid biosynthesis may account for this observation. This stage was defined as rapid lipid accumulation stage. After 40 h, cells entered a stationary phase and kept a constant biomass with little lipid accumulation. This stage was called the late lipid accumulation stage. Changes of fatty acid proportion in batch cultivation GC-MS analysis showed that 90% of total fatty acids extracted from Schizochytrium sp. HX-308 were tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), docosapentenoic acid (C22:5), and docosahexaenoic acid (C22:6). Table 1 shows the variation in the percentage of these four fatty acids during fermentation. The DHA proportion of total fatty acids did not increase gradually with time as expected. This proportion was 37% when first detected at 8 h and kept constant until 32 h when it reduced to 28%, and then the ratio increased to 35% at the end of the fermentation. The proportion of other saturated fatty acids such as C14:0 and C16:0 showed the opposite trend. The proportion of C14:0 was increased from 6% at 8 h to Table 1 Percent of all kinds of fatty acids (%, w/w) during fermentation Time (h)

Percent of group I fatty acids (%)a C14:0

C16:0

C22:5

C22:6

8

5.95

21.02

24.61

36.91

16

8.07

18.26

23.02

37.53

24

7.01

19.64

22.81

37.22

32

13.13

28.34

19.24

28.86

40

19.84

25.09

18.63

27.94

48

15.34

17.59

23.61

35.39

Percent of group II fatty acids (%)

Fig. 2 Fermentation profiles of cell growth, glucose consumption, and total lipid production during batch cultivation of Schizochytrium sp. HX-308. The culture was grown at 25 °C, and agitated at 160 rpm

b

C18:0

C16:1

C18:3

C18:1

C20:5

Others

8

1.12

2.28

0.24

3.43

3.21

1.23

16

1.55

1.94

0.22

5.41

2.01

1.99

24

1.34

2.11

0.23

4.92

2.72

2

32

1.29

1.74

0.41

1.77

2.46

2.76

40

1.22

1.77

0.38

1.01

2.73

1.39

48

1.49

1.37

0.3

0.92

2.19

1.8

a

Group I represents fatty acids which exceed 10%

b

Group II represents fatty acids which below 10%

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Bioprocess Biosyst Eng (2009) 32:837–843

20% at 40 h, and the proportion of C16:0 was maintained at about 20% during the first 24 h and then increased to 28% by 32 h. The possible reason for the reduction of DHA proportion and the increase of saturated fatty acids at the rapid lipid accumulation phase might be the insufficient NADPH supply. The proportion of other fatty acids such as palmitic acid (C16:1), oleinic acid (18:1), linolenic acid (C18:3), ecosapeatanolic acid (C20:5) was combined not more than 6% during the fermentation stages, and remained constant except C18:1, which dropped from 5% at 16 h to 1% at 48 h (Table 1). Enzyme activity change during lipid accumulation ME, G6PDH, ACL activities were estimated in the cellfree extract of Schizochytrium sp.HX-308 culture broth every 8 h during fermentation. All the assays were carried out in duplicate and the average values were reported. Figure 3 showed the changes of the three key enzymatic activities with cultivation time. ACL activity was 35 U mg-1 protein when first detected at 8 h and reached a maximum of 301 U mg-1 protein at 40 h. It then decreased to 35 U mg-1 protein at the end of fermentation. This tendency was consistent with the total lipid accumulation change. Lower ACL activity at the late lipid accumulation phase led to lower amount of acetylCoA, which hindered lipid biosynthesis, and finally induced lower lipid accumulation at this period. Accordingly, reinforcing acetyl-CoA supply at the late lipid accumulation stage was extraordinary. The summation of ME and G6PDH activity (SMG) was used to reflect NADPH supply information as shown in Fig. 3. In the early lipid accumulation stage, there was a clear increase in SMG. The activity of G6PDH increased sharply with constant ME activity. G6PDH was the main source of NADPH for lipid accumulation at this stage. In the rapid lipid accumulation stage, SMG kept at a relative constant level except for a slight decrease at 24 h. G6PDH activity showed a clearly downward tread at the same time ME activity increased sharply from 252 to 463 U mg-1 protein. In this stage, both the enzymes played a crucial role in NADPH supply, and the role of ME became more and more obvious. Most lipids were synthesized in this stage and needed much more NADPH to convert saturated fatty acids to polyunsaturated fatty acids. The decrease of SMG from 531 U mg-1 protein at 32 h to 484 U mg-1 protein at 40 h weakened the ability of PUFA synthesis. Therefore, reinforcing NADPH supply at this stage could effectively enhance DHA production. In the late lipid accumulation stage, these three enzyme activities were all diminished with much higher ME activity. Apparently, NADPH supply at this stage comes

