Genome Shuffling for Rapid Improvement of L-Ornithine Production by Corynebacterium glutamicum

Abstract

Abstract: Genome shuffling was applied to Corynebacterium glutamicum ATCC13032 to achieve rapid improvement of L-ornithine production. Six mutant strains with subtle improvements in L-ornithine production were obtained by treatment with UV light, nitrosoguanidine or ethyl methane sulfonate and subjected to recursive protoplast fusion. The effects of medium components on the protoplast regeneration rate were investigated. High-yield strains were screened using plates containing different concentrations of sulfaguanidine and NaF. After two rounds of genome shuffling, two shuffled strains producing L-ornithine at high level with good genetic stability were selected and named as F2-6 and F2-29. After 72h of shake-flask fermentation, the L-ornithine yield of strain F2-6 reached 2.99 g/L, which was 13.6 times higher than that of the original strain.

Key words: genome shuffling, Corynebacterium glutamicum, L-ornithine, production

CLC Number:

Q939.97

Jiazhou LI. Genome Shuffling for Rapid Improvement of L-Ornithine Production by Corynebacterium glutamicum[J]. FOOD SCIENCE, 2012, 33(15): 206-209.

References

[1] Morris S M. Regulation of enzymes of the urea cycle and arginine metabolism[J]. Annu Rev Nutr, 2002, 22: 87-105.
[2] 汪江波, 邹玉玲, 薛海燕. L-鸟氨酸的生物功能及生产研究[J]. 食品研究与开发, 2007, 28(3): 166-169.
[3] Jalan R, Wright G, Davies N A, et al. L-Ornithine phenylacetate (OP): a novel treatment for hyperammonemia and hepatic encephalopathy[J]. Med Hypotheses, 2007, 69: 1064-1069.
[4] Shi H P, Fishel R S, Efron D T, et al. Effect of supplemental ornithine on wound healing[J]. J Surg Res, 2002, 106: 299-302.
[5] Gotoh T, Kikuchi K I, Kagaya A. Direct production of L-ornithine from casein by commercial digestive enzymes and in situ activated arginase[J]. Bioprocess Biosyst Eng, 2010, 33: 773–777.
[6] Hwang J H, Hwang G H, Cho J Y. Effect of increased glutamate availability on L-ornithine production in Corynebacterium glutamicum[J]. J Microbiol Biotechnol, 2008, 18(4): 704–710.
[7] Lee S Y, Cho J Y, Lee H J, et al. Enhancement of ornithine production in proline-supplemented Corynebacterium glutamicum by ornithine cyclodeaminase[J]. J Microbiol Biotechnol, 2010, 20(1): 127–131.
[8] Lee Y J, Cho J Y. Genetic manipulation of a primary metabolic pathway for L-ornithine production in Escherichia coli[J]. Biotechnol Lett, 2006, 28: 1849-1856.
[9] Zhang Y X, Perry K, Vinci V A, et al. Genome shuffling leads to rapid phenotypic improvement in bacteria[J]. Nature, 2002, 415: 644?646.
[10] Kang J X, Chen X J, Chen W R, et al. Enhanced production of pullulan in Aureobasidium pullulans by a new process of genome shuffling[J]. Process Biochem, 2011, 46: 792–795.
[11] 于雷, 雷霆, 裴晓林, 等. 应用基因组改组选育耐糖L-乳酸高产菌[J]. 食品科学, 2007, 28(9): 369-373.
[12] 王立梅, 齐斌. 基因组改组技术对L-乳酸产生菌耐热性的影响[J]. 食品科学, 2008, 29(10): 395-398.
[13] John R P，Gangadharan D，Nampoothiri K M． Genome shuffling of Lactobacillus delbrueckii mutant and Bacillus amyloliquefaciens through protoplasmic fusion for L-lactic acid production from starchy wastes[J]. Bioresource Technol, 2008, 99( 17): 8008-8015．
[14] Wang Mingzi, Liu Shaosong, Li Yaoyao, et al. Protoplast mutation and genome shuffling induce the endophytic fungus Tubercularia sp. TF5 to produce new compounds[J]. Curr Microbiol, 2010, 61: 254–260.
[15] Zhang Ying, Liu Jianzhong, HuangJunsheng, et al. Genome shuffling of Propionibacterium shermanii for improving vitamin B12 production and comparative proteome analysis[J]. J Biotechnol, 2010, 148: 139-143.
[16] 张媛媛, 刘建忠，江文明. 定向诱变谷氨酸棒杆菌 13032产L-鸟氨酸的研究[J]. 现代食品科技, 2010, 26(12): 1330-1334.
[17] 张克旭, 陈宁, 张永志, 等. 用原生质体融合技术选育谷氨酸高产菌[J]. 微生物学报, 1991, 31(2): 108-114.
[18] 陈涛, 陈洵, 王靖宇，等. DNA及基因组改组在代谢工程中的应用[J]. 化工学报, 2004, 55(11): 1753-1758.
[19] 邹玉玲, 薛海燕, 汪江波, 等. 钴60与紫外线复合诱变选育L－鸟氨酸高产菌株[J]. 华西药学杂志，2007, 22(3)：263-265.
[20] 王春平, 韦强, 鲍国连, 等. 微生物原生质体融合技术研究进展[J]. 动物医学进展, 2008, 29(5): 64-67.