Production of Cadaverine in Recombinant Corynebacterium Glutamicum Overexpressing Lysine Decarboxylase (ldcC) and Response Regulator dr1558

In this study, the response regulator DR1558 from Deinococcus radiodurans was overexpressed in recombinant Corynebacterium glutamicum with lysine decarboxylase (ldcC). The recombinant C. glutamicum strain overexpressing dr1558 and ldcC produced 5.9 g/L of cadaverine by ask cultivation, whereas the control strain overexpressing only ldcC produced 4.5 g/L of cadaverine. To investigate the mechanism underlying the effect of DR1558, the expression levels of genes related to central metabolism and lysine-biosynthesis were analyzed by quantitative-real time polymerase chain reaction. The results showed that phosphoenolpyruvate carboxykinase (pck) was downregulated, and pyruvate kinase (pyk) and other lysine biosynthesis genes were upregulated. Furthermore, in fed-batch fermentation, C. glutamicum coexpressing dr1558 produced 25.14 g/L of cadaverine, a 1.25-fold increase in concentration relative to the control. These results suggested that the heterologous expression of dr1558 may improve the production of biorenery products by recombinant C. glutamicum.

CadA, encoded by the cadA gene in the cadBA operon, is induced by low external pH, excess lysine, and low oxygen levels [15]. CadA functions only under acidic conditions, with an optimum pH of 5.7. In contrast, LdcC encoded by the ldcC is expressed constitutively, independent of pH changes [15]. LdcC is active across a relatively wide pH range, and the optimum pH for activity is 7.5. Here, LdcC was used for cadaverine biosynthesis because it is functional under fermentation conditions without pH manipulation.
Corynebacterium glutamicum is an industrial bacterial strain known for its ability to produce amino acids, such as L-lysine and glutamate, as well as organic acids, nucleotides, diamines, and polymers. However, C. glutamicum strains used to produce lysine, a precursor of cadaverine, are exposed to various environmental stresses during fermentation. These stresses, such as high osmotic pressure, accumulation of metabolites, and oxidation that occur during cultivation, have negative effects on cell growth and chemical productivity [16][17][18][19]. Many recent studies have reported that stress resistance could be improved by changing the physiological characteristics of the microorganisms [20][21][22].
Deinococcus radiodurans is a bacterium known for its resistance to various abiotic stresses, such as γradiation, reactive oxygen species, and oxidants [23]. DR1558, a response regulator of D. radiodurans, has been reported to improve stress resistance and cell growth when overexpressed in Escherichia coli [24].
Recently, the dr1558 gene was introduced into C. glutamicum to improve L-lysine production [25]. Since DR1558 increased the production of L-lysine, a precursor of cadaverine, it is expected to also increase cadaverine biosynthesis in C. glutamicum.
In this study, we examined the effect of plasmid-based heterologous coexpression of ldcC and dr1558 on cadaverine production by C. glutamicum. Cell growth, glucose consumption, and cadaverine production of the recombinant C. glutamicum strain were analyzed in comparison to a control strain expressing only ldcC. In addition, the expression levels of the genes involved in cadaverine biosynthesis were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) to determine the effect of DR1558 on the metabolic processes related to cadaverine biosynthesis in recombinant C. glutamicum.

Materials And Methods
Bacterial Strains, Plasmids, and Culture Medium All bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli XL1-Blue (Stratagene Cloning Systems, La Jolla, CA, USA) was used for gene cloning. Corynebacterium glutamicum KCTC 1857 (Korean Collection for Type Cultures, Joengeup, Republic of Korea), a mutant strain of C. glutamicum ATCC 13032, was used as the host strain for cadaverine production [27]. E. coli ldcC and D. radiodurans dr1558 were expressed under a synthetic P H30 promoter. Plasmids pCES208H30EcLdcC and pCES208H30EcLdcCdr1558 harboring the E. coli ldcC gene and both the E. coli ldcC gene and D. radiodurans dr1558 were constructed as follows, respectively. All DNA manipulations were performed according to standard procedures. Corynebacterium glutamicum KCTC 1857 was transformed with pCES208H30EcLdcC [26] to generate C. glutamicum(pCES208H30EcLdcC) (Cg_ldcC). The response regulator DR1558 was cloned into the plasmid pCES208H30EcLdcC. The dr1558 gene was ampli ed by PCR using forward (attacccgggtaccaaagtaacttttcggt) and reverse primers (attacccgcgaattggagctccaccgcg) and was inserted into the BstZI71 site of pCES208H30EcLdcC. The nal constructed strain, C. glutamicum(pCES208H30EcLdcCdr1558) (Cg_ldcC+dr1558), was used to investigate the effect of DR1558 on cadaverine biosynthesis.

