Effects of 20 Standard Amino Acids on the Growth, Total FattyAcids Production, and c-Linolenic Acid Yield in Mucorcircinelloides

Xin Tang • Huaiyuan Zhang • Haiqin Chen •

Yong Q. Chen • Wei Chen • Yuanda Song

Received: 7 February 2014 / Accepted: 21 June 2014

� Springer Science+Business Media New York 2014

Abstract Twenty standard amino acids were examined as

single nitrogen source on the growth, total fatty acids

production, and yield of c-linolenic acid (GLA) in Mucor

circinelloides. Of the amino acids, tyrosine gave the

highest biomass and lipid accumulation and thus resulted in

a high GLA yield with respective values of 17.8 g/L, 23 %

(w/w, dry cell weight, DCW), and 0.81 g/L, which were

36, 25, and 72 % higher than when the fungus was grown

with ammonium tartrate. To find out the potential mecha-

nism underlying the increased lipid accumulation of M.

circinelloides when grown on tyrosine, the activity of lip-

ogenic enzymes of the fungus during lipid accumulation

phase was measured. The enzyme activities of glucose

6-phosphate dehydrogenase, 6-phosphogluconate dehy-

drogenase, and ATP-citrate lyase were up-regulated, while

NADP-isocitrate dehydrogenase was down-regulated by

tyrosine during the lipid accumulation phase of the fungus

which suggested that these enzymes may be involved in the

increased lipid biosynthesis by tyrosine in this fungus.

Introduction

Microorganisms have often been considered to produce the

oils containing nutritionally important polyunsaturated

fatty acids (PUFAs) or the lipid presenting composition

similarities with exotic fats (e.g., the cocoa butter) [31, 32,

35, 41]. c-Linolenic acid (GLA, 18:3; cis-6,9,12-octadec-

atrienoic acid), as a critical PUFA, is experimentally pro-

ven that it has beneficial effects for prevention and

treatment of inflammatory disorders, diabetes, cardiovas-

cular disorders, cancers, and some other diseases [5]. GLA

is found in a relatively small number of plant seed oils,

including borage oil, evening primrose oil, and blackcur-

rant seed oil, with GLA contents of 22, 8, and 16 %,

respectively [6, 41]. Compared with these traditional plant

GLA-rich oils, microorganisms have several advantages

such as high growth rate, simple cultural conditions whose

yields are not subject to variations in the climate or weather

[44]. Thus, production of GLA from microorganisms is a

promising alternative to plants. Mucor circinelloides, as an

oleaginous fungus, was widely used to investigate GLA

production [1–4], and it had been selected as a model

microbe to produce GLA in last century [40].

Nitrogen sources are critical components of the growth

media for microorganisms, and many researchers have

reported that nitrogen sources play important roles in the

growth and the production of active compounds in micro-

organisms [27, 30]. The effects of nitrogen sources on lipid

accumulation and production of unsaturated fatty acids in

oleaginous microorganisms have been widely investigated.

In Neochloris oleoabundans, sodium nitrate not only

prompted cell growth but also enhanced lipid accumulation

[28]. Mortierella alpina accumulated twice as much ara-

chidonic acid when soybean meal was used as nitrogen

source in the medium as when yeast extract was used as

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00284-014-0671-z) contains supplementarymaterial, which is available to authorized users.

X. Tang � H. Zhang � H. Chen � Y. Q. Chen � W. Chen �Y. Song (&)

State Key Laboratory of Food Science and Technology, School

of Food Science and Technology, Jiangnan University, 1800

LiHu Road, Wuxi 214122, Jiangsu, People’s Republic of China

e-mail: ysong@jiangnan.edu.cn

X. Tang

e-mail: tangxin_801@163.com

H. Chen � Y. Q. Chen � W. Chen

Synergistic Innovation Center for Food Safety and Nutrition,

Wuxi 214122, People’s Republic of China

123

Curr Microbiol

DOI 10.1007/s00284-014-0671-z

nitrogen source due to their different effects on the

mycelial morphology [37]. Organic nitrogen compounds

are more favorable for both cell growth and lipid accu-

mulation than inorganic nitrogen sources in Mort. alpina

[29]. Furthermore, amino acids, as nitrogen sources, can

differentially regulate genes expression in Saccharomyces

cerevisiae [15]. However, no study has been carried out to

investigate the individual effects of each of the 20 amino

acids found in proteins on lipid biosynthesis in any ole-

aginous microorganism.

