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Bioprocess and Biosystems Engineering

, Volume 41, Issue 8, pp 1143–1151 | Cite as

Metabolic analyses of the improved ε-poly-l-lysine productivity using a glucose–glycerol mixed carbon source in chemostat cultures

  • Jian-Hua Zhang
  • Xin Zeng
  • Xu-Sheng Chen
  • Zhong-Gui Mao
Research Paper

Abstract

The glucose–glycerol mixed carbon source remarkably reduced the batch fermentation time of ε-poly-l-lysine (ε-PL) production, leading to higher productivity of both biomass and ε-PL, which was of great significance in industrial microbial fermentation. Our previous study confirmed the positive influence of fast cell growth on the ε-PL biosynthesis, while the direct influence of mixed carbon source on ε-PL production was still unknown. In this work, chemostat culture was employed to study the capacity of ε-PL biosynthesis in different carbon sources at a same dilution rate of 0.05 h−1. The results indicated that the mixed carbon source could enhance the ε-PL productivity besides the rapid cell growth. Analysis of key enzymes demonstrated that the activities of phosphoenolpyruvate carboxylase, citrate synthase, aspartokinase and ε-PL synthetase were all increased in chemostat culture with the mixed carbon source. In addition, the carbon fluxes were also improved in the mixed carbon source in terms of tricarboxylic acid cycle, anaplerotic and diaminopimelate pathway. Moreover, the mixed carbon source also accelerated the energy metabolism, leading to higher levels of energy charge and NADH/NAD+ ratio. The overall improvements of primary metabolism in chemostat culture with glucose–glycerol combination provided sufficient carbon skeletons and ATP for ε-PL biosynthesis. Therefore, the significantly higher ε-PL productivity in the mixed carbon source was a combined effect of both superior substrate group and rapid cell growth.

Keywords

ε-Poly-l-lysine Mixed carbon source Chemostat culture Metabolic analyses 

Introduction

ε-Poly-l-lysine (ε-PL) is a basic cationic biopolymer composed of 25 to 35 l-lysine residues with isopeptide bonds between α-carboxyl group and ε-amino group. It is a secondary metabolite mainly produced through microbial fermentation by Streptomyces species. Good safety and wide spectrum of antimicrobial activity made ε-PL to be successfully used as a natural food preservative in Japan, the US as well as South Korea for many years [1]. Moreover, it has also been applied in pharmaceutical industry as drug carrier, liposomes, interferon inducer, lipase inhibitor, etc. [2, 3]. Therefore, it is of great significance to promote the commercial production of ε-PL.

At present, microbial fermentation is the only approach to commercially produce ε-PL. To improve ε-PL production, strain improvement, optimization of culture medium and regulation of fermentation process have been carried out [4, 5, 6, 7, 8]. Since carbon source provides carbon skeletons and energy for the cells’ metabolism, it plays a crucial role in medium composition. Glucose and glycerol are usually used as carbon sources in most ε-PL fermentation. Recently, we have found that Streptomyces albulus M-Z18 could simultaneously use both glucose and glycerol, leading to significant reduction of batch culture time [9]. The essence lies in the improvements of ε-PL productivity (1.84- and 1.35-folds of that in glucose or glycerol, respectively) and cell growth rate (1.21- and 1.30-folds of that in glucose or glycerol, individually). Interestingly, similar improvements of both productivity and biomass has been also observed in other fermentations with multiple substrates [10, 11, 12], highlighting the great significance of mixed carbon sources in industrial microbial fermentation. In our previous study, the physiological basis of the high ε-PL productivity in batch fermentation using glucose–glycerol mixed carbon source are investigated in terms of key enzymes activities and energy cofactors [13]. However, whether the improvement of ε-PL productivity is attributed to the mixed carbon source or the rapid cell growth rate is unknown. Subsequent study has confirmed the positive correlation between the cell growth rate and ε-PL productivity [14], indicating that accelerated cell growth rate in the mixed carbon source contributes to the enhancement of ε-PL productivity. However, it is still unknown that whether the mixed carbon source could directly improve the ε-PL productivity.

