Optimization of ʟ-ornithine production in recombinant Corynebacterium glutamicum S9114 by cg3035 overexpression and manipulating the central metabolic pathway
© The Author(s) 2018
Received: 6 March 2018
Accepted: 8 June 2018
Published: 13 June 2018
ʟ-Ornithine is an important amino acid with broad applications in pharmaceutical and food industries. Despite lagging ʟ-ornithine productivity and cost reduction, microbial fermentation is a promising route for sustainable ʟ-ornithine production and thus development of robust microbial strains with high stability and productivity is essential.
Previously, we systematically developed a new strain, SO1 originate from Corynebacterium glutamicum S9114, for ʟ-ornithine production. In this work, overexpression of cg3035 encoding N-acetylglutamate synthase (NAGS) using a plasmid or by inserting a strong P tac promoter into the chromosome was found to increase ʟ-ornithine production in the engineered C. glutamicum SO1. The genome-based cg3035 modulated strain was further engineered by attenuating the expression of pta and cat, inserting a strong P eftu promoter in the upstream region of glycolytic enzymes such as pfkA, gap, and pyk, and redirecting carbon flux to the pentose phosphate pathway. The final strain with all the exploratory metabolic engineering manipulations produced 32.3 g/L of ʟ-ornithine, a yield of 0.395 g ornithine per g glucose, which was 35.7% higher than that produced by the original strain (23.8 g/L).
These results clearly demonstrated that enhancing the expression of NAGS promoted ʟ-ornithine production and provide a promising alternative systematic blueprint for developing ʟ-ornithine-producing C. glutamicum strains.
Chemicals produced by metabolically engineered strains have gained major importance in industrial biotechnology, which is developing as a stimulating field due to the advantages of providing environmentally friendly products to replace petrochemicals . With the development of gene manipulation tools, there is an increasing number of strains such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis and others that have been modified to produce economically valuable products. C. glutamicum is a favored industrial microorganism with promising applications in the industrial production of amino acids such as ʟ-glutamate and ʟ-lysine at a million-ton scale in the past decades [2, 3]. Recently, increasing studies have used C. glutamicum for producing several products including diamines [4, 5], terpene , diols , and amino acids such as ʟ-isoleucine, ʟ-arginine, and ʟ-ornithine . ʟ-Ornithine is an intermediate metabolite in the urea cycle, a key precursor for the biosynthesis of ʟ-citrulline, ʟ-proline, and polyamines, and a non-essential amino acid that plays a critical role in post-traumatic treatment, liver protection  and treatment of liver disease, strengthening the heart, and maintaining the working of the immune system .
Due to the importance of ʟ-ornithine in human health-promoting activities, economical and efficient production of ornithine has received much attention in the past years. The major strategy for producing ʟ-ornithine is focused on the enzymatic action of arginase on arginine and microbial fermentation. Metabolic engineering of microorganisms to produce ʟ-ornithine is an attractive alternative due to the high economic cost pressures of using ʟ-arginine for the enzymatic reaction. Recently, several reports have focused on the development of metabolically engineered strains that rapidly convert high concentrations of simple sugars to ʟ-ornithine. Jensen et al. constructed a metabolically engineered C. glutamicum strain that could produce 0.524 g ʟ-ornithine per g glucose in CgXII medium through disruption of argFRG and overexpression of gdh and argCJBD . Hwang and Cho modulated the NADPH supply to ʟ-ornithine biosynthesis by inactivating three putative glucose dehydrogenases, which improved the yield of ʟ-ornithine up to 14 g/L . Kim et al. developed a high ʟ-ornithine producing C. glutamicum strain by disrupting argF, argR, and proB, and overexpressing the operon argCJBD from C. glutamicum ATCC 21831. This strain produced 51.5 g/L ʟ-ornithine from glucose in a fed-batch culture in a 6.6-L fermenter . Jiang et al. engineered a C. glutamicum ATCC 13032-derived strain that produced 24.1 g/L ʟ-ornithine in shake flask cultures by genetic modulation and adaptive evolution .