In the present study, the effects of the 20 standard amino

acids, as single nitrogen source, on the growth, glucose

utilization, lipid accumulation, and GLA yield in M. cir-

cinelloides were investigated. Furthermore, the molecular

mechanism of the effect of tyrosine on lipid production was

analyzed. To our knowledge, this is the first report of the

study of the effect of all 20 standard amino acids on lipid

accumulation in oleaginous fungus.

Materials and Methods

Microorganism and Cultivation

M. circinelloides CBS108.16 was used in this study.

100 lL spore suspension whose concentration was

approximately 107 spores/ml of M. circinelloides was used

to inoculate 150 ml K & R medium [22] held in 1 L baffled

flasks equipped with baffles to improve aeration. The cul-

ture was incubated for 24 h at 30 �C with shaking at

150 rpm and then used at 10 % (v/v) to inoculate 1 L

baffled flasks containing 150 ml modified K & R (N-lim-

iting) medium containing 80 g glucose/1, 0.5 g nitrogen/L,

and no yeast extract. Ammonium tartrate (control) and 20

amino acids were used as single nitrogen sources, and each

medium contained 0.5 g nitrogen/L (e.g., 3.29 g ammo-

nium tartrate/L, or 2.68 g glycine/L, or 3.19 g alanine/L,

equivalent to 0.5 g nitrogen/L). The culture was incubated

for 4 days at 30 �C with shaking at 150 rpm.

Determination of Dry Cell Weight and Glucose,

Ethanol, and Organic Acids Concentration

Biomass was harvested by filtration through a Buchner

funnel under reduced pressure, washed three times with

distilled water, and dried at 105 �C to a constant weight.

Glucose concentration in the culture was measured using a

glucose oxidase Perid-test kit (Boehringer Mannheim)

according to the manufacturer’s instructions. Ethanol

concentration in the culture was determined according to

Lubbehusen et al. [24]. Organic acids (lactic acid, malic

acid, acetic acid, citric acid, succinic acid, fumaric acid, a-

ketoglutaric acid, and pyruvic acid) concentration in the

culture was analyzed by HPLC (Agilent 1100, USA)

equipped with a Diamonsil C18 (4.6 9 250 mm, Dikma,

China), and the mixture of methanol/water/phosphoric acid

(5/95/0.05, v/v) was used as the mobile phase. Column

temperature and flow rate were set at 30 �C and 0.8 ml/

min, respectively. The wavelength of the UV detector was

set at 210 nm, and organic acids were identified and

quantified by comparison of their retention times and peak

areas with standards.

Analysis of Cell Lipid and Fatty Acid Profile

Biomass was collected by filtration under reduced pressure,

rapidly frozen, and then freeze dried. Pentadecanoic acid

(15:0, Sigma) was added into the freeze-dried cell as an

internal standard, and cell lipid was extracted with chlo-

roform/methanol (2:1, v/v) [13]. The extracted cell lipid

was methylated, and the fatty acyl composition was ana-

lyzed by GC using standard procedures.

Preparation of Cell Extracts and Determination

of Enzyme Activities

Biomass was harvested by filtration and washed three times

with distilled water. The harvested cells were disrupted using

a mortar in liquid N2 and suspended in extraction buffer

[100 mM KH2PO4/KOH, pH 7.5, containing 20 % (w/v)

glycerol, 1 mM benzamidine�HCl, and 1 mM DTT]. The

disrupted cell suspensions were centrifuged at 10000g for

10 min at 4 �C and then the supernatants were used for

enzyme analysis. Protein concentrations were determined

using the method of Bradford with BSA as a standard. The

determination of individual enzyme activities was performed

in the supernatant fraction by the following established

methods: ATP: citrate lyase (ACL) (EC 4.1.3.8) [45], malic

enzyme (ME) (EC1.1.1.40) [19], glucose 6-phosphate dehy-

drogenase (G6PD) (EC 1.1.1.49) [26], 6-phosphogluconate

dehydrogenase (PGD) (EC 1.1.1.44) [39], and NADP-isoci-

trate dehydrogenase (NADP-ICD) (EC 1.1.1.42) [23]. Each

enzyme activity was measured at three biological replicates to

assess reproducibility.

