Introduction

Lactic acid bacteria (LAB) are a heterogeneous group of low-GC, nonsporulating Gram-positive bacteria, which ferment a range of carbon sources primarily to lactic acid (Gaspar et al. 2013). In food industry, LAB are mainly used for food and beverage fermentation, production of add-in ingredients, bacteriocins, exopolysaccharides (Zhu et al. 2009; Table 1). In addition, LAB are also used to produce bulk and fine chemicals including organic acids, polyols, and vitamins (Gaspar et al. 2013; Table 1). However, as cell factories, LAB encounter various stress conditions during the industrial production and in the gastrointestinal tract. Among various environmental stresses, acid stress is one of the most important survival challenges, and acid tolerance is one of the criterias to select potential probiotics (Parvez et al. 2006). During fermentation, the growth of LAB is accompanied by lactic acid production leading to acidification of the media, arrest of cell growth, and possibly cell death due to the entrance of undissociated form of lactic acid into the cytoplasm by simple diffusion (Serrazanetti et al. 2009). Intracellular lactic acid dissociates, changes the intracellular pH, and disrupts the cytoplasm anion pool, which affects the integrity of purine bases and results in denaturing of essential enzymes inside the cells (Warnecke and Gill 2005). Thus, improving the acid stress resistance is important for the industrial application of LAB.

Table 1 Application of lactic acid bacteria
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In response to acid stress, LAB have evolved stress-sensing systems and employed numerous mechanisms to withstand harsh conditions and sudden environmental changes, including the maintenance of intracellular pH homeostasis, cell membrane functionality, and upregulation of stress response proteins (Lebeer et al. 2008; Wu et al. 2012b; O'Sullivan and Condon 1997; Fig. 1). In addition, acid tolerance response (ATR) appears to confer protection against environmental stresses by prior exposure of cells to moderately acidic conditions (De Angelis et al. 2001). Meanwhile, omics methods combined with molecular techniques have contributed to the understanding and validation of the molecular mechanisms involved in acid tolerance, and many feasible strategies (engineering general stress response proteins, maintaining cell membrane functionality, and regulating amino acid metabolism) have been proposed to improve the acid stress resistance of LAB (Wu et al. 2012a, 2013b; Trip et al. 2012). Thus, it is necessary to timely summarize the progress to further stimulate the research interest in this field. In this review, we provide an overview of the recent progress in engineering acid stress resistance of LAB, with emphasis on engineering intracellular microenvironment (engineering amino acid metabolism, introduction of exogenous biosynthetic capacity, and overproduction of stress response proteins) and maintaining cell membrane functionality based on physiological and omics analysis.

Fig. 1
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Responses of lactic acid bacteria as a cell factory to acid stress. S substrate, M intermediate, P product

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Engineering intracellular microenvironment of LAB

Regulation of intracellular amino acid metabolism

Regulation of intracellular amino acid metabolism is a common mechanism utilized by LAB upon environmental stresses. Arginine deiminase (ADI) system is a widely reported regulative system in LAB during acid stress. This system converts arginine and subsequently leads to the production of NH3, CO2, and ATP. The generation of ATP enables extrusion of cytoplasmic protons by H+-ATPase (Burne and Marquis 2000). Previous research demonstrated acid stress induced the accumulation of arginine in Lactobacillus casei, and Streptococcus faecium could degrade arginine at extremely low initial pH of 2.5 and raise the pH to nearly 8.0 with 80 mM NH3 accumulation (Wu et al. 2012a). Moreover, the addition of arginine improved the survival of L. casei during acid stress by increasing the activity of H+-ATPase and intracellular ATP levels (Zhang et al. 2012b). An example with Streptococcus suis showed that knockout of arcABC encoding genes involved in the ADI system resulted in decreased ammonia production and decreased cell growth during acidic conditions (Fulde et al. 2011).

Intracellular accumulation of aspartate is another response induced by LAB during acid stress, and the biomass and survival at low pH were significantly improved in the presence of aspartate (Wu et al. 2013a). In addition, an aspartate-dependent acid survival system was also characterized in Yersinia pseudotuberculosis. The expression of aspartase (AspA), which catalyzed the deamination of aspartate to form fumarate and ammonia, increased acid survival of Y. pseudotuberculosis (Hu et al. 2010).

