Comparative Metabolomic-Based Metabolic Mechanism Hypothesis

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Comparative Metabolomic-Based Metabolic Mechanism Hypothesis for Microbial Mixed Cultures Utilizing Cane Molasses Wastewater for Higher 2‑Phenylethanol Production Xinrong Pan,§ Haishan Qi,§ Li Mu, Jianping Wen,* and Xiaoqiang Jia Key Laboratory of Systems Bioengineering (Ministry of Education) and SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China S Supporting Information *

ABSTRACT: The mixed microbes coculture method in cane molasses wastewater (CMW) was adopted to produce 2phenylethanol (2-PE). Comparative metabolomics combined with multivariate statistical analysis was performed to profile the differences of overall intracellular metabolites concentration for the mixed microbes cocultured under two different fermentation conditions with low and high 2-PE production. In total 102 intracellular metabolites were identified, and 17 of them involved in six pathways were responsible for 2-PE biosynthesis. After further analysis of metabolites and verification by feeding experiment, an overall metabolic mechanism hypothesis for the microbial mixed cultures (MMC) utilizing CMW for higher 2-PE production was presented. The results demonstrated that the branches of intracellular pyruvate metabolic flux, as well as the flux of phenylalanine, tyrosine, tryptophan, glutamate, proline, leucine, threonine, and oleic acid, were closely related to 2-PE production and cell growth, which provided theoretical guidance for domestication and selection of species as well as medium optimization for MMC metabolizing CMW to enhance 2-PE yield. KEYWORDS: mixed microbes, cane molasses wastewater, 2-phenyethanol, comparative metabolomics, metabolic mechanism

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INTRODUCTION 2-Phenylethanol (2-PE) is a higher aromatic alcohol with a roselike odor, naturally existing in the essential oils of many flowers and plants, with very high commercial price and widely used in cosmetic, perfume, and food industries.1 2-PE can be biosynthesized from L-phenylalanine (Phe) through the Ehrlich pathway in some microorganisms. Most yeasts such as Saccharomyces cerevisiae, Kloeckera saturnus, and Kluyveromyces marxianus have been reported to have the ability to synthesize 2-PE via de novo synthesis or the Ehrlich pathway.2 Some fungi, such as Phellinus ignarius, Phellinus laevigatus, and Phellinus tremulae,3 Hericium erinaceus and Nigroporus durus,4 Ischnoderma benzoinum,5 and Aspergillus niger,6 have also been shown to produce 2-PE. In previous reports, strain improvement7 and medium composition and culture condition optimization,8 as well as in situ hybridization technology,1 have been applied to increase 2-PE production. However, all those 2-PE bioconversions are carried out under pure culture fermentation based on refined substrate and sterilization, generating a high cost.9 To lower production cost, it has been currently shifted to the process of utilizing potential low-value raw materials and microbial mixed cultures (MMC).10,11 Among the low-value raw materials, cane molasses wastewater (CMW) is characterized by high organic load and unpleasant smell. Some physicochemical treatments, such as adsorption, coagulation and flocculation, and oxidation, as well as membrane filtration, have been used to dispose of CMW;12 these methods are just for reducing organic pollutants but result in resource waste. Therefore, a better method to recycle valuable components at © 2014 American Chemical Society

lowest cost becomes significant. The MMC method has advantages of no sterilization requirements, adaptive capacity owing to microbial diversity, capacity of using mixed substrates, and the possibility of a continuous process.11 Wool-scour wastewater has been effectively utilized to produce esterase by Pseudomonas aeruginosa and Acinetobacter calcoaceticus separated from a microbial mixed culture, which effectively reduced the organic load of wastewater and achieved a high value-added product.13 Thus, the use of mixed microbes to metabolize cane molasses wastewater to produce 2-PE was investigated in this study. Some characteristics of yeast pure culture to obtain 2-PE have been illuminated.9 However, the mechanisms of MMC utilizing cane molasses wastewater to produce 2-PE are still ambiguous, which limits further improvement of 2-PE production. Metabolomics can provide insights into the metabolism of the cell by comprehensive metabolite analysis in a biological system,14 by which unexpected metabolic relationships and metabolite responses may be discovered, generating a relevant hypothesis.15 Differences in intracellular metabolites between mutant and original strains were analyzed by the comparative metabolomics method, and the metabolic mechanism of Rhizopus oryzae with high-yielding fumaric acid was elucidated by Yu et al.16 and Wang et al.,17 which provided theoretical guidance to enhance fumaric production. Therefore, a full Received: Revised: Accepted: Published: 9927

