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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (7): 29-39    DOI: 10.13523/j.cb.20180705
    
Producing L-ornithine by Heterologous Expression of N-acetyl-L-ornithine Deacetylase in Corynebacterium crenatum
Qun-feng SHU,Mei-juan XU(),Jing LI,Xian ZHANG,Tao-wei YANG,Zheng-hong XU,Zhi-ming RAO()
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
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Abstract  

Objective: The metabolic pathway engineering of Corynebacterium crenatum SYPA5-5/△proB/△argF (SYPO-1) has been performed to further enhance the pathway flux of L-ornithine biosynthesis. Firstly, the four genes encoding N-acetyl-L-ornithine deacetylase (NAOD) from different bacterial sources were screened, cloned and expressed in Escherichia coli BL21 (DE3). Then the recombinant NAODs were purified and characterized. The argE gene from Serratia marcescens Y213 was overexpressed in the L-ornithine producing strain C. crenatum SYPO-1 to increase the L-ornithine production. Methods: The genes from different sources were sub-cloned into the pDXW10 plasmid and expressed under the tacM promoter in E. coli BL21(DE3). Then the recombinant N-acetyl-L-ornithine deacetylation were purified and their characterization were studied. The optimal N-acetyl-L-ornithine deacetylase was expressed in recombinant C. crenatum. The parameters of the recombinant strains during fermentation were also investigated. Results: The recombinant argE coding NAOD enzyme from S. marcescens showed a very higher activity than the other NAOD enzymes from E. coli BL21(DE3), K. pneumoniae and B. subtilis, the activity was 798.98U / mg, the optimum pH and temperature were 7℃ and 37℃ respectively. SmNAOD was expressed in C. crenatum, and the activity was 128.4U/ml, which was significantly increasing intracellular acetyl cycle levels. At the end of fermentation, L-ornithine yield increased to 38.5g/L with the overall productivity of 0.401g/(L·h) in the recombinant SYPO-2, which was approximately 21.3% and 33.2% higher than that of SYPO-1 and SYPO-3, respectively. Conclusion: The N-acetyl-L-ornithine deacetylase from S. marcescens Y213 has been screened and overexpressed in the L-ornithine producing strain C. crenatum SYPO-1, which could promote the rapid consumption of L-ornithine precursors and achieve L-ornithine accumulation. A huge potential of C. crenatum to overproduce not only L-ornithine but also L-citrulline, L-arginine from renewable resources such as glucose were demonstrated.

Key words： L-ornithine      Corynebacterium crenatum      N-acetyl-L-ornithine deacetylase      argE     
Received: 27 January 2018      Published: 13 August 2018
ZTFLH:  Q819  
Corresponding Authors: Mei-juan XU,Zhi-ming RAO     E-mail: xumeijuan@jiangnan.edu.cn;raozhm@jiangnan.edu.cn
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Qun-feng SHU
Mei-juan XU
Jing LI
Xian ZHANG
Tao-wei YANG
Zheng-hong XU
Zhi-ming RAO
Cite this article:

Qun-feng SHU,Mei-juan XU,Jing LI,Xian ZHANG,Tao-wei YANG,Zheng-hong XU,Zhi-ming RAO. Producing L-ornithine by Heterologous Expression of N-acetyl-L-ornithine Deacetylase in Corynebacterium crenatum. China Biotechnology, 2018, 38(7): 29-39.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180705     OR     https://manu60.magtech.com.cn/biotech/Y2018/V38/I7/29