Regulation of branched-chain amino acids (BCAA) leucine, isoleucine, and valine was also an ATR in LAB. During acid stress, the enzymes (IlvA, IlvC2, IlvD, and IlvE) involved in BCAA metabolism were overproduced, and deamination of BCAA was postulated as a mechanism to maintain the internal pH of the cells (Sánchez et al. 2007; Ganesan and Weimer 2004). Knockout of the ilvE gene led to decreased F0F1-ATPase activity and acid tolerance in Streptococcus mutans (Santiago et al. 2012). In addition, decarboxylation was also reported to protect cells against acid damage by generation of ATP and consumption of a single proton (Higuchi et al. 1997). Trip et al. (2012) heterologously expressed the histidine decarboxylation pathway in L. lactis, and this pathway enabled cells to survive at low pH in the presence of histidine.

Introduction of exogenous biosynthetic pathway

Nowadays, a vast genetic toolbox for the regulation of LAB gene expression levels is available, allowing the manipulation of acid tolerance through metabolic engineering (Table 2). Previous researches showed that glutathione can protect LAB against a variety of environmental stresses (acid, oxygen, cold and salt stresses; Kim et al. 2012; Zhang et al. 2010a, b, 2012a; Li et al. 2003). To further investigate the protective roles of glutathione during stressed conditions, two genes gshA and gshB, encoding γ-glutamylcysteine synthetase and glutathione synthetase, respectively, from Escherichia coli, were expressed in L. lactis NZ9000. As expected, the recombinant strain exhibited higher resistance to acid stress compared to the control strain (Zhang et al. 2007; Fu et al. 2006).

Table 2 Improving acid stress resistance of lactic acid bacteria by metabolic engineering
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Previous study showed that acid stress led to substantial accumulation of trehalose in Propionibacterium freudenreichii during acid stress (Cardoso et al. 2004). Inspired by this observation, Carvalho et al. (2011) introduced the P. freudenreichii trehalose de novo biosynthetic pathway into L. lactis to investigate the effect of trehalose production on the tolerance of host strain to acid stress. As expected, the mutant exhibited higher tolerance to acid (pH 3.0) and cold (4 °C) shock, as well as to heat stress (45 °C; Carvalho et al. 2011).

Overproduction of stress response proteins by genetic modification

With the development of genome sequencing and other high-throughput technologies, it has enabled us to engineer the robustness of industrial microbes at a global or systems biology levels (Zhu et al. 2012). At present, several systems biology approaches (e.g., genomics, transcriptomics, proteomics, metabolomics), combined with the molecular techniques have been employed to further understand the physiological mechanisms of LAB, and based on these, strategies to improve the physiological functions and engineer stress tolerance of LAB were proposed (Fig. 2). For example, Broadbent et al. (2010) investigated the acid stress response of L. casei ATCC334 during acid stress by transcriptional analysis, and the results showed that the two genes involved in malolactic fermentation (mleS, malolactic enzyme; mleP, malate/lactate antiporter) and eight genes cluster for histidine biosynthesis (LSEI_1426–1434) were significantly upregulated. To further validate the microarray data, 30 mM malate or 30 mM histidine were supplemented into the acid challenge medium, and the presence of either malate or histidine in the medium at pH 2.5 resulted in a more than 100-fold increase in cell survival after 60-min incubation, and greater than 107-fold improvement after 2 h (Broadbent et al. 2010).

Fig. 2
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Schematic representation of the approach to identify and validate stress-related genes via the omics-based technologies

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Generally, bacteria maintain protein homeostasis under normal or stressed conditions using various mechanisms including the action of a group of regulatory proteins. Likely, LAB upregulated the expression of general stress response proteins in response to environmental stress (Wu et al. 2011, 2012a), among which molecular chaperones and DNA repair proteins were widely investigated (Table 3). Heterologous expression of dnaK from E. coli in L. lactis NZ9000 resulted in improved tolerance to lactic acid, NaCl, and ethanol stresses (Abdullah-Al-Mahin et al. 2010). Tian et al. (2012) expressed a small shock protein (shsp) gene from Streptococcus thermophilus in L. lactis, and the recombinant strain displayed significantly higher survival rate under acid, heat, ethanol, bile salt, and H2O2 stresses. In addition, comparative proteomic analysis with L. casei parental strain and its acid-resistant mutant demonstrated that higher expression of DNA repair proteins (e.g., MutL, MutS2, UvrC, RecO) were observed in the mutant. Engineering the overproduction of DNA repair protein RecO in L. lactis NZ9000 was carried out, and the recombinant strain exhibited higher tolerance to lactic acid, salt, and H2O2 stresses (Wu et al. 2013b).