May 12, 2014 September 4, 2014 September 8, 2014 September 8, 2014 dx.doi.org/10.1021/jf502239d | J. Agric. Food Chem. 2014, 62, 9927−9935

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Figure 1. Time course profiles of key fermentation parameters (dry cell weight, total sugar, and 2-PE concentration) with 2 L airlift loop reactors. Red triangles represent dry cell weight, black inverted triangles represent 2-PE production, and blue circles represent total sugar. Solid and open symbols represent C1 and C2 conditions, respectively. Values shown represent the means of five independent experiments, and error bars represent standard deviations of five values. Meyerhof−Parnas (EMP) pathway, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), shikimate pathway, amino acid metabolism, and fatty acid metabolism. Airlift loop reactors with 2 L volume were used in the experiments, and 1.2 L of fermentation broth was transferred into each reactor. The initial operating conditions (C1) were set as temperature 30 °C, pH 5.0 ± 0.1, and air flow rate 0.4 vvm. The optimized operating conditions (C2) were kept as the temperature 35 °C, pH 7.0 ± 0.1, and air flow 0.8 vvm. Analysis Method. The reactor operated for 96 h in batch cultivation mode. Five parallel cultures were sampled with 10 mL aliquots regularly. Biomass, calculated by dry cell weight, was measured by filtering through a preweighed 45 μm pore size filter, washing with 0.9% NaCl (w/w), and then drying to a constant weight at 80 °C.19 Total sugar concentration in the fermentation broth was determined by anthrone colorimetry.20 2-PE was quantified by gas chromatography (GC, Agilent 6890N); conditions were set as PEG-20 M capillary column (30 m, 0.32 mm internal diameter, 0.25 μm film thickness, China) at 210 °C with a nitrogen carrier gas flow rate of 90 mL/min. A flame ionization detector was employed with hydrogen flow rate of 500 mL/min and air flow rate of 50 mL/min. The detection and sample temperatures were 210 and 240 °C, respectively. Sampling, Quenching, and Extraction of Intracellular Metabolites. Samples (10 mL) for metabolic profiling were harvested at 24, 40, 64, and 80 h from the initial inoculation. The mixed cells were quenched and extracted as in previous reports16,21 with slight modifications. For metabolite extraction, 1 mL of cold methanol− water solution (50% v/v, −40 °C) was added into cells, which were then frozen in liquid nitrogen for 1 min. The freeze−thaw cycle was repeated three times. After centrifugation at 5000g for 3 min, the supernatant was collected. Another 2.5 mL of cold methanol−water solution (60% v/v, −40 °C) was added into the precipitate for further extraction. After mixing and centrifugation at 10000g for 3 min, the extract was pooled with the former one, and the combined sample was centrifuged at 10000g for 5 min again. Finally, 100 μL extracts, mixed with 10 μL of succinic acid-d4 (0.1 mg/mL) as internal standard, were lyophilized (D307 Christ, Germany) at −40 °C for 3−4 h and stored at −80 °C for further processing. Determination of Intracellular Metabolites. Intracellular metabolites were determined by gas chromatography−mass spectrometry (GC-MS) and liquid chromatography−tandem mass spectrometry (LC-MS/MS). The apparatus, specific process, and parameter selection were performed according to Xia et al.19 with slight changes

understanding of the influences of intracellular metabolites on 2-PE production, as well as revealing the mechanism of MMC high-producing 2-PE at a systematic level, becomes increasingly important. Here, the MMC utilizing CMW were employed to produce 2-PE under different conditions. Differences in 2-PE production and cell growth are presented, and comparative metabolomics was applied to analyze the differences for overall intracellular metabolites of microbes. Feeding experiments were further carried out to verify the analysis results, and the metabolic mechanism hypothesis of the MMC high-producing 2-PE was proposed eventually.

