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Ann Microbiol (2017) 67:383–389 DOI 10.1007/s13213-017-1268-0 ORIGINAL ARTICLE GC-MS based metabolomics analysis reveals the effects of different agitation speeds on the level of proteinogenic amino acids in Lactococcus lactis subsp. cremoris MG1363 1 1 1 Shuhaila Sharif & Kamalrul Azlan Azizan & Syarul Nataqain Baharum Received: 2 January 2017 /Accepted: 19 April 2017 /Published online: 2 May 2017 Springer-Verlag Berlin Heidelberg and the University of Milan 2017 Abstract Lactococcus lactis subsp. cremoris MG1363 is an Heatmap analysis showed that the levels of pyruvate-, opportunistic lactic acid bacterium (LAB) that has emerged as glutamate- and aspartate-based amino acids were varied under one of the most promising candidate cell factories. The avail- the different agitation conditions. The time-series analysis ability of genome-level information and U.S. Federal Drug showed an increment of lysine when L. lactis’ cells were cul- tured with shaking at 50, 100 and 200 rpm. Taken together, administration’sdesignation of ‘generally recognized as safe’ (GRAS) are two of the more important key factors for its these results highlight the changes in the levels of PAAs in wide-ranging applications in numerous biotechnological pro- L. lactis cells in response to agitation. In addition, the collect- cesses. Several studies have shown that various physiological ed dataset will be useful for optimization of C-labeling conditions, such as temperature, salinity and pH, can influence based experiments in L. lactis. the physiological growth of L. lactis; agitation, in particular, . . can increase the production of amino acids and fermentation Keywords Microbial metabolomics GC-MS L. lactis by-products. However, the effect of different agitation speeds MG1363 Proteinogenic amino acids on the growth of L. lactis’ has rarely been examined. In the study reported here, we used a gas chromatography–mass spectrometry-based metabolomics approach to investigate Introduction the effects of different agitation speeds on the production of proteinogenic amino acids (PAAs) by L. lactis MG1363. Lactococcus lactis is a Gram-positive, mesophilic and Lactococcus lactis MG1363 was grown under four different microaerophilic lactic acid bacterium (LAB) that is of great agitation speeds (50, 100, 150 and 200 rpm) at a constant importance in the fermentation industry (Miyoshi et al. 2003; temperature of 30 °C, and the differences in the specific Lahtvee et al. 2011). In fermentation processes, L. lactis growth rate and levels of PAAs were determined. strains are used as starter culture and can contribute to the taste Approximately 15 PAAs with concentrations ranging from 0 and flavor of the fermented end-products by producing organ- to 50 mmol/L were detected under all conditions. Partial least oleptic properties (Brandsma et al. 2012; Flahaut et al. 2013; squares discriminant analysis (PLS-DA) revealed a distinct Dijkstra et al. 2014; van de Bunt et al. 2014). Lactococcus difference when L. lactis was incubated at 100 and 150 rpm. lactis is known to be auxotrophic for several amino acids, with the specific amino acids varying among strains. It has been Electronic supplementary material The online version of this article shown that L. lactis strains require six essential amino acids, (doi:10.1007/s13213-017-1268-0) contains supplementary material, namely, methionine (Met), glutamate (Glu), valine (Val), his- which is available to authorized users. tidine (His), leucine (Leu) and isoleucine (Ile), for growth (van Niel and Hahn-Hagerdal 1999; Oliveira et al. 2005). Like * Syarul Nataqain Baharum other LAB, L. lactis uses amino acids to perform physiologi- firstname.lastname@example.org cal functions, such as intracellular pH control, the generation 1 of metabolic energy or redox power and stress resistance Metabolomics Research Laboratory, Institute of Systems Biology (Fernández and Zúñiga 2006). Moreover, amino acids play (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia important roles in the central metabolic pathway of L. lactis. 384 Ann Microbiol (2017) 67:383–389 For example, amino acids such as alanine (Ala), cysteine that agitation speed plays an important role in increasing the (Cys), glycine (Gly), serine (Ser), aspartate (Asp) and threo- viability of L. lactis when subjected to environmental nine (Thr) are responsible either directly or indirectly for the perturbations. production of pyruvate (Pyr), which then leads to fermentation by-product metabolites of lactate, acetate and ethanol (Adamberg et al. 2012). Materials and methods Consequently, during fermentation processes, L. lactis strains are exposed to a number of stresses that affect the Bacterial cultivation growth and performance of L. lactis starters (Dijkstra et al. 2014). Several studies have reported physiological changes in The Lactococcus lactis subsp. cremoris MG1363 used in this L. lactis when exposed to stresses such as pH (Carvalho et al. study was provided by Raha Abd. Rahim from Universiti 2013;deJongetal. 