Strain/plasmid Characteristic Source
Strains
E. coli JOM109 recA1, endA1, gyrA96, thi, hsdR17, supE44, relA1, Δ(lac-proAB), [F’traD36, proAB+, lac Iq, lacZ ΔM15] Invitrogen
E. coli BL21(DE3) F- ompT gal dcm lon hsdSB (rB- mB-)λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) Invitrogen
C.crenatum SYPA5-5 A hyper arginine production strain, His-, SGr, D-Argr, H-Argr Our lab
SYPO-1 C.crenatum SYPA5-5 with proB and argF deletion, the positive mutation E19Y of CcNAGK into the chromosome of SYPA5-5 Our lab
SYPO-2 SYPO-1 with pDXW10-SmargE This study
SYPO-3 SYPO-1 with pDXW10-CcargJ This study
Plasmids
pMD-18T E. coli clone plasmid Ampr, Col E origin TaKaRa
T-CcargJ A derivative of pMD18-T, Ampr, harboring argJ gene from C. crenatum SYPA5-5 This study
T-argE A derivative of pMD18-T, Ampr, harboring argE gene This study
pDXW10 A shutter expression vector, KmR, Ptac promoter [17]
pDXW10-CcargJ A derivative of pDXW10, harboring argJ gene from C. crenatum SYPA5-5 under its native promoter This study
pDXW10-EcargE A derivative of pDXW10, harboring argE gene from E. coli BL21(DE3) under its native promoter This study
pDXW10-KpargE A derivative of pDXW10, harboring argE gene from Klebsiella pneumoniae P01 under its native promoter This study
pDXW10-BsargE A derivative of pDXW10, harboring argE gene from Bacillus subtilis subsp. subtilis strain 168G under its native promoter This study
pDXW10-SmargE A derivative of pDXW10, harboring argE gene from Serratia marcescens Y213 under its native promoter This study
Table 1 Strains and plasmid used in this study
Name DNA Sequence (5' - 3') Restriction site
P1 F CCGGAATTCAAAGGAGGGAAATCATGAAAAACAAATTACCGCCATT EcoR I
P1 R CGAGCTCTTAGTGGTGGTGGTGGTGGTGATGCCAGCAAAAATGGG Sac I
P2 F CCGGAATTCAAAGGAGGGAAATCATGACCGCGACCCTTGAGCT EcoR I
P2 R CGAGCTCTTAGTGGTGGTGGTGGTGGTGCTCATTTTTTACTTTCG Sac I
P3 F CCGGAATTCAAAGGAGGGAAATCATGCCGTTGCCGACGCTG EcoR I
P3 R CGAGCTCTTAGTGGTGGTGGTGGTGGTGGTGCAGGCAATAGTGCCG Sac I
P4 F CCGGAATTCAAAGGAGGGAAATCGTGAAGATGAAATTACCTC EcoR I
P4 R CGAGCTCTTAGTGGTGGTGGTGGTGGTGCTGCCGGCAAAAGTGAT Sac I
P5 F CGCGTCGACAAAGGAGGGAAATCATGCACCACCACCACCACCACATGGCAGAAA AAGGCATTAC Sal I
P5 R CGCGAATTCTTAAGAGCTGTACGCGGAGTTG EcoR I
Table 2 Primers used in this study
Fig.1 The L-ornithine biosynthesis pathway in Corynebacterium crenatum SYPO-1
Fig.2 PCR results of argE genes and identification of pDXW10-argE plasmids and empty pDXW10 plasmid by single and double enzyme digestion (a) PCR results of different argE gene amplification M: DL2000; Lane 1: BsargE; Lane : SmargE; Lane 3: KpargE; Lane 4: EcargE (b) Identification of pDXW10-argE plasmids and empty pDXW10 plasmid by single and double enzyme digestion M: DL10000 maker; Lane 1,2; Results of single and double enzyme digestion of pDXW10-BsargE; Lane 3,4: Results of single and double enzyme digestion of pDXW10-SmargE; Lane 5,6: Results of single and double enzyme digestion of pDXW10-KpargE; Lane 4: Results of single and double enzymes digestion of pDXW10-EcargE; Lane 9: Result of double enzyme digestion of empty pDXW10
Enzyme Organism Specific
activity(U/mg)		 pH optimum Temperature
optimum (℃)		 Metals
EcNAOD Escherichia coli BL21(DE3) 416.26 7.0 37 Mg2+, Li+ (0.1mmol/L a litter promote to activity)