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Fig. 3 Profiles of ME activity, G6PDH activity, ACL activity during batch fermentation of Schizochytrium sp. HX-308. Each datum is the mean value of three identical samples. SMG means the summation of ME and G6PDH activity

from the reaction catalyzed by ME. From the change tendency of ME activity, there was a peak value at 48 h when approached to the end of fermentation. The early appearance of this peak value might solve the insufficient NADPH supply at the rapid lipid accumulation stage. Improvement of DHA production by reinforcing acetyl-CoA supply Ethanol and sodium acetate were introduced to the fermentation system at the late lipid accumulation stage as a result of the analysis of acetyl-CoA supply information at different lipid accumulation phases in ‘‘Enzyme activity change during lipid accumulation’’. This may solve the insufficient acetyl-CoA supply problem at this stage. Figure 4a and b shows changes of biomass, total lipids and DHA percentage of total fatty acids resulting from addition of different concentrations of ethanol (2, 4, 6, 8 mL/50 mL broth) and sodium acetate (2, 4, 6, 8 g/L), respectively at the late lipid accumulation stage. While adding 40 mL ethanol/L broth to the broth at late fermentation stage, algae could stand up to the effect of ethanol, and total lipids content showed an increase of 35% from 14 to 18 g/L with a slight increase of DHA percentage from 35 to 38%. This phenomenon agrees with Sijtsma L’s standpoint that ethanol can be converted to acetyl-CoA directly and in its metabolism might generate additional reducing power, NADPH, for lipogenesis [24].Both the biomass and lipid content decreased at higher ethanol concentration. A slight inhibition on biomass synthesis was observed at 80 mL ethanol/L broth, but DHA percentage increased from 35 to 40%, which might be explained by additional reducing power, NADPH, generation during ethanol metabolism [24]. The total lipid content was nearly

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Figure 5 shows changes of biomass, total lipid concentration and DHA percentage of total fatty acids with different concentration (1, 2, 4, 6 g/L) of malic acid at the rapid lipid accumulation stage. As shown in Fig. 5, adding malic acid at all four concentrations enhanced DHA percentage of total fatty acids, but showed no significant impact on cell growth and total fatty acids. Total lipids showed a minor increase of 15% when adding 4 g/L malic acid at rapid lipid accumulation stage, meanwhile, the final DHA content of total fatty acids was increased from 35 to 60% (Fig. 6). The DHA percentage enhancement could be explained by Randolph T. Wedding’ statements that malic acid could induce the structure change of malic enzyme, from the dimer to the more active tetramer or ocatamer forms [14], which enhanced the NADPH generating reaction from malic acid to pyruvate and ensure enough NADPH for DHA production. This phenomenon illustrated that NADPH could change fatty acid composition effectively, but has little effect on total lipids accumulation, so combining this strategy with other enhancing lipids accumulation strategy, such as ethanol addition regulation, might enhance lipids and DHA percentage simultaneously.

Conclusion

Fig. 4 Changes of biomass, total lipids, DHA percentage of total fatty acids by adding different concentrations of ethanol/sodium acetate at rapid lipid accumulation stage. a Adding different concentrations (40, 80, 120, 160 mL ethanol/L broth) of ethanol at rapid lipid accumulation stage. b Adding different concentrations (2, 4, 6, 8 g/L) of sodium acetate at rapid lipid accumulation stage. Control means no ethanol or sodium acetate addition

Acetyl-CoA and NADPH supply systems of Schizochytrium sp. HX-308 during the lipid accumulation phase have been analyzed in this paper. According to this information, a regulation strategy was proposed reinforcing acetyl-CoA and NADPH supply at a specific fermentation stage. Lipid concentration increased by 35% and reached 19 g/L by

zero at 160 mL ethanol/L broth. Perhaps high ethanol concentrations killed the algae releasing enzymes which decomposed the lipids. A similar phenomenon appeared when sodium acetate was added. Both these addition of substrates improved the lipid concentration at a proper concentration with slight effect on the DHA percentage of total fatty acids. Improvement of DHA production by reinforcing NADPH supply In response to the analysis of NADPH supply information in ‘‘Enzyme activity change during lipid accumulation’’, malic acid was introduced to the fermentation system at the rapid lipid accumulation stage to activate malic enzyme activity and solve the problem of insufficient NADPH supply at this stage.

Fig. 5 Changes of biomass, total lipids, DHA percentage of total fatty acids by adding different concentrations (1, 2, 4, 6 g/L) of malic acid at rapid lipid accumulation stage. Control means no malic acid addition

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Fig. 6 Changes of GC-MS results before adding 4 g/L malic acid at rapid lipid accumulation stage. a GC-MS results before adding malic acid, b GC-MS results after adding malic acid

adding ethanol and DHA percentage was increased from 35 to 60% by adding malic acid. This work gives us solid scientific evidence and guidance to improve lipid accumulation from the perspective of physiology and biochemistry. Although this study has proposed two regulation strategies to enhance lipid accumulation and DHA percentage, respectively, the detailed regulation mechanism should be further studied. In addition, combination of the regulation methods should also be attempted to enhance DHA production. Acknowledgments This work was financed by the National Natural Science Foundation of China (No. 20606018), the Ministry of Science and Technology of China (National Basic Research Program of China (No. 2007CB707805)) and Scientific Research Project for Postgraduate in Jiangsu Province of China (No. CX07s_032z). We also thank Prof. Walt Johnson for his language assistance with this paper.

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