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MATERIALS AND METHODS

Wastewater. Cane molasses wastewater (CMW), discharged in the pretreatment of cane molasses and alcohol fermentation process, was obtained from a cane sugar refinery in Nanning, Guangxi Province, China. CMW was centrifuged at 10000g for 10 min, the insoluble impurities were removed, and then an appropriate concentration of ammonium sulfate and sodium dihydrogen phosphate at the ratio chemical oxygen demand (COD):N:P = 100:2:1, as well as 6 g/L Phe, were added into the supernatant. Chemicals. Succinic acid was purchased from Fluka (Buchs, Switzerland). Spectroscopic-grade methanol, acetonitrile, and trichloroacetic acid were purchased from Concord (Tianjin, China). N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was purchased from J&K Scientific. Pyridine, methoxyamine hydrochloride, glucose 6-phosphate, ribulose 5-phosphate, ribose 5-phosphate, and fructose 6-phosphate were obtained from Sigma−Aldrich (St. Louis, MO). Distilled water was purified by use of the Milli-Q20 system (Millipore, Billerica, MA). Microbes and Culture Conditions. The strains used in this study were derived from Mu et al.18 in our previous work. They were identified as Bacillus sp., Pseudomonas sp., Candida tropicalis, A. niger, S. cerevisiae, Kluyveromyces hubeiensis, Rhodotorula glutinis, and Issatchenkia orientalis. All the fungi in this study were proposed as main 2-PE producers.2,6 The eight strains could tolerate high concentrations of 2PE, and mutual cooperation between the strains could well decolorize the wastewater and achieve resource ultilization.18According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, each of the microbes contains six basic metabolic pathways: Embden− 9928

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Figure 2. (a) PCA score and (b) loading plots. in parameter settings. Derivatized sample (1 μL) was injected with a split ratio of 1:10, and scan range was set as 50−800 m/z to acquire mass spectra in GC-MS. High-performance liquid chromatography−electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) analysis was performed to quantify CoA esters. Extraction and analysis of intracellular CoA esters was carried out according to the method described by Park et al.22 Parameters of selective ion scan of ester coenzyme A (m/z parent > m/z daughter) were monitored as a series of chromatograms: acetyl-CoA for 810/303, succinyl-CoA for 868/ 361. The area of each metabolite peak was normalized with the internal standard and cell biomass on the same chromatograph. The generated normalized peak areas (variables) were imported into SIMCA-P v 11.0 (Umetrics, Umea, Sweden) for multivariate statistical analysis. Principal component analysis (PCA) and partial least-squares (PLS) discriminant analysis were carried out to investigate intracellular metabolite profiles. PCA, an unsupervised method, can gain insight into the nature of multivariate data. The PCA score plot was used to judge the similarities and differences among the samples by similar scores clustering together and different scores scattering. In the loading plot, each data point represented a unique metabolite, which was important to discriminate metabolic differences.19 PLS analysis, a supervised method, was subsequently applied to evaluate the relationship between metabolites and products.23 The variable importance in the projection (VIP) index was introduced to estimate the contribution of each metabolite variable to the pattern recognition quantitatively. Generally, a metabolite with VIP value greater than 1 illustrates a significant contribution to the separation of sample groups within the PLS model, and the higher the VIP value of a metabolite, the greater contribution it has.19 Statistical Analysis. Two-tailed t-tests were performed on specific metabolites by Microsoft Excel. At least a P value < 0.01 was considered statistically different. Five replicates were used to perform multivariate analysis for each sample. Data were shown as the mean ± standard deviations for each analyzed sample.

and synthesis of some enzymes were being activated.24 The biomass and total sugar consumption under the two conditions showed no significant difference. The biomass increased to 0.18 (C1) and 0.19 (C2) g/L. Little 2-PE was detected (Figure 1). In logarithmic phase, the highest biomass was achieved, up to 6.8 and 9.4 g/L under C1 and C2 conditions, respectively. The total sugar concentration decreased from 45 to 18.8 (C1) and 16.5 (C2) g/L. The production of 2-PE accumulated up to 175 (C1) and 361 (C2) mg/L, beginning to present significant differences. But it was not the main phase of 2-PE biosynthesis (Figure 1). During this period, some preferable nitrogen source ammonium sulfate existing in wastewater was first absorbed to facilitate the rapid growth of MMC but contributed little to 2PE biosynthesis. When ammonium sulfate was depleted, the uptake of Phe was begun to synthesize 2-PE.9 In stationary phase (phase III), cell growth stopped. As the main nitrogen source, Phe was utilized for 2-PE synthesis. During 40−64 h, the productivity of 2-PE was 0.01 (C1) and 0.02 (C2) g·L−1· h−1. The 2-PE concentration reached 459 (C1) and 910 (C2) mg/L, 2.62- and 2.52-fold higher compared to phase II, respectively. However, during 64−96 h, the production of 2-PE remained invariable with extremely low substrate consumption. The final concentration of 2-PE under C2 conditions (987 mg/ L) was 2.07-fold higher than that under C1 conditions (477 mg/L). Therefore, based on the variation of 2-PE biosynthesis, phase III was divided into early stationary phase (40−64 h) and later stationary phase (64−96 h) for discussion below. Results indicated that the biosynthesis of 2-PE under two conditions mainly occurred in early stationary phase, and the MMC presented a perfect performance in production of 2-PE under C2 conditions. Taken together, the MMC exhibited different fermentation behaviors under C1 and C2 conditions. To explore the reason why microbes had higher production under C2 conditions, it is necessary to study the intracellular metabolite profile of the mixed microbes. Hence, the changing of MMC intracellular metabolites would be profiled, and the metabolic mechanism hypothesis of MMC high-producing 2-PE could be proposed eventually. Identification of Key Metabolites and Metabolic Pathways Closely Associated with 2-PE Biosynthesis. In total, 102 intracellular metabolites were identified by GC-

