Effect of Temperature on Β-Phenylethanol Anabolism in .... Cheng Lei.pdf · spicery labeled...

Romanian Biotechnological Letters Vol. 21, No. 3, 2016 Copyright © 2016 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

11550 Romanian Biotechnological Letters, Vol. 21, No. 3, 2016

Effect of Temperature on Β-Phenylethanol Anabolism

in Saccharomyces Cerevisiae and its Recombinant Strains

Received for publication, November 20, 2014 Accepted, November 30, 2014

CHENG LEI 1, 2, YIN SHENG 1, 2, LIANG JINGRU 2, LIU LI 2, SUN BAOGUO 1, 2, WANG CHENGTAO 1, 2 *, WEN YANJUN 3

1 Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology & Business University (BTBU) and China Agricultural University (CAU), Beijing 100048, China;

2 Beijing Engineering and Technology Research Center of Food Additives, BTBU, Beijing 100048, China;

3 Henan Zhongda Biological Engineering Co., Ltd.，Zhengzhou 451162, China * Address correspondence to: No.33, Fucheng Road, Haidian District, Beijing 100048,

China, WANG CHENGTAO (Wang C.T.) , Tel. (fax): +86-10-68984547; Email: [email protected]

Abstract

β-Phenylethanol is an important flavor component of a variety of fermented foods with rose scent, and it is commonly used to make many types of flavoring essence. In this study, effects of fermentation temperature on β-phenylethanol anabolism and related enzyme activities were investigated for Saccharomyces cerevisiae S288C and its engineered strains ARO-8, ARO-9, ARO-10, ARO 8-10, and ARO 9-10. Compared with the wild type strain S288C, the engineered strains showed overexpression of aminotransferase gene ARO8 and decarboxylase gene ARO10, and both ARO-8 and ARO 8-10 strains had significantly elevated activities of aminotransferase and decarboxylase (P≤0.01); all the engineered strains with single or coupled overexpression displayed improved production of β-phenylethanol than the strain S288C; ARO 8-10 had significantly increased β-phenylethanol production, L-phenylalanine conversion, and glucose utilization at 0.98 g/L, 38.14%, and 61.60%, respectively, with 30 ℃ fermentation, which were 24.6%, 5.1%, and 68.3% higher, respectively, than the wild type. Our results indicate that production of β-phenylethanol and the metabolic flux distribution of Ehrlich pathway were significantly enhanced by coexpression of the gene ARO8 and ARO10 in S. cerevisiae.

Keywords: Saccharomyces cerevisiae, gene overexpression, engineered strains, fermentation

temperature, β-phenylethanol, key metabolic enzymes, aminotransferase, decarboxylase

1. Introduction β-Phenethylalcohol, also known as 2-phenylethanol (2-PE), is a type of alcohol with

long-lasting, elegant rose aroma ( SUN [1]). It is the second most used flavor material after vanillic aldehyde, widely used in foods, cosmetics, tobacco, and daily chemical products. 2-PE can also be used in pharmaceutical industry because of its bactericidal and sedative refreshing effects.

Natural β-phenylethanol is mainly found in the essential oil extracted from roses, which does not meet the market demand due to resource and cost constraints and high prices. Currently, tens of thousands of tons of β-phenylethanol is demanded globally each year. It is produced mainly by chemical synthesis, which generates significant pollution and toxic byproducts (WANG & al [2]). As required by U.S. Federal Regulations and EU legislation, all

Effect of Temperature on Β-Phenylethanol Anabolism in Saccharomyces Cerevisiae and its Recombinant Strains

Romanian Biotechnological Letters, Vol. 21, No. 3, 2015 11551

spicery labeled "natural" must be produced naturally, i.e. through physical extraction from plants or animals (CHEN & al [3]; HUANG & al [4]); fragrant substances obtained through microbiological or biological enzymatic conversion are called natural-identical spices, which may be considered as natural spices. Therefore, natural spice production through microbiological conversion has become the focus of attention of recent natural spice research (CHEN & al [3]; HUANG & al [4]; ESHKOL & al [5]; GAO & al [6]).

