Enhanced Cadaverine Production by Recombinant Corynebacterium Glutamicum with Response Regulator DR1558 at low pH Conditions


 Background: Corynebacterium glutamicum is used industrially to produce various bio-based organic acids. However, it is often cultivated under abiotic stress conditions, such as low pH, which can reduce both cell growth and the yield of the target compound. Here, a response regulator from Deinococcus radiodurans, DR1558, was introduced into a recombinant C. glutamicum strain expressing lysine decarboxylase (cadA) to enhance cadaverine production at acidic pHs.Results: During batch cultivation under acidic conditions, 6.4 g/L of cadaverine was produced by the recombinant C. glutamicum strain expressing cadA and dr1558; this yield was 1.7-fold higher than that produced by a recombinant C. glutamicum strain expressing only cadA. Transcriptional analysis revealed altered expression levels of stress defense- and cadaverine biosynthesis-related genes in the recombinant C. glutamicum strain expressing dr1558. During fed-batch cultivation, the recombinant C. glutamicum strain expressing cadA and dr1558 showed a 2.4-fold increase in cadaverine production compared to that produced by the recombinant C. glutamicum strain expressing only cadA. The cell growth of C. glutamicum expressing both cadA and dr1558 increased markedly during fed-batch cultivation at acidic pH.Conclusion: These results indicated that the response regulator dr1558 altered the expression of genes involved in metabolic pathways and stress defense mechanisms in C. glutamicum. Furthermore, C. glutamicum expressing the D. radiodurans dr1558 can be used to produce bio-based organic acids by fermentation in processes requiring acidic conditions.

When generating a target product through microbial cultivation, the microbial strain may be exposed to various stresses, depending on the requirements for target production, including high osmotic pressure, high temperature, and an unfavorable pH [7][8][9]. Among these stresses, low pH is a common factor that can reduce the yield of bio-based compounds produced by fermentation [10]. The acidic pH required for enzyme activity during the production of cadaverine using CadA leads to decreased cell growth and lower productivity. Recently, a response regulator from Deinococcus radiodurans, DR1558, was introduced into a recombinant Escherichia coli strain to minimize the effects of stress under acidic conditions [11]. D. radiodurans is highly resistant to abiotic stresses, including gamma radiation, reactive oxygen species (ROS), and oxidants [11][12], and dr1558 is one of the genes responsible for its remarkable resistance. Introduction of dr1558 improved the stress resistance of E. coli [11]. The dr1558 gene has also been introduced into recombinant E. coli to improve the production of succinate, polyhydroxybutyrate, γaminobutyric acid, and 2,3-butanediol [13][14][15][16]. Since the expression of dr1558 increased the tolerance of E. coli cells to low pH, it was expected that it would also increase the productivity of cadaverine, especially at an acidic pH.
In this study, a recombinant plasmid that expressed both E. coli cadA and D. radiodurans dr1558 was constructed and transformed into C. glutamicum. Cadaverine biosynthesis by the C. glutamicum strain expressing cadA and dr1558 and a strain expressing only cadA was compared. To investigate the metabolic changes induced by introducing dr1558 into C. glutamicum, the changes in the transcriptome of recombinant C. glutamicum expressing dr1558 was analyzed. Finally, cadaverine production by the recombinant C. glutamicum strain expressing cadA and dr1558 was assessed in fed-batch cultivation at acidic pH.

Materials And Methods
Strains, plasmids, and culture media All bacterial strains and plasmids used in this study are listed in Table 1. E. coli XL1-Blue (Stratagene, La Jolla, CA, USA) was used for general cloning. C. glutamicum KCTC 1857 was obtained from the Korean Collection for Type Cultures (KCTC; Joengeup, Republic of Korea). The plasmids used for the expression of the E. coli cadA and D. radiodurans dr1558 genes under the control of the synthetic H30 promoter were constructed as described below. Recombinant C. glutamicum KCTC 1857 strains were constructed to express cadA with or without dr1558. E. coli XL1-Blue was cultured at 37°C in Lysogeny-broth medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl). C. glutamicum was grown in CG-50 medium (50 g/L glucose, 15 g/L yeast extract, 15 g/L (NH 4 ) 2 SO 4 ·7H 2 O, 0. Fed-batch fermentations were carried out at 30°C and an initial agitation speed of 200 rpm in 2.5-L jar fermenters (BioCNS) initially containing 500 mL of CG-100 medium. USA). The agitation speed was increased to maintain the dissolved oxygen (DO) level above 10%. Cell growth was monitored by measuring the optical density of the culture broth at 600 nm (OD 600 ).