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RESULTS AND DISCUSSION Performance of MMC Fermentation under C1 and C2 Conditions. Fermentation performance under C1 and C2 conditions is shown in Figure 1. According to the change of biomass, the entire fermentation process could be divided into lag phase (phase I, 0−16 h), logarithmic phase (phase II, 16− 40 h), and stationary phase (phase III, 40−96 h). In lag phase, the mixed microbes were adapting themselves to the substrates, and the utilization of corresponding substrates 9929

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Figure 3. Results of PLS analysis: (a) PLS score plot t[1]/u[1]; (b) VIP plot.

Figure 4. Schematic diagram of main metabolites in key pathways.

Initially, PCA (R2 = 0.91, Q2 = 0.94) was performed. A tight clustering of five parallels for individual time points and a clear separation between the different conditions revealed quite clear discrimination between samples under different culture conditions and fermentation times (Figure 2a). The result

MS, LC-MS/MS, and HPLC-ESI-MS/MS, including sugars, organic acids, alcohols, amino acids, fatty acids, amines, and other compounds (see Table S1 in Supporting Information). The intracellular metabolic differences under C1 and C2 conditions were analyzed by multivariate statistical analysis. 9930

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declined from 2 (24 h) to 0.95 (40 h), then rose slightly up to 1.25 (64 h), and eventually fell to 0.35 (80 h). The change of 3PG was closely related to levels of its derivatives such as serine, glycine, and cysteine (Figure 4). During 24−40 h, with 3PG being consumed rapidly, amino acids were quickly synthesized under C2 conditions. During stationary phase, synthetic rates of amino acids slowed down with the accumulation of 3PG; afterward 3PG was consumed for maintaining the survival of microbes. The relative abundance of pyruvate under C2 conditions was obviously higher than that under C1 conditions at 24 and 40 h. Then it dropped sharply in the entire fermentation process, falling from 3.3 to 0.75. But no significant change was found for C1. However, lactate and acetic acid under C1 conditions were accumulated, and the relative abundance reached 0.75 and 0.53, respectively, at 80 h. Conversely, the relative abundance of lactate and acetate under C2 conditions was extremely low at 80 h: 0.043 and 0.13, respectively. According to the carbon metabolic network of microorganisms, pyruvate flux could be toward lactate, acetate, amino acids, or acetyl-CoA. These metabolic branches competed with each other for carbon flux, and the flux distribution determined the metabolic state of MMC.26 Results indicated that some pyruvate metabolic flows under C1 conditions distributed to lactate and acetate, resulting in accumulation of lactate and acetate during stationary phase. However, under C2 conditions, pyruvate was mainly expended in anabolism or converted to acetyl-CoA, and intracellular lactate and acetate were utilized. The high VIP from PLS suggested that lactate (VIP = 2.3), acetate (VIP = 2.5), and pyruvate (VIP = 4.37) were significant relevant to 2-PE biosynthesis. Therefore, feeding experiments were carried out to prove the analysis above, under the premise that carbon flux distribution to acetyl-CoA under C2 conditions was still insufficient. Pyruvate (5 g/L), lactate (2 g/L), and acetate (0.5 g/L) were added at 16 h under C2 conditions, since their high concentration would make the pH drop and cause microorganism overload.27 It was found that 2-PE production was increased to 1077, 1204, and 1180 mg/L, respectively, suggesting that an improvement of carbon flux distribution to acetyl-CoA would benefit the accumulation of 2-PE. Tricarboxylic Acid Cycle. TCA cycle metabolite levels under C2 conditions were apparently higher than those under C1 conditions at 24 and 40 h (Figure 6). It demonstrated that the intracellular metabolites NADH and ATP under C2 conditions were higher at 24 and 40 h, providing more material, reduction−oxidation, and energy to maintain microbe energetics and keep the 2-PE synthesis system highly efficient.28 The levels of OAA, AKG, SUCC−COA, and FUM dropped at 64 and 80 h compared to those at 24 h. Furthermore, the relative abundance of intracellular OAA under C2 conditions was lower than that under C1 at 64 and 80 h, reflecting the weak TCA cycle under C2 conditions during stationary phase. No obvious differences were found for FUM and CIT between C1 and C2. However, AKG and SUCC−COA under C2 conditions were much higher than under C1 conditions. Since AKG and SUCC−COA were correlated with TCA cycle as well as amino acid (Glu and Pro) metabolism (Figure 4), higher AKG and SUCC−COA level as well as lower citric acid level indirectly reflected the higher catabolism of Glu and Pro. Furthermore, not only could AKG provide NADH for microbes when AKG converted to SUCC−COA, but it could also be regarded as the ammonia receptor in the deamination process of Phe. Therefore, a higher intracellular AKG level under C2