2-PE is an important flavor component of many fermented foods, such as wine, cider, sake, soy sauce, cheese, and breads (WANG & al [2]; HUANG & al [4]). For example, Japanese sake contains 20-80 mg/L 2-PE, and Chinese rice wine has about 100 mg/L 2-PE, both through microbiological fermentation. It has been shown that many microorganisms have the ability to synthesize β-phenylethanol (ESHKOL & al [5]; ETSCHMANN & al [7]), such as Saccharomyces cererisiae, Rhodotorula glutinis, Kluyveromyces marxianus, K. lactis, Pichia fermentans, Zygosaccharomyces rouxii, Torulopsis utilis, and Hansenula anomala. As shown in Figure 1, S. cerevisiae’s glucose metabolism and L-Phe metabolism contain many metabolic pathways, such as Shikimate Pathway, Glycolysis (EMP), Pentose Phosphate Pathway (PPP), Tricarboxylic Acid cycle (TCA), and Ehrlich pathway, which affect each other and form the 2-PE anabolic network(ESHKOL & al [5]; ETSCHMANN & al [7]; HUA & al [8]; SICARD & al [9]). S. cerevisiae can produce 2-PE de novo from glucose through the Shikimate pathway, however, it involves many branching metabolic pathways, leading to very low 2-PE yield (ESHKOL & al [5]; HUA & al [8]). Etschmann et al. (ETSCHMANN & al [7]) found that L-phenylalanine (L-Phe) is an excellent precursor for 2-PE biosynthesis, and addition of L-Phe in fermentation medium can significantly increase 2-PE yield. S. cerevisiae metabolizes L-phe through Ehrlich pathway, a rapid way to generate 2-PE; it requires transamination, decarboxylation, and alcohol dehydrogenation, thus is regulated by phenylalanine transaminase (EC 2.6.1.58), pyruvate decarboxylase (EC 4.1.1.1), and alcohol dehydrogenase (EC 1.1.1.1) (HAZELWOOD & al [10]; HAZELWOOD & al [11]). Transamination requires α-ketoglutarate, an intermediate of TCA. Therefore, the metabolism for yeast growth is closely related to Ehrlich pathway, Shikimic acid pathway, EMP, PPP, and TCA, all of which form the β-phenylethanol anabolic network with a variety of interactions among different metabolic enzymes, such as Glucose-6-phosphate dehydrogenase (EC:1.1.1.49, G6PDH), Isocitrate dehydrogenase (EC 1.1.1.42, IDH) (SICARD & al [9]; HAZELWOOD & al [10]; LIANG & al [12]).

S. cerevisiae’s 2-PE synthesis is affected by many environmental factors, including temperature, pH, and dissolved oxygen (DO). In this study, the effects of temperature on β-phenylethanol anabolism and related enzymes’ activity were investigated using previously generated S. cerevisiae strains ARO-8, ARO-9, ARO-10, ARO 8-10, and ARO 9-10 with single or coupled over-expression of aminotransferase gene ARO8, aminotransferase gene ARO9, decarboxylase gene ARO10, in order to provide theoretical basis and data support for 2-PE metabolism regulation and industrial production.

2. Materials and Methods 2.1 Materials and equipment Methanol (HPLC grade), β-phenylethanol and antibiotics G418 were purchased from Fisher

Scientific; 2,4-dinitrobenzene hydrazine (DNPH), alpha-ketone-gamma (methylmercapto)-butyric acid (alpha-KMBA), methyl mercaptan (PLP), 5,5'-disulfide double (2-nitro benzoic acid) (DTNB) were purchased from Sigma- Aldrich Co. LLC(China); other reagents were of analytical

