沈观宇,吴绵斌,林建平,杨立荣

• 1:浙江大学 生物质化工教育部重点实验室，浙江大学 化学工程与生物工程学院，浙江 杭州 310027

摘要(Abstract)：

目前氨基酸生产菌已广泛应用于氨基酸的工业化生产中，而生产菌的胞内氨基酸浓度过高往往不利于发酵生产，研究加快胞内氨基酸向外转运能很好地解决这一问题，并增加氨基酸的发酵产量。细菌中氨基酸外向转运蛋白负责将胞内氨基酸转运至胞外，因此氨基酸外向转运蛋白的研究是氨基酸发酵生产的潜在研究方向。作者根据近年来的相关报道，总结了氨基酸生产菌中氨基酸外向转运蛋白的研究现状，介绍了氨基酸外向转运蛋白在发酵生产中的最新应用。同时，对外向转运蛋白研究中采用的主要策略和方法进行了概括，并结合最新的技术进展，对今后的研究方向进行了展望。

关键词(KeyWords)： 外向转运蛋白;氨基酸;代谢工程;通道蛋白

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 沈观宇,吴绵斌,林建平,杨立荣

DOI: 10.3969/j.issn.1003-9015.2018.06.002

参考文献(References)：

• [1] Shiio I, Ujigawa K. Enzymes of the glutamate and aspartate synthetic pathways in a glutamate-producing bacterium, Brevibacterium flavum[J]. Journal of Biochemistry, 1978, 84(3):647-657.
  [2] Shimizu H, Hirasawa T. Production of glutamate and glutamate-related amino acids:molecular mechanism analysis and metabolic engineering[M]. Qerlin:Springer Berlin Heidelberg, 2006,5:1-38
  [3] Radmacher E, Stansen K C, Besra G S, et al. Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis, elicits L-glutamate efflux of Corynebacterium glutamicum[J]. Microbiology, 2005, 151(5):1359-1368.
  [4] Park S, Imlay J A. High levels of intracellular cysteine promote oxidative DNA damage by driving the fenton reaction[J]. Journal of Bacteriology, 2003, 185(6):1942-1950.
  [5] Kim S, Ihara K, Katsube S, et al. Characterization of the L-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of L-alanine and its derivatives[J]. MicrobiologyOpen, 2015, 4(4):632-643.
  [6] Stefan B, Reinhard K. Lysine excretion by Corynebacterium glutamicum[J]. European Journal of Biochemistry, 1991, 202(1):137-143.
  [7] Vrljic M, Sahm H, Eggeling L. A new type of transporter with a new type of cellular function:L-lysine export from Corynebacterium glutamicum[J]. Molecular Microbiology, 1996, 22(5):815-826.
  [8] Li Z H, Chen N. Study on the directional breeding of high glutamic-acid production strain GTS190 and the control on its fermentation[J]. Advanced Materials Research, 2012, 573(10):1029-1033.
  [9] SU Yue-wen (苏跃稳). Modification of L-threonine-producing strain and optimization of fermentation process (L-苏氨酸基因工程菌的改造及发酵过程的优化)[D]. Changchun (长春):Jilin University (吉林大学), 2017.
  [10] YAO Li-pin (姚丽萍). Metabolic modification of glycometabolism for sucrose to increase L-serine production by Corynobacterium glutamicum(蔗糖利用途径改造促进谷氨酸棒杆菌合成L-丝氨酸)[D]. Wuxi (无锡):Jiangnan University (江南大学), 2016.
  [11] ZHANG Xin-wu(张新武), YANG Xiao-ming(杨晓明), LIAN Na-na(廉娜娜), et al. Research on fermentation process with L-tryptophan auxotrophic strains of high-yield (L-色氨酸营养缺陷型高产菌株的发酵工艺研究)[J]. Journal of Food Safety and Quality(食品安全质量检测学报), 2014, 5(12):4056-4062.
  [12] Milton H. Saier, Jr. Families of transmembrane transporters selective for amino acids and their derivatives[J]. Microbiology, 2000, 146(8):1775-1795.
  [13] Sasaki H, Sato D, Oshima A. Importance of the high-expression of proline transporter PutP to the adaptation of Escherichia coli to high salinity[J]. Biocontrol Science, 2017, 22(2):121-124.
  [14] Nakamura J, Hirano S, Ito H, et al. Mutations of the Corynebactetium glutamicum NCgl1221 gene, encoding a mechanosensitive channel hornolog, induce L-glutarnic acid production[J]. Applied and Environmental Microbiology, 2007, 73(14):4491-4498.
  [15] Wang Y, Cao G, Xu D, et al. A novel L-glutamate exporter of Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2018, 84(6):1-15.
