Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production

L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L−1 L-arginine with yield up to 0.431 g L-arginine g−1 glucose in fed-batch fermentation.

In PCR4, the amplified DNA-fragment upstream of argG gene from PCR1 and the amplified DNA-fragment of the eftu-promoter from PCR2 were fused in an overlap-extension PCR using primers argG-1 and Peftu-2. In PCR5, the amplified DNA fragment of the upstream region of argG gene from PCR3 and the amplified DNA fragment from PCR4 were fused in an overlap-extension PCR using primers argG-1 and argG-4. The resulting DNA fragment contained the DNA fragment upstream of argG gene, the sequence of eftu-promoter and the upstream region of argG gene from upstream to downstream, as well as recognition sites EcoRI and HindIII which were added by argG-1 and argG-4, respectively. This fragment was digested with the restriction enzymes EcoRI and HindIII and ligated into the equally digested vector pK18, resulting plasmid pK18-P eftu argGH. This plasmid was used to transform CcMB-P eftu argCJBDFR using electroporation, which results in the first recombination. The selected colonies were subjected to the second recombination on the plate with LBG medium containing 10% (w/v) sucrose. The modified genotype was investigated by PCR analysis. A positively identified mutant, in the following referred to as Cc1, was used for subsequent genetic modifications.
For construction of pK18-2pfk, the pfk gene was amplified by PCR with primers pfk-1, pfk-2, pfk-3 and pfk-4. In PCR1 and PCR2 the complete pfk sequence was amplified together with flanking regions upstream and downstream of the pfk gene using the primer combinations pfk-1/pfk-2 and pfk-3/pfk-4, respectively, from genomic DNA of C. crenatum SYPA5-5. In the next step, the amplified DNA fragments were fused in an overlap-extension PCR using primers pfk-1 and pfk-4.
The resulting DNA fragment contained two complete pfk genes each flanked with upstream and downstream sequences, as well as recognition sites EcoRI and BamHI which were added by pfk-1 and pfk-4, respectively. This fragment was digested with the restriction enzymes EcoRI and BamHI and ligated into the equally digested vector pK18, resulting plasmid pK18-2pfk. This plasmid was used to transform Cc1 using electroporation, which results in the first recombination. The selected colonies were subjected to the second recombination on the plate with LBG medium containing 10% (w/v) sucrose. The modified genotype was investigated by PCR analysis and determination of enzyme activity. A positively identified mutant, in the following referred to as Cc1-2pfk, was used for subsequent genetic modifications.
For construction of pK18-rbspgi, the DNA fragment upstream of pgi gene and the upstream region of pgi gene were amplified using the primer combinations pgi-1/pgi-2 and pgi-3/pgi-4, respectively, from genomic DNA of C. crenatum  In the next step, the amplified DNA fragments were fused in an overlap-extension PCR using primers pgi-1 and pgi-4. In the resulting DNA fragments the sequence of natural RBS of pgi gene was replaced by the sequences of synthetic RBSs with strengths of 4000 au, 5000 au, 6500 au and 8500 au which were added by the primer pgi-3, respectively, as well as two recognition sites EcoRI and HindIII which were added by pgi-1 and pgi-4, respectively. These fragments were digested with the restriction enzymes EcoRI and HindIII and ligated into the equally digested vector pK18, resulting plasmids pK18-4000rbspgi, pK18-5000rbspgi, pK18-6500rbspgi, pK18-8500rbspgi, respectively. These plasmids were used to transform Cc1 using electroporation, which result in the first recombination. The selected colonies were subjected to the second recombination on the plates with LBG medium containing 10% (w/v) sucrose. The modified genotypes were investigated by PCR and DNA sequencing analysis and determination of enzyme activity. A positively identified mutant, in the following referred to as Cc2, was used for subsequent genetic modifications.
In PCR4, the amplified DNA-fragment upstream of ptsG gene from PCR1 and the amplified DNA-fragment of the sod-promoter from PCR2 were fused in an overlap-extension PCR using primers ptsG-1 and Psod-2. In PCR5, the amplified DNA fragment of the upstream region of ptsG gene from PCR3 and the amplified DNA fragment from PCR4 were fused in an overlap-extension PCR using primers ptsG-1 and ptsG-4. The resulting DNA fragment contained the DNA fragment upstream of ptsG gene, the sequence of sod-promoter and the upstream region of ptsG gene from upstream to downstream, as well as recognition sites EcoRI and HindIII which were added by ptsG-1 and ptsG-4, respectively. This fragment was digested with the restriction enzymes EcoRI and HindIII and ligated into the equally digested vector pK18, resulting plasmid pK18-P sod ptsG. This plasmid was used to transform Cc2-6500 using electroporation, which results in the first recombination. The selected colonies were subjected to the second recombination on the plate with LBG medium containing 10% (w/v) sucrose. The modified genotype was investigated by PCR analysis. A positively identified mutant, in the following referred to as Cc2-G sod , was used for subsequent genetic modifications.
