Oxidative stress response ofBlakeslea trispora induced by H2

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J Ind Microbiol Biotechnol

DOI 10.1007/s10295-013-1392-1

Oxidative stress response of Blakeslea trispora induced by H2O2 during β?carotene biosynthesis

Hong?Bo Wang · Jun Luo · Xiao?Yan Huang · Ming?Bo Lu · Long?Jiang Yu

received: 20 august 2013 / accepted: 5 December 2013

© Society for Industrial Microbiology and Biotechnology 2013

Abstract the cellular response of Blakeslea trispora to oxidative stress induced by H2O2 in shake flask culture was investigated in this study. a mild oxidative stress was created by adding 40 μm of H2O2 into the medium after 3 days of the fermentation. the production of β-carotene increased nearly 38 % after a 6-day culture. under the oxi-dative stress induced by H2O2, the expressions of hmgr, ipi, carG, carRA, and carB involving the β-carotene biosyn-thetic pathway all increased in 3 h. the aerobic metabolism of glucose remarkably accelerated within 24 h. In addition, the specific activities of superoxide dismutase and catalase were significantly increased. these changes of B. trispora were responses for reducing cell injury, and the reasons for increasing β-carotene production caused by H2O2.Keywords Blakeslea trispora · Oxidative stress ·

β-carotene · H2O2 · Synthetic medium · Shake flask cultureIntroduction

Studies on the function of β-carotene and lycopene have drawn significant attention because of their biological functions in human wellness, which lead to their com-mercialization in functional food, medicine, and cosmetic

H.-B. Wang · J. luo · X.-Y. Huang · M.-B. lu · l.-J. Yu (*) Department of Biotechnology, College of life Science and technology, Institute of resource Biology

and Biotechnology, Huazhong university of Science and technology, Wuhan 430074, Chinae-mail: yulongjiang@http://wendang.chazidian.com

H.-B. Wang · J. luo · X.-Y. Huang · M.-B. lu · l.-J. Yu

Key laboratory of Molecular Biophysics, Ministry of education, Wuhan 430074, China

industries. Compared with other microorganisms, Blakes-lea trispora exhibits higher potential for β-carotene produc-tion [10]. at present, B. trispora is the main microbiologi-cal subject for industrial production of β-carotene.

the improvement of carotenoid production using B. trispora induced by oxidative stress has become a research hotspot. Mantzouridou et al. [11] reported that oxygen transfer rate, H2O2 accumulation, and β-carotene synthesis reveal a positive relationship under oxidative stress in B. trispora. nanou et al. [14] indicated that enhanced aeration changes the oxidative stress of B. trispora and increases β-carotene production in a bubble column reactor. Xu et al. [25] studied the production of β-carotene and lycopene by combining B. trispora with oxygen vectors (n-hexane or n-dodecane) to increase dissolved oxygen concentration. the oxidative stress responses of B. trispora induced by butylated hydroxytoluene (BHt), H2O2, liquid paraffin, and iron ions during β-carotene production in shake flask culture were studied [7, 8, 15, 17]. at present, studies have shown that oxidative stress can increase the production of carotenes in B. trispora [16, 19]. However, the effects of oxidative stress on carotenoid biosynthesis are mainly con-fined to the changes of mycelium morphology and scaveng-ing enzyme activity (SOD and Cat) in B. trispora [18]. the synthesis of carotenes is closely related with oxidative stress; however, the mechanisms have been rarely reported.Oxidative stress has been considered as the disturbance in pro-oxidant/anit-oxidant balance, resulting in potential cell damage. aerobic organisms consume molecular oxygen for aerobic metabolism and energy supply. at the same time, a large amount of molecular oxygen can cause the toxic pro-duction of reactive oxygen species (rOS), such as superox-ide radicals, hydroxyl radicals, and H2O2, highly damaging to cellular constituents, including enzymes, protein, lipids, and Dna injury [2]. aerobic organisms have both enzymatic

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and non-enzymatic defense systems to protect the cells from oxidative damage. these systems act as radical scavengers, being oxidized by rOS, removing oxidants from the cells [6, 17]. the enzymatic defense system includes catalase (Cat) and superoxide dismutase (SOD) [5]. the non-enzymatic defense system includes antioxidants, such as carotenes.