Table 3 Protectants used for improving acid stress resistance of lactic acid bacteria
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The whole-genome sequencing has yielded increasing numbers of completed genomes of LAB, many of which are publicly available on the Internet. This allows us to characterize their gene expression profiles and to identify the genes during environmental stresses. In addition, it provides an effective platform for us to engineer LAB with improved robustness. However, it should be noted that the strains obtained by genetic engineering may be hampered by legal issues and the general public opinion during industrial application. Therefore, further efforts should be made to ensure the acceptability of recombinant LAB in industrial manufacture, especially in food industry.

Engineering cell membrane functionality

As the first barrier of the cell, cell membrane separates cells from their environments and is a primary target for damage during environmental stresses. Changes in the cell membrane can protect the cell from environmental damage by modifying the physicochemical properties of membrane (Mykytczuk et al. 2007). Upon acid stress, L. casei increased the fluidity of cell membrane and increased the proportions of monounsaturated fatty acids, as well as mean chain length (Wu et al. 2012b). Therefore, engineering the production of unsaturated fatty acids could be a potential method to increase the acid tolerance of LAB. FabM, a novel enzyme, responsible for the production of monounsaturated fatty acids, was identified in S. mutans, and the FabM-defective mutant was extremely sensitive to acid stress compared with the wild type (Fozo and Quivey 2004). However, the acid-sensitive phenotype was relieved by growth in the presence of monounsaturated fatty acids or through genetic complementation (Fozo and Quivey 2004). In addition, production of cyclopropane fatty acids (CFA) was also a general stress response to acid stress (Broadbent et al. 2010; Wu et al. 2012b). Previous work with E. coli demonstrated that the CFA-defective mutant exhibited decreased resistance to acid stress, and the acid tolerance was restored by incorporation of a functional cfa gene (Chang and Cronan 1999). Conversely, recent work with L. lactis subsp. cremoris wild-type strain, the cfa mutant, and the complemented strain showed that the cyclopropanation of unsaturated fatty acids was not essential for survival under acidic conditions (To et al. 2011). Thus, further investigation concerning the detailed protective mechanisms of CFA during acid stress is necessary.

Adaptive evolution

Adaptive evolution, as a convenient approach to study many microbial phenomena, such as the emergence of new pathogens and the acquisition of environmental resistance factors, can address fundamental question on adaptation to selection pressures and evolution, and it has also become a widely used tool for biotechnological applications, improving yields and reducing costs in industrial settings (Fig. 3; Portnoy et al. 2011; Fong et al. 2005; Wang et al. 2011). Recently, adaptive evolution has been used with great success to gain insight into the genetic basis and dynamics of adaptation (Teusink et al. 2009). An example with L. casei, Zhang et al. (2012b) isolated acid-resistant mutant lb-2 by adaptive evolution for 70 days, and the evolved mutant exhibited higher biomass and survival during acid stress. Analysis of the intracellular microenvironments showed that the acid tolerance mutant displayed higher intracellular pH and NH4 + concentration in acidic conditions. In addition, at least 40.0 % and 23.9 % higher contents of intracellular arginine and aspartate were observed in the mutant compared with that in the parental strain (Zhang et al. 2012b). Moreover, proteomic analysis showed that higher expressions of many proteins including chaperonin (groEL), DNA repair protein (RecO) were observed in the evolved strain, and the overproduction of RecO in L. lactis led to increased tolerance to acid and NaCl stresses (Wu et al. 2012a, 2013b). These results suggest that adaptive evolution might be a useful method to engineer robust LAB.