Strains and plasmids
Strains and plasmids used in this study
E. coli DH5ɑ
Clone host strain
C. glutamicum S9114 with deletion of argF, ncgl1221, argR, putP, attenuation of odhA, proB, ncgl2228, overexpression of lysE, gdh, argCJBD
SO1 carrying expression vector pEC-XK99E
SO1 carrying expression vector pEC-cg3035
SO1 with P tac promoter inserted in front of cg3035
SO1 with terminator inserted in front of pta
SO1 with terminator inserted in front of cat
SO1 with P eftu promoter inserted in front of pfkA
SO1 with P eftu promoter inserted in front of gap
SO1 with P eftu promoter inserted in front of pyk
SO1 with P eftu promoter inserted in front of pfkA and P tac promoter inserted in front of cg3035
SO1 with P eftu promoter inserted in front of pfkA, P tac promoter inserted in front of cg3035 and a terminator inserted in front of pta
SO1 with P eftu promoter inserted in front of pfkA, P tac promoter inserted in front of cg3035 and a terminator inserted in front of cat
SO1 with P eftu promoter inserted in front of pfkA, P tac promoter inserted in front of cg3035 and terminator inserted in front of pta and cat
SO12 with an AAV degradation label inserted in the C terminal of pgi
SO14 with synthetic RBS and A1G replacement in front of zwf.
SO15 with P tac promoter inserted in front of tkt operon.
Mobilizable vector, allows for selection of double crossover in C. glutamicum, KmR, sacB
A shuttle expression vector, KmR
A derivative of pEC-XK99E, harboring cg3035 gene from C. glutamicum S9114 under its native promoter
pK18-P tac -cg3035
A derivative of pK18mobsacB, harboring P tac -cg3035 fragment
A derivative of pK18mobsacB, harboring T-pta fragment
A derivative of pK18mobsacB, harboring T-cat fragment
pK18-P eftu -pfkA
A derivative of pK18mobsacB, harboring P eftu -pfkA fragment
pK18-P eftu -gap
A derivative of pK18mobsacB, harboring P eftu -gap fragment
pK18-P eftu -pyk
A derivative of pK18mobsacB, harboring P eftu -pyk fragment
A derivative of pK18mobsacB, harboring pgi-AAV fragment
A derivative of pK18mobsacB, harboring SB-zwf fragment
pK18-P tac -tkt
A derivative of pK18mobsacB, harboring P tac -tkt fragment
DNA manipulation and strain construction
To construct the recombinant strains, the genomic DNA of strain C. glutamicum S9114 was isolated using a genomic DNA extraction kit (Tiangen, Beijing, China) and employed for DNA fragment amplification. The PCR product and vectors were obtained using the PCR products Purification kit and the mini-plasmid isolation kit (Tiangen, Beijing, China), respectively. The primers used in this study are listed in Additional file 1: Table S1. For introducing modifications into the chromosome of C. glutamicum S9114, the suicide vector pK18mobsacB containing the sucrose lethal gene sacB from Bacillus subtilis was employed by double crossover recombination, as described previously [16, 17].
For cg3035 overexpression in the high ʟ-ornithine producing strain SO1, a widely used strong P sod promoter and the open reading frame of cg3035 was amplified and spliced using PCR. The overlapped fragment was ligated into the expression vector pEC-XK99E by Gibson assembly to construct a constitute expression cassette pEC-cg3035. After isolation from E. coli, this recombinant plasmid and empty vector pEC-XK99E were transformed into SO1 by electroporation. The positive transformants were pointed out and confirmed by colony PCR.