  [16] Nandineni M G J. Evidence for an arginine exporter encoded by yggA (argO) that is regulated by the LysR-type transcriptional regulator ArgP in Escherichia coli[J]. Journal of Bacteriology, 2004, 186(11):3539-3546.
  [17] Pathania A, Sardesai A A. Distinct paths for basic amino acid export in Escherichia coli:YbjE (LysO) mediates export of L-lysine[J]. Journal of Bacteriology, 2015, 197(12):2036-2047.
  [18] Lubitz D, Jorge J M, PacRez-GarcãA F, et al. Roles of export genes cgmA and lysE for the production of L-arginine and L-citrulline by Corynebacterium glutamicum[J]. Applied Microbiology and Biotechnology, 2016, 100(19):8465-8474.
  [19] Kutukova E A, Livshits V A, Altman I P, et al. The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression[J]. Febs Letters, 2005, 579(21):4629-4634.
  [20] Doroshenko V, Airich L M, Kolokolova A, et al. YddG from Escherichia coli promotes export of aromatic amino acids[J]. Fems Microbiology Letters, 2007, 275(2):312-318.
  [21] Kennerknecht N, Sahm H, Yen M R, et al. Export of L-isoleucine from Corynebacterium glutamicum:a two-gene-encoded member of a new translocator family[J]. Journal of Bacteriology, 2002, 184(14):3947-3956.
  [22] Trötschel C, Deutenberg D, Bathe B, et al. Characterization of methionine export in Corynebacterium glutamicum[J]. Journal of Bacteriology, 2005, 187(11):3786-3794.
  [23] Park J H, Lee K H, Kim T Y, et al. Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(19):7797-7802.
  [24] Park J H, Oh J E, Lee K H, et al. Rational design of Escherichia coli for L-isoleucine production[J]. Acs Synthetic Biology, 2012, 1(11):532-540.
  [25] Liu Q, Liang Y, Zhang Y, et al. YjeH is a novel exporter of l-methionine and branched-chain amino acids in Escherichia coli[J]. Applied and Environmental Microbiology, 2015, 81(22):7753-7766.
  [26] Dassler T, Maier T, Winterhalter C, et al. Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway[J]. Molecular Microbiology, 2000, 36(5):1101-1112.
  [27] Franke I, Resch A, Dassler T, et al. YfiK from Escherichia coli promotes export of O-acetyl serine and cysteine[J]. Journal of Bacteriology. 2003, 185(4):1161-1166.
  [28] Yamada S, Awano N, Inubushi K, et al. Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli[J]. Applied and Environmental Microbiology, 2006, 72(7):4735-4742.
  [29] Pittman M S, Corker H, Wu G, et al. Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter required for cytochrome assembly[J]. Journal of Biological Chemistry, 2002, 277(51):49841-49849.
  [30] Wiriyathanawudhiwong N, Ohtsu I, Li Z D, et al. The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli[J]. Applied Microbiology and Biotechnology, 2009, 81(5):903-913.
  [31] Hori H, Yoneyama H, Tobe R, et al. Inducible l-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia coli[J]. Applied and Environmental Microbiology, 2011, 77(12):4027-4034.
  [32] Livshits V A, Zakataeva N P, Aleshin V V, et al. Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli[J]. Research in Microbiology, 2003, 154(2):123-135.
  [33] Zakataeva N P, Tokmakova I L, Troshin P V, et al. The novel transmembrane Escherichia coli proteins involved in the amino acid efflux[J]. Febs Letters, 1999, 452(3):228-232.
  [34] Simic P, Sahm H, Eggeling L. L-Threonine export:use of peptides to identify a new translocator from Corynebacterium glutamicum[J]. Journal of Bacteriology, 2001, 183(18):5317-5324.