Chemostat culture has been widely used to establish steady-state as a basis to investigate the physiology of microorganisms in specific growth rate [15, 16, 17]. To study the direct effects of the mixed carbon source on ε-PL production, in this study, comparison of fermentation parameters with a same dilution rate was carried out in chemostat cultures using glucose, glycerol and glucose–glycerol combination as the carbon sources. In addition, metabolic fluxes analysis was performed at steady-state to understand the corresponding cell physiology in chemostat cultures using different carbon sources. Furthermore, the results were confirmed by activity determination of multiple key enzymes. Finally, intracellular energy cofactors were analyzed to study the energy metabolism in different carbon sources. The results further proved that the improvement of ε-PL productivity in the mixed carbon source was a combined effect of both rapid cell growth and superior substrate group.

Materials and methods

Microorganism and culture media

Streptomyces albulus Z-18, isolated from the soil as described by Nishikawa et al. [18], has been registered in Chinese General Microbiological Culture Collection Center (CGMCC 10479). S. albulus M-Z18 was used throughout this study, which is a mutagenesis from S. albulus Z-18. Culture media were prepared as follows. Agar slant media for spores’ generation (g L−1): 10 glucose, 5 yeast extract, 5 peptone and 20 agar with initial pH value of 7.5 by addition of 2 M NaOH. The M3G media for seeds pre-culture (g L−1): 50 glucose, 10 yeast extract, 10 (NH4)2SO4, 1.36 KH2PO4, 0.8 K2HPO4, 0.5 MgSO4·7H2O, 0.04 ZnSO4·7H2O, 0.03 FeSO4·7H2O with initial pH of 6.8 by addition of 6 M NaOH. Carbon source and nitrogen source provide energy and precursor l-lysine for ε-PL production, which are not suitable as the growth-limited substrates in chemostat cultures. Therefore, to decrease additional influence on ε-PL biosynthesis, phosphorus was selected as the growth-limited substrate for the chemostat cultures in this study. The media for chemostat culture contained (g L−1): 10 beef extract, 10 (NH4)2SO4, 0.4 KH2PO4, 0.8 MgSO4·7H2O and 0.05 FeSO4·7H2O with initial pH of 6.8 by addition of sterilized 12.5% (v v−1) NH3·H2O. The carbon sources for chemostat cultures were prepared in three groups: (a) 120 g L−1 glucose; (b) 120 g L−1 glycerol; (c) 60 g L−1 glucose + 60 g L−1 glycerol (glucose:glycerol = 1:1, w w−1). Media sterilization was performed at 121 °C for 20 min by an autoclave and the glucose in these media was separately autoclaved to prevent Maillard reaction.

Culture conditions

Spores of S. albulus M-Z18 were obtained by cultivation on agar slant media in a constant temperature incubator at 30 °C for 7 days. For the pre-culture, two loops of spores (2 × 105 L−1) were inoculated in 80 mL of M3G media and incubated in 500 mL flasks at 200 rpm and 30 °C for 24 h in a rotary shaker. 5-L fermenters (Baoxing Corp., Shanghai, China) equipped with one 6-blade Rushton turbine impeller were used for chemostat cultures in this study. Seeds broth after pre-culture was transferred into 5-L fermenters (Baoxing Corp., Shanghai, China) with 1.5-L working volume and 8% inoculum size for 30-h batch culture. Chemostat culture was carried out by nutrient feeding and equal volume of withdrawn broth with a peristaltic pump (Longer Corp., Baoding, China). A same dilution rate of 0.05 h−1 was carried out in three different carbon sources and higher dilution rate could not reached in chemostat culture using single glycerol [14]. The cultivation temperature was maintained at a constant temperature of 30 °C, and the dissolved oxygen (DO) remained above 30% with 1.0 vvm aeration and 550 rpm stirring rate. The fermentation pH was maintained at 4.0 with 12.5% (v v−1) NH3·H2O solution. After 4–5 cultivation volume replacements, no significant change was observed in DO, biomass density as well as ε-PL concentration, which could be considered as steady-state. Broth samples were withdrawn at this time for analyses of fermentation parameters and cell physiological status.