Rna Extraction And Quantitative Real-time Pcr (Qrt-pcr)
A transcriptional analysis was performed to evaluate the gene expression changes in the C. glutamicum strain expressing dr1558. Cells were harvested by centrifugation (14,000 g, 10 min, 4°C) at 10 h during batch fermentation, and total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Venlo, Netherlands), according to the manufacturer's protocol. The qRT-PCR analysis was performed using TB Green Premix ExTaq (TaKaRa Bio, Shiga, Japan) under the following cycling conditions: 40 cycles of 95°C for 10 s and 58°C for 30 s. The primers used are listed in Supplement Table 1. Data were analyzed using the 2 −ΔΔCt method, and 16S rRNA was used as an internal control [19]. The experiment was performed in triplicate using an applied Eco™ Real-Time PCR System (Illumina, Inc., San Diego, CA, USA).

Analytical Procedures
The concentrations of organic acids and glucose were determined by high-performance liquid chromatography using an In nity 1260 system (Agilent Technologies, Santa Clara, CA, USA). The glucose concentration was determined using an Aminex HPX-87H ion exclusion column (Bio-Rad, Hercules, CA, USA). The mobile phase was 5 mM H 2 SO 4 , the ow rate was 0.6 mL/min, and the column was maintained at 50°C. The concentrations of cadaverine and lysine were determined using an ZORBAX SB-C18 column (Agilent Technologies). The mobile phase was 25 mM sodium acetate buffer (pH 4) and 1 M acetonitrile, the ow rate was 1 mL/min, and the column was maintained at 35°C. The concentrations of cadaverine and lysine were measured after diethyl ethoxymethylenemalonate (DEEMM) derivatization [20].