Results and Discussion

Effects of 20 Amino Acids on the Growth and Glucose

Utilization of M. circinelloides

When M. circinelloides was cultivated in the modified K &

R fermentation medium for 4 days, the cell growth with

different nitrogen sources was determined (Fig. 1). Com-

pared to ammonium tartrate, proline, serine, asparagine,

glutamic acid, and especially tyrosine and aspartic acid

X. Tang et al.: Effects of 20 Standard Amino Acids on the Growth

123

stimulated the growth of the fungus. When tyrosine and

aspartic acid were used as the single nitrogen source, the

DCW reached up to 17.8 and 16.9 g/L, respectively, which

were 36 and 30 % higher than that of ammonium tartrate,

respectively, and significantly higher than that of all the

other nitrogen sources except asparagine. On the other

hand, the biomass production of the fungus grown on

valine, phenylalanine, tryptophan, threonine, cysteine,

glutamine, and lysine was markedly lower than that of

ammonium tartrate, suggesting that these amino acids were

not good nitrogen sources for the growth of the fungus. In

another fungus, Cunninghamella echinulata, it was also

found that glutamine and lysine were not good for the cell

growth compared with the ammonium salt [9]. In sum-

mary, the 20 standard amino acids differentially affected

the growth of M. circinelloides, among which tyrosine and

aspartic acid appeared to be favorable nitrogen sources for

cell growth.

The carbon substrates (e.g., glucose, glycerol, or

hydrophobic substrates) are critical to cell growth of

microorganisms, and the utilization of carbon substrates

was associated with the cell growth and lipid accumulation

[35]. In our study, glucose was used as the carbon sub-

strates. Lipid accumulation in microorganisms grown on

the glucose was regulated by fatty acid de novo synthesis,

which was completely different with that of growth on fatty

substrates [31, 32]. Since the growth of microorganism

might be associated with the utilization of glucose, the

residual glucose concentration was determined after culti-

vation for 4 days in M. circinelloides. As shown in Fig. 2,

the residual glucose concentrations of the cultures grown

on phenylalanine, tryptophan, glutamine, and lysine were

significantly higher than that of ammonium tartrate, and the

biomass production grown on these amino acids was cor-

respondently lower than that of ammonium tartrate

(Fig. 1). While the residual glucose concentrations of the

cultures grown on glycine, valine, proline, tyrosine, serine,

cysteine, asparagine, aspartic acid, glutamic acid, and

arginine were markedly lower than that of ammonium

tartrate, and the growth on these amino acid was better than

that of ammonium tartrate except valine and cysteine. It

was reported that M. circinelloides was a Crabtree-positive

and dimorphic microorganism and could use glucose to

produce ethanol [24]. Indeed, we found that significant

amount of ethanol and organic acids (lactic acid and malic

acid) was produced when the fungus was grown on valine

or cysteine. This indicates that glucose was partially fer-

mented to produce ethanol and other organic acids rather

than to be used for biomass synthesis, which resulted in the

poor cell growth on valine and cysteine despite that the

glucose was well consumed (Table S1). The main result

suggested that the high biomass production of the fungus

grown on tyrosine and aspartic acid may be connected with

the good utilization of glucose.

Effects of 20 Amino Acids on Total Fatty Acids

Production, Fatty Acid Compostion, and Yield of GLA

in M. circinelloides

To investigate the influence of amino acids on lipid

accumulation in M. circinelloides, the lipid analysis of the

fungus grown in the same conditions was carried out

(Fig. 3). Only tyrosine significantly increased the lipid

production, most amino acids such as glycine, valine,

Fig. 1 Dry cells weight (DCW)

of M. circinelloides CBS 108.16

grown on different nitrogen

sources for 4 days. Ammonium

tartrate was used as the control

nitrogen source. Values are

mean of three biological

replicates. Error bars represent

the average standard deviations

of these replicates. Values

which do not share common

superscripts were significantly

different to each other

(P \ 0.05) 61 9 43 mm

(600 9 600 DPI)

X. Tang et al.: Effects of 20 Standard Amino Acids on the Growth

123

isoleucine, proline, serine, threonine, cysteine, asparagine,

aspartic acid, glutamic acid, and lysine decreased lipid

production compared with ammonium tartrate. While

alanine, leucine, phenylalanine, tryptophan, methionine,

glutamine, arginine, and histidine had no significant effect

on lipid production compared with ammonium tartrate.

