葡萄糖和木糖高效共利用代谢工程研究进展
作者:
基金项目:

国家自然科学基金(22161142008);中国科学院大连化学物理研究所创新基金(DICP I202210)


Metabolic engineering for the efficient co-utilization of glucose and xylose
Author:
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [64]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    摘要:

    利用可再生生物质高效生产化学品是可持续生物制造的重要途径。木质纤维素来源广泛且可再生,被认为是极具潜力的第二代生物炼制原料,木质纤维素水解物中混合糖的高效共发酵被认为是降低产品成本的关键途径之一。然而,大多数微生物由于碳源阻遏效应会优先消耗葡萄糖而非木糖,严重制约了木质纤维素转化效率。因此,开发能够同时利用葡萄糖和木糖的微生物平台对工业规模生产至关重要。本文综述了基于代谢工程策略促进微生物高效共利用葡萄糖和木糖的关键方法和研究实例,包括葡萄糖抑制缓解、木糖跨膜转运途径改造、木糖代谢途径构建和定向进化等策略。

    Abstract:

    Microbial production of chemicals from renewable biomass has emerged as a crucial route for sustainable bio-manufacturing. Lignocellulose with a renewable property and wide sources is supposed to be a promising feedstock for the second-generation biorefinery. The efficient co-utilization of mixed sugars from lignocellulosic hydrolysates represents one of the key challenges in reducing the production cost. However, most microorganisms prefer glucose over xylose due to carbon catabolite repression, which constrains the efficiency of lignocellulosic conversion. Therefore, developing the microbial platforms capable of simultaneously utilizing glucose and xylose is paramount for economically viable industrial-scale production. This article reviews the key strategies and studies of metabolic engineering for promoting efficient co-utilization of glucose and xylose by microorganisms. The representative strategies include relieving glucose repression, enhancing xylose transport, constructing xylose metabolic pathways, and directed evolution.

    参考文献
    [1] ZHENG XJ, XIAN XL, HU L, TAO SH, ZHANG XD, LIU Y, LIN XQ. Efficient short-time hydrothermal depolymerization of sugarcane bagasse in one-pot for cellulosic ethanol production without solid-liquid separation, water washing, and detoxification[J]. Bioresource Technology, 2021, 339: 125575.
    [2] WANG L, YORK SW, INGRAM LO, SHANMUGAM KT. Simultaneous fermentation of biomass-derived sugars to ethanol by a co-culture of an engineered Escherichia coli and Saccharomyces cerevisiae[J]. Bioresource Technology, 2019, 273: 269-276.
    [3] ASEMOLOYE MD, BELLO TS, OLADOYE PO, REMILEKUN GBADAMOSI M, BABARINDE SO, EBENEZER ADEBAMI G, OLOWE OM, TEMPORITI MEE, WANEK W, MARCHISIO MA. Engineered yeasts and lignocellulosic biomaterials: shaping a new dimension for biorefinery and global bioeconomy[J]. Bioengineered, 2023, 14(1): 2269328.
    [4] MOYSÉS DN, REIS VCB, de ALMEIDA JRM, de MORAES LMP, TORRES FAG. Xylose fermentation by Saccharomyces cerevisiae: challenges and prospects[J]. International Journal of Molecular Sciences, 2016, 17(3): 207.
    [5] HOU J, QIU CX, SHEN Y, LI HX, BAO XM. Engineering of Saccharomyces cerevisiae for the efficient co-utilization of glucose and xylose[J]. FEMS Yeast Research, 2017, 17(4): fox034.
    [6] BUIJS NA, SIEWERS V, NIELSEN J. Advanced biofuel production by the yeast Saccharomyces cerevisiae[J]. Current Opinion in Chemical Biology, 2013, 17(3): 480-488.
    [7] PARK JM, VINUSELVI P, LEE SK. The mechanism of sugar-mediated catabolite repression of the propionate catabolic genes in Escherichia coli[J]. Gene, 2012, 504(1): 116-121.
    [8] CHANDUKISHORE T, DAS S, DAS P, VEERANKI VD, PRABHU AA. Re-routing the hemicellulosic fraction of lignocellulosic biomass toward value added products: a pragmatic bio refinery approach[J]. Journal of Environmental Chemical Engineering, 2024, 12(2): 111971.