Analytical methods

The broth was centrifuged at 4500×g for 10 min to obtain precipitate and supernatant. For biomass determination, the precipitate was collected and washed twice, filtered by pre-weighted filter paper and dried at 105 °C to constant weight. The supernatant was used for determination of ε-PL concentration as described by Kahar et al. [8]. The concentrations of glucose and glycerol were measured using a HPLC system (DIONEX, U-3000, US) with refractive index detector (Shodex RI-101, Japan) and ion exchange column (Aminex HPX-87H, 300 × 7.8 mm; Hercules, CA, USA). The flow rate of mobile phase (5 mM H2SO4) was set at 0.6 mL min−1, and the column temperature was maintained at 60 °C. Assays were performed at least in triplicate. To determine the amino acids uptakes/releases of cells, in the chemostat culture, feeding media and culture broth were withdrawn and centrifuged at 4500×g for 10 min. The supernatants were treated with 6 M HCl for 22 h in evacuated glass envelopes. After treatment of acid hydrolysis, the samples were filtered by filter paper, and the clear liquid was centrifuged at 12,000×g for 10 min. The supernatant was used for measurement of hydrolyzed amino acids by HPLC as described by Fountoulakis [19].

Analysis of metabolic fluxes

The genome of S. albulus ZPM is the most comprehensive-mapped genome in ε-PL-producing strains, and the task of gap closing was achieved. The metabolic reaction model was established based on the complete genome of S. albulus ZPM (CP006871). The model contained complete pentose phosphate (PPP) pathway, embden meyerhof (EMP) pathway, tricarboxylic acid (TCA) cycle, anaplerotic metabolic pathway, diaminopimelate (DAP) pathway and ε-PL biosynthesis process. The primary metabolisms of glucose and glycerol were also included. The metabolic reaction model was shown in Fig. 2. Assumptions were set out as follows: (1) NADPH was used for synthesis of amino acids and methyltetrahydrofolate (MTHF), and there was no interconversion between NADPH and NADH; (2) the fluxes for biomass production were calculated based on precursors requirements for biomass synthesis in Streptoverticillium mobaraense for a close genetic relationship [20]; (3) there was no changes in biomass composition in chemostat culture with different carbon sources; (4) metabolic reactions without metabolic bypass were regarded as one reaction; (5) the flux of cell maintenance and unknown metabolites synthesis was incorporated into flux of biomass synthesis; (6) once the steady-state was achieved, there was no changes in the concentration of intracellular intermediate metabolites according to the pseudo-steady-state assumption.

Metabolic flux analysis

As shown in Online Resources 1, the model was composed of 55 metabolic reaction rates and 35 metabolites. Negative value of reaction rate represented reverse chemical reaction. Based on the principle of chemometrics, calculations were carried out by the following equation:
$${r_i}\left( t \right)=~\mathop \sum \limits_{m} {a_i}{x_m}\left( t \right) - \mathop \sum \limits_{n} {a_n}{x_n}\left( t \right),$$
(1)
where ri(t) represented rate of specific metabolic reaction, ai and ak represented stoichiometric coefficient, xm(t) and xn(t) represented the specific formation rate and specific consumption rate of metabolites, respectively. Therefore, equations concerning node metabolites in the model could be established and merged to matrix as follows:
$$Ax\left( t \right)=r(t).$$
(2)

When the chemostat culture reached steady-state, the values of r(t) were zero. According to the numbers of metabolic reaction rates and metabolites, 35 metabolic equations and 55 unknown numbers were set out. The calculation of specific ε-PL formation rate, specific consumption rates of glucose and glycerol, specific consumption/release rates of amino acids were carried out based on concentration assay of ε-PL, carbon sources and amino acids in broth and feeding media, generating 20 reaction rates values. Therefore, the unknown number reduced to 35, which was in consistence with the number of metabolic equations. Finally, the metabolic reaction matrixes could be solely solved using software Matlab 7.0 (The MathWorks, Natick, MA, USA) in personal computer. The abbreviations in metabolic reactions were shown in Online Resources 2.