  [35] Hashimoto K, Murata J, Konishi T, et al. Glutamate is excreted across the cytoplasmic membrane through the NCgl1221 channel of Corynebacterium glutamicum by passive diffusion[J]. Biosci Biotechnol Biochem, 2012, 76(7):1422-1424.
  [36] Becker M, Krämer R. MscCG from Corynebacterium glutamicum:functional significance of the C-terminal domain[J]. European Biophysics Journal, 2015, 44(7):577-588.
  [37] Yamashita C, Hashimoto K, Kumagai K, et al. L-Glutamate secretion by the N-terminal domain of the Corynebacterium glutamicum NCgl1221 mechanosensitive channel[J]. Biosci Biotechnol Biochem, 2013, 77(5):1008-1013.
  [38] Hashimoto K I, Nakamura K, Kuroda T, et al. The protein encoded by NCgl1221 in Corynebacterium glutamicum functions as a mechanosensitive channel[J]. Biosci Biotechnol Biochem, 2010, 74(12):2546-2549.
  [39] Park S H, Kim H U, Kim T Y, et al. Metabolic engineering of Corynebacterium glutamicum for L-arginine production[J]. Nature Communications, 2014, 5(8):4618-4618.
  [40] Zhang B, Yu M, Zhou Y, et al. Systematic pathway engineering of Corynebacterium glutamicum S9114 for L-ornithine production[J]. Microbial Cell Factories, 2017, 16(1):158-158.
  [41] Eggeling L, Sahm H. l -Glutamate and l -lysine:traditional products with impetuous developments[J]. Applied Microbiology and Biotechnology, 1999, 52(2):146-153.
  [42] Eggeling L, Bott M. A giant market and a powerful metabolism:L-lysine provided by Corynebacterium glutamicum[J]. Applied Microbiology and Biotechnology, 2015, 99(8):3387-3394.
  [43] Bellmann A, Vrljić M, Pátek M, et al. Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum[J]. Microbiology, 2001, 147(7):1765-1774.
  [44] Ishikawa K, Toda-Murakoshi Y, Ohnishi F, et al. Medium composition suitable for L-lysine production by Methylophilus methylotrophus in fed-batch cultivation[J]. Journal of Bioscience and Bioengineering, 2008, 106(6):574-579.
  [45] Xu M, Rao Z, Yang J, et al. The effect of a LYSE exporter overexpression on l-arginine production in Corynebacterium crenatum[J]. Current Microbiology, 2013, 67(3):271-278.
  [46] Ginesy M, Belotserkovsky J, Enman J, et al. Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis[J]. Microbial Cell Factories, 2015, 14(1):29-29.
  [47] Tsuchiya H, Doki S, Takemoto M, et al. Structural basis for amino acid export by DMT superfamily transporter YddG[J]. Nature, 2016, 534(7607):417-420.
  [48] Liu Q, Cheng Y, Xie X, et al. Modification of tryptophan transport system and its impact on production of L-tryptophan in Escherichia coli[J]. Bioresource Technology, 2012, 114(2):549-554.
  [49] Wang J, Cheng L K, Wang J, et al. Genetic engineering of Escherichia coli to enhance production of L-tryptophan[J]. Applied Microbiology and Biotechnology, 2013, 97(17):7587-7596.
  [50] Vogt M, Haas S, Klaffl S, et al. Pushing product formation to its limit:metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction[J]. Metabolic Engineering, 2014, 22(3):40-52.
  [51] QIN Tian-yu (秦天宇). Metabolic engineering of Corynebacterium glutamicum ATCC 13032 to produce L-methionine (代谢工程改造谷氨酸棒杆菌生产L-甲硫氨酸)[D]. Wuxi (无锡):Jiangnan University (江南大学), 2014.
  [52] Yin L, Shi F, Hu X, et al. Increasing l-isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two-component export system BrnFE[J]. Journal of Applied Microbiology, 2013, 114(5):1369-1377.