CHENG LEI, YIN SHENG, LIANG JINGRU, LIU LI, SUN BAOGUO, WANG CHENGTAO, WEN YANJUN


grade and purchased from Sinopharm Chemical Reagent Beijing Co. Ltd. S. cerevisiae wild type strain S288C, stains ARO-8, ARO-9, and ARO-10 with single

overexpression of aminotransferase gene ARO8, aminotransferase gene ARO9, and decarboxylase gene ARO10, respectively, and strains ARO 8-10 and ARO 9-10 with combinatorial overexpression of gene ARO8-ARO10 and gene ARO9-ARO10, respectively, were selected, bred, and preserved in our laboratory(LIANG & al [12]; YIN & al [13]; YIN & al [14]).

Figure 1. 2-PE biosynthetic pathways and related enzymes in S. cerevisiae using their substrates L-

phenylalanine and glucose

The YPD medium (w/v) was prepared with 2% peptone, 1% yeast extract, 2% glucose, 1.5% agar, and 115 ℃ sterilization for 20 min. The fermentation medium I (g/L) was prepared with 1.8 g/L non-amino acid nitrogen source YNB, 40 g/L glucose, 7 g/L L-phenylalanine and 0.5 g/L Na2HPO4 at pH 5.5 in 100 mL liquid volume (250 mL flask) with 121℃ sterilization for 30min. The 2,4-dinitrophenyl hydrazine (DNPH) solution was prepared with 22 mg 2,4-dinitrophenylhydrazine dissolved in 10 mL concentrated hydrochloric acid, diluted with distilled water to 100 mL, filtered, and saved for further experiments. The GPT matrix solution was prepared with 29.2 mg α-ketoglutarate and 1.78 g L-phenylalanine dissolved in pH7.4 phosphate buffer with a final volume of 100 mL (pH was adjusted to 7.4 using 1 mol/L NaOH solution).

The major instruments and apparatuses used in this study were mettler electronic balance (0.1mg), high performance liquid chromatograph (Shimadzu LC-20A), UV spectrophotometer (Shimadzu 2550), continuous wavelength microplate reader (USA MD Spectra Max i3), refrigerated centrifuge (Germany Sigma 3-16KL), precision digital pH meter ADR100012, laminar flow hood, and shaker (Taizhou Laboratory value Co., Ltd, China).



2.2. Methods 2.2.1. Biomass determination Five milliliters of culture after 48 h fermentation at 26-36 ℃ was centrifuged at 8,000

rpm for 10 min. The pellet was dried in oven at 105℃ until it reached constant weight as cell dry weight (Cdw). Two milliliters fermentation yeast suspension was diluted with distilled water until OD600 was 0.2~0.8 (OD600 = yeast suspension OD600 × dilution factor).

2.2.2. Detection of β-phenylethanol and L-phenylalanine (LIANG & al [12]) Engineered strains ARO-8, ARO-9, ARO-10, ARO 8-10 and ARO 9-10 and the wild

type strain S288C were cultured in fermentation medium I with 5% inoculation at 26-36℃ with 200 rpm shaking for 48 h. The culture was centrifuged at 8,000 rpm for 10 min, and the supernatant was filtered with 0.45 μm membrane. The contents of β-phenylethanol and L-phenylalanine were determined by using Shimadzu LC-20A liquid chromatography, RP-C18 column (4.6×150 mm, 5 μm), diode array detector; the injection volume was 10 μL, and the flow matching ratio of methanol to water was 50:50 (V:V); the column temperature was 30℃; the detection wavelength was 215 nm, and the flow rate was 1.0 mL/min. A standard curve was generated for the calculation of β-phenylethanol and L-phenylalanine levels. L-phenylalanine utilization rate = L-phenylalanine in the reaction / the initial amount of L-phenylalanine) × 100%.

2.2.3 Detection of glucose and alcohol Shimadzu LC-20A HPLC, Aminex HPX-87H column (300 mm × 7.8 mm, 9 μm),

differential refractive index detector, 45℃ column temperature, 0.5 mM H2SO4 as the mobile phase, flow rate of 0.5 mL/min, and injection volume of 20 μL. Glucose utilization rate = (amount of glucose in the reaction / the initial amount of glucose) × 100 (%).