    [9] DONZELLA L, VARELA JA, SOUSA MJ, MORRISSEY JP. Identification of novel pentose transporters in Kluyveromyces marxianus using a new screening platform[J]. FEMS Yeast Research, 2021, 21(4): foab026.
    [10] CHUPAZA MH, PARK YR, KIM SH, YANG JW, JEONG GT, KIM SK. Bioethanol production from Azolla filiculoides by Saccharomyces cerevisiae, Pichia stipitis, Candida lusitaniae, and Kluyveromyces marxianus[J]. Applied Biochemistry and Biotechnology, 2021, 193(2): 502-514.
    [11] FUJIWARA R, NODA S, TANAKA T, KONDO A. Metabolic engineering of Escherichia coli for shikimate pathway derivative production from glucose-xylose co-substrate[J]. Nature Communications, 2020, 11: 279.
    [12] BRAT D, BOLES E, WIEDEMANN B. Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae[J]. Applied and Environmental Microbiology, 2009, 75(8): 2304-2311.
    [13] HO NWY, CHEN ZD, BRAINARD AP. Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose[J]. Applied and Environmental Microbiology, 1998, 64(5): 1852-1859.
    [14] SUN LL, WU B, ZHANG ZQ, YAN J, LIU PT, SONG C, SHABBIR S, ZHU QL, YANG SH, PENG N, HE MX, TAN FR. Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis[J]. Biotechnology for Biofuels, 2021, 14(1): 221.
    [15] LI XW, CHEN Y, NIELSEN J. Harnessing xylose pathways for biofuels production[J]. Current Opinion in Biotechnology, 2019, 57: 56-65.
    [16] HOSSAIN SA, ŠVEC D, MRŠA V, TEPARIĆ R. Overview of catalytic properties of fungal xylose reductases and molecular engineering approaches for improved xylose utilisation in yeast[J]. Applied Food Biotechnology, 2018, 5(2): 47-58.
    [17] VALDEHUESA KNG, RAMOS KRM, NISOLA GM, BAÑARES AB, CABULONG RB, LEE WK, LIU HW, CHUNG WJ. Everyone loves an underdog: metabolic engineering of the xylose oxidative pathway in recombinant microorganisms[J]. Applied Microbiology and Biotechnology, 2018, 102(18): 7703-7716.
    [18] de PAULA RG, ANTONIÊTO ACC, RIBEIRO LFC, SRIVASTAVA N, O’DONOVAN A, MISHRA PK, GUPTA VK, SILVA RN. Engineered microbial host selection for value-added bioproducts from lignocellulose[J]. Biotechnology Advances, 2019, 37(6): 107347.
    [19] STEPHENS C, CHRISTEN B, FUCHS T, SUNDARAM V, WATANABE K, JENAL U. Genetic analysis of a novel pathway for d-xylose metabolism in Caulobacter crescentus[J]. Journal of Bacteriology, 2007, 189(5): 2181-2185.
    [20] ZHAO Z, XIAN M, LIU M, ZHAO G. Biochemical routes for uptake and conversion of xylose by microorganisms[J]. Biotechnology for Biofuels, 2020, 13: 21.
    [21] LIU DF, ZHANG YL, LI JG, SUN WL, YAO YH, TIAN CG. The Weimberg pathway: an alternative for Myceliophthora thermophila to utilize d-xylose[J]. Biotechnology for Biofuels and Bioproducts, 2023, 16(1): 13.
    [22] GÖRKE B, STÜLKE J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients[J]. Nature Reviews Microbiology, 2008, 6: 613-624.
    [23] STASYK OG, STASYK OV. Glucose sensing and regulation in yeasts[M]//Non-conventional Yeasts: from Basic Research to Application. Cham: Springer, 2019: 477-519.
    [24] KIM SM, CHOI BY, RYU YS, JUNG SH, PARK JM, KIM GH, LEE SK. Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli[J]. Metabolic Engineering, 2015, 30: 141-148.