For P tac promoter insertion in front of cg3035, the upstream region and the coding region of cg3035 was amplified. A strong P tac promoter was introduced between the upstream region and coding region by primers. The overlapping fragment containing the upstream region, P tac promoter, and the coding region was cloned into the Hind III/Xba I sites in the suicide vector pK18mobsacB by Gibson assembly. The engineered plasmid was then transferred into strain SO1 by electroporation. After double crossover recombination, the positive recombinant strain was detected by colony PCR. Similarly, the insertion of the P eftu promoter in front of pfkA, gap and pyk or insertion of P tac in front of tkt was also performed using these procedures.
To attenuate the expression of genes, strategy described in our previous study were carried out . To attenuate the expression of pta and cat, a transcription terminator was introduced into the right upstream of the genes pta and cat which lines between the upstream fragment and downstream fragment by PCR. The recombinant fragment was then cloned into the Hind III/Xba I sites in the suicide vector pK18mobsacB by Gibson assembly. After extraction from E. coli, this engineered vector was transformed into SO1 by electroporation. After double crossover recombination, the mutant strains with a terminator inserted in the upstream region of pta and cat were determined by colony PCR. When constructing the mutant strains, an AAV taq was inserted in the C terminal of pgi with a synthetic RBS and G1A replacement in zwf. The RBS sequence with the predicted translation start strength of 50000 au was designed using the RBS Calculator (https://www.denovodna.com/software/doLogin) and is listed in Additional file 1: Table S2.
Fermentation in shake flasks
Fermentation with recombinant strains was performed in batch culture in shake flasks as described in our previous work [14, 15]. A single clone of the mutants was activated on LB agar plate for two cycles of 12 h. Subsequently, a ring of bacteria was inoculated into 10 mL of seed medium in a 100-mL normal shake flask. The seed medium consisted of (per liter) 25 g glucose, 10 g yeast extract, 10 g corn steep liquor, 15 g (NH4)2SO4, 2.5 g MgSO4·7H2O, 1 g KH2PO4, 0.5 g K2HPO4, 0.5 g Na2HPO4, and 10 g CaCO3. After 11 h of cultivation at 32 °C and 220 rpm, the appropriate amount of culture was transferred to 24 mL of fermentation medium in a 250-mL baffle shake flask. Initial OD600 of the fermentation culture was adjusted to one. The fermentation medium consisted of (per liter) 100.0 g glucose, 20.0 g corn steep liquor, 50.0 g (NH4)2SO4, 2.5 g MgSO4·7H2O, 1.0 g KH2PO4, 0.5 g K2HPO4, 0.5 g Na2HPO4, 0.02 g FeSO4·7H2O, 0.02 g MnSO4·4H2O, and 10 g CaCO3. The initial pH was adjusted to 7.0. All cultures were grown at 32 °C and 250 rpm, and 200-µL samples were collected every 12 h to measure l-ornithine concentration, cell density, and residual glucose concentration. If necessary, 50 mg/L kanamycin was used to cultivate E. coli and 12.5 mg/L kanamycin was used to cultivate C. glutamicum.
Measurement of NAGS enzyme activity
For NAGS enzyme activity analysis, 20 mL of fermentation samples were collected at 10 h by centrifugation (at 5000 rpm, 4 °C, and 10 min) and washed twice with 100 mM Tris–HCl (pH 7.5) which was supplemented with 20 μM PMSF. Following the pure cells were incubated in 5 mL of 100 mM Tris–HCl (pH 7.5) containing 30% (v/v) glycerol and 10 mg/mL lysozyme at 37 °C for 3 h and then disrupted by sonication. After removing cell debris by centrifugation, the supernatant was collected as crude enzyme, and the protein concentration was determined by bicinchoninic acid (BCA) assay using bovine serum albumin as the standard. NAGS specific activity assay was performed as described previously .
For RNA analysis, 500 μL of fermentation samples were collected at 12 h. RNA extraction and RT-PCR assays were performed as described in our early report .
