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 confirmed that the recombinant strain Cg-cadA + dr1558 showed significantly 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 specific growth rate and specific 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 specific growth rate and specific 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 specific 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 findings, 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 flux 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 flux of oxaloacetate is also increased; ultimately, this enhanced the synthesis of lysine, which is a precursor of cadaverine.
In the terminal pathway, no significant 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 significantly higher (3.39-fold) than that in the control strain. The lysine-dependent acid resistance (LDAR) system, which consists of lysine and the inducible lysine decarboxylase CadA , operates most efficiently 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 . 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 [24–26]. 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 . 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)  and KatA (catalase) is cooperatively regulated by intracellular ROS scavenging, and these proteins are required for resistance to low pH stress in C. glutamicum . qRT-PCR analysis of Cg-cadA + dr1558 confirmed 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 .
In C. glutamicum, mycothiol peroxidase (MPx), mycothiol disulfide 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 1.2-fold. 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 . Inhibition of the sulfur anabolic pathway by McbR has been shown to contribute to a reduction in L-cysteine accumulation and have a beneficial effect on cell growth under acidic pH conditions .
The iron storage protein ferritin, which is encoded by ftn , was upregulated in Cg-cadA + dr1558. To protect the reducing environment of the cells from unwanted Fe3+/Fe2+ redox cycling, intracellular levels of free Fe2+ are maintained by both limiting external iron absorption and enhancing intracellular iron storage . 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 trafficking . This cytoplasmic protein functions to specifically 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 confirmed 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 log2 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 log2 fold change of 1, was used for the analysis in this study.
These findings indicate that the expression of dr1558 in C. glutamicum influences 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 fed-batch 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 profiles of cell growth and the concentrations of glucose, lysine, and cadaverine during the fed-batch 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 OD600 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.