2.2.4 Related metabolic enzyme activity detection Thirty milliliters 48 h fermentation broth was centrifuged at 8,000 rpm for 10 min. The

pellet was washed twice with pH 6.0 0.2 M phosphate buffer, and yeast cells were re-suspended in phosphate buffered solution, sonicated in ice bath (200-400 W) 45 times with 10 s intervals, and centrifuged at 8,000 rpm for 10 min. The supernatant crude enzyme solution was used for the following enzyme activity assays.

2.2.5 Determination of glucose-6-phosphate dehydrogenase (G6PDH) activity (ROSSI & al [15])

The reaction was carried out with 0.1 M pH7.5 Tris-HCl, 10 mM MgCl2, 5 mM glucose-6-phosphate, 0.8 mM NADP+, 1 mM DTT, and 10 μL crude enzyme supernatant. The reduction rate of NADP+ was measured at 340 nm. One unit of enzyme activity is defined as the amount of enzyme needed to catalyze the reduction of 1 μmol NADP+ per min.

2.2.6 Determination of L-phenylalanine transaminase activity (ZHOU & al [16]) Eighty microliters GPT matrix liquid, 80 μL 2,4-dinitrophenylhydrazine solution, and

20 μL supernatant crude enzyme solution (blank control wells: 20 μL water) were incubated in microtiter plates at 37℃ for 10min. The reaction was terminated by adding 20 μL 1 mol/L NaOH solution, and the absorbance of each well was measured at 340 nm. Absorbance-time curves were generated for the calculation of enzyme activity. At 37℃ and pH7.4, one activity unit (U) is defined as the generation of 1 μmol amino acid per minute.



2.2.7 Determination of pyruvate decarboxylase activity It was modified from the method of Gao (GAO & al [17]). 176 μL pH6.0 0.2 mol/L

citrate buffer, 7 μL 1 mol/L sodium pyruvate aqueous solution, 3.5 μL 6.4 mmol/L β-NADH, 3.5 μL 200 U/mL alcohol dehydrogenase solution, and 10 μL supernatant crude enzyme solution were added into each well of microtiter plates (blank control: 183 μL 0.2 mol/L citrate buffer, 7 μL 1 mol/L sodium pyruvate solution, 10 μL supernatant crude enzyme solution) and mixed for the measurement of absorbance at 340 nm for the calculation of enzyme activity. One unit was defined as the amount of enzyme needed to converse 1.0 μmol pyruvate to acetaldehyde per minute at 37℃ and pH6.0.

2.2.8 Determination of isocitrate dehydrogenase (ALINKEEL & al [18]) The reaction was carried out with 0.1 M pH7.4 Tris-HCl, 5 mM MnCl2, 4 mM sodium

iso-citrate, 0.2 mM NADP+, 1 mM EDTA, and 20 μL supernatant crude enzyme solution. The reduction rate of NADP+ was measured at 340 nm. One activity unit (U) was defined as the amount of enzyme needed to reduce 1.0 μmol NADP+ per minute.

3. Results and Conclusions

3.1 Effect of fermentation temperature on the biomass of wild-type and engineered strains

As shown in Figure 2 (a) and (b), S. cerevisiae wild type strain S288C and its 5 engineered strains had basically the same biomasses after 48 h fermentation at 26-36℃, as revealed by their similar dry weights and OD600 values. The growth of engineered strains ARO-8, ARO-9, ARO-10, ARO 8-10, and ARO 9-10 did not show significant changes with changed temperatures, and the wild type strain S288C displayed the best growth at 28℃.

Figure 2. Effect of fermentation temperature on S288C and its engineered strains’ biomass.