Measurement of glucose consumption, optical density, and metabolite analysis
After dissolving CaCO3 in 0.125 mol/L HCl, cell growth was monitored by measuring the OD600 using a microplate reader (BioTek Instruments, Winooski, VT, USA). ʟ-Ornithine concentrations were determined by colorimetry using ninhydrin, as described previously [21, 22]. l-Ornithine standard curve of colorimetric assay was listed in Additional file 1: Figure S1. The fermentation supernatant was passed through a 0.22-µm filter and analyzed for glucose levels, using a SBA-40C biosensor (developed by Biology Institute of Shandong Academy of Sciences). Acetic acid were analyzed as described previously . All experiments were conducted in triplicate; the data were averaged and presented as mean ± standard deviation (SD).
Overexpression of cg3035 exerts a positive effect on ʟ-ornithine production
Acetic acid concentration in 72 h fermentation broth
Relative NAGS specific activity
Acetic acid concentration (g/L)
1 ± 0.13
3.85 ± 0.14
6.63 ± 0.07
4.62 ± 0.21
1.05 ± 0.05
1.34 ± 0.06
Improvement of ʟ-ornithine production by attenuating the acetate synthesis pathway
Effect of overexpressing enzymes of the glycolytic pathway on ʟ-ornithine production
Combination of Ptac-cg3035, Peftu-pfkA, T-pta, and T-cat targets and its influence on ʟ-ornithine production
Enhancement of ʟ-ornithine production by redirecting the metabolic flux into pentose phosphate pathway
In this study, we demonstrate system-wide engineering and optimization of cellular metabolism in C. glutamicum SO1 to further improve strain performance with respect to ʟ-ornithine biosynthesis. Depending on the current knowledge about ʟ-ornithine biosynthesis pathway, cg3035 was overexpressed using a plasmid that significantly improved ʟ-ornithine production. This confirmed our speculation that glutamate acetylation is a rate-limiting step for ʟ-ornithine accumulation after removing the feedback inhibition of ArgR on the argCJBDF operon. In the incipient fermentation period, the improvement of ʟ-ornithine production was not obvious probably due to slow glucose utilization rate. In addition, we induced genome-based cg3035 overexpression to ensure strain stability, which also promote ʟ-ornithine accumulation. The improved percentage in ornithine production by genome-based cg3035 overexpression was lower than plasmid based cg3035 overexpression probably due to the not superior NAGS enzyme activity. We believe that this intriguing target could be extended to ʟ-arginine and ʟ-citrulline production.
Notably, N-acetylglutamate biosynthesis also requires acetyl-CoA as a precursor in addition to N-acetylglutamate synthase. Therefore, we attenuated the acetate biosynthesis pathway to reduce acetyl-CoA consumption and improved ʟ-ornithine production titer, thus indicated that acetyl-CoA supplementation is an important factor for ʟ-ornithine production, which further confirmed previous reports that deletion of dtsR1 encoding a subunit of acetyl-CoA carboxylase is beneficial for ʟ-glutamate  and ʟ-arginine accumulation .This result is also consistent with previous work reported that block the acetate biosynthesis pathway promote the production of several compounds [27–30]. Attenuating acetate biosynthesis also conserves carbon flux into the glycolytic pathway, which may also account for the high ʟ-ornithine production performance. This encouraged us to explore the glycolytic pathways. Overexpression of pfkA by the insertion of a strong P eftu promoter tremendously promoted ʟ-ornithine production, whereas insertion of a strong P eftu promoter in front of gap or pyk did not contribute to ʟ-ornithine accumulation, which is in contrast with previous results . The differences in genetic background between C. glutamicum ATCC 13032 and C. glutamicum S9114 may account for these diverse outcomes. There is a one percent gap in the genomes of those two strains. In addition, C. glutamicum S9114, a mutant strain derived from Brevibacterium tianjinese T6–13, processed a faster cell growth than C. glutamicum ATCC 13032. Modulations in pyk and gap slightly affected cell growth and glucose consumption in C. glutamicum S9114 probably acting as the main reason for the reduced ʟ-ornithine yield. Based on the aforementioned results, P tac -cg3035, P eftu -pfkA, T-pta, and T-cat were confirmed to independently increase ʟ-ornithine accumulation. To further improve the yield of ʟ-ornithine, these targets were assembled. The highest performance was obtained in strain SO12 with P tac -cg3035, P eftu -pfkA, and T-pta, but not in strain SO13 with P tac -cg3035, P eftu -pfkA, T-pta, and T-cat, which illustrated that attenuating pta and cat simultaneously can affect ʟ-ornithine production by interfering with normal physiological metabolism in engineered strains.