indicated that metabolomics was suitable to monitor culture processes and metabolic characteristics under different conditions.25 In the loading plot (Figure 2b), each data point represented a unique metabolite and reflected the contribution of a specific component to the loading value. It demonstrated that the intermediate metabolites (represented by red circles in Figure 2b) from the EMP pathway, TCA cycle, PPP, shikimate pathway, amino acid metabolism, and fatty acid metabolism contributed most to the separate clustering. In addition, PLS was applied to further verify the differences and identify the metabolites mainly responsible for discrimination between the two conditions. Owing to relatively high 2PE production under C2 conditions, the samples were located in the upper right quadrant at 64 and 80 h, while the other samples were located in the lower left quadrant of the plot (Figure 3a). The significant discrimination on the PLS score plot profiling reflected the MMC metabolic differences under different conditions and stages. The contribution of each intracellular metabolite to 2-PE was shown by VIP score. Metabolites correlated to the first component (VIP score >1) were selected for further investigation. A total of 17 identified metabolites including lactate (LAC), pyruvate (PYR), acetate (ACE), oxaloacetate (OAA), succinyl-CoA (SUCC−COA), αketoglutarate (AKG), sedoheptulose-7-phosphate (S7P), erythrose 4-phosphate (E4P), L-tyrosine (Tyr), L-tryptophan (Trp), L-phenylalanine (Phe), L-glutamate (Glu), L-proline (Pro), Lleucine (Leu), L-threonine (Thr), stearic acid (SFA18), and oleic acid (USFA18) were found to be the key metabolites that were responsible for differentiation between the two conditions (Figure 3b). Those metabolites were involved in six basic metabolic pathways stated above (Figure 4) and will be discussed below in detail. Analysis of Six Basic Metabolic Pathways. Embden− Meyerhof−Parnas Pathway. As illustrated in Figure 5, EMP

Figure 5. Relative abundance of intracellular metabolites in EMP pathway under (left) C1 and (right) C2 conditions. F6P, fructose 6phosphate; 3PG, 3-phosphoglyceric acid; PYR, pyruvate; LAC, lactate; ACE, acetate.

metabolite levels under C2 conditions were higher than those under C1 conditions at 24 and 40 h. However, few differences could be seen in the EMP pathway at 40 and 80 h. In the whole fermentation process, 3-phosphoglyceric acid (3PG) presented different trends between C1 and C2. The relative abundance of 3PG under C1 conditions rose from 0.75 (24 h) to 1.55 (40 h) and then decreased to 0.6 (80 h). But under C2 conditions, it 9931

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accumulation during early stationary phase. It could be concluded that S7P and E4P influenced 2-PE biosynthesis by influencing the metabolism of the shikimate pathway indirectly. Shikimate Pathway. The relative abundance of shikimic acid presented similar variation under both conditions. It first increased from 0.45 (24 h) to 0.82 (40 h) and then gradually decreased to 0.35 (80 h) under C2 conditions, and it increased from 0.23 (24 h) to 0.41 (40 h) and then decreased to 0.33 (80 h) under C1 conditions (Figure 8). It can be seen that shikimic

Figure 6. Relative abundance of TCA cycle intracellular metabolites under (left) C1 and (right) C2 conditions. AC−COA, acetyl-CoA; OAA, oxaloacetate; CIT, citrate; AKG, α-ketoglutarate; SUCC−COA, succinyl-CoA; FUM, fumarate.