2013), temperature (Ibrahim et al. 2010; Putra Malaysia, Serdang, Malaysia, courtesy of K. Azizan et al. 2012), oxidative stress or aeration (Nordkvist Leenhouts from University of Groningen, Groningen, et al. 2003; Ibrahim et al. 2010;Cretenet etal. 2014; Dijkstra The Netherlands. All cultivations were performed as batch et al. 2014) and nutrient starvation (Dressaire et al. 2011). cultivations with working volumes of 100 mL. Cells were More importantly, these studies were mainly conducted under grown in M17 medium (ascorbic acid, 0.5 g/L; MgSO , anaerobic conditions to mimic the fermentation process condi- 0.25 g/L; disodium glycerophosphate, 19 g/L; tryptone, 5 g/ tions. As facultative anaerobes, L. lactis is able to grow under L; soytone, 5 g/L; beef extract, 2.5 g/L; yeast extract, 2.5 g/L; aerobic conditions, and it has an oxygen resistance mechanism Oxoid Ltd. Hampshire, UK) media with glucose (0.5 g/L) as that enables it to adapt to certain levels of toxic substances in the carbon source, with a minimum of three biological repli- the environment, such as superoxide (Pedersen et al. 2012). Its cates. Incubation was performed at 30 °C with shaking at ability to tolerate oxygen at an early stage of growth is a great agitation speeds of 50, 100, 150 and 200 rpm, respectively. advantage in terms of maintaining cell viability during early The control was L. lactis cells grown at 30 °C without agita- cultivation. It has been reported that aeration during fermenta- tion. Cell growth was monitored by measuring optical density tion triggers resistance against heat stress, whereas high tem- at 600 nm (OD ) on a spectrophotometer (model DU 800; perature leads to robustness to oxidative stress (Cretenet et al. Beckman Coulter, Brea, CA). The specific growth rate (μ) 2014). Ibrahim et al. (2010) reported that recombinant L. lactis was calculated using a linear regression of the plots of responds differently when grown at different agitation speeds ln(OD ) versus time during the exponential growth phase. and temperatures. The results of a previous study by our group suggest that L. lactis MG1363 produces various low- Extraction of PAAs molecular-weight metabolites when grown under agitation (Azizan et al. 2012). In that study, levels of organic acids pri- Cellular extracts for all experiments were obtained by harvest- mary alcohols, aldehydes, amino acids and fatty acids in- ing the cells at different time points (3, 5, 6, 7 h) during the creased when L. lactis MG1363 was grown at 30 °C and an exponential and stationary phases. More specifically, the bac- agitation speed of 150 rpm (Azizan et al. 2012). terial culture was centrifuged at 13000 rpm for 10 min at 4 °C, In order to understand the association between amino acid following which the pellet was washed twice with 1 mL phos- levels in L. lactis and agitation speed, it would be useful to phate buffer saline solution and transferred into a new glass analyze and measure the metabolism patterns of proteinogenic vial. Approximately 200 μL of 6 M HCl was then added to the amino acids (PAAs) found in L. lactis. PAAs are relatively cell pellet, and the cell pellet subjected to cell hydrolysis by stable and abundant inside the cell (Szyperski 1995; heating at 105 °C for 16 h. The cell hydrolysate was then Zambonietal. 2009). Through analysis of these protein- further dried at 95 °C for 6 h. bound amino acids, the response of metabolic pathways inside the cell to environmental stresses can be analyzed in more Detection and quantification of PAAs detail, specifically, central carbon and amino acid metabolism (Szyperski 1995). The derivative technique was used as described by Zamboni et al. In this study, we used a gas chromatography mass spec- (2009). Cell hydrolysates were heated for 10 min at 105 °C to dry trometry (GC–MS)-based metabolomics approach to investi- the samples completely prior to derivatization. The dried samples gate the effects of different agitation speeds on the production were then dissolved in dimethylformamide and derivatized with of PAAs by L. lactis MG1363. Briefly, L. lactis was cultivated N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% at 