Compared with other compounds (iron ions, BHt, and liquid paraffin) and oxygen carrier (n-hexane or n-dode-cane) induced oxidative stress, Hadvantages, including low cost, non-environmental pollu-2O2 possesses significant tion, and suitability for mass production. therefore, this study investigates the response of B. trispora induced by H2O2 during β-carotene biosynthesis.Materials and methodsChemicals and reagents

all chemicals, reagents, and solvents used in this study were of analytical or high-performance liquid chromatog-raphy (HPlC) grade.Microorganisms

the microorganisms used in this study consisted of B. tris-pora atCC 14060 mating type (+) and B. trispora atCC 14059 mating type (?). Both strains were purchased from China Center for type Culture Collection.Culture conditions

the strains were grown on PDa at 28 °C for 3 days, and the spores were used for the inoculation of the culture medium. the spores suspension contained 1.0 × 106 and 2.0 × 106 spores/ml of the strains 14,060 (+) and 14,059 (?), respectively.Fermentation conditions

the spores suspension was inoculated into 250 ml erlen-meyer flasks containing 100 ml sterile medium for β-carotene production. Fermentation was conducted at 28 °C on a rotary shaker (200 rpm) for 7 days. the com-position of the fermentation medium was as follows (g/l):

Span 20 20, glucose 100, yeast extract 12, naKH2HPO4 1.5, 2PO4 3, and MgSO4 0.5. the flasks were inoculated

with 2 %, v/v of the spores suspension.

Carotenes extraction

the wet biomass underwent vacuum freeze-drying for 48 h, weighed the cell dry weight and ruptured with liquid 1 3

J Ind Microbiol Biotechnol

nitrogen by manual grinding until complete cell disrup-tion [12]. the samples were then subjected thrice to stir-ring-assisted extraction with petroleum ether at room temperature.

Analytical methodsanalysis of carotenes

the HPlC methods reported by Bononi [3] have been modified was used to analyze the carotenoid content. β-Carotene was separated using an agilent HPlC sys-tem equipped with an ascentis rP-column (rP-amide, 15 cm × 2.1 mm × 5 μm). the operating conditions were as follows: elution solvent, acetonitrile; flow rate, 0.4 ml/min; injection volume, 10 μl. β-Carotene absorption was measured at 450 nm.

analysis of glucose concentration and glucose metabolism rate

the 3, 5-dinitrosalicylic acid was used for the glucose analysis to test the reducing sugar [13]. the rate of glucose metabolism was calculated using the following equation: change of glucose concentration (g l?1)/time (hours).analysis of SOD and Cat enzyme activity

Wet cells (1 g) were disrupted by liquid nitrogen and the enzymes were extracted with 10 ml of prechilled physiolog-ical saline (0.9 % naCl), then centrifuged for 15 min at 4 °C and the supernatant was collected for the analysis of SOD and Cat enzyme activity. analysis of the enzyme activities was according to the manual of the reagent kits (purchased from nanjing Jiancheng Bioengineering Institute) [7].Calculation of specific oxygen uptake rates

Dissolved oxygen concentration was determined with a

microprocessor oximeter. Specific oxygen uptake rate

(SOur) is the ratio of oxygen consumption rate (Our) and dry biomass. Our was determined using the method reported by Casas lópez et al. [4].

real-time PCrthe real-time polymerase chain reaction (PCr) primers are presented in table 1. the primers of hmgr, ipi, carG, carRA, and carB were used according to the report by Sun et al. [24]. the real-time PCr cycling conditions were as follows: 95 °C

for 3 min followed by 40 cycles at 95 °C for 15 s, 58 °C for 20 s, and 72 °C for 20 s. Measurements were performed in

J Ind Microbiol Biotechnol Table 1 real-time PCr primers used in this study

genes18sIPIcarGhmgRcarRAcarB

Forward and reverse primers (5′ → 3′)18S-F18S-ripi-Fipi-rcarg-Fcarg-rhmgr-Fhmgr-rcarra-Fcarra-rcarB-FcarB-r

tgCtggCgaCggtCtaCtCgCtgCCttCCttggatgtg

tCtCaCCCCttaaataCagCagatgCtCggtgCCaaataatgaataCgaattgttttggCgtgaCaCCttCagttCCCgattgaCtagCttCttaaaCgatggattgaaCaagagggtagaCtagaCgaCCggCaagagCCtaaagCCgtttCaCtCaCagCaaCaagtaggaCagtaCCaCCaagCgagaCCtagtaCCaaggattCCaCaaagaaCgataggaaCaCCagtaCCtg

amplicon length (bp)14916112911312992

triplicate. all results were normalized to the 18S gene of B. trispora (gene Bank: aF157124.1) and are expressed rela-tive to expression of the corresponding control group (non-H2O2-added; value = 1), using the comparative method of livak and Schmittgen [9]. Values are the mean ± standard error of three independent experiments. Values >1 represent over expression compared with the control group.Statistical analysis

the means of three independent experiments are presented in Figs. 1, 2, 3 and 4. each data point represents the mean and the standard error. Data was analyzed using student’s t test. a p value between 0.01 and 0.05 was considered as significant (*), and p value <0.01 was considered as very significant (**).