Results And Discussion
Production of cadaverine by the recombinant C. glutamicum strain expressing cadA and dr1558 during batch fermentation To investigate the effects of the D. radiodurans response regulatory gene dr1558 on C. glutamicum, cell growth, glucose consumption, and lysine and cadaverine production were compared between the recombinant strain expressing both cadA and dr1558 (Cg-cadA + dr1558) and a recombinant strain expressing only cadA (Cg-cadA; control) ( Fig. 1) during batch cultivation.
The results of the batch fermentation con rmed that the recombinant strain Cg-cadA + dr1558 showed signi cantly increased rates of cell growth and glucose consumption in batch fermentation, compared to those of Cg-cadA. Even after the pH of the medium was adjusted to 5.7, the growth rate of Cg-cadA + dr1558 continued to increase, whereas the growth rate of Cg-cadA decreased. Additionally, despite the acidic conditions, Cg-cadA + dr1558 consumed 100 g/L of glucose within 20 h. In contrast, glucose remained detectable in the medium of the control fermentation at 24 h. The amounts of cadaverine produced in the fermentations were 3.29 g/L for Cg-cadA and 6.39 g/L for Cg-cadA + dr1558, representing a 1.9-fold increase in cadaverine production. From Fig. 1, the maximum speci c growth rate and speci c cadaverine productivity of Cg-cadA + dr1558 were 0.302 g cells/L/h and 2.697 mg cadaverine/g cells/L/h, respectively. However, the maximum speci c growth rate and speci c cadaverine productivity of Cg-cadA were decreased to 0.0653 g cells/L/h and 1.071 mg cadaverine/g cells/L/h, respectively. This indicates that the speci c growth rate was greatly increased by the expression of dr1558 at acidic pH conditions.
It was previously reported that E. coli expressing dr1558 showed greater resistance to acidic conditions as well as the enhanced production of polyhydroxybutyrate and 2,3-butanediol due to the altered expression of genes in metabolic pathways [14,15]. Based on these ndings, we expected changes in the expression levels of genes related to cadaverine production and glucose consumption. To investigate the alterations in the expression of these genes in the recombinant C. glutamicum strain expressing dr1558, a transcriptional analysis was carried out.
Transcriptional analysis of the C. glutamicum strain expressing cadA and dr1558 A transcriptional analysis of Cg-cadA + dr1558 and Cg-cadA was performed to investigate the reason for the observed increases in cadaverine production, cell density, and glucose consumption rate under acidic conditions. The analysis included 37 metabolism-related genes and 25 genes related to acid stress resistance (Figs. 2 and 3).
In Cg-cadA + dr1558, upregulation of the glycolysis-related genes pfkA, eno, and pyk may improve the carbon ux of the phosphotransferase system (PTS), thereby increasing glucose uptake. In the TCA cycle, changes in the expression levels of genes involved in the biosynthesis of oxaloacetate were also observed; pck was upregulated by 2.71-fold and pyc, ppc, and pyk were upregulated by 2.70-, 2.26-, and 1.5-fold, respectively. Thus, in the recombinant strain expressing dr1558, the ux of oxaloacetate is also increased; ultimately, this enhanced the synthesis of lysine, which is a precursor of cadaverine.
In the terminal pathway, no signi cant changes were observed in the expression levels of dapB, dapD, dapC, dapE, dapF, and ddh, which are directly involved in cadaverine biosynthesis. However, the expression of cadA was signi cantly higher (3.39-fold) than that in the control strain. The lysinedependent acid resistance (LDAR) system, which consists of lysine and the inducible lysine decarboxylase CadA [21], operates most e ciently under mild acid stress conditions [22,23]. The LDAR system is a proton consumption-dependent system. The cadA expression was upregulated in the presence of dr1558; this enhanced the acid resistance of the dr1558-expressing C. glutamicum strain and promoted the conversion of lysine to cadaverine. The function of D. radiodurans dr1558 was investigated in E. coli [11]. It was reported that the foreign regulator DR1558 bound to the promoter regions of some sigma factors and modulated their expression levels. However, although the effect of dr1558 expression in Corynebacterium has not been investigated, DR1558 could alter the expression levels of several regulators and may indirectly increase the expression of cadA.
We investigated the expression of genes involved in pH homeostasis, which enables C. glutamicum to respond to, and survive under, acidic pH conditions. Recent studies revealed the physiological and biochemical processes involved in the defense mechanism against low pH in C. glutamicum [24][25][26]. A previous comprehensive analysis of pH homeostasis in C. glutamicum demonstrated a functional link between the pH response, oxidative stress, iron homeostasis, and metabolic shift [27]. Therefore, the changes in the expression levels of key genes related to the intracellular defense against acidic conditions were investigated. The expression levels of 25 genes related to acid resistance were examined via transcriptome analysis (Fig. 3). The expression of DNA-binding Proteins from Starved cells (Dps) [9] and KatA (catalase) is cooperatively regulated by intracellular ROS scavenging, and these proteins are required for resistance to low pH stress in C. glutamicum [28]. qRT-PCR analysis of Cg-cadA + dr1558 con rmed that dps expression was increased by about 1.5-fold. Given that an external acidic environment can lead to an accumulation of ROS in cells, elimination of ROS is a promising way to confer acid resistance [27].
In C. glutamicum, mycothiol peroxidase (MPx), mycothiol disul de reductase (Mtr), and mycothiol glycosyltransferase (MshA) have been shown to promote adaptation to acid stress by regulating ROS homeostasis [29,30]. qRT-PCR analysis showed that expression of the mtr gene was upregulated by 1.2fold. ROS accumulation in the cells induced by the acidic conditions was likely effectively reduced by the upregulation of mtr, and like the upregulated levels of dps, may contribute to the increased growth that was observed under acidic conditions.
The qRT-PCR analysis also revealed that the mRNA expression of mcbR, which encodes a TetR-type transcriptional inhibitor of sulfur metabolism, was approximately 1.1-fold higher in Cg-cadA + dr1558 than in Cg-cadA. The accumulation of certain sulfur-containing intermediates, such as cysteine, can disrupt intracellular thiol homeostasis and cause oxidative damage by driving the Fenton reaction [31]. Inhibition of the sulfur anabolic pathway by McbR has been shown to contribute to a reduction in Lcysteine accumulation and have a bene cial effect on cell growth under acidic pH conditions [27].
The iron storage protein ferritin, which is encoded by ftn [32], was upregulated in Cg-cadA + dr1558. To protect the reducing environment of the cells from unwanted Fe 3+ /Fe 2+ redox cycling, intracellular levels of free Fe 2+ are maintained by both limiting external iron absorption and enhancing intracellular iron storage [28]. Thus, this increase in ftn expression may help protect cells from iron-mediated oxidative stress.
Cg1328, which encodes a copper chaperone, has been implicated in copper metabolism and tra cking [33]. This cytoplasmic protein functions to speci cally deliver copper to copper proteins in plant, bacterial, yeast, and animal cells. The cg1328 gene also promotes cell survival under acid stress conditions, which is consistent with the interplay between acid stress and copper toxicity reported in some bacteria. In this study, qRT-PCR analysis con rmed that the expression of the cg1328 gene was upregulated. Thus, intracellular acid resistance may also involve enhanced intracellular copper metabolism and transport. In addition, slight upregulation of sucE, a putative succinate exporter that has not yet been functionally characterized, was also observed. In addition, the expression of cglK, which was reported to encode a protein that is essential for pH homeostasis in the presence of acidic pHs in the absence of K + , was downregulated. However, since potassium was added to the culture medium, the function of the putative channel protein CglK may not be important. Most researchers consider a log 2 fold change of 2 in expression as the cutoff for a differentially expressed gene. However, to consider all the changes in gene expression to understand the mechanism underlying the enhanced cadaverine production and cell growth, a less strict condition, i.e., a log 2 fold change of 1, was used for the analysis in this study.
These ndings indicate that the expression of dr1558 in C. glutamicum in uences the expression of metabolic pathway-related genes and genes related to the defense against acidic stress. These changes in gene expression enhance pH homeostasis, leading to increases in the cell growth rate and cadaverine production.
Fed-batch fermentation for the production of cadaverine by recombinant C. glutamicum expressing dr1558 and cadA at an acidic pH Cadaverine production by the recombinant C. glutamicum strain expressing dr1558 was enhanced in batch fermentation. To further investigate the effect of DR1558 on the production of cadaverine, a fedbatch fermentation was carried out. When the glucose concentration in the broth decreased to below 1 g/L, an appropriate amount of feeding solution was added to adjust the glucose concentration to 50 g/L. The time pro les of cell growth and the concentrations of glucose, lysine, and cadaverine during the fedbatch fermentation of Cg-cadA + dr1558 and Cg-cadA are shown in Fig. 4.
During the culture of Cg-cadA + dr1558, the pH was adjusted from an initial value of 7.1 to 5.7 when the OD 600 of the culture reached 50. Even at this acidic pH, additional glucose was consumed, and at the end of the fermentation (35 h), 10.3 g/L of cadaverine was produced (Fig. 4). Cell growth also continued for 35 h, even after the pH was adjusted to 5.7. In contrast, the control strain Cg-cadA, which did not express dr1558, displayed lower rates of glucose consumption and cell growth at the acidic pH (Fig. 4). The strain expressing dr1558 and cadA showed a 1.5-fold increase in cadaverine production, compared to that of the control strain after 35 h.