    [25] 丁小云, 顾健健, 王永泽, 赵锦芳, 王金华, 赵筱. 产d-乳酸重组大肠杆菌ptsG基因的敲除及其混合糖同步发酵[J]. 生物技术通报, 2015, 31(12): 221-226. DING XY, GU JJ, WANG YZ, ZHAO JF, WANG JH, ZHAO X. The knockout of gene ptsG of recombinant Escherichia coli producing d-lactic acid and the simultaneous fermentation of mixed sugars[J]. Biotechnology Bulletin, 2015, 31(12): 221-226 (in Chinese).
    [26] SAINI M, LIN LJ, CHIANG CJ, CHAO YP. Synthetic consortium of Escherichia coli for n-butanol production by fermentation of the glucose-xylose mixture[J]. Journal of Agricultural and Food Chemistry, 2017, 65(46): 10040-10047.
    [27] YUAN XS, TU S, LIN JP, YANG LR, SHEN HH, WU MB. Combination of the CRP mutation and ptsG deletion in Escherichia coli to efficiently synthesize xylitol from corncob hydrolysates[J]. Applied Microbiology and Biotechnology, 2020, 104(5): 2039-2050.
    [28] 蔡艳青, 齐显尼, 齐奇, 蔺玉萍, 王正祥, 王钦宏. 敲除MIG1SNF1基因对酿酒酵母共利用葡萄糖和木糖的影响[J]. 生物工程学报, 2018, 34(1): 54-67. CAI YQ, QI XN, QI Q, LIN YP, WANG ZX, WANG QH. Effect of MIG1 and SNF1 deletion on simultaneous utilization of glucose and xylose by Saccharomyces cerevisiae[J]. Chinese Journal of Biotechnology, 2018, 34(1): 54-67 (in Chinese).
    [29] HUA Y, WANG JC, ZHU YL, ZHANG B, KONG X, LI WJ, WANG DM, HONG J. Release of glucose repression on xylose utilization in Kluyveromyces marxianus to enhance glucose-xylose co-utilization and xylitol production from corncob hydrolysate[J]. Microbial Cell Factories, 2019, 18(1): 24.
    [30] SEMKIV MV, RUCHALA J, TSARUK AY, ZAZULYA AZ, VASYLYSHYN RV, DMYTRUK OV, ZUO MX, KANG YQ, DMYTRUK KV, SIBIRNY AA. The role of hexose transporter-like sensor hxs1 and transcription activator involved in carbohydrate sensing azf1 in xylose and glucose fermentation in the thermotolerant yeast Ogataea polymorpha[J]. Microbial Cell Factories, 2022, 21(1): 162.
    [31] KIM SR, HA SJ, WEI N, OH EJ, JIN YS. Simultaneous co-fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol[J]. Trends in Biotechnology, 2012, 30(5): 274-282.
    [32] KATAHIRA S, MIZUIKE A, FUKUDA H, KONDO A. Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide- assimilating yeast strain[J]. Applied Microbiology and Biotechnology, 2006, 72(6): 1136-1143.
    [33] NAKAMURA N, YAMADA R, KATAHIRA S, TANAKA T, FUKUDA H, KONDO A. Effective xylose/cellobiose co-fermentation and ethanol production by xylose-assimilating S. cerevisiae via expression of β-glucosidase on its cell surface[J]. Enzyme and Microbial Technology, 2008, 43(3): 233-236.
    [34] PARISUTHAM V, CHANDRAN SP, MUKHOPADHYAY A, LEE SK, KEASLING JD. Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries[J]. Bioresource Technology, 2017, 239: 496-506.
    [35] HA SJ, GALAZKA JM, KIM SR, CHOI JH, YANG XM, SEO JH, GLASS NL, CATE JHD, JIN YS. Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(2): 504-509.
    [36] OH EJ, HA SJ, KIM SR, LEE WH, GALAZKA JM, CATE JHD, JIN YS. Enhanced xylitol production through simultaneous co-utilization of cellobiose and xylose by engineered Saccharomyces cerevisiae[J]. Metabolic Engineering, 2013, 15: 226-234.
    [37] CABULONG RB, BAÑARES AB, NISOLA GM, LEE WK, CHUNG WJ. Enhanced glycolic acid yield through xylose and cellobiose utilization by metabolically engineered Escherichia coli[J]. Bioprocess and Biosystems Engineering, 2021, 44(6): 1081-1091.