Cell extract preparation

For the measurement of key enzymes activities and energy cofactors concentration, the samples of cell extraction were separately prepared as described in our previous study [13]. All procedures of cell extract preparation were carried out at 4 °C. Protein content of cell extraction was determined by Super-Bradford Protein Assay Kit as defined in the specification.

Assay of key enzymes activities

The activities of phosphoenolpyruvate carboxylase (PEPC), citrate synthase (CS), aspartokinase (Ask) and ε-PL synthetase (Pls) were measured as described previously [13]. For the determination of PEPC, 300 µL assay mixture contained 30 µL 40 mM MnSO4, 30 µL 100 mM NaHCO3, 30 µL 1.5 mM NADH, 6 µL 500 U mL−1 malate dehydrogenase, 15 µL 1 mM acetyl CoA, 10 µL 40 mM MgCl2, 12 µL 20 mM phosphoenolpyruvate, 117 µL 100 mM Tris–HCl buffer (pH 7.5), 50 µL cell extract. One unit of PEPC activity was defined as the amount of enzyme which catalyzed the oxidation of 1 µmol of NADH per minute in the assay at 30 °C. For the determination of CS, 300 µL assay mixture contained 5 µL 200 mM oxaloacetate, 10 µL 6.7 mM DTNB, 25 µL 1 mM acetyl CoA, 210 µL 100 mM Tris–HEPES buffer (pH 7.4), 50 µL cell extract. One unit of CS activity was defined as the amount of enzyme which catalyzed the formation of 1 µmol of citryl-CoA per minute in the assay at 30 °C. The Ask and Pls were determined as described by Chen et al. [21]. One unit of Ask activity was defined as the amount of enzyme which catalyzed the formation of 1 µmol of aspartyl-β-hydroxamate per minute in the assay at 30 °C. And, one unit of Pls activity was defined as the amount of enzyme which catalyzed the consumption of 1 pmol of l-Lys per second in the assay at 30 °C. Assays were performed at least in triplicate.

Assay of intracellular energy cofactors

The concentration of intracellular ATP, ADP, AMP, NADH and NAD+ were measured as described previously [13]. Samples were measured by HPLC (Agilent 1200, USA) using a spectrophotometer at 254 nm and a LAChrom C18-AQ (250 × 4.6 mm) column at 25 °C. The mobile phase was composed of 5% acetonitrile, 95% 0.2 M Na2HPO4–NaH2PO4 buffer (pH 7.0) as well as 10 mM tetrabutyl ammonium bromide. The flow rate was remained at 1.0 mL min−1 and a 10 µL of inject volume was used in this study. Assays were performed in triplicate.

Calculation

Once the steady-state was achieved, fermentation parameters including dilution rate (D, h−1), specific cell growth rate (µ, h−1), ε-PL-to-substrate yield (YP/S, g g−1), biomass-to-substrate yield (YX/S, g g−1), specific ε-PL formation rate (QP, h−1) and specific carbon source consumption rate (QS, h−1) of different carbon sources were calculated as follows:
$$D=~~\frac{F}{V},$$
(3)
$$\mu ~=~~D,$$
(4)
$${Y_{X/S}}=~\frac{{c(X)}}{{c({S_0}) - c(S)}}~,$$
(5)
$${Y_{P/S}}=~\frac{{c\left( P \right)}}{{c\left( {{S_0}} \right) - c\left( S \right)}}~,$$
(6)
$${Y_{X/P}}=\frac{{c(X)}}{{c(P)}}~,$$
(7)
$${Q_P}~=~~\frac{D}{{{Y_{X/P}}}}~,$$
(8)
$${Q_S}~=~~\frac{D}{{{Y_{X/S}}}}~,$$
(9)