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
Transcriptional analysis was performed to analyze the changes in gene expression levels in the recombinant C. glutamicum strain overexpressing the dr1558gene. Cells were harvested during the midexponential growth phase (12 h) by centrifugation (16,000 × g, 10 min, 4°C). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions. RNA was reverse-transcribed using a rst-strand cDNA synthesis kit (TaKaRa, Kusatsu, Japan). The synthesized cDNA was subjected to qRT-PCR to determine the expression levels of the genes of interest and the housekeeping genes 16SrRNAand sigA. Real-time PCR (Illumina, San Diego, CA, USA) was performed using speci c primers (Supplement Table 1

Analytical Procedures
Cell growth was monitored by measuring the OD 600 using a UV/Vis spectrophotometer (Molecular Devices, San Jose, CA, USA). The concentrations of organic acids and glucose were determined by highperformance liquid chromatography using an Agilent In nity 1260 system (Agilent Technologies, Santa Clara, CA, USA). The glucose concentration was determined using an Agilent In nity 1260 System equipped with an Aminex HPX-87H ion exclusion column (BioRad, Hercules, CA, USA). The mobile phase was 5 mM H 2 SO 4 at a ow rate of 0.6 mL/min, and the column was maintained at 50°C. The concentrations of cadaverine and lysine were determined using an Agilent In nity 1260 System equipped with a ZORBAX SB-C18 column (Agilent Technologies). The mobile phase consisted of 25 mM sodium acetate buffer (pH 4) and 1 M acetonitrile solution at a ow rate of 1 mL/min, and the column was maintained at 35°C. The concentrations of amino acids were measured after diethyl ethoxymethylenemalonate derivatization [29].

Results And Discussion
Cadaverine Production in Recombinant C. glutamicum with dr1558 and ldcC To determine the effect of dr1558 expression in C. glutamicum on cadaverine production, the constructed recombinant strains with only ldcC (Cg_ldcC) and with both ldcC and dr1558 (Cg_ldcC + dr1558) were cultivated in asks for comparison (Fig. 1).
Both Cg_ldcC and Cg_ldcC + dr1558 recombinant strains grew similarly, but the growth rate of Cg_ldcC + dr1558 was higher than that of Cg_ldcC during the exponential phase. Additionally, the glucose consumption rate was higher in Cg_ldcC + dr1558. Glucose in the medium was completely consumed within 24 h of cultivation for both strains. Cadaverine was produced rapidly during the exponential phase in both strains. However, after 24 h, during the stationary phase, the growth of the Cg_ldcC quickly decreased, whereas that of Cg_ldcC + dr1558 gradually decreased. The amount of lysine remaining in the medium was similar for both strains. The lysine concentration was below 1 g/L during cultivation. However, nal cadaverine production was increased by 15% (from 4.5 to 6.1 g/L) in Cg_ldcC + dr1558 compared with that in Cg_ladC. Therefore, it was shown that dr1558 overexpression led to enhanced cell growth and cadaverine production. To investigate the metabolic changes caused by dr1558 expression in cadaverine-producing recombinant C. glutamicum, the expression levels of several genes involved in central and cadaverine biosynthesis pathways were compared.