    [38] ZHENG ZJ, JIANG T, ZOU LH, OUYANG SP, ZHOU J, LIN X, HE Q, WANG LM, YU B, XU HJ, OUYANG J. Simultaneous consumption of cellobiose and xylose by Bacillus coagulans to circumvent glucose repression and identification of its cellobiose-assimilating operons[J]. Biotechnology for Biofuels, 2018, 11: 320.
    [39] DIEN BS, NICHOLS NN, BOTHAST RJ. Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of l-lactic acid[J]. Journal of Industrial Microbiology and Biotechnology, 2002, 29(5): 221-227.
    [40] YOUNG EM, TONG A, BUI H, SPOFFORD C, ALPER HS. Rewiring yeast sugar transporter preference through modifying a conserved protein motif[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(1): 131-136.
    [41] LEANDRO MJ, FONSECA C, GONÇALVES P. Hexose and pentose transport in ascomycetous yeasts: an overview[J]. FEMS Yeast Research, 2009, 9(4): 511-525.
    [42] HAMACHER T, BECKER J, GÁRDONYI M, HAHN-HÄGERDAL B, BOLES E. Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization[J]. Microbiology, 2002, 148(Pt 9): 2783-2788.
    [43] JEFFRIES TW, JIN YS. Metabolic engineering for improved fermentation of pentoses by yeasts[J]. Applied Microbiology and Biotechnology, 2004, 63(5): 495-509.
    [44] YOUNG E, LEE SM, ALPER H. Optimizing pentose utilization in yeast: the need for novel tools and approaches[J]. Biotechnology for Biofuels, 2010, 3: 24.
    [45] JOJIMA T, OMUMASABA CA, INUI M, YUKAWA H. Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook[J]. Applied Microbiology and Biotechnology, 2010, 85(3): 471-480.
    [46] WEISSER P, KRÄMER R, SPRENGER GA. Expression of the Escherichia coli pmi gene, encoding phosphomannose-isomerase in Zymomonas mobilis, leads to utilization of mannose as a novel growth substrate, which can be used as a selective marker[J]. Applied and Environmental Microbiology, 1996, 62(11): 4155-4161.
    [47] DUNN KL, RAO CV. Expression of a xylose-specific transporter improves ethanol production by metabolically engineered Zymomonas mobilis[J]. Applied Microbiology and Biotechnology, 2014, 98(15): 6897-6905.
    [48] YOUNG E, POUCHER A, COMER A, BAILEY A, ALPER H. Functional survey for heterologous sugar transport proteins, using Saccharomyces cerevisiae as a host[J]. Applied and Environmental Microbiology, 2011, 77(10): 3311-3319.
    [49] SALOHEIMO A, RAUTA J, STASYK OV, SIBIRNY AA, PENTTILÄ M, RUOHONEN L. Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases[J]. Applied Microbiology and Biotechnology, 2007, 74(5): 1041-1052.
    [50] LEANDRO MJ, GONÇALVES P, SPENCER-MARTINS I. Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter[J]. The Biochemical Journal, 2006, 395(3): 543-549.
    [51] WEIERSTALL T, HOLLENBERG CP, BOLES E. Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipitis[J]. Molecular Microbiology, 1999, 31(3): 871-883.
    [52] DU J, LI SJ, ZHAO HM. Discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis[J]. Molecular BioSystems, 2010, 6(11): 2150-2156.
    [53] HECTOR RE, QURESHI N, HUGHES SR, COTTA MA. Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption[J]. Applied Microbiology and Biotechnology, 2008, 80(4): 675-684.
    [54] RUNQUIST D, HAHN-HÄGERDAL B, RÅDSTRÖM P. Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2010, 3: 5.
    [55] LI HB, SCHMITZ O, ALPER HS. Enabling glucose/xylose co-transport in yeast through the directed evolution of a sugar transporter[J]. Applied Microbiology and Biotechnology, 2016, 100(23): 10215-10223.