Total fatty acids production of the fungus grown on

tyrosine was 23 % (w/w) of DCW, which was the highest

among all these nitrogen sources and 1.25 fold of that of

ammonium tartrate, suggesting that tyrosine is the optimal

nitrogen source for total lipid accumulation in M.

circinelloides.

The fatty acid composition of oleaginous microorgan-

isms was reported to be influenced by the carbon sub-

strates, initial sugar concentration, the fermentation time,

the physiological state, and initial molar ratio C/N [11, 12,

21, 33, 51]. In order to investigate the effects of 20 amino

acids on the fatty acids composition of M. circinelloides,

the fungus was grown on glucose as carbon source and

each of the 20 amino acids as nitrogen source with same

C/N ratio and at the same culture condition. The fatty acid

composition of the fungus grown on different amino acids

is shown in Table 1. Oleic acid (18:1) was the predominant

fatty acid grown on each amino acid. Among the 20 amino

Fig. 2 The residual glucose

concentration of the culture with

different amino acids as

nitrogen sources at the 4th day.

Ammonium tartrate was used as

a control nitrogen source, and

the initial glucose concentration

was 80 g/L. Values are mean of

three biological replicates.

Error bars represent the average

standard deviations of these

replicates. Values which do not

share common superscripts were

significantly different to each

other (P \ 0.05) 61 9 43 mm

(600 9 600 DPI)

Fig. 3 Total fatty acids (TFAs)

production of M. circinelloides

CBS 108.16 grown on different

nitrogen sources for 4 days.

Ammonium tartrate was used as

a control nitrogen source.

Values are mean of three

biological replicates. Error bars

represent the average standard

deviations of these replicates.

Values which do not share

common superscripts were

significantly different to each

other (P \ 0.05) 61 9 43 mm

(600 9 600 DPI)

X. Tang et al.: Effects of 20 Standard Amino Acids on the Growth

123

acids, tryptophan prominently enhanced the stearic acid

(18:0) concentration of total fatty acids by 4.7 fold com-

pared to ammonium tartrate. Leucine, tryptophan, and

methionine greatly decreased the GLA concentration of the

lipids by 40, 39, and 33 %, respectively, compared with

ammonium tartrate. A possible explanation for this is that

the relevant desaturases (e.g., D 6 desaturase) for GLA

synthesis were inhibited when the fungus grown on these

amino acids. Although valine strongly increased the GLA

concentration of the total fatty acids by 20 %, the cell

growth and lipid accumulation were poor, and therefore it

is not a favorable nitrogen source for GLA production.

The effect of nitrogen sources on the GLA content of

DCW in M. circinelloides is shown in Fig. 4a. Most amino

acids markedly decreased the GLA content of DCW when

compared to ammonium tartrate. Two amino acids, alanine

and asparagine, had no prominent effect on the GLA

content of DCW, and only tyrosine significantly increased

the GLA content of DCW among the 20 amino acids. As

shown in Fig. 4b, asparagine and aspartic acid slightly

increased the GLA yield, and the maximum yield of GLA

(0.81 g/L) was obtained when grown on tyrosine, which

was 72 % higher than that of ammonium tartrate (Fig. 4b).

This maximum yield of GLA was remarkably higher than

that obtained from other species of Mucor and some other

Zygomycetes such as M. circinelloides CBS 172-27

(0.22 g/L) [3], M. circinelloides CBS 203.28 (0.51 g/L)

[20], Mucor rouxii CBS 416.77 (0.32 g/L) [17], M. mucedo

CCF-1384 (0.38 g/L) [8], M. sp. LGAM 366 (0.18 g/L)

[47], Mort. ramanniana CBS 478.63 (0.44 g/L) [16], C.

echinulata CCF-103 (0.37 g/L) [8], and C. sp. LGAM (9)2

(0.26 g/L) [21]. Mort. isabellina ATHUM 2935 accumu-

lated 0.80 g/L of GLA when cultivated on high glucose

content media [34], whereas growth on xylose and lactose-

enriched cheese whey was accompanied by the GLA yield

of 0.25 and 0.30 g/L, respectively [12, 47]. However, some

Zygomycetes may have higher capacity to produce GLA

than Mucor species. C. echinulata CCRC 31840 produced

significant amounts of GLA (up to 1.35 g/L) after optimi-

zation of the growth conditions and inoculation [10].