SmNAOD* Serratia marcescens Y213 798.98 7.0 37 Mn2+,Li+,Mg2+ (0.1mmol/L activity increases more than 50%)
KpNAOD Klebsiella pneumoniae P01 460.06 7.5 37 Mn2+ (0.1mmol/L activity increases more than 50%)
BsNAOD Bacillus subtilis subsp. subtilis strain168G 62.03 8.0 50 Mn2+ (0.1mmol/L 57% inhibition)
Table 3 Enzymatic properties of recombinant enzymes used in this study
Fig.3 SDS-PAGE analysis of crude proteins and NAOD purification M: Unstained protein ladder; Lane1: Crude enzyme of E. coli BL21 with plasmid pDXW10(+); Lane 2,6: Crude enzyme of E. coli BL21 with plasmid pDXW10(+)-KpargE, purified enzyme of E. coli BL21 with plasmid pDXW10(+)-KpargE; Lane 3, 7: Crude enzyme of E. coli BL21 with plasmid pDXW10(+)-EcargE, purified enzyme of E. coli BL21 with plasmid pDXW10(+)-KpargE; Lane 4, 8: Crude enzyme of E. coli BL21 with plasmid pDXW10(+)-BsargE, purified enzyme of E. coli BL21 with plasmid pDXW10(+)-BsargE; Lane 5,9: Crude enzyme of E. coli BL21 with plasmid pDXW10(+)-SmargE, purified enzyme of E. coli BL21 with plasmid pDXW10(+)-SmargE
Fig. 4 Thermal and pH stability of N-acetyl-L-ornithine deacetylase (a)Under 30℃, thermal stability of N-acetyl-L-ornithine deacetylase (b) Under pH7, pH stability of N-acetyl-L-ornithine deacetylase
Fig. 5 SDS-PAGE analysis of the overexpression of SmNAOD and CcOATase in recombinant Corynebacterium crenatum SYPO-1 M: Unstained protein ladder;Lane 1: SYPO-1/pDXW10(+);Lane 2: SYPO-1/pDXW10(+)-CcargJ;Lane 3: SYPO-1/pDXW10(+)-SmargE
Crude enzyme activity Total activity
of NAOD
(U/ml)		 Total activity
of OATase
(U/ml)
C. crenatum SYPO-1 0 5.9
SYPO-1/pDXW10-argJ 0 50.8
SYPO-1/pDXW10-argE 128.4 12.5
Table 4 Crude enzyme activities for NAOD and OATase in C. crenatum
Fig.6 Comparison of L-ornithine production between C. crenatum SYPO-1, recombinant SYPO-2 and SYPO-3 (a) Dry cell weight and glucose concentration (b) L-ornithine concentration and L-ornithine productivity C. crenatum SYPO-1 (filled up triangles); Recombinant SYPO-2 (filled squares) and recombinant SYPO-3 (filled circles) (all values the mean of at least three independent experiments, and error bars represent the standard deviations of the biological replicates)
Amino acid/strains SYPO-1 (g/L) SYPO-1/argJ (g/L) SYPO-1/argE (g/L)
Asp 0.028±0.000 2 0.026±0.000 3 0.023±0.000 2
Glu 0.15±0.001 0.14±0.009 0.16±0.009
Ser 0.004±0.000 2 0.005±0.000 3 0.004±0.000 2
His 0.022±0.000 3 0.023±0.000 1 0.024±0.000 2
Gly 0.058±0.000 5 0.054±0.000 21 0.059±0.000 43
Thr 0.008±0.000 06 0.005±0.000 02 0.004±0.000 03
Arg 0.005±0.000 2 0.004±0.000 1 0.004±0.000 3
Ala 0.706±0.004 3 0.659±0.003 2 0.694±0.004 5
Tyr 0.196±0.001 2 0.210±0.002 6 0.187±0.001 5
Cys 0.008±0.000 2 0.012±0.000 3 0.009±0.000 2
Val 0.985±0.003 0.598±0.002 0.241±0.001
Met 0.035±0.000 1 0.034±0.000 2 0.032±0.000 1
Phe 0.042±0.000 3 0.040±0.000 2 0.032±0.000 2
Ile 3.528±0.032 3.112±0.027 2.432±0.018
Leu 0.769±0.006 5 0.654±0.004 3 0.712±0.005 9
Lys 3.947±0.043 3.017±0.037 2.192±0.015
Pro 0.0007±0.000 03 0.000 4±0.000 02 0.000 2±0.000 01
Trp 0.008±0.000 3 0.006±0.000 5 0.005±0.000 3
Table 5 Concentration of 18 standard amino acid in the 5L fermentation supernatants of the mutant strains
Strain L-ornithine
titer(g/L)		 Cultivation
method		 Medium Key
engineering		 Reference
C. crenatum SYPO-3 38.5 Bioreactor;
batch		 Semi-defined ?Deletion of argF, argR, and proB