In PCR4, the amplified DNA-fragment upstream of iolT1 gene from PCR1 and the amplified DNA-fragment of the sod-promoter from PCR2 were fused in an overlap-extension PCR using primers iolT1-1 and Psod-2. In PCR5, the amplified DNA fragment of the upstream region of iolT1 gene from PCR3 and the amplified DNA fragment from PCR4 were fused in an overlap-extension PCR using primers iolT1-1 and iolT1-4. The resulting DNA fragment contained the DNA fragment upstream of iolT1 gene, the sequence of sod-promoter and the upstream region of iolT1 gene from upstream to downstream, as well as recognition sites EcoRI and HindIII which were added by iolT1-1 and iolT1-4, respectively. This fragment was digested with the restriction enzymes EcoRI and HindIII and ligated into the equally digested vector pK18, resulting plasmid pK18-P sod iolT1. This plasmid was used to transform Cc2-G sod using electroporation, which results in the first recombination.
The selected colonies were subjected to the second recombination on the plate with LBG medium containing 10% (w/v) sucrose. The modified genotype was investigated by PCR analysis. A positively identified mutant, in the following referred to as Cc2-G sod -P sod iolT1, was used for subsequent genetic modifications.
In PCR4, the amplified DNA-fragment upstream of ppgk gene from PCR1 and the amplified DNA-fragment of the sod-promoter from PCR2 were fused in an overlap-extension PCR using primers ppgk-1 and Psod-2. In PCR5, the amplified DNA fragment of the upstream region of ppgk gene from PCR3 and the amplified DNA fragment from PCR4 were fused in an overlap-extension PCR using primers ppgk-1 and ppgk-4. The resulting DNA fragment contained the DNA fragment upstream of ppgk gene, the sequence of sod-promoter and the upstream region of ppgk gene from upstream to downstream, as well as recognition sites EcoRI and HindIII which were added by ppgk-1 and ppgk-4, respectively. This fragment was digested with the restriction enzymes EcoRI and HindIII and ligated into the equally digested vector pK18, resulting plasmid pK18-P sod ppgk. This plasmid was used to transform Cc2-G sod -P sod iolT1 using electroporation, which results in the first recombination. The selected colonies were subjected to the second recombination on the plate with LBG medium containing 10% (w/v) sucrose. The modified genotype was investigated by PCR analysis. A positively identified mutant, in the following referred to as Cc3, was used for subsequent genetic modifications.
For construction of pK18-ATGpyc, the DNA fragment upstream of pyc gene and the upstream region of pyc gene were amplified using the primer combinations pyc-1/pyc-2 and pyc-3/pyc-4, respectively, from genomic DNA of C. crenatum SYPA5-5. In the next step, the amplified DNA fragments were fused in an overlap-extension PCR using primers pyc-1 and pyc-4. In the resulting DNA fragments the natural start codon GTG of pyc gene was replaced by ATG through the primer pyc-3, as well as two recognition sites EcoRI and HindIII which were added by pyc-1 and pyc-4, respectively. This fragment was digested with the restriction enzymes EcoRI and HindIII and ligated into the equally digested vector pK18, resulting plasmid pK18-ATGpyc. This plasmid was used to transform Cc3 using electroporation, which result in the first recombination. The selected colonies were subjected to the second recombination on the plates with LBG medium containing 10% (w/v) sucrose. The modified genotypes were investigated by PCR and DNA sequencing analysis and determination of enzyme activity. A positively identified mutant, in the following referred to as Cc3-pyc G1A , was used for subsequent genetic modifications.
For construction of pK18-2gltA, the gltA gene was amplified by PCR with primers gltA-1, gltA-2, gltA-3 and gltA-4. In PCR1 and PCR2 the complete gltA sequence was amplified together with flanking regions upstream and downstream of the gltA gene using the primer combinations gltA-1/gltA-2 and gltA-3/gltA-4, respectively, from genomic DNA of C. crenatum SYPA5-5. In the next step, the amplified DNA fragments were fused in an overlap-extension PCR using primers gltA-1 and gltA-4. The resulting DNA fragment contained two complete gltA genes each flanked with upstream and downstream sequences, as well as recognition sites EcoRI and HindIII which were added by gltA-1 and gltA-4, respectively. This fragment was digested with the restriction enzymes EcoRI and HindIII and ligated into the equally digested vector pK18, resulting plasmid pK18-2gltA. This plasmid was used to transform Cc3-pyc G1A using electroporation, which results in the first recombination. The selected colonies were subjected to the second recombination on the plate with LBG medium containing 10% (w/v) sucrose. The modified genotype was investigated by PCR analysis and determination of enzyme activity. A positively identified mutant, in the following referred to as Cc4, was used for subsequent genetic modifications.