Another stain, C. echinulata ATHUM 4411, produced

0.72 g GLA per liter culture medium when cultivated on

glucose [14], and growth on potato starch was accompa-

nied by the GLA yield of 0.54 g/L [33]. However, a higher

yield of GLA (1.12 g/L) was obtained when the strain was

grown on xylose [12]. Thamnidium elegans CCF-1465

produced significantly high GLA yield (1.01 g/L) when

cultivated on glucose [51]. The highest yield of GLA

Table 1 Effect of nitrogen

sources on fatty acid

composition of M. circinelloides

CBS 108.16 grown at 30 �C for

4 days

All values are mean of three

biological replicates ± standard

deviations

N-source Fatty acid composition (%)

14:0 16:0 16:1 18:0 18:1 18:2 18:3

(GLA)

Ammonium

tartrate

1.8 ± 0.1 18.3 ± 0.7 1.5 ± 0.3 3.7 ± 0.1 44.7 ± 0.1 10.3 ± 0.1 19.6 ± 0.4

Glycine 2.0 ± 0.2 17.5 ± 0.4 1.7 ± 0.1 5.4 ± 0.4 42.4 ± 3.1 10.2 ± 0.4 20.5 ± 1.3

Alanine 2.0 ± 0.2 15.7 ± 0.9 1.7 ± 0.3 3.3 ± 0.5 47.6 ± 1.3 10.3 ± 0.5 19.1 ± 0.1

Valine 1.7 ± 0.4 11.4 ± 1.2 2.1 ± 0.2 2.0 ± 0.1 44.8 ± 3.7 14.3 ± 1.9 23.5 ± 1.1

Leucine 2.0 ± 0.1 16.4 ± 0.2 1.5 ± 0.1 3.2 ± 0.1 55.3 ± 0.9 9.5 ± 0.1 11.8 ± 0.5

Isoleucine 2.2 ± 0.1 13.7 ± 0.3 1.8 ± 0.2 2.4 ± 0.3 48.3 ± 2.0 11.9 ± 0.2 19.5 ± 1.1

Proline 3.0 ± 0.5 19.6 ± 0.1 1.5 ± 0.1 9.9 ± 2.0 35.2 ± 2.2 9.8 ± 0.4 20.4 ± 1.1

Phenylalanine 1.5 ± 0.2 13.5 ± 0.5 1.5 ± 0.1 3.1 ± 0.5 51.9 ± 0.8 11.4 ± 0.4 17.1 ± 0.3

Tyrosine 1.4 ± 0.1 15.3 ± 0.4 1.4 ± 0.2 3.7 ± 0.1 49.5 ± 1.0 8.8 ± 0.4 19.8 ± 0.1

Tryptophan 1.8 ± 0.1 19.6 ± 0.5 0.7 ± 0.1 21.1 ± 0.8 33.8 ± 0.3 10.8 ± 0.8 12.0 ± 0.3

Serine 1.7 ± 0.1 15.7 ± 0.1 1.6 ± 0.2 3.1 ± 0.1 47.1 ± 0.6 9.9 ± 0.1 20.9 ± 0.3

Threonine 2.0 ± 0.1 13.8 ± 0.2 1.6 ± 0.1 2.4 ± 0.2 49.5 ± 0.8 9.6 ± 0.1 21.1 ± 0.5

Cysteine 2.1 ± 0.1 12.4 ± 0.2 2.3 ± 0.1 1.7 ± 0.2 49.3 ± 0.8 9.6 ± 0.4 22.4 ± 0.3

Methionine 1.8 ± 0.1 17.6 ± 0.2 1.9 ± 0.3 2.7 ± 0.1 52.2 ± 0.4 10.7 ± 0.3 13.1 ± 0.1