30 °C under four different agitation speeds, and the PAAs tert-butyldimethylchlorosilane for 1 h at 85 °C. The derivatized were extracted from the cell pellets and quantified. The results samples were then transferred into new GC vials and analyzed by indicate notable differences in the levels of PAAs when GC–MS (Zamboni et al. 2009; Azizan et al. 2012). The PAAs L. lactis was exposed to different agitation speeds, suggesting detected were then quantified using an external calibration curve. Ann Microbiol (2017) 67:383–389 385 GC-MS parameters Results An aliquot of approximately 1 μL was injected into an Elite- Growth characteristics of L. lactis MG1363 at different 5MS capillary column coated with 5 % diphenyl 95 % dimeth- agitation speeds yl polysiloxane (ID 30 × 0.25 mm, thickness 0.25 μm) (PerkinElmer, Waltham, MA). The injection temperature Cultures of strain MG1363 were grown at four different agi- was set to 250 °C, and the ion source was set to 200 °C. The tation speeds (50, 100, 150, and 200 rpm) at 30 °C with a GC system was set to range from 70 to 300 °C with a constant limited supply of glucose, resulting in variation in growth −1 helium gas flow at 1.1 min . Measurements were performed characteristics, including specific growth rate (μ) and optical in a scan mode of m/z 180–550. Analysis was performed using density (OD ). Incubation at an agitation speed of 100 rpm −1 the PerkinElmer TurboMass Clarus 600 spectrometer coupled resulted in the highest growth rate (0.890 h ), and incubation to a quadruple mass selective detector with electron ionization at an agitation speed of 200 rpm resulted in the lowest growth −1 operated at 70 eV. rate (0.686 h ). Overall, the growth rate was lower at 200 rpm than at 50, 100, and 150 rpm [Electronic Supplementary Material (ESM) Table 1].The maximum OD at the end of Multivariate statistical analysis of GC-MS data incubation period ranged from 0.45 to 0.47 (Fig. 1). There was no difference in the duration of lag phase of cultures grown at The general approach used for data analysis and validation the different agitation speeds and, in addition, the different was performed according to Villas-Bôas et al. (2006)and agitation speeds had no effect on the growth profiles of Smart et al. (2010). In summary, detected metabolites were L. lactis. Before this bacterium entered the exponential phase identified using an in-house TMS library of derivatized pure it had a lag phase of 2 h. The mid-exponential phase of standards and the 2008 National Institute of Standards and L. lactis was reached after 3–4 h of incubation, while the Technology (NIST) mass spectral database library (NIST, stationary phase started after 6 h of incubation. Gaithersburg, MD) with a similarity cut-off of 90%. The area of the peak was used to represent the abundance of the detect- ed metabolites. The values were normalized by sample medi- PAAs and multivariate statistical analysis an followed by log transformation. One-way analysis of var- iance (ANOVA) followed by comparison using Fisher’sleast Specifically, 16 PAAs were identified using GC-MS, as significant difference and two-way ANOVA was used where shown in ESM Fig. 1. The area of each PAAs was then further necessary to statistically validate the values and method with a processed using multivariate statistical analysis (Fig. 2). significance level of P ≤ 0.05. Visualization of the clean, val- Except for proline, all PAAs displayed p <0.05. idated data was then conducted using principal component PCA was first used to show treatment-to-treatment varia- analysis and partial least squares-discriminant analysis (PLS- tion and to identify variations in the level of PAAs when the DA) in Simca-P+ version 12.0 (Umetrics AB, Ume, Sweden) culture was grown at different agitation speeds (Fig. 2a, b), for group classification and discrimination analysis (Azizan and PLS-DA was then performed to provide a better visuali- et al. 2012). zation of the treatment clustering found by PCA. As shown in Fig. 1 Growth profiles of Lactococcus lactis during incubation at 30 °C and at different agitation speeds. Lightly- shaded long-dashed line, filled diamond 0 rpm, short-dashed/ dotted line, filled circle 50 rpm, short-dashed line, filled square 100 rpm, dotted line, filled triangle 150 rpm, dark long- dashed line, X 200 rpm 386 Ann Microbiol (2017) 67:383–389 Fig. 2 Principal component analysis (PCA) score scatter plot (a)and 29.39 and 18.87%, respectively. d PLS-DA-derived loading scatter plot loading scatter plot (b) of proteinogenic amino acid (PAA) profiles that analysis of profiled metabolites. PLS-DA