Several previous works have demonstrated that NADPH availability is strongly correlated with ʟ-ornithine production. In this work, ʟ-ornithine production was improved by suppositional redirecting the carbon flux to the pentose phosphate pathway according to previous study , which further demonstrated the importance of NADPH supplementation in developing ʟ-ornithine producing strains. This is consistent with previous work by Kim et al., where the pentose phosphate pathway was reinforced by replacing the native promoter of the tkt operon with a strong sod promoter and changing the start codons of zwf and pgi .
Comparison of other C. glutamicum strains engineered for ʟ-ornithine production
Strains (C. glutamicum)
ʟ-Ornithine titer (g/L)
ʟ-Ornithine yield (g/g glucose)
Shake flask; batch
Deletion of argF, ncgl1221, argR, and putP; attenuation of odhA, proB, pta, cat and ncgl2228; overexpression of lysE, gdh, cg3035, pfkA, pyk, tkt, and argCJBD
Deletion of argF, argR, and proB; Reinforcement of the PPP pathway flux; The use of a feedback-resistant enzyme
Shake flask; batch
Deletion of argF, argR, and argG; overexpression of argB M ; attenuation of pgi.
Shake flask; batch
Deletion of argF, argR, and proB; Blocking gluconate biosynthesis
Deletion of argF, argR, and proB; Adaptive evolution in presence of ʟ-ornithine
Shake flask; batch
Deletion of argR; overexpression of argJ.
Redesigning and engineering strains for use in the industrial production of ʟ-ornithine has significant potential application for reducing the economic cost pressures of using ʟ-arginine for enzymatic reaction and extending the fermentation organism. C. glutamicum has been extensively studied for its ability to produce ʟ-ornithine, though its yield and productivity are still low compared to other strategies. Very recently, we reported a C. glutamicum S9114 derived recombinant strain with high ʟ-ornithine production titer. Here, we have further enhanced the ʟ-ornithine yield through genome-based cg3035 overexpression and systematic manipulation of central metabolic pathways including glycolysis, acetate metabolism, and pentose phosphate pathway, consequently suggesting that improved ʟ-ornithine production can be obtained by these modulations. C. glutamicum is an excellent producer for producing ʟ-glutamate and ʟ-glutamate-derived products including ʟ-ornithine, ʟ-citrulline, and ʟ-arginine. We supposed that the metabolic engineering strategies reported in this work can be applied to constructing strains producing such products.
BZ planned and conducted the experiments, analyzed and interpreted data, and wrote the manuscript; MY and W-PW participated in the research; B-CY supervised the research and finalized the manuscript. All authors read and approved the final manuscript.
We thank Dr. Xue-Lan Chen for providing pk18mobsacB. We thank Dr. Zhong-Gui Mao and Dr. Li-Ming Liu for providing strains Corynebacterium glutamicum S9114. Thank my wife Yan Jiang for giving great support and encouragement during my research.
The authors declare that they have no competing interests.
Availability of data and materials
Gene sequences used in this project are from Genbank (http://www.ncbi.nlm.nih.gov/) and the material and data supporting their findings can be found in the main paper and in additional file.
Ethics approval and consent to participate
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in the study.
This work was supported by grants from the National Natural Science Foundation of China (31730004 and 21575089).
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