Conclusions
In this study, enhanced cadaverine production was observed in a recombinant C. glutamicum strain coexpressing dr1558 and cadA. The addition of the dr1558 gene altered the expression levels of metabolism-related genes under acidic conditions. The metabolic changes induced in the recombinant C. glutamicum strain as a result of dr1558 expression are summarized in Fig. 5. The expression levels of genes related to glycolysis, the TCA cycle, and terminal pathways were altered. Some genes involved in defense mechanisms, including dps, mcbR, mtr, cg1328, and ftn, were also upregulated during cultivation at acidic pH. These genes, which are related to mechanisms underlying the defense against low pH, may be associated with the positive effects on cell growth. The exact mechanisms underlying the upregulation of the cadA gene and other stress-related genes following overexpression of dr1558 are still under investigation. However, dr1558 might bind to some regulator genes and thus change the expression level of genes involved in cadaverine biosynthesis and acid tolerance. Furthermore, these results suggest the possible application of dr1558 for the enhanced production of biochemicals under acidic conditions. Figure 1 Time pro les of cell growth, the production of cadaverine and lysine, and glucose consumption during batch cultivation by (A) a recombinant C. glutamicum strain expressing cadA and dr1558 (Cg-cadA+dr1558) and (B) a recombinant C. glutamicum strain expressing cadA (Cg-cadA). The cells were harvested for mRNA preparation at 10 h (indicated by arrows).