(a) Cdw, (b) OD600

3.2 Effect of fermentation temperature on the activity of β-phenylethanol anabolic pathway enzymes

3.2.1 Effect of fermentation temperature on transaminase activity The aminotransferation that converses L-phenylalanine to β-phenylethanol is an important

regulatory point in Ehrlich pathway, and the ARO8 and ARO9 genes encode aminotransferase (WANG & al [19]; IRAQUI & al [20]). As shown in Figure 3(a), fermentation temperature had



significant effects on the activity of aminotransferase in ARO8-overexpression strain ARO-8 and ARO8-ARO10 coupled- overexpressing strain ARO 8-10, while other strains were not sensitive to fermentation temperature. The activity of aminotransferase of engineered strains ARO-8 and ARO 8-10 was significantly higher than that of wild-type strain S288C and other engineered strains (P≤0.01), indicating that the ARO8 gene plays an important regulatory role in β-phenylethanol metabolism, and that enhancing its expression can significantly increase Ehrlich pathway’s metabolic flow. On the contrary, overexpression the ARO9 gene in strain ARO-9 failed to significantly increase aminotransferase activity, indicating a weak regulatory effect of the ARO9 gene and its expression product.

Figure 3. Effect of fermentation temperature on the activities of (a) transaminase, (b) decarboxylase, (c) G6PDH, (d) isocitrate dehydrogenase

3.2.2 Effect of fermentation temperature on the activity of pyruvate decarboxylase The catalysis of conversion of phenylpyruvic acid to phenylacetaldehyde by pyruvate

decarboxylase is the second step of Ehrlich pathway, and the ARO10 gene encodes pyruvate decarboxylase (BOLAT & al [21]; VURALHAN & al [22]). As shown in Figure 3(b), each strain’s decarboxylase activity was gradually increasing with increasing fermentation temperature; the engineered strains ARO-10, ARO 8-10, and ARO 9-10 all had significantly higher decarboxylase activity than the wild-type strain S288C (P≤0.01), indicating that overexpression of ARO-10 improves decarboxylase activity and enhances β-phenylethanol metabolic flux.

3.2.3 Effect of fermentation temperature on the activity of glucose-6-phosphate

dehydrogenase Glucose-6-phosphate dehydrogenase (EC: 1.1.1.49, G6PDH) promotes the flow of

glucose metabolism to non-glycolysis pathways (PPP). The dehydrogenation reaction is

(a) (b)

(c) (d)



accompanied with the reduction of NADP+ to generate NADPH (BOLAT & al [23]), which is important to evaluate the effects of sugar metabolism on Ehrlich pathway. As shown in Figure 3(c), in the range of 26-36℃ fermentation temperature, each engineered strain and wild-type strain S288C’s G6PDH enzyme activity was enhanced gradually with increasing fermentation temperature, along with the tendency of glucose metabolic flux towards pentose phosphate pathway. Particularly, when the fermentation temperature was ≥32 ℃, all strains’ activity of G6PDH was significantly enhanced, indicating largely enhanced metabolic flux of the pentose phosphate pathway.

3.2.4 Effect of fermentation temperature on the activity of isocitrate dehydrogenase Isocitrate dehydrogenase (IDH) is the key rate-limiting enzyme of the TCA cycle,

catalyzing the conversion of isocitrate to oxaloacetic acid, which is then decarboxylated into α-ketoglutarate that participates in Ehrlich pathway’s aminotransfer (HAZELWOOD & al [11]; ZHANG & al [24]). As shown in Figure 3(d), increasing fermentation temperature led to elevated IDH activity in wild type and engineered strains, with the most increase in dehydrogenase activity being found in strain ARO 8-10 (P≤0.01). This indicated that appropriate increases in fermentation temperature enhanced the activity of TCA’s key enzymes, facilitating more production of ATP and its precursors to improve fermentation metabolic rates.