conditions during stationary phase contributed to 2-PE biosynthesis. Pentose Phosphate Pathway. PPP metabolites under C2 conditions were at higher levels than under C1 conditions at 24 h (Figure 7). However, the relative abundance of 6-

Figure 8. Relative abundance of intracellular metabolites in shikimate pathway under (left) C1 and (right) C2 conditions. Ski, shikimic acid; Phe, L-phenylalanine; Tyr, L-tyrosine; Trp, L-tryptophan.

acid was rapidly synthesized during logarithmic phase, which was confirmed by the simultaneous increment of intracellular aromatic amino acids. During stationary phase, the anabolic rate of shikimic acid declined, which could be seen from the decline of intracellular shikimic acid concentration. Meanwhile, the relative abundance of aromatic amino acids Tyr and Trp slowly decreased under both C1 and C2 conditions. It might be that accumulation of excessive aromatic amino acids at 40 h generated feedback inhibition on the activity of key enzymes in the shikimic acid anabolic pathway.29 As a result, shikimic acid synthesis was repressed and the metabolism of aromatic amino acids was shifted from anabolism to catabolism, promoting 2PE biosynthesis. The relative abundance of Phe under C2 conditions declined notably from 6.3 at 40 h to 0.14 at 80 h. Phe under C1 conditions also declined in some degree, from 2.7 to 1.4. It was thought that the Ehrlich pathway was activated, leading to rapid decline of Phe as well as biosynthesis of 2-PE during the process.30 At 40 h, the intracellular Phe level under C2 was higher than that under C1. It may be because metabolism of MMC under C2 conditions was more active, and more nitrogen (Phe) uptake from the environment was needed.9,31 Subsequently, intracellular Phe was consumed to biosynthesize 2-PE and thus declined sharply.32 To prove the analysis above, feeding experiments were performed, with the assumption that the uptake of amino acids from the environment by cells did not saturate. Phe (12 g/L), Tyr (1 g/L), and Trp (1 g/L) were supplied at 32 h under C2 conditions, since a high concentration of Tyr and Trp could compete with Phe for catabolism, which would reduce 2-PE production.33 The results showed that 2-PE production was increased to 1341, 1200, and 1169 mg/L, for enhancement of 36%, 21.6%, and 18.4%, respectively. This suggested that the appropriate accumulation of intracellular Tyr and Trp could

Figure 7. Relative abundance of intracellular metabolites in PPP under (left) C1 and (right) C2 conditions. 6PG, glucose 6-phosphate; RL5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; S7P, sedoheptulose 7phosphate; E4P, erythrose 4-phosphate.

phosphogluconic acid (6PG), ribulose 5-phosphate (RL5P), and ribose 5-phosphate (R5P) under both conditions declined in the entire metabolic process. As precursors of nucleotide synthesis, 6PG, RL5P, and R5P were rapidly consumed in nucleotide synthesis. The level of sedoheptulose 7-phosphate (S7P) and erythrose 4-phosphate (E4P) under C2 conditions declined at 40 h and then slightly increased at 64 h. E4P is a precursor of aromatic amino acid biosynthesis. The level change of S7P and E4P was opposite to that of intracellular aromatic amino acids. This phenomenon might be related to the process where anabolism of aromatic amino acids was repressed and turned into catabolism.29 It was accompanied by rapid catabolism of Phe and biosynthesis of 2-PE. Therefore, S7P and E4P declined during logarithmic phase, with a slight 9932

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level of EMP pathway and metabolic flux to fatty acid pathway. At 40 h, the relative abundance of Leu was up to 2.8 under C2 and 1.7 under C1, demonstrating that intracellular metabolic flux of EMP pathway was higher under C2 during logarithmic phase. However, the relative abundance of Leu under C2 decreased to 1.6 at 80 h. The sharp decline of Leu under C2 conditions might correlate with the precursor supplement of fatty acid synthesis and promote the synthesis of membrane lipid composition to tolerate 2-PE toxicity.37 Therefore, feeding those amino acids in the early stage could provide precursors for metabolism. However, as the adding time was delayed, those amino acids, regarded as nitrogen, could compete with Phe for nitrogen utilization,32 which would result in the drop of 2-PE production. Complex amino acids (2 g/L Glu, Pro, Thr, and Leu, mixed in equal proportion) were added to wastewater at 16 h under C2 conditions. The 2-PE production was increased to 1133 mg/L, demonstrating that Glu, Pro, Thr, and Leu would affect 2-PE biosynthesis. Fatty Acid Metabolism. In fatty acid metabolism, the levels of metabolites such as eicosanoic acid (SFA20), stearic acid (SFA18), heptadecanoic acid (SFA17), and palmitic acid (SFA16) under C2 conditions were significantly lower than under C1 conditions (Figure 10). However, the levels of