    [56] KIM H, LEE HS, PARK H, LEE DH, BOLES E, CHUNG D, PARK YC. Enhanced production of xylitol from xylose by expression of Bacillus subtilis arabinose: H+ symporter and Scheffersomyces stipitis xylose reductase in recombinant Saccharomyces cerevisiae[J]. Enzyme and Microbial Technology, 2017, 107: 7-14.
    [57] SLOOTHAAK J, TAMAYO-RAMOS JA, ODONI DI, LAOTHANACHAREON T, DERNTL C, MACH- AIGNER AR, MARTINS dos SANTOS VAP, SCHAAP PJ. Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei[J]. Biotechnology for Biofuels, 2016, 9: 148.
    [58] ROJAS SAT, SCHADEWEG V, KIRCHNER F, BOLES E, OREB M. Identification of a glucose-insensitive variant of Gal2 from Saccharomyces cerevisiae exhibiting a high pentose transport capacity[J]. Scientific Reports, 2021, 11: 24404.
    [59] JIANG Y, SHEN Y, GU LC, WANG ZZ, SU N, NIU KL, GUO W, HOU SL, BAO XM, TIAN CG, FANG X. Identification and characterization of an efficient d-xylose transporter in Saccharomyces cerevisiae[J]. Journal of Agricultural and Food Chemistry, 2020, 68(9): 2702-2710.
    [60] FARWICK A, BRUDER S, SCHADEWEG V, OREB M, BOLES E. Engineering of yeast hexose transporters to transport d-xylose without inhibition by d-glucose[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(14): 5159-5164.
    [61] ZHU XN, FAN FY, QIU HN, SHAO MY, LI D, YU Y, BI CH, ZHANG XL. New xylose transporters support the simultaneous consumption of glucose and xylose in Escherichia coli[J]. mLife, 2022, 1(2): 156-170.
    [62] UTRILLA J, LICONA-CASSANI C, MARCELLIN E, GOSSET G, NIELSEN LK, MARTINEZ A. Engineering and adaptive evolution of Escherichia coli for d-lactate fermentation reveals GatC as a xylose transporter[J]. Metabolic Engineering, 2012, 14(5): 469-476.