Asparagine 1.7 ± 0.1 15.8 ± 0.2 1.7 ± 0.3 3.5 ± 0.1 47.5 ± 1.1 9.8 ± 0.4 19.9 ± 0.8

Glutamine 1.8 ± 0.2 19.1 ± 1.1 1.8 ± 0.5 5.0 ± 0.3 45.9 ± 0.7 9.5 ± 0.1 16.7 ± 0.3

Aspartic acid 1.6 ± 0.1 14.7 ± 1.0 1.7 ± 0.3 3.2 ± 0.4 47.0 ± 1.1 10.5 ± 0.4 21.2 ± 0.2

Glutamic acid 1.7 ± 0.1 16.4 ± 1.2 1.7 ± 0.4 3.7 ± 0.3 46.3 ± 1.3 10.4 ± 0.3 19.5 ± 0.2

Lysine 1.5 ± 0.1 23.1 ± 0.1 1.0 ± 0.1 6.5 ± 0.1 41.3 ± 1.3 9.9 ± 0.2 16.8 ± 0.5

Arginine 1.7 ± 0.1 16.4 ± 0.6 1.9 ± 0.3 4.3 ± 0.1 47.6 ± 0.5 10.1 ± 0.2 18.0 ± 0.1

Histidine 1.6 ± 0.1 20.4 ± 1.0 1.7 ± 0.3 4.6 ± 0.4 46.3 ± 1.5 8.6 ± 0.3 16.6 ± 0.4

X. Tang et al.: Effects of 20 Standard Amino Acids on the Growth

123

reported in the literature has been obtained by the mutant

of the fungus Mort. ramanniana MM15-1 cultivated in a

specific type of bioreactor and corresponded to the value of

5.54 g/L [18].

Taken together, apart from amino acids, other nutrition

such as carbon source and fermentation conditions may

greatly affect GLA production. Although GLA production

in M. circinelloides grown on tyrosine is the highest in

Mucor, there is still much room to further increase GLA

production using combined strategy of carbon source

optimization, nitrogen source optimization, and fermenta-

tion technology. The current advancement of GLA pro-

duction in oleaginous microorganisms of Zygomycetes is

summarized in Table 2.

It is clear that tyrosine was the most favorable amino

acid nitrogen source for GLA production in M. circi-

nelloides. Compared to ammonium tartrate, tyrosine not

only stimulated cell growth but also promoted lipid

accumulation, although the GLA content of the total

fatty acids production in the fungus grown on tyrosine

was not significantly different from that of ammonium

tartrate. In order to know whether it is a general rule for

M. circinelloides, the biomass, lipid, and GLA accu-

mulation were measured in the other M. circinelloides

strain CBS 277.49 when grown on tyrosine and

ammonium tartrate. The results showed that tyrosine

also increased the cell growth, lipid accumulation of the

strain CBS 277.49 by 24 and 23 %, respectively, and

Fig. 4 a Effect of nitrogen

sources on the GLA content of

DCW in M. circinelloides CBS

108.16 grown at 30 �C for

4 days. Ammonium tartrate was

used as a control nitrogen

source. Values are mean of

three biological replicates.

Error bars represent the average

standard deviations of these

replicates. Values which do not

share common superscripts were

significantly different to each

other (P \ 0.05) 61 9 43 mm

(600 9 600 DPI). b Effect of

nitrogen sources on the yield of

GLA in M. circinelloides CBS

108.16 grown at 30 �C for

4 days. Ammonium tartrate was

used as a control nitrogen

source. Values are mean of

three biological replicates.

Error bars represent the average

standard deviations of these

replicates. Values which do not

share common superscripts were

significantly different to each

other (P \ 0.05) 61 9 43 mm

(600 9 600 DPI)

X. Tang et al.: Effects of 20 Standard Amino Acids on the Growth

123

then resulted in an increase of GLA yield by 57 %

compared to ammonium tartrate (Table S2), which were

slightly lower than that of the strain CBS 108.16.

Therefore, the effect of tyrosine on cell growth and

lipid accumulation of the stain CBS 277.49 was similar

with the stain CBS 108.16. Thus, it is likely to be a

general rule that tyrosine could stimulate cell growth

and promote lipid accumulation.