facilitate the shift of aromatic amino acids from anabolism to catabolism, and high Phe concentration could promote 2-PE biosynthesis through the Ehrlich pathway at the end of logarithmic phase. Amino Acid Metabolism. For amino acid metabolism, changes of various amino acids are shown in Figure 9. These

Figure 9. Relative abundance of intracellular metabolites in amino acid metabolic pathway under (left) C1 and (right) C2 conditions. Glu, Lglutamate; Pro, L-proline; Thr, L-threonine; Leu, L-leucine; Val, Lvaline; Ile, L-isoleucine; Ser, L-serine; Gly, L-glycine; Cys, L-cysteine.

detected amino acids required biosynthetic precursors involved in central carbon metabolism (EMP pathway, TCA cycle, and PPP) and were linked to the synthesis of new cellular components or energy sources needed in fermentation at the expense of the amino acid pool. For example, Gly, Ser, Cys, Leu, and Ile could derive from the EMP pathway. Glu, Pro, and Val could flow to TCA, and Thr could flow to the EMP pathway. PLS analysis demonstrated that Glu, Pro, Thr, and Leu, with VIP values greater than 1, were correlated with 2-PE biosynthesis. Glu and Pro are important carbon skeletons for AKG by catabolism (Figure 4).34 The relative abundance of Glu and Pro under C2 conditions was 4.2 and 3.3 at 40 h and then significantly fell to 0.4 and 0.23, respectively. However, the maximum relative abundance of Glu and Pro under C1 conditions was just 1.7 and 1.5 and subsequently declined to 0.6 and 0.6, respectively. It could be inferred that intracellular Glu and Pro under C2 conditions were consumed rapidly during the 2-PE biosynthesis period. Apart from supporting protein synthesis and cell growth, Glu and Pro are also catabolized for AKG regeneration, which is consistent with the analysis result of TCA. Thr is an important amino acid for pyruvate synthesis. The alteration of Thr level directly reflected its metabolic flux state toward the EMP pathway.35 Under C1 conditions, relative abundance of Thr remained below 1, without obvious fluctuation. On the contrary, under C2 conditions, the abundance of Thr changed dramatically from 1.7 at 24 h to 0.2 at 80 h, indicating that Thr was consumed to provide some metabolites related to the EMP pathway and maintain cells’ normal metabolism during stationary phase. The sharp decline of Thr under C2 conditions reflected the urgent need of microbes for catabolism, and indirectly influenced 2-PE biosynthesis. Leu could be derived from pyruvate and degraded to acetyl-CoA, which was closely associated with fatty acid synthesis (Figure 4).36 The abundance of Leu may reflect the

Figure 10. Relative abundance of intracellular metabolites in fatty acid metabolic pathway under (left) C1 and (right) C2 conditions. USFA16, palmitoleic acid; USFA18, oleic acid; SFA16, palmitic acid; SFA17, heptadecanoic acid; SFA18, stearic acid; SFA20, eicosanoic acid.

palmitoleic acid (USFA16) and oleic acid (USFA18) were higher than under C1 conditions. During the whole fermentation process, the average relative abundance of SFA18 under C2 conditions was only 0.49-fold of that under C1, and USFA18 was 2.7-fold of that under C1 conditions. The ratio of unsaturated fatty acids (USFA) to saturated fatty acids (SFA) under C2 conditions was obviously higher than that under C1 conditions. A high USFA/SFA ratio is conducive to the cell membrane gaining a strong capacity to resist the tough outside environment.38 It is the main mechanism for microbes to adapt to environmental stress. 2-PE was toxic to microbes. One of the toxicity mechanisms is that aromatic alcohol could change the liquidity of the membrane, leading to easy permeability. Then the transport systems of sugars and amino acids in cytoplasmic membrane are affected, the passive diffusion of ions and small metabolites through the membrane 9933