analysis with agitation speed as distinguished L. lactis grown under non-agitated and agitated conditions. the y variable was used to identify the metabolites that distinguished The proportions of variance between spectra explained by principal L. lactis grown under non-agitated and agitated conditions. Open box components 1 and 2 were 27.45 and 22.95 %, respectively. c Partial (d) Metabolites with variable importance for projection (VIP) values of least squares-discriminant analysis (PLS-DA)-derived score plot >1 were highlighted in red box. Agitation speeds: Cross 0rpm, X 50 rpm, analysis of metabolite profiles with latent variable (LV) 1 and 2 of filled diamond 100 rpm, filled triangle 150 rpm, filled square 200 rpm Fig. 2c, d, good clustering was observed in the score plot of Pattern of PAA changes in response to time PLS-DA, with a total variance of 48 % (LV1 29.39%, LV2 18.87%). The score plot showed clear discrimination of be- To further characterize the metabolic changes at the level of tween cells grown at 100 and 150 rpm. The loading plot sug- PAAs in response to different agitation speeds, we harvested gested that Ala, Gly and Met were the PAAs which most cell pellets at four different time points (3, 5, 6 and 7 h of contributed towards the separation of clusters at 100 and incubation) and compared the PAA profiles. As shown in 150 rpm. To evaluate the metabolic changes in the levels of Fig. 4, the levels of the majority of PAAs decreased from 3 PAAs, a heatmap was constructed to compare the overall to 7 h of incubation. However, notable increases in the levels levels of PAAs in response to different agitation speeds. of some PAAs were observed at the end of the incubation The heatmap in Fig. 3 revealed differences in the levels of period of 8 h, with the level of Lys increasing by 0.9, 0.3 PAAs detected when L. lactis was grown at the four different and 0.25-fold at 50, 100, and 200 rpm, respectively. In addi- agitation speeds. The levels of Ala and Gly were higher at an tion, the level of ornithine (Orn) increased by 0.82-fold from 3 agitation speed of 100 rpm and lower at 150 and 200 rpm, to 6 h and decreased slightly by 0.1-fold at 7 h of incubation at while those of Leu, Ile and lysine (Lys) were higher at 50 and 200 rpm. 200 rpm but lower at 150 rpm. The heatmap showed that the levels of branched-chain amino acids (Ile, Leu) were higher at 200 rpm, at which agitation speed the culture showed a low Discussion specific growth rate. However, a comparison between cultures grown under agitation and the control showed that the levels The variations in the levels of PAAs shown in Fig. 3 may of Leu, Ile, tyrosine (Tyr) and Met were higher when grown suggest the response mechanisms of central metabolism of without agitation. L. lactis towards agitation perturbations. The results may also Ann Microbiol (2017) 67:383–389 387 we compared the levels of PAAs in L. lactis MG1363 cells cultured at different agitation speeds. The level of Orn was the highest at 200 rpm despite a low specific growth rate. Our findings are similar to those of Dressaire et al. (2008) and Lahtvee et al. (2011) who suggested that genes encoding en- zymes in the Orn biosynthesis pathway through the arginine deiminase pathway and in Glu metabolism are upregulated at the lowest specific growth rate. These findings highlight the importance of Orn metabolism in the growth rate adaptation of L. lactis MG1363. Concomitantly, in our study a high specific growth rate was associated with a decrease in amino acid metabolism and low levels of Orn, which was also reported by Dressaire et al. (2011). In our study the level of Lys increased throughout the ex- Fig. 3 Heatmap of PAAs detected at different agitation speeds. Amino ponential phase until the early stationary phase. The observed acids in bold font act as a precursor to the other amino acids in the same increase in the level of Lys might be due in part to the lack of a group. Color coding: Red Relatively high abundance, black median Lys transport system in L. lactis subsp. cremoris (Dreissen abundance, green relatively low abundance. PYR Pyruvate, 3PG 3- phosphoglyceric acid et al. 1989). Lys and arginine (Arg) are used by some lactococcal strains as cell-wall constituents. In the basic amino provide an overview of the production and consumption acid transport system, Arg is degraded into Orn through the of amino acids by L. lactis MG1363 at different agitation arginine deiminase pathway, which yields ATP. This ATP is speeds. then used to generate proton-motive force, which acts as a To further demonstrate the relation between the specific