Above parameters are averaged values performed at steady-state in chemostat cultures, where F is feeding rate (L h−1), V is the working volume (1.5 L), and c(X), c(P) and c(S) are the biomass concentration (g L−1), ε-PL concentration (g L−1) and the total carbon source concentration (g L−1) at the steady-state, respectively. c(S0) is the total carbon source concentration (g L−1) of the feeding media.

Statistical analysis

All these data were treated with OriginPro (version 8.5, Northampton, MA, USA). All statistical analyses were performed using ANOVA. Results at p < 0.05 were considered statistically significant.

Results

Direct effects of the mixed carbon source on the ε-PL production in chemostat cultures

The glucose–glycerol mixed carbon source could greatly enhance the ε-PL productivity in batch fermentation. Our previous study found that the mixed carbon source could accelerate the cell growth, which was beneficial for ε-PL production [14]. However, whether the mixed carbon source exerted direct influence on ε-PL biosynthesis was still unknown. To re-evaluate the direct effects of the glucose–glycerol mixed carbon source on the production of ε-PL, chemostat cultures were employed in glucose, glycerol and the glucose–glycerol mixed carbon source at a same dilution rate of 0.05 h−1. The fermentation profiles of chemostat cultures were shown in Online Resource 3 and 4 and important parameters were calculated and depicted in Fig. 1. As shown in Fig. 1a, the QP in the mixed carbon source showed 3.1- and 2.5-folds higher than that in single glucose and glycerol, respectively. Meanwhile, Fig. 1b showed that the mixed carbon source could be more quickly used by microorganisms, and it also led to higher ε-PL yield than single glucose (Fig. 1c). Above results indicated that, besides the positive effect of rapid cell growth [14], the glucose–glycerol mixed carbon source could also directly promote the ε-PL production.

Fig. 1

Effect of carbon sources on ε-PL fermentation kinetic parameters under phosphorus-limited chemostat cultures using the glucose–glycerol mixed carbon source by S. albulus M-Z18. a Specific ε-PL formation rate (qP); b specific total carbon source consumption rate (qS); c ε-PL-to-substrate yield (YP/S). Error bars data range about mean (n ≥ 3) and correspond to the SD. Statistical significance is denoted by different letters for the same strategy (p < 0.05)

Comparison of metabolic fluxes in chemostat cultures using glucose, glycerol and glucose–glycerol combination as carbon sources

The formation/release rates of carbon sources, amino acids, ε-PL and biomass were shown in Table 1, which could be used for the calculation of 35 unknown reaction rates. The unique solution of the 35 reaction rates in metabolic fluxes was shown in Fig. 2. In phosphorus-limited chemostat cultures, glycerol showed much lower specific consumption rate than glucose. Metabolic fluxes analysis revealed that the fluxes through TCA cycle in glycerol were greatly reduced than that in glucose. However, the fluxes of DAP and anaplerotic pathway were much higher than those in glucose, implying that glycerol was more efficient for l-lysine biosynthesis. Interestingly, compared to those in single carbon sources, much higher fluxes through TCA cycle, DAP pathway as well as anaplerotic metabolic pathway were observed in the mixed carbon source. The carbon fluxes from aspartate to aspartic β-semialdehyde were 3.5- and 2.5-folds higher than that in single glucose and glycerol, respectively, while fluxes from aspartic β-semialdehyde to l-lysine were 3.2- and 2.0-folds higher than that in single glucose and glycerol, individually. Moreover, the mixed carbon source showed higher fluxes from phosphoenolpyruvate to oxaloacetate than that in single carbon sources. Therefore, in the mixed carbon source, both the oxaloacetate for l-lysine synthesis and l-lysine for ε-PL production could be quickly compensated, which guaranteed sufficient carbon skeletons for ε-PL biosynthesis.