?Heterologous expression of N-acetyl-L-ornithine deacetylase		 This study
C. glutamicum YW06
(pSY223)		 51.5 Bioreactor;
fed-batch		 Semi-defined ?Deletion of argF, argR, and proB
?Reinforcement of the pp pathway flux
?The use of a feedback-resistant enzyme		 [8]
E. coli SJ7055 0.009 Shake flask;
batch		 Semi-defined ?Deletion of argF, argR, argI, speF and proB
?The use of feedback-resistant argG		 [3]
C. glutamicum SJ8074
(pEK-CJBD)		 0.179 Shake flask;
batch		 Semi-defined ?Deletion of argF, argR, and proB [7]
C. glutamicum
△APE6937R42		 24.1 Shake flask;
batch		 Semi-defined ?Deletion of argF, argR, and proB
?Adaptive evolution in presence of L-ornithine		 [5]
Corynebacterium glutamicum S9114 18.4 Shake flask;
batch		 Semi-defined ?Inactivation of argF, ncgl1221, argR, and putP, attenuation of odhA
?Overexpression of LysE		 [9]
Table 6 Comparison of various strains for the production of L-ornithine
[1]   Han P S, Fishel R S, Efron D T , et al. Effect of supplemental ornithine on wound healing. Journal of Surgical Research, 2002,106(2):299.
doi: 10.1006/jsre.2002.6471 pmid: 12175982
[2]   卢冬梅 . 微生物合成鸟氨酸的代谢工程研究进展. 微生物学通报, 2015,42(7):1391-1399.
doi: 10.13344/j.microbiol.china.140716
[2]   Lu D M . Progress in metabolic engineering for microbial synthesis of ornithine. Microbiology China, 2015,42(7):1391-1399.
doi: 10.13344/j.microbiol.china.140716
[3]   Lee Y J, Cho J Y . Genetic manipulation of a primary metabolic pathway for L-ornithine production in Escherichia coli. Biotechnol Lett, 2006,28(22):1849-1856.
doi: 10.1007/s10529-006-9163-y
[4]   Sakanyan V, Petrosyan P, Lecocq M , et al. Genes and enzymes of the acetyl cycle of arginine biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early steps of the arginine pathway. Microbiology, 1996,142(Pt 1):99-108.
doi: 10.1099/13500872-142-1-99
[5]   Jiang L Y, Chen S G, Zhang Y Y , et al. Metabolic evolution of Corynebacterium glutamicum for increased production of L-ornithine. BMC Biotechnology, 2013,13(1):47.
doi: 10.1186/1472-6750-13-47 pmid: 3681597
[6]   Dou W, Xu M, Cai D , et al. Improvement of L-arginine production by overexpression of a bifunctional ornithine acetyltransferase in Corynebacterium crenatum. Appl Biochem Biotechnol, 2011,165(3-4):845-855.
doi: 10.1007/s12010-011-9302-3
[7]   Hwang J H, Hwang G H, Cho J Y . Effect of increased glutamate availability on L-ornithine production in Corynebacterium glutamicum. Journal of Microbiology & Biotechnology, 2008,18(4):704.
[8]   Kim S Y, Lee J, Lee S Y . Metabolic engineering of Corynebacterium glutamicum for the production of L-ornithine. Biotechnol Bioeng, 2015,112(2):416-421.
doi: 10.1002/bit.v112.2
[9]   Zhang B, Yu M, Zhou Y , et al. Systematic pathway engineering of Corynebacterium glutamicum S9114 for L-ornithine production. Microb Cell Fact, 2017,16(1):158.
doi: 10.1186/s12934-017-0776-8
[10]   Xu M, Rao Z, Dou W , et al. The role of ARGR repressor regulation on L-arginine production in Corynebacterium crenatum. Appl Biochem Biotechnol, 2013,170(3):587-597.
doi: 10.1007/s12010-013-0212-4 pmid: 23564434
[11]   Man Z, Xu M, Rao Z , et al. Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production. Scientific Reports, 2016,6:28629.
doi: 10.1038/srep28629 pmid: 27338253
[12]   赵芹芹, 罗玉常, 窦文芳 , 等. 钝齿棒杆菌的代谢改造:L-鸟氨酸与L-瓜氨酸合成菌株的构建. 微生物学通报, 2014,41(10):1941-1947.