3.3 Effect of fermentation temperature on the metabolism of wild-type strain and

engineered strains 3.3.1 Effect of fermentation temperature on β-phenylethanol biosynthesis in S.

cerevisiae When the fermentation temperature was 26-36℃, the synthesis amount of β-phenylethanol

of each strain showed a first increasing but then decreasing trend (Figure 4(a)). All five engineered strains’ β-phenylethanol yields were somewhat higher than that of the wild-type strain. Particularly, the β-phenylethanol yield reached 0.98 g/L with strain ARO 8-10 overexpressing both ARO8 and ARO10, a 24.6% increase than the wild-type strain, indicating that ARO8, ARO9, and ARO10 genes are all important enzyme genes in β-phenylethanol anabolism in S. cerevisiae. Therefore, enhancing the expression of these three genes may facilitate the metabolic flux and production of β-phenylethanol of S. cerevisiae.

3.3.2 Effect of fermentation temperature on L-phenylalanine conversion in S. cerevisiae As shown in Figure 4(b), the conversion rates of L-phenylalanine reached the maximum

at 37.71%, 36.71%, 37.86%, 38.14%, and 37.29% in the engineered strains ARO-8, ARO-9, ARO-10, ARO 8-10, and ARO 9-10, respectively. Each engineered strain had higher L-phenylalanine conversion rate than the wild-type strain (36.29%), consistent with the results of β-phenylethanol yields.

3.3.3 Effect of fermentation temperature on glucose utilization rate in S. cerevisiae. Glucose is the main source of the carbon skeleton of S. cerevisiae. It can be converted

into shikimic acid through EMP and PPP pathways, and shikimic acid is used to synthesize β-phenylethanol de novo. As shown in Figure 4(c), fermentation at 30℃ led to the highest utilization rates of glucose in both wild-type and engineered strains. All engineered strains showed higher glucose utilization rates than the wild type strain (36.60%), with strain ARO 8-10 having the highest at 61.60%, followed by ARO-10, ARO-8, ARO 9-10, and ARO-9. In



combination with the results shown in Figure 3(a), single or combinatorial overexpression of the ARO8, ARO9, and ARO10 genes in the engineered strains had significantly more active glucose metabolism; a certain amount of glucose can be used to synthesize β-phenylethanol de novo through shikimic acid pathway, which further enhanced the anabolic flux of β-phenylethanol.

Figure 4. Effect of fermentation temperature on the metabolism of S288C and its engineered strains (a) β-phenylethanol biosynthesis, (b) L-phenylalanine conversion rates, (c) glucose utilization rates, (d) ethanol

biosynthesis

3.3.4 Effect of fermentation temperature on S. cerevisiae ethanol fermentation With increasing fermentation temperature, each strain’s ethanol fermentation was

gradually increasing as revealed by the ethanol contents after 48 h culture (Figure 4(d)). Strains ARO 8-10 and ARO 9-10’s ethanol yields were the most and statistically significantly higher than that of the wild type strain S288C (P≤0.01), indicating that single or coupled overexpression of the ARO8, ARO9, and ARO10 genes can also enhance ethanol metabolic flux.

4. Conclusions

The recombinant strains showed overexpression of aminotransferase gene ARO8 and decarboxylase gene ARO10, and both ARO-8 and ARO 8-10 strains had significantly elevated activities of aminotransferase and decarboxylase (P≤0.01). The ARO8 and ARO10 genes play important regulatory roles in β-phenylethanol anabolism of S. cerevisiae by enhancing their expression, which as a result facilitates β-phenylethanol production and its metabolic flux distribution.

(a) (b)

(c) (d)



5. Acknowledgements This work was financially supported by support programs for the Beijing Municipal Natural

Science Foundation (No. 5122008), National Natural Science Foundation of China (NSFC, No. 31571801), Beijing Municipal Science and Technology Project (Z151100001215008), Beijing Innovation Service Ability Construction--Science and Technology Achievement Transformation-Promotion Project (PXM2016-014213-000034) and National High Technology Research and Development Program of China (863 Program, 2012AA021502).

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