    [63] SARKAR P, GOSWAMI G, MUKHERJEE M, DAS D. Heterologous expression of xylose specific transporter improves xylose utilization by recombinant Zymomonas mobilis strain in presence of glucose[J]. Process Biochemistry, 2021, 102: 190-198.
    [64] ELIASSON A, CHRISTENSSON C, WAHLBOM CF, HAHN-HÄGERDAL B. Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying<癩椀猀椀愀攀??椀??猀琀爀愀椀渀猀?挀愀爀爀礀椀渀最?琀栀攀?圀攀椀洀戀攀爀最?瀀愀琀栀眀愀礀嬀?崀???攀琀愀戀漀氀椀挀??渀最椀渀攀攀爀椀渀最??? ??????????????戀爀?嬀? 崀?????匀??????????伀????一一??????一???刀??倀唀吀刀??匀倀???唀?唀匀?????????唀甀洀氀???匀???????一???????漀洀瀀愀爀椀猀漀渀?漀昀?椀猀漀洀攀爀愀猀攀?愀渀搀?眀攀椀洀戀攀爀最?瀀愀琀栀眀愀礀?昀漀爀???倀???瀀爀漀搀甀挀琀椀漀渀?昀爀漀洀?砀礀氀漀猀攀?戀礀?攀渀最椀渀攀攀爀攀搀??椀??愀挀椀氀氀甀猀?猀甀戀琀椀氀椀猀??椀?嬀?崀???爀漀渀琀椀攀爀猀?椀渀??椀漀攀渀最椀渀攀攀爀椀渀最?愀渀搀??椀漀琀攀挀栀渀漀氀漀最礀??? ? ??????????戀爀?嬀??崀?吀?圀?刀??刀??匀?吀??匀??倀刀??唀????????夀??????匀???匀椀洀甀氀琀愀渀攀漀甀猀?甀琀椀氀椀稀愀琀椀漀渀?漀昀?最氀甀挀漀猀攀?愀渀搀?砀礀氀漀猀攀?戀礀?洀攀琀愀戀漀氀椀挀愀氀氀礀?攀渀最椀渀攀攀爀攀搀??椀?倀猀攀甀搀漀洀漀渀愀猀?瀀甀琀椀搀愀??椀??昀漀爀?琀栀攀?瀀爀漀搀甀挀琀椀漀渀?漀昀???栀礀搀爀漀砀礀瀀爀漀瀀椀漀渀椀挀?愀挀椀搀嬀?崀???椀漀爀攀猀漀甀爀挀攀?吀攀挀栀渀漀氀漀最礀??? ??????????? ?????戀爀?嬀??崀?匀??唀匀???甀洀氀?刀嘀?????吀伀?嘀?刀?????嘀???伀???甀洀氀?????????伀?嘀?匀吀伀?一?一?伀???伀?娀?吀?????一???????甀洀氀?????倀?一吀吀????甀洀氀?????刀唀伀?伀一?一????倀爀漀搀甀挀琀椀漀渀?漀昀?攀琀栀礀氀攀渀攀?最氀礀挀漀氀?漀爀?最氀礀挀漀氀椀挀?愀挀椀搀?昀爀漀洀?搀?砀礀氀漀猀攀?椀渀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀?嬀?崀???瀀瀀氀椀攀搀??椀挀爀漀戀椀漀氀漀最礀?愀渀搀??椀漀琀攀挀栀渀漀氀漀最礀??? ????? ??????????????????戀爀?嬀??崀???伀??匀夀??????圀???夀唀?匀???倀?刀??匀??????匀???????匀夀???渀最椀渀攀攀爀椀渀最?琀栀攀?砀礀氀漀猀攀?挀愀琀愀戀漀氀椀稀椀渀最??愀栀洀猀?瀀愀琀栀眀愀礀?昀漀爀?瀀爀漀搀甀挀琀椀漀渀?漀昀?瀀漀氀礀?搀?氀愀挀琀愀琀攀?挀漀?最氀礀挀漀氀愀琀攀??愀渀搀?瀀漀氀礀?搀?氀愀挀琀愀琀攀?挀漀?最氀礀挀漀氀愀琀攀?挀漀?搀???栀礀搀爀漀砀礀戀甀琀礀爀愀琀攀??椀渀??椀??猀挀栀攀爀椀挀栀椀愀?挀漀氀椀??椀?嬀?崀???椀挀爀漀戀椀愀氀??椀漀琀攀挀栀渀漀氀漀最礀??? ????? ????????????????戀爀?嬀??崀??圀???匀????一?夀匀??倀爀漀搀甀挀琀椀漀渀?漀昀?昀甀攀氀猀?愀渀搀?挀栀攀洀椀挀愀氀猀?昀爀漀洀?砀礀氀漀猀攀?戀礀?攀渀最椀渀攀攀爀攀搀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀???愀?爀攀瘀椀攀眀?愀渀搀?瀀攀爀猀瀀攀挀琀椀瘀攀嬀?崀???椀挀爀漀戀椀愀氀??攀氀氀??愀挀琀漀爀椀攀猀??? ???????????????