Transcriptional Analysis of Cadaverine Biosynthesis Metabolism in Recombinant C. glutamicum
Comparative transcriptional analysis of Cg_ldcC + dr1558 and Cg_ldcC (the control strain) was performed to investigate the mechanism underlying the increased cadaverine production. Quantitative real-time PCR of 39 genes in three pathways, namely glycolysis, the tricarboxylic acid (TCA) cycle, and lysinebiosynthesis pathway, was performed (Fig. 2) [30,31].
As expected, changes in the expression patterns of pyc and pyk, involved in oxaloacetate metabolism, were observed during the mid-exponential growth phase. The expression of pyc, which converts pyruvate to oxaloacetate, was upregulated 2.40-fold, while the expression of pck, which converts oxaloacetate to phosphoenolpyruvate, was downregulated 3.40-fold. The increase in pyc expression increased the ux into oxaloacetate, thereby increasing lysine-biosynthesis [32]. Furthermore, ppc, encoding phosphoenolpyruvate carboxylase, was slightly upregulated in Cg_ldcC + dr1558. Increasing the synthesis and reducing consumption of precursor were important factors because precursor availability was essential for producing the desired product. Cadaverine is synthesized from oxaloacetate produced in the TCA cycle [33][34][35]. Therefore, upregulation of genes in oxaloacetate biosynthesis would increase cadaverine production in recombinant C. glutamicum [33].
Other genes related to the TCA cycle, including odhA, sdhA, sdhB, fumH, and mqO, were upregulated, which would, in turn, increase the carbon ux into oxaloacetate in the TCA cycle. On the other hand, the upregulation of these genes was previously observed in a recombinant C. glutamicum strain with increased oxidative stress resistance [16]. In E. coli, it has been reported that dr1558 bound to the promoter region of sigma factor rpoS and modulated its expression. Because the RpoS regulated various stress resistance-related genes, recombinant E. coli with dr1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H 2 O 2 . The function of dr1558 in Corynebacterium was not exhaustively investigated, but it may be possible that dr1558 expression could improve oxidative stress tolerance and increase cell growth during cultivation by minimizing cell damage.
Therefore, the improvement of tolerance could sustain the growth of Cg_ldcC + dr1558 during the stationary phase.
The expression levels of genes involved in the lysine-biosynthesis pathway were also investigated. In the lysine pathway, lysine is synthesized from oxaloacetate through seven reactions catalyzed by the products of the aspC, lysC, asd, dapA, dapB, ddh, and lysA genes [35] (Fig. 3). In the present study, the expression levels of lysC, asd, dapA, dapB, ddh, and lysA were upregulated. The changes in the expression levels of these genes could increase the oxaloacetate ux into lysine synthesis. There was a decrease of ldcC expression level in Cg_ldcC + dr1558, but the difference was statistically insigni cant. Figure 3 summarizes the metabolic changes that occurred in the recombinant C. glutamicum strain overexpressing dr1558. Among the metabolic pathways of C. glutamicum, expression of the response regulator DR1558 could affect the TCA cycle and the lysine-biosynthesis pathway; speci cally, genes related to intracellular oxaloacetate supply in the TCA cycle and those related to lysine-biosynthesis were upregulated. However, the exact reason for the up-regulation of these genes was not thoroughly investigated, and further study of DR1558 in Corynebacterium at the molecular level is necessary.

Enhanced Cadaverine Production by Recombinant C. glutamicum in Fed-batch Culture
From the ask cultures, it was shown that a recombinant C. glutamicum strain, co-expressing DR1558, produced more cadaverine than the control strain. After con rming the expression patterns of genes related to cadaverine synthesis in C. glutamicum through qRT-PCR, cadaverine production by fed-batch cultivation was conducted. Previous studies have reported increased production of succinate [36], poly (3hydroxybutyrate) [37], and 2,3-butanediol [38] in E. coli expressing DR1558. In particular, succinate production was increased in large-scale cultivation. Therefore, cadaverine production by the fed-batch cultivation of recombinant C. glutamicum coexpressing DR1558 was conducted. Fed-batch fermentation for cadaverine production was compared between Cg_ldcC + dr1558 and Cg_ldcC. The cell growth, glucose consumption, extracellular lysine concentration, and cadaverine concentration obtained during fed-batch fermentation are shown in Fig. 4. The results were similar to those of the ask cultures. Higher cell growth was observed with Cg_ldcC + dr1558 than with Cg_ldcC. Furthermore, after 45 h of cultivation, Cg_ldcC + dr1558 produced 25.1 g/L cadaverine (125% compared with that of Cg_ldcC). Similar lysine levels were accumulated in the culture medium with both strains, and no other byproducts were detected.

Conclusion
In the present study, the lysine-producing host strain C. glutamicum KCTC 1857 was genetically modi ed to co-express the E. coli lysine decarboxylase gene (ldcC) and D. radiodurans response regulator DR1558 (dr1558) to obtain the enhanced cadaverine production. According to the results, dr1558 expression improved the metabolic synthesis of cadaverine by improving oxaloacetate ux. Furthermore, dr1558 expression resulted in a 1.25-fold increase in cadaverine production relative to the control strain during fed-batch fermentation. Therefore, the application of DR1558 may be of potential use for improving the yield of target products generated using microbial fermentation.

Declarations Ethical Approval
Not applicable.
Consent to Participate I con rm that the nal manuscript has been seen and approved by all the authors. The undersigned author transfers all copyright ownership of the manuscript to Applied Biochemistry and Biotechnology in the event the work is published.   Relative expression levels of metabolic pathway genes in recombinant C. glutamicum expressing dr1558. The analyzed genes included the glycolytic pathway genes pgi, tpi, pfkA, fbp, gapA, pgk, gpmA, eno, and pyk, the TCA cycle genes pyc, ppc, pck, gltA, aceE, acn, icd, odhA, sucC, sucD, sdhA, sdhB, fum, mdh, mqO, and aceB, and the terminal pathway genes lysC, asd, dapA, dapB, dapD dapC, dapE, dapF, ddh, lysA, lysE, ldcC, and cg2893. The data were analyzed by the 2−ΔΔCt method. The histogram shows the mean of three biological replicates, and the error bars show the standard deviations.

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