Effect of Tyrosine on the Activity of Lipogenic

Enzymes in M. circinelloides

To find out the potential mechanism underlying the

increased lipid accumulation of M. circinelloides when

grown on tyrosine, the activity of lipogenic enzymes of the

fungus during lipid accumulation phase was measured

(Fig. 5). The ammonium tartrate was used as the control,

Table 2 Comparative results of GLA production among different studies by Zygomycetes

Microorganisms Nitrogen sources Carbon substrates GLA yield (g/L) Reference

M. circinelloides CBS 172-27 NH4? Glucose 0.22 [3]

M. circinelloides CBS 203.28 NH4?, yeast extract Acetic acid 0.51 [20]

M. rouxii CBS 416.77 NH4?, yeast extract Glucose 0.32 [17]

M. mucedo CCF-1384 Cornsteep Glucose, sunflower oil 0.38 [8]

M. sp. LGAM 366 Cheese whey, NH4? Cheese whey, lactose 0.18 [47]

Mort. ramanniana CBS 478.63 NH4?, yeast extract Glucose 0.44 [16]

Mort. isabellina ATHUM 2935 NH4?, yeast extract Glucose 0.80 [34]

Mort. isabellina ATHUM 2935 NH4?, yeast extract Xylose 0.25 [12]

Mort. isabellina ATHUM 2935 Cheese whey, NH4? Cheese whey, lactose 0.30 [47]

Mort. ramanniana MM15-1 (with a special bioreactor) Urea Glucose 5.54 [18]

C. echinulata CCRC 31840 NH4NO3, yeast extract Starch 1.35 [10]

C. echinulata CCF-103 Cornsteep Glucose, 0.37 [8]

sunflower oil

C. sp. LGAM (9)2 NH4?, yeast extract Glucose 0.26 [21]

C. echinulata ATHUM 4411 NH4? Glucose 0.72 [14]

C. echinulata ATHUM 4411 NH4?, yeast extract Starch 0.54 [33]

C. echinulata ATHUM 4411 NH4?, yeast extract Xylose 1.12 [12]

T. elegans CCF-1465 NH4?, yeast extract, Glucose 1.01 [51]

M. circinelloides CBS 108.16 Tyrosine Glucose 0.81 This study

Fig. 5 Effect of tyrosine on

lipogenic enzymes of M.

circinelloides CBS 108.16

grown at 30 �C for 4 days.

Values are mean of three

biological replicates. Error bars

represent the average standard

deviations of these replicates.

Values which do not share

common superscripts were

significantly different to each

other in each enzyme

(P \ 0.05) 61 9 42 mm

(600 9 600 DPI)

X. Tang et al.: Effects of 20 Standard Amino Acids on the Growth

123

and isoleucine which gave the lowest lipid accumulation

among the 20 amino acids was used as the negative control.

Tyrosine increased the activity of ACL, which provides

acetyl-CoA for fatty acid biosynthesis, slightly but signif-

icantly. Among the four NADP-generating enzymes (ME,

G6PD, PGD, NADP-ICD), ME activity was not signifi-

cantly affected, both activities of G6PD and PGD were

significantly increased, while NADP-ICD activity was

decreased by tyrosine. In contrast, isoleucine, which

inhibited lipid accumulation, decreased ACL, G6PD, and

PGD activity while increased NADP-ICD activity. These

results suggested that ACL, NADP-ICD, and especially

G6PD and PGD were associated with increased lipid

accumulation in M. circinelloides when grown on tyrosine.

In the lipid accumulation phase, acetyl-CoA and

NADPH are essential for fatty acids biosynthesis in ole-

aginous microorganisms [41]. Acetyl-CoA, as a precursor

for lipid accumulation, is produced in oleaginous micro-

organisms via ACL [7]. In Aspergillus oryzae, the fatty

acids production was increased when the expression of the

ACL gene was enhanced [46]. In this study, the level of

ACL activity is correlated with the amount of lipid being

produced in M. circinelloides grown on different amino

acids, tyrosine which increased lipid accumulation,

induced ACL activity, while isoleucine decreased lipid

accumulation, inhibited ACL activity, compared to

ammonium tartrate. This indicates that more acetyl-CoA,

substrate for fatty acids synthesis, was provided when the

fungus grown on tyrosine, which may lead to the increased

lipid production. ME was considered to be important in the

provision of NADPH for lipid accumulation and disap-

pearance of ME activity thus appeared to cause the ces-

sation of lipid accumulation in M. circinelloides and Mort.

alpina [48]. In Aspergillus nidulans, ME was considered as

a major source of NADPH for the lipid synthesis [49].