doi: 10.13344/j.microbiol.china.140065
[12]   Zhao Q Q, Luo Y C, Dou W F , et al., Engineering Corynebacterium crenatum for construction of L-ornithine and L-citrulline producers. Microbiology China, 2014,41(10):1941-1947.
doi: 10.13344/j.microbiol.china.140065
[13]   Xu M, Rao Z, Yang J , et al. Heterologous and homologous expression of the arginine biosynthetic argC-H cluster from Corynebacterium crenatum for improvement of (L) -arginine production. J Ind Microbiol Biotechnol, 2012,39(3):495-502.
doi: 10.1007/s10295-011-1042-4
[14]   Zhao Q, Luo Y, Dou W , et al. Controlling the transcription levels of argGH redistributed L-arginine metabolic flux in N-acetylglutamate kinase and argR-deregulated Corynebacterium crenatum. Journal of Industrial Microbiology & Biotechnology, 2016,43(1):55-66.
[15]   李子武, 张显, 徐美娟 , 等. 一株产灵菌红素黏质沙雷氏菌的筛选、鉴定及发酵条件. 食品与生物技术学报, 2012,31(10):1018-1024.
doi: 10.3969/j.issn.1673-1689.2012.10.002
[15]   Li Z W, Zhang X, Xu M J , et al. Screening and identification a Serratia marcescen Strain producing red-pigment and preliminary study of the fermentation conditions. Journal of Food Science and Biotechbology, 2012,31(10):1018-1024.
doi: 10.3969/j.issn.1673-1689.2012.10.002
[16]   徐美娟, 杨套伟, 饶志明 , 等. 克雷伯氏菌甘油脱氢酶dhaD的克隆表达、纯化及酶学性质研究. 中国生物工程杂志, 2008,28(12):30-35.
doi: 10.3321/j.issn:1002-6630.2008.06.045
[16]   Xu M, Rao Z, Yang W , et al. Expression, purification and enzymatic characterization of Klebsiella sp. glycerol dehydrogenase in E. coli. Chinese Journal of Biotechnology, 2008,28(12):30-35.
doi: 10.3321/j.issn:1002-6630.2008.06.045
[17]   Xu D, Tan Y, Shi F , et al. An improved shuttle vector constructed for metabolic engineering research in Corynebacterium glutamicum. Plasmid, 2010,64(2):85.
doi: 10.1016/j.plasmid.2010.05.004
[18]   徐美娟, 张显, 饶志明 , 等. 钝齿棒杆菌N-乙酰鸟氨酸转氨酶的克隆表达分析及其重组菌的精氨酸发酵. 生物工程学报, 2011,27(7):1013-1023.
[18]   Xu M J, Zhang X, Rao Z M , et al, Expression, cloning, expression and characterization of N-acetylornithine aminotransferase from Corynebacterium crenatum and its effects on L-arginine fermentation. Chinese Journal of Biotechnology, 2011,27(7):1013-1023.
[19]   Schã Gger H .Tricine-SDS-PAGE. Nature Protocols, 2006,1(1):16-22.
doi: 10.1038/nprot.2006.4
[20]   Sambrook J, Fritsch E F, Maniatis T . Molecular cloning. a laboratory manual. Analytical Biochemistry, 2001,186(1):182-183.
[21]   Bradford M M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976,72(s 1-2):248-254.
doi: 10.1016/0003-2697(76)90527-3
[22]   Javidmajd F, Blanchard J S . Mechanistic analysis of the argE-encoded N-acetylornithine deacetylase. Biochemistry, 2000,39(6):1285.
doi: 10.1021/bi992177f
[23]   Marc F, Weigel P, Legrain C , et al. An invariant threonine is involved in self-catalyzed cleavage of the precursor protein for ornithine acetyltransferase. J Biol Chem, 2001,276(27):25404-25410.
doi: 10.1074/jbc.M100392200
[24]   Tauch A, Kirchner O, Löffler B , et al. Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. Current Microbiology, 2002,45(5):362-367.
doi: 10.1007/s00284-002-3728-3
[25]   Xu M, Rao Z, Dou W , et al. Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production. Amino Acids, 2012,43(1):255-266.
doi: 10.1007/s00726-011-1069-x
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