Table 1

Formation/release rate of metabolites under phosphorus-limited chemostat cultures using glucose, glycerol and glucose–glycerol combination as the carbon source

Reactions

Reactions number

Glucose

Glycerol

Glucose + glycerol

Glucose ⟺ G6P

r1

1.353

0.000

0.950

Glycerol ⟺ DHAP

r19

0.000

1.294

1.107

Alaex ⟺ Ala

r37

0.015

− 0.032

− 0.016

Valex ⟺ Val

r38

0.013

0.014

− 0.005

Leuex ⟺ Leu

r39

0.030

0.020

− 0.001

Gluex ⟺ Glu

r40

0.028

− 0.060

0.007

Argex ⟺ Arg

r41

0.014

0.011

0.009

Aspex ⟺ Asp

r42

− 0.010

− 0.014

− 0.015

Lysex ⟺ Lys

r43

− 0.002

− 0.001

− 0.008

Ileex ⟺ Ile

r44

0.001

0.005

− 0.004

Threx ⟺ Thr

r45

0.000

0.002

− 0.001

Metex ⟺ Met

r46

0.001

0.003

0.003

Hisex ⟺ His

r47

− 0.003

− 0.001

− 0.006

Pheex ⟺ Phe

r48

− 0.004

0.022

0.016

Tyrex ⟺ Tyr

r49

0.003

0.003

0.002

Serex ⟺ Ser

r50

0.001

− 0.011

0.000

Cysex ⟺ Cys

r51

− 0.067

− 0.096

− 0.006

Glyex ⟺ Gly

r52

0.037

0.008

0.002

Lys ⟺ ε-PL

r54

0.001

0.002

0.004

Precursors ⟺ biomass

r55

0.002

0.002

0.002

The rates of reactions were identified as mM h−1 g−1 DCW

Fig. 2

Metabolic flux distribution under phosphorus-limited chemostat cultures using different carbon sources. a glucose; b glycerol; c glucose–glycerol mixed carbon source

Metabolic activity validation of important reactions involved in ε-PL production in chemostat culture using the mixed carbon source

TCA cycle, anaplerotic metabolic pathway, DAP pathway as well as ε-PL assembly were crucial in ε-PL production. To validate the results of metabolic fluxes analysis, activities of key enzymes in above pathways were measured and compared among chemostat cultures with three different carbon sources (Fig. 3). Compared with that in glucose, the glycerol showed lower activity of CS, while it showed higher Ask activity and exhibited 4.4- and 1.6-folds higher activities of PEPC and Pls, individually. Therefore, glycerol could enhance the metabolism of DAP pathway in chemostat culture, which provided more carbon skeletons for the biosynthesis of oxaloacetate, aspartate, lysine and ε-PL. With a combined effect of both glucose and glycerol, the activities of CS, PEPC, Ask as well as Pls were all enhanced. The mixed carbon source performed 5.2- and 1.2-folds higher PEPC activity than glucose and glycerol, respectively; it also showed 1.2- and 1.8-folds higher CS activity than glucose and glycerol, individually. The mixed carbon source also inherited higher Pls activity as in glycerol. Moreover, it significantly improved the activity of Ask (2.7-folds of that in glucose and 2.0-folds of that in glycerol), which was beneficial for l-lysine production.