However, the activity of ME in M. circinelloides was not

affected by either tyrosine or isoleucine in the present

study, indicating that lipid accumulation of this fungus

grown on amino acids is not regulated by ME activity.

Another recent study also pointed in the same direction

which indicated ME is not the only bottleneck in lipid

accumulation in M. circinelloides [42].

Although it has been shown that NADPH generated

from the pentose phosphate pathway plays a key role in

fatty acid biosynthesis in plants and microalga [38, 50], it is

not clear whether the pentose phosphate pathway may also

provide NADPH for fatty acid biosynthesis in fungi.

Nevertheless, a previous report showed that activated

pentose phosphate pathway by glutamate enhanced the

biomass and ARA biosynthesis in Mort. alpina [25]. In our

study, the activity of G6PD and PGD was both increased

significantly by tyrosine and decreased by isoleucine

compared to ammonium tartrate. This result suggests that

tyrosine can specifically induce the pentose phosphate

pathway to provide more NADPH for fatty acid biosyn-

thesis in this fungus. For another NADPH-generating

enzyme NADP-ICD, both cytosolic and mitochondrial

forms exist, and the cytosolic form of the enzyme has been

shown to contribute to lipid accumulation in oleaginous

yeasts [43]. Lipid accumulation was initiated by the

attenuation of NAD-ICD and NADP-ICD activity in C.

echinulata and Mort. isabellina [36]. The genomic infor-

mation showed that NADP-ICD in M. circinelloides is

located in the mitochondria (http://www.ncbi.nlm.nih.gov/

protein/?term=isocitrate?dehydrogenase?NADP?Mucor?

circinelloides?1006PhL); therefore, it is very unlikely that

this enzyme may contribute NADPH for lipid biosynthesis,

and indeed, in our study, the activity of this enzyme was

inhibited by tyrosine. The decreased activity of this mito-

chondrial NADP-ICD may down-regulate the tricarboxylic

acid cycle, leading to the carbon flux to acetyl-CoA syn-

thesis, and therefore increased the fatty acids biosynthesis.

In contrast, isoleucine which decreased lipid accumulation

enhanced the activity of NADP-ICD, which up-regulate the

tricarboxylic acid cycle, lead to the reduced carbon flux to

acetyl-CoA, and thus decreased the fatty acid biosynthesis.

Conclusions

The present study showed that different amino acids can

affect the cell growth, fatty acids production, and yield of

GLA in M. circinelloides. Tyrosine appeared to be the most

favorable amino acid nitrogen source for the cell growth

and total fatty acids production, which lead to the highest

yield of GLA among the 20 amino acids. The potential

molecular mechanism of increased lipid accumulation of

the fungus grown on tyrosine could be as follows: (1) the

pentose phosphate pathway was induced by tyrosine, and

the increased activity of G6PD and PGD may provide more

reducing power NADPH for fatty acid biosynthesis. (2) the

increased activity of ACL, which provides more acetyl-

CoA and decreased activity of mitochondrial NADP-ICD,

which down-regulate the TCA activity and provide more

substrate for ACL, these two synergic effects may increase

the production of acetyl-CoA.

Acknowledgments The work was supported by the National Nat-

ural Science Foundation of China (31271812, 21276108), the

National High Technology Research and Development Program of

China (863 Program 2012AA022105C), Strategic Merieux Research

Grant, the Program for New Century Excellent Talents (NCET-13-

0831), the National Science Fund for Distinguished Young Scholars

(31125021), and the Fundamental Research Funds for the Central

Universities (No. JUSRP51320B). We thank Professor Colin Ratledge

for his critical comments to our manuscript.

X. Tang et al.: Effects of 20 Standard Amino Acids on the Growth

123

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