For construction of pK18-2icd, the icd gene was amplified by PCR with primers icd-1, icd-2, icd-3 and icd-4. In PCR1 and PCR2 the complete icd sequence was amplified together with flanking regions upstream and downstream of the icd gene using the primer combinations icd-1/icd-2 and icd-3/icd-4, respectively, from genomic DNA of C. crenatum SYPA5-5. In the next step, the amplified DNA fragments were fused in an overlap-extension PCR using primers icd-1 and icd-4. The resulting DNA fragment contained two complete icd genes each flanked with upstream and downstream sequences as well as recognition sites EcoRI and XbaI which were added by icd-1 and icd-4, respectively. This fragment was digested with the restriction enzymes EcoRI and XbaI and ligated into the equally digested vector pK18, resulting plasmid pK18-2icd. This plasmid was used to transform Cc4 using electroporation, which results in the first recombination. The selected colonies were subjected to the second recombination on the plate with LBG medium containing 10% (w/v) sucrose.
The modified genotype was investigated by PCR analysis and determination of enzyme activity. A positively identified mutant, in the following referred to as Cc4-2icd, was used for subsequent genetic modifications.
For construction of pK18-2gdh, the gdh gene was amplified by PCR with primers gdh-1, gdh-2, gdh-3 and gdh-4. In PCR1 and PCR2 the complete gdh sequence was amplified together with flanking regions upstream and downstream of the gdh gene using the primer combinations gdh-1/gdh-2 and gdh-3/gdh-4, respectively, from genomic DNA of C. crenatum SYPA5-5. In the next step, the amplified DNA fragments were fused in an overlap-extension PCR using primers gdh-1 and gdh-4. The resulting DNA fragment contained two complete gdh genes each flanked with upstream and downstream sequences as well as recognition sites EcoRI and HindIII which were added by gdh-1 and gdh-4, respectively. This fragment was digested with the restriction enzymes EcoRI and HindIII and ligated into the equally digested vector pK18, resulting plasmid pK18-2gdh. This plasmid was used to transform Cc4-2icd using electroporation, which results in the first recombination.
The selected colonies were subjected to the second recombination on the plate with LBG medium containing 10% (w/v) sucrose. The modified genotype was investigated by PCR analysis and determination of enzyme activity. A positively identified mutant, in the following referred to as Cc4-2icd-2gdh, was used for subsequent genetic modifications.
For construction of pK18-rbsodhA, the DNA fragment upstream of odhA gene and the upstream region of odhA gene were amplified using the primer combinations odhA-1/odhA-2 and odhA-3/odhA-4, respectively, from genomic DNA of C.

Microbial production of L-arginine
Batch fermentations in shake flasks were performed as follows. A stock culture was maintained and revived on agar slants containing 10 g L -1 peptone, 10 g L -1 beef extract, 5 g L -1 yeast extract, 5 g L -1 NaCl, 20 g L -1 agar. One loop of colonies from agar slants was inoculated into 20 mL seed medium containing 30 g L -1 glucose, 20 g L -1 yeast extract, 20 g L -1 (NH 4 ) 2 SO 4 , 1 g L -1 KH 2 PO 4 , 0.5 g L -1 MgSO 4 · 7H 2 O, and Fed-batch fermentations were carried out in 5 L stirred fermenters (BIOTECH-5BG, Baoxing Co., China). A stock culture was maintained and revived on agar slants containing 10 g L -1 peptone, 10 g L -1 beef extract, 5 g L -1 yeast extract, 5 g L -1 NaCl, 20 g L -1 agar. One loop of colonies from agar slants was inoculated into 50 mL seed medium containing 30 g L -1 glucose, 20 g L -1 yeast extract, 20 g L -1 (NH 4 ) 2 SO 4 , 1 g L -1 KH 2 PO 4 , 0.  SDs based on three biologically independent experiments. SDs based on three biologically independent experiments. SDs based on three biologically independent experiments. SDs based on three biologically independent experiments. SDs based on three biologically independent experiments. SDs based on three biologically independent experiments. SDs based on three biologically independent experiments. SDs based on three biologically independent experiments. SDs based on three biologically independent experiments.

Arginine (g L -1 ) Time (h)
Residual glucose (g L -1 ) Yield (g g -1 ) DCW (g L -1 ) SDs based on three biologically independent experiments.   The price of food-grade L-arginine produced by keratin hydrolysis method is 55,000-60,000 RMB ton -1 in China now.