Fig. 1 effect of added amount of H2O2 on biomass and β-carotene production

Results and discussion

effect of added amount of H2O2 on biomass and β-carotene production

the effect of H2O2 on β-carotene synthesis and grown biomass were examined after adding different concentra-tions in all cases (0, 5, 10, 20, 40, 60, 80, and 100 μM) to the 3-day old cultures of B. trispora (Fig. 1). the control (0 μM added) was the culture to which H2O2 was not added during cultivation. after 6 days of culturing, dry biomass was slightly decreased from 17.11 to 16.23 g/l when the H2O2 concentration ranged from 0 to 60 μM, but remarka-bly decreased from 16.23 to 14.79 g/l when the H2O2 con-centration increased from 60 to 100 μM. this result is due

to the toxicity of high H2O2 concentration, resulting to cell damage [18]. this phenomenon indicates that the addition of H2O2 at low concentration (below 60 μM) to the 3-day old cultures of B. trispora leads to mild oxidative stress.

the production of β-carotene increased significantly from 140.1 to 193.5 mg/l with the increase in the added amount of H2O2 from 0 to 40 μM. therefore, carotenes syn-thesis may be increased by oxidative stress. Jeong et al. [8] reported that the addition of H2O2 (10 μM) to 1.5-day old cultures of B. trispora resulted to a 46 % higher β-carotene than that without addition. However, reports on the reasons of increasing the production of β-carotene by mild oxida-tive stress are rare. the production of β-carotene decreased dramatically from 193.5 to 59.9 mg/l when the H2O2 con-centration increased from 40 to 100 μM. Carotenes in B. trispora served as major antioxidants that protect against cellular injury by quenching the active oxygen species under wild oxidative stress (H2O2 concentration above 40 μM). this phenomenon is consistent with the research that the external addition of H2O2, in combination with high aera-tion rates, led to wild oxidative stress and caused significant decrease in β-carotene concentration [14].

Overall, low added concentrations of H2O2 (below 40 μM) increased the production of β-carotene, whereas

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Fig. 2 Fold change of gene

transcription after H2O2

addition for 3 and 6 h (a).

β-Carotene biosynthetic path-

way (b

)J Ind Microbiol Biotechnol

Fig. 3 effect of H2O2 on glu-

cose metabolism (a), SOur (b),

cell grown (c), and β-carotene

production (d) during carotene

production by B. trispora in

shake flask culture. the cycle

and square represent H2O2-

added and non-H2O2

-added

Fig. 4 effect of H2O2 on the

specific activity of Cat (a) and

SOD (b) during β-carotene pro-

duction by B. trispora in shake

flask culture. the circle and

square represent H2O2-added

and non-H2O2

-added

high added concentrations of H2O2 (above 40 μM) con-

sumed β-carotene acting as antioxidants to prevent oxi-

dative stress and decreased the production of β-carotene.

thus, 40 μM is an optimal added concentration for enhanc-

ing the production of β-carotene.effect of H2O2 on the expressions of five genes involving the β-carotene biosynthetic pathwaythe growth of fungal strains in presence of H2O2 shows

clear signs of increased oxidative stress [2]. to investigate

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the molecular mechanisms of the 3-day-old B. trispora under oxidative stress induced by Hhmgr, ipi, carG2O, 2, the gene expres-sion of five enzymes (carRA, and carB) involving the β-carotene synthesis was analyzed by real-time PCr. the result of transcription level is shown in Fig. 2a, and the β-carotene synthesis pathway is shown in Fig. 2b.