Fig. 3

Activities of key enzymes in ε-PL production under phosphorus-limited chemostat cultures using glucose, glycerol and glucose–glycerol combination as the carbon sources

Analysis of energy metabolism in chemostat cultures using the mixed carbon source

Adenylylation of l-lysine is the premise of ε-PL biosynthesis, which consumes large amount of ATP. To evaluate the energy metabolism of cells in chemostat cultures with mixed carbon source, the ratio of NADH/NAD+ and intracellular energy charge were calculated and compared among three different carbon sources (Fig. 4). As shown in Fig. 4b, under phosphorus-limited condition, single glucose and glycerol showed similar levels of intracellular energy charge. However, higher ratio of NADH/NAD+ was observed in single glycerol rather than glucose. Notably, the mixed carbon source showed 2.1- and 1.5-folds higher energy charge levels than that in single glucose and glycerol, respectively; it also exhibited 2.2- and 1.6-folds higher NADH/NAD+ ratio than that in single glucose and glycerol, individually. The results revealed that the cells in mixed carbon source was more proficient in NADH production as well as ATP biosynthesis through aerobic respiration.

Fig. 4

Energy metabolism of cells in phosphorus-limited chemostat cultures using glucose, glycerol and glucose–glycerol combination as the carbon sources. a NADH/NAD+; b energy charge. Error bars data range about mean (n ≥ 3) and correond to the SD. Statistical significance is denoted by different letters for the same strategy (p < 0.05)

Discussion

The glucose–glycerol mixed carbon source performed higher cell growth rate and ε-PL productivity in batch fermentation, which was of great significance in industrial fermentation. How could mixed carbon source induce such an improvement has attracted extensive attention from both academia and industry. Our previous study indicated that, in the batch culture, the mixed carbon source could enhance the activities of key enzymes in EMP, TCA cycle, anaplerotic metabolic pathway, diaminopimelate. However, these improvements were observed in rapid-growing cells. Subsequent investigation found that accelerated cell growth exerted positive influence on ε-PL production [14]. Whether the mixed carbon source could directly influence the ε-PL productivity is still unknown. In this study, chemostat cultures at a same dilution rate of 0.05 h−1 were employed to prevent the influence from various cell growth rate in different carbon sources. In single carbon sources, glucose was easily used by S. albulus (Fig. 1b), whereas it was difficult to be converted into ε-PL (Fig. 1c). In contrast, glycerol was relatively less consumed by microorganisms, while it led to higher conversion rate from substrate to ε-PL. The mixed carbon source combined the advantages of these two carbon sources and showed higher substrate uptake rate and conversion rate (Fig. 1b, c). Moreover, the mixed carbon source could directly improve the ε-PL productivity (Fig. 1a).

To understand the underlying mechanism, the cell physiological basis was further investigated in terms of carbon metabolic fluxes, activities of key enzymes as well as intracellular energy status. Chemostat culture was also employed for the analysis of carbon metabolic fluxes. Organic nitrogen source was important in ε-PL fermentation, which could not be replaced with (NH4)2SO4. Hence, besides of the glucose and glycerol, amino acids were also served as sources of carbon skeletons in this study. The uptake/release rates of amino acids were calculated based on the determination of amino acids in feeding media and culture supernatant after acid-hydrolyzation treatment. Results of carbon metabolic fluxes analysis demonstrated that the single glucose could increase the fluxes through TCA cycle, while the single glycerol could enhance the fluxes through DAP and anaplerotic pathway (Fig. 2a, b). Interestingly, in the glucose–glycerol mixed carbon source, a complementary effect was observed. The fluxes from phosphoenolpyruvate to oxaloacetate and from oxaloacetate to l-lysine were all improved in the mixed carbon source (Fig. 2c), which provided sufficient carbon skeletons for the synthesis of oxaloacetate and precursor l-lysine and finally led to much higher ε-PL productivity.

The results of metabolic fluxes analysis were further validated by activities measurement of key enzymes (Fig. 3). The glycerol showed significantly higher activities of PEPC and Pls, which was beneficial to ε-PL assembly and quickly compensated the carbon skeletons exhausted through DAP pathway. On the contrary, the glucose exhibited higher activity of CS, which increased the metabolic capacity of TCA cycle. Notably, the mixed carbon source performed higher activities of PEPC, Ask and Pls than that in single glucose, meanwhile it exhibited higher activities of CS and Ask than that in single glycerol. Hence, the enzyme activity assay further confirmed that the mixed carbon source could directly enhance the metabolic capability for biosynthesis of precursor l-lysine and ε-PL, which was in good accordance with the results of metabolic fluxes analysis.