  [53] CHEN Cheng (陈诚). Metabolic engineering of Corynebacterium glutamicum for L-valine production (代谢工程改造谷氨酸棒状杆菌生产L-缬氨酸)[D]. Wuxi (无锡):Jiangnan University (江南大学), 2015.
  [54] Hoffmann C, Leis A, Niederweis M, et al. Disclosure of the mycobacterial outer membrane:cryo-electron tomography and vitreous sections reveal the lipid bilayer structure[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(10):3963-3967.
  [55] Liu H, Fang G, Wu H, et al. L-Cysteine Production in Escherichia coli based on rational metabolic engineering and modular strategy[J]. Biotechnology Journal, 2018, 13(5):e1700695.
  [56] Stolz M, Peterswendisch P, Etterich H, et al. Reduced folate supply as a key to enhanced L-serine production by Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2007, 73(3):750-755.
  [57] Ihara K, Sato K, Hori H, et al. Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli[J]. Journal of Bioscience and Bioengineering, 2017, 123(4):444-450.
  [58] Kim S, Ihara K, Katsube S, et al. Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli, on the L-alanine export[J]. Archives of Microbiology, 2017, 199(1):105-114.
  [59] ZHANG Xue-li(张学礼), ZHANG Dong-zhu(张冬竹). Construction and application of a strain with high yield of L-alanine(一种高产L-丙氨酸的xz-a26菌株及构建方法与应用):CN, 102329765 A[P]. 2012-01-25.
  [60] Kruse D, Krämer R, Eggeling L, et al. Influence of threonine exporters on threonine production in Escherichia coli[J]. Applied Microbiology and Biotechnology, 2002, 59(2-3):205-210.
  [61] Simic P, Willuhn J, Sahm H, et al. Identification of glyA (encoding serine hydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2002, 68(7):3321-3327.
  [62] Kutukova E A, Zakataeva N P, Livshits V A. Expression of genes encoding the RhtB family proteins depends on the global regulator lrp[J]. Molecular Biology, 2005, 39(3):374-378.
  [63] Diesveld R, Tietze N, Fürst O, et al. Activity of exporters of Escherichia coli in Corynebacterium glutamicum, and their use to increase L-threonine production[J]. Journal of Molecular Microbiology and Biotechnology, 2009, 16(3-4):198-207.
  [64] Lee J H, Sung B H, Mi S K, et al. Metabolic engineering of a reduced-genome strain of Escherichia coli for L-threonine production[J]. Microbial Cell Factories, 2009, 8(1):2-2.
  [65] Zhen K, Yang W, Qiang W, et al. Metabolic engineering to improve 5-aminolevulinic acid production[J]. Bioengineered Bugs, 2011, 2(6):342-345.
  [66] Lee K H, Park J H, Kim T Y, et al. Systems metabolic engineering of Escherichia coli for L-threonine production[J]. Molecular Systems Biology. 2007, 3(1):149-149.
  [67] Gu P, Yang F, Li F, et al. Knocking out analysis of tryptophan permeases in Escherichia coli for improving L-tryptophan production[J]. Applied Microbiology and Biotechnology, 2013, 97(15):6677-6683.
  [68] Kawakami M, Juge N, Kato Y, et al. Efficient mass spectral analysis of active transporters overexpressed in Escherichia coli[J]. Journal of Proteome Research, 2018, 17(3):1108-1119.
  [69] Yan Z, Meng Y, Xu D, et al. Structural insights into the secretin translocation channel in the type Ⅱ secretion system[J]. Nature Structural and Molecular Biology, 2017, 24(2):177-183.
  [70] Rouse S L, Hawthorne W J, Lambert S, et al. Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter[J]. Acta Crystallographica, 2016, 72(12):892-896.