HMg-Coa (3-hydroxy-3-methylglutaryl-coenzyme a) reductase is a rate-limiting enzyme in the mevalonate path-way [21]. Compared with the control group (non-Hadded), the transcription of hmgR increased by 2.51-fold 3 2O2-in hours (P < 0.01) and 2.23-fold in 6 h (P < 0.01) after the addition of H2O2. therefore, the expression of hmgR in the cultures remained stable after oxidative stress was induced by H2O2. IPP (isopentenyl pyrophosphate) isomerase cata-lyzes the formation of more reactive DMaPP (dimethylal-lyl pyrophosphate) from the relatively unreactive IPP [1]. the transcription level of ipi improved by 1.43-fold in 3 h (P < 0.01) and 1.19-fold in 6 h (P > 0.05) after Hipi in the cultures slightly 2O2 treatment. the expression of increased after Hipi2O was not sensitive to the oxidative stress 2 treatment. the results show that the transcription of caused by Hsynthase catalyzes the formation of FPP (farnesyl pyroph-2O2. ggPP (geranylgeranyl pyrophosphate) osphate). the expression of carG increased by 2.29-fold in 3 h (P < 0.01) and 2.03-fold in 6 h (P < 0.01) after the addition of H2O2. the expression of carG in the cultures remained stable after H2O2 treatment. CarRA and CarB genes have been previously identified to be involved in β-carotene biosynthesis in B. trispora [20]. CarRA encodes a double-functional enzyme with phytoene synthase and lycopene cyclase activity. CarB encodes the phytoene dehydrogenase. the expression of carRA and carB both increased by Ha 6.45-fold improvement at 3 h (2O2 treatment. the addition of HP < 0.01) and 5.57-fold 2O2 led to enhancement at 6 h (P < 0.01) in the transcripts of carRA, suggesting that carRA transcription was sensitive to oxi-dative stress by Hβ-carotene with increasing expression of 2O2. H2O2 may increase the production of carRA. the expression of CarB increased by 1.65-fold at 3 h (P < 0.01) and 1.20-fold at 6 h (P < 0.01) after H2O2 treatment.

In summary, five genes in the β-carotene biosynthetic pathway (hmgr, ipi, carG, carRA, and carB) have increased expression after 3 h under Hwas the most sensitive to oxidative stress by H2O2 treatment, in which carRA these five genes. this result demonstrated that the meta-2O2 among bolic flow of β-carotene was increased by HOxidative stress induced by HO2O2 treatment. production of β-carotene through transcriptional induction 22 appears to increase the of their biosynthetic genes. the increased transcription of five genes is a response for oxidative stress by H2O2 treat-ment in B. trispora.

effect of Hβ-carotene production

2O2 on glucose metabolism, SOur, cell growth, and the effects on cell growth (a), β-carotene production (b), glucose metabolism (c), and SOur (d) are shown in Fig. 3, wherein the phases of cell growth and β-carotene biosyn-thesis were not synchronized. according to the report by nanou, the process of β-carotene production was divided into three phases: the growth state (0–3 days), production state (3–6 days), and the consumption state (6–7 days), respectively [18].

In the growth state, dry biomass reached up to 15 g/l, and small amount of β-carotene were synthesized. In addi-tion, the metabolic rate of glucose was the highest in the three states, reaching 615 mg/l h, because the rapid metab-olism of glucose supplied enough energy for the growth of B. trispora in the logarithmic growth phase. SOur sig-nificantly decreased from 297.3 mg O2/h g dry biomass to 81.8 mg O2/h g dry biomass for 1-day and 3-day old myceliums, respectively. Mycelium in the early growth state needs large energy consumption for growth, which is closely related to higher glucose metabolism and oxygen consumption.

In the early production state (3–4 days), biomass and β-carotene were constant, the metabolic rates of glucose were 163 mg/l h and 404 mg/l h for non-H2Oand H2-added 2O2-added, respectively. thus, the glucose metabolic rate increased by 148 % under oxidative stress induced by H2O2. Furthermore, SOur was significantly increased, indicating that the aerobic metabolism of glucose was enhanced, but the biomass and β-carotene remained con-stant. these phenomena showed that increasing the glu-cose aerobic metabolism is a cellular response to oxidative stress for supplying enough reduced form of nicotinamide-adenine dinucleotide (naDH) or reduced nicotinamide adenine dinucleotide phosphate (naDPH), which may be conducive for alleviating cellular oxidative stress. naDPH was pivotal to the cellular anti-oxidative defense strategies in most organisms [22]. naDPH acts as a key component in cellular antioxidation systems [26]. In Pseudomonas fluorescence, Singh demonstrated a metabolic network pro-moting naDPH production and limiting naDH synthesis as a consequence of an oxidative stress [23]. the remark-able improvement in the glucose metabolic rate may due to the pentose phosphate pathway shunting the glucose metabolism to supply enough naDPH. therefore, the aer-obic metabolism of glucose was significantly increased for supplying enough naDPH to protect B. trispora from oxi-dative stress. Meanwhile, the strengthening of the aerobic metabolism of glucose may increase intracellular adenosine triphosphate (atP) and naDPH content. atP and naDPH are two important cofactors for β-carotene production [27],

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