Besides the l-lysine provision, ε-PL biosynthesis was also influenced by intracellular energy status. It was reported that the biosynthesis of ε-PL consumed a large amount of ATP [22] and the Pls activity was highly regulated by intracellular ATP concentration [23]. In this work, the activity of energy metabolism in the mixed carbon source was investigated by determination of intracellular energy cofactors. In phosphorus-limited chemostat cultures, similar levels of energy charge were observed in the single glucose and glycerol at the same dilution rate of 0.05 h−1, while the single glycerol showed higher ratio of NADH/NAD+ than that in glucose. In fact, glycerol was believed as a more reductive substrate than glucose [24]. One mole of glycerol produces two more NADH molecules than equal mole of glucose do, which was beneficial for ATP production. Nevertheless, higher fluxes through TCA cycle in single glucose (Fig. 2a) provided more NADH for ATP biosynthesis, which relieved the relative energy shortage (Fig. 4b). In addition, phosphorus-limited environment also reduced the ATP production in single glycerol and finally resulted in the similar levels of energy charge in single glucose and glycerol. Even though, the mixed carbon source showed superiority in energy metabolism. The highest carbon fluxes through TCA cycle generated a large amount of NADH (Fig. 2c), which subsequently was used for ATP biosynthesis by cell respiration. With little phosphorus in broth, the production of ATP was relatively limited in the mixed carbon source. Therefore, on the energy metabolism, the advantage of mixed carbon source was mainly observed on much higher NADH/NAD+ ratio rather than the level of energy charge. Besides, it was worthy to notice that the high concentration of glucose/glycerol might inhibit the ε-PL production. Even though, as the same concentration of total carbon source (120 g·L−1), the mixed carbon source (glucose:glycerol = 60:60) could minimize the stress of single carbon source on the ε-PL-producing strain, which could be one of the benefits of the mixed carbon source.

In general, the advantages of glucose–glycerol mixed carbon source in batch culture mainly lies in fast cell growth and high ε-PL productivity. Our previous study confirmed the positive influence of fast cell growth on the ε-PL biosynthesis, while chemostat culture was employed in this study to understand the direct influence of mixed carbon source on ε-PL production. Metabolic analysis proved the positive effect of the glucose–glycerol combination on the ε-PL biosynthesis. The mixed carbon source performed a combined effect of both glucose and glycerol. It exhibited higher activities of key enzymes in TCA cycle, anaplerotic and DAP pathway, which supplied sufficient carbon skeletons for the biosynthesis of oxaloacetate, aspartate as well as the precursor l-lysine. Moreover, higher fluxes through TCA cycle guaranteed a rapid generation of NADH, which could subsequently be used for ATP biosynthesis. Furthermore, the Pls activity was also enhanced in the mixed carbon source compared with that in single glucose. Finally, higher levels of precursor, Pls activity and energy status synergistically promoted the ε-PL production. Therefore, the high ε-PL productivity was a combined effect of both rapid cell growth and the mixed carbon source itself.

Notes

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (31671846, 31301556), the Science and Technology Department of Jiangsu Province (BY2016022-25), the Fundamental Research Funds for the Central Universities (JUSRP51504), the Open Project Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (KLIBKF201302), and the Jiangsu Province Collaborative Innovation Center for Advanced Industrial Fermentation Industry Development Program.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

449_2018_1943_MOESM1_ESM.doc (1.2 mb)
Supplementary material 1 (DOC 1220 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jian-Hua Zhang
    • 1
  • Xin Zeng
    • 2
  • Xu-Sheng Chen
    • 1
  • Zhong-Gui Mao
    • 1
  1. 1.The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina
  2. 2.College of Life SciencesHuaibei Normal UniversityHuaibeiChina

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