Fig. 3
figure 3

Application of adaptive evolution. a Investigation of the mechanisms to environmental adaptation. b Engineering cellular metabolism for enhanced biosynthetic capacity of desired product (a) and decreased amount of by-product (b). c Improved robustness to environmental stresses. d Schematic representation of the procedure for adaptive evolution

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Pre-adaptation and cross-protection

In response to environmental stress, LAB employ sophisticated mechanisms to combat stress. In many cases, similar responses were induced during different stressful conditions (heat, acid, oxygen, and cold), and as such, these mechanisms of resistance were interconnected. In this respect, preadaptation (pretreatment of a strain to a sublethal level can improve its resistance toward a potential severe stress) and cross-protection (one kind of stress tolerance confers protection against other stresses). Notably, Broadbent et al. (Broadbent et al. 2010) enhanced the survival of L. casei ATCC 334 to severe acidic conditions (pH 2.0) by prior exposure of the cells at pH 4.5 for 20 min. Moreover, a dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by pre-exposing the L. lactis subsp. lactis cells for 30 min to a mildly acid shock at pH 5.5 (Hartke et al. 1996). Cross-protection was also reported as an effective approach to increase the acid stress resistance of LAB. For example, L. plantarum pre-exposed to sublethal heat treatment displayed enhanced growth at pH 5.0 (De Angelis et al. 2004). Generally, pre-exposure to mild acidic condition induced an ATR, which protected cells against multiple-environmental stresses. Pre-exposure of L. lactis subsp. cremoris to sublethal acid treatment displayed enhanced tolerance to acid, heat, NaCl, H2O2, and ethanol stresses (O'Sullivan and Condon 1997). In conclusion, pre-adaptation and cross-protection led to significant improvement of LAB to acid stress. However, the exact molecular mechanisms involved in pre-adaptation and cross-protection are not fully understood, and further exploration is also needed.

Exogenous addition of protectants

Exogenous addition of protectants is a relatively straightforward way to protect cells against acid stress or improve acid tolerance of LAB. Recently, numerous protectants have been developed to protect LAB against acid stress including amino acids, fatty acids, and saccharides (Table 3). For example, the addition of arginine increased the survival of L. casei Zhang at low pH (Zhang et al. 2012b). Physiological analysis showed that the exogenous arginine improved the viability of cells during acid stress by increasing the H+-ATPase activity and intracellular ATP level (Zhang et al. 2012b). Generally, regulation of ADI system is a widely reported ATR in a variety of bacteria including LAB, E. coli, S. mutans, Listeria monocytogenes, and Bacillus spp. (Zhao and Houry 2010; Senouci-Rezkallah et al. 2011; Matsui and Cvitkovitch 2010; Ryan et al. 2009). In another study with L. casei, aspartate was supplemented into the MRS media, and this led to the increment of the biomass and survival during acid stress (Wu et al. 2013a). The subsequent analysis of the intracellular microenvironment revealed that higher concentrations of intermediates involved in glycolysis and tricarboxylic acid cycle were observed, and L. casei shifted the metabolic pathway by increasing the flux from aspartate to arginine (ADI system) and decreasing the flux from aspartate to asparagine (Wu et al. 2012a, 2013a). Yet another example revealed that the addition of Tween-80 to the growth medium of L. rhamnosus resulted in 1,000-fold higher survival during exposure to gastric juice (Corcoran et al. 2007). Analysis of the fatty acids composition of L. rhamnosus revealed a 55-fold higher oleic acid content and a significantly higher unsaturated/saturated fatty acids ratio in the membrane of cells in the presence of Tween-80 (Corcoran et al. 2007). These results suggest that exogenous addition of protectants could be a feasible strategy to improve acid stress resistance of LAB.

Concluding remarks

Acid stress is a common environmental challenge to LAB, and improving the acid stress resistance is crucial to the application of LAB as probiotic. The advent of genome sequencing has increase our understanding of the molecular biology of LAB, and based on this, many post-genomic approaches (such as omics method) have accelerated the identification of genes/proteins involved in stress response and tolerance. This knowledge contributes to the design of rational approaches to engineering LAB with increased robustness. Moreover, specific fermentation conditions may be employed on the basis of the understanding of characterization of LAB to increase the stress tolerance. In conclusion, these systems biology approaches combined with the molecular techniques have provided us more opportunities to engineering LAB with improved robustness and industrial functionality.