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Funding

is accelerated, and the gradient of transmembrane protons is disturbed, thus leading to leakage of intracellular ions and reduced intake of amino acids and sugars.1 It was reported that oleic acid could prevent alcohol poisoning of cells by regulating the liquidity of the cell membrane.39 Thus, unsaturated fatty acid played a significant role in improving the capacity of cell membrane to resist the toxicity of 2-PE. However, it also had been reported that a high concentration of oleic acid could inhibit the growth of microorganisms.40 During early growth phase, oleic acid existing in the medium would be toxic to microbes.41 Therefore, 15% (v/v) oleic acid was added to the CMW at 40 h under C2 conditions. Finally, 2-PE production was elevated to 1378 mg/L, increased by 39.6% compared to the control group without oleic acid supplement. The results suggested that cell membranes of 2-PE high-producers had a strong capacity to resist outside toxicity by increasing the ratio between intracellular unsaturated and saturated fatty acid concentrations. Metabolic Mechanism Hypothesis for High 2-PE Production by MMC Utilizing CMW. From the analysis above, it was found that EMP pathway, TCA cycle, PPP, shikimate pathway, and amino acid pathway presented high metabolic levels during logarithmic phase, which was beneficial for absorbing nutrition for cell growth and promoting Phe uptake from the wastewater. During stationary phase, the metabolic levels of EMP and TCA cycle weakened. To maintain basic metabolism, the catabolism of intracellular amino acids was accelerated and the intracellular Phe was predominantly used to transaminate and synthesize 2-PE through the Ehrlich pathway. On the basis of metabolic profiling analysis, during logarithmic phase, the metabolic flux of intracellular pyruvate, lactate, and acetate for high-producing MMC was converted to acetyl-CoA, improving the metabolic flux related to acetyl-CoA, which facilitated the uptake of Phe by cells for 2-PE biosynthesis. In the shikimate pathway, an appropriate accumulation of intracellular Tyr and Trp as well as an increase of intracellular Phe for microbes at the end of logarithmic phase could inhibit de novo synthesis of 2-PE, leading to 2-PE biosynthesized in quantity through the Ehrlich pathway. In amino acid metabolism, Glu, Pro, Thr, and Leu, associated with central carbon metabolism and fatty acid metabolism, indirectly reflected the cell metabolic state. In fatty acid metabolism, the resistance of cell membrane to 2-PE toxicity could be strengthened by increasing USFA/SFA. Those findings provided theoretical guidance for species domestication and selection as well as medium optimization when MMC utilized CMW to produce 2-PE.

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This research was financially supported by the National 973 Project of China (2013CB733600), the Key Program of National Natural Science Foundation of China (21236005), and the Program of Introducing Talents of Discipline to Universities (B06006). Notes

The authors declare no competing financial interest.

ABBREVIATIONS

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REFERENCES

MMC, microbial mixed cultures; CMW, cane molasses wastewater; 2-PE, 2-phenyethanol; Phe, phenylalanine; Tyr, tyrosine; Thr, tryptophan; LAC, lactate; PYR, pyruvate; ACE, acetate; SUCC−COA, succinyl-CoA; Glu, glutamate; Pro, proline; Leu, leucine; Thr, threonine; Ser, serine; Gly, glycine; Cys, cysteine; Val, valine; Ile, isoleucine; E4P, erythrose 4phosphate; S7P, sedoheptulose 7-phosphate; 3PG, 3-phosphoglyceric acid; OAA, oxaloacetate; AKG, α-ketoglutarate; SFA18, stearic acid; USFA18, oleic acid; USFA16, palmitoleic acid; SFA17, heptadecanoic acid; SFA16, palmitic acid; SFA20, eicosanoic acid; USFA, unsaturated fatty acids; SFA, saturated fatty acids; EMP, Embden−Meyerhof−Parnas pathway; TCA, tricarboxylic acid cycle; PPP, pentose phosphate pathway; C1, initial operating conditions; C2, optimized operating conditions; COD, chemical oxygen demand; PCA, principal component analysis; PLS, partial least-squares method; VIP, variable importance in projection; GC-MS, gas chromatography−mass spectrometry; LC-MS/MS, liquid chromatography−mass spectrometry/mass spectrometry; HPLC-ESI-MS/ MS, high-performance liquid chromatography−electrospray ionization tandem mass spectrometry

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ASSOCIATED CONTENT

S Supporting Information *

One table listing intracellular metabolites and VIP score order of PLS analysis under C1 and C2 conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone and fax +86-22-27892061; e-mail [email protected]. cn. Author Contributions §

X.P. and H.Q. contributed equally to this work. 9934

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