driving force for Lys accumulation. Since L. lactis subsp. growth rates of L. lactis MG1363 and the PAAs produced, cremoris lacks the Arg:Orn antiporter, which somehow helps Fig. 4 Levels of PAAs in the amino acid pathways. Each column of each section of each graph represents the growth phases studied (left to right incubation time: 3, 5, 6, 7 h). Patterns of bars as indicated in the Legends box indicate agitation speed. The pathway is adapted from the KEGG database. Glu Glucose, ACoA acetyl-CoA, CIT citrate, AKG α-ketoglutarate, OAA oxaloacetate, TCA tricarboxylic acid. Asterisk PAAs showing a significant change at P ≤ 0.05 388 Ann Microbiol (2017) 67:383–389 to counterflow the uptake of Lys and Arg, Lys is accumulated L. lactis varied at the different agitation speeds. Thee levels of inside the cell. This interpretation is supported by the increase amino acids originating from the TCA cycle at 0 rpm were in the level of Lys detected inside the cell during the incuba- expected to be low—but were instead high. This result strong- tion period. ly suggests that these amino acids may be produced from other In L. lactis, branched-chain amino acids (BCAAs), namely, precursors or pathways other than the TCA cycle; for exam- Val, Leu and Ile, are essential for protein synthesis and act as ple, Thr can be derived from Gly (Thr < − >Gly + acetalde- precursors for volatile compounds. BCAAs are degraded into hyde). This notion is supported by Oliveira et al.’s(2005) α-keto acids by branched chain aminotransferase (BcaT) and study. The ability to perform biphasic fermentative fermenta- aromatic aminotransferase (AraT). To produce volatile com- tion and respiration helps L. lactis to maintain better long-term pounds, they are further converted to aldehydes and esters survival if exposed to oxygen (Wegmann et al. 2007). (Dressaire et al. 2008, 2011; García-Cayuela et al. 2012). Furthermore, under aerobic conditions and limited carbon The biosynthesis of BCAAs is regulated by the CodY repres- sources, Pyr is converted into acetyl-CoA by pyruvate dehy- sor protein.CodY ismediatedbyIle,sothatanexcessofIle drogenase (Oliveira et al. 2005) and produces mixed-acid fer- leads to growth inhibition by blocking CodY-dependent path- mentation products. Thus, L. lactis MG1363 can switch to ways. In our study, high levels of Ile were detected at 200 rpm, hetero-fermentative fermentation to generate more ATP than accompanied by lower specific growth rates of L. lactis,as the homolactic pathway, even though the end-products of the also demonstrated by Guédon et al. (2005). mixed-acid pathway are more inhibitory than lactate to the We observed that the level of Ala was higher when L. lactis bacterium itself (Price et al. 2012). This result strongly sug- cells were cultivated at 100 rpm but lower at 150 rpm. Ala is a gests that agitation will affect the production of TCA cycle- Pyr-based amino acid which is produced through the reduc- derived amino acids by supplying more dissolved oxygen to tion of Pyr by alanine dehydrogenase (Hols et al. 1999). Ala is the medium, as demonstrated by Chen et al. (2013). also used by LAB as a cell-wall constituent (Chapot-Chartier and Kulakauskas 2014). This amino acid is of great interest to both the food and pharmaceutical industries in the context of Conclusion bioengineering activity which would turn Lactococcus into a cell factory to produce Ala instead of lactate (Papagianni In this study, we demonstrated metabolic changes in PAAs in 2012). We found that the level of Gly was high at agitation response to different agitation speeds. The PLS-DA score plot speeds of 50, 100, and 150 rpm. This finding is in agreement showed that Met, Ala and Gly were the PAAs which most with a study conducted by Lahtvee et al. (2011) in which the contributed towards the separation of clusters at 100 and consumption of Gly was observed to increase with increasing 150 rpm. The time-series analysis showed an increase of Lys growth rate. We found different levels of Met at the different at 50, 100 and 200 rpm. These results highlight the importance agitation speeds. This sulfur-containing amino acid is known of PAA inter-relationships as an adaption response to environ- to be one of the essential amino acids for L. lactis growth, and mental perturbation. Moreover, the results are useful for future it is an important component of flavor formation. optimization of C–labeling-based experiments in L. lactis. Interestingly, Met can be used to regenerate NAD when ar- ginine is used as the energy source (Brandsma et al. 2012). 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