戀爀?嬀??崀???吀???刀??匀???唀刀??伀吀伀?一???伀刀?夀??匀??一??唀刀??刀??吀????一??夀?匀唀吀?一??一??伀??唀??????伀一?匀???吀??吀伀?唀??刀伀????匀挀爀攀攀渀椀渀最?愀渀搀?攀瘀漀氀甀琀椀漀渀?漀昀?愀?渀漀瘀攀氀?瀀爀漀琀椀猀琀?砀礀氀漀猀攀?椀猀漀洀攀爀愀猀攀?昀爀漀洀?琀栀攀?琀攀爀洀椀琀攀??椀?刀攀琀椀挀甀氀椀琀攀爀洀攀猀?猀瀀攀爀愀琀甀猀??椀??昀漀爀?攀昀昀椀挀椀攀渀琀?砀礀氀漀猀攀?昀攀爀洀攀渀琀愀琀椀漀渀?椀渀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀?嬀?崀???椀漀琀攀挀栀渀漀氀漀最礀?昀漀爀??椀漀昀甀攀氀猀??? ????? ??? ???戀爀?嬀??崀??伀唀????匀??一?夀?????伀????????刀???娀??一??堀?????伀?堀????栀愀爀愀挀琀攀爀椀稀愀琀椀漀渀?愀渀搀?攀瘀漀氀甀琀椀漀渀?漀昀?砀礀氀漀猀攀?椀猀漀洀攀爀愀猀攀?猀挀爀攀攀渀攀搀?昀爀漀洀?琀栀攀?戀漀瘀椀渀攀?爀甀洀攀渀?洀攀琀愀最攀渀漀洀攀?椀渀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀?嬀?崀???漀甀爀渀愀氀?漀昀??椀漀猀挀椀攀渀挀攀?愀渀搀??椀漀攀渀最椀渀攀攀爀椀渀最??? ?????????????? ??????戀爀?嬀??崀?搀攀?匀伀唀娀???伀?伀??伀????嘀??一????一??匀????搀攀??伀刀??匀?匀伀唀吀伀???????嘀?匀???刀刀?吀伀?????匀匀?匀?匀?刀刀???????刀刀??刀??一伀刀伀一??????匀?伀刀唀倀??倀?刀????一?一???伀刀??刀??搀攀??????????刀????刀刀?娀?儀唀?刀?一伀?????搀攀渀琀椀昀椀挀愀琀椀漀渀?愀渀搀?昀甀渀挀琀椀漀渀愀氀?攀砀瀀爀攀猀猀椀漀渀?漀昀?愀?渀攀眀?砀礀氀漀猀攀?椀猀漀洀攀爀愀猀攀?昀爀漀洀?琀栀攀?最漀愀琀?爀甀洀攀渀?洀椀挀爀漀戀椀漀洀攀?椀渀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀?嬀?崀???攀琀琀攀爀猀?椀渀??瀀瀀氀椀攀搀??椀挀爀漀戀椀漀氀漀最礀??? ????????????????????戀爀?嬀??崀????一?匀吀??堀唀?娀堀????一???一??娀??一??夀圀????唀?匀???????????????娀?????????????一??????椀最?搀愀琀愀?洀椀渀椀渀最??爀愀琀椀漀渀愀氀?洀漀搀椀昀椀挀愀琀椀漀渀??愀渀搀?愀渀挀攀猀琀爀愀氀?猀攀焀甀攀渀挀攀?爀攀挀漀渀猀琀爀甀挀琀椀漀渀?椀渀昀攀爀爀攀搀?洀甀氀琀椀瀀氀攀?砀礀氀漀猀攀?椀猀漀洀攀爀愀猀攀猀?昀漀爀?戀椀漀爀攀昀椀渀攀爀礀嬀?崀??匀挀椀攀渀挀攀??搀瘀愀渀挀攀猀??? ??????????攀愀搀搀??????戀爀?嬀??崀???伀??儀??夀唀?圀?????夀堀????一?????夀?伀????娀?伀唀?夀????渀最椀渀攀攀爀椀渀最?挀漀?甀琀椀氀椀稀愀琀椀漀渀?漀昀?最氀甀挀漀猀攀?愀渀搀?砀礀氀漀猀攀?昀漀爀?挀栀攀洀椀挀愀氀?漀瘀攀爀瀀爀漀搀甀挀琀椀漀渀?昀爀漀洀?氀椀最渀漀挀攀氀氀甀氀漀猀攀嬀?崀??一愀琀甀爀攀??栀攀洀椀挀愀氀??椀漀氀漀最礀??? ???????????????????戀爀?嬀? 崀???唀?????娀?一?堀夀??吀?一??堀??娀??伀??一?????一??儀?????一?夀儀?????一?圀??匀伀一??夀???吀栀攀?爀漀氀攀?漀昀?愀?砀礀氀漀猀攀?椀猀漀洀攀爀愀猀攀?瀀愀琀栀眀愀礀?椀渀?琀栀攀?挀漀渀瘀攀爀猀椀漀渀?漀昀?砀礀氀漀猀攀?琀漀?氀椀瀀椀搀?椀渀??椀??甀挀漀爀?挀椀爀挀椀渀攀氀氀漀椀搀攀猀??椀?嬀?崀??刀匀???搀瘀愀渀挀攀猀??? ????????????????????????戀爀?嬀??崀???一?刀???????刀?????????唀?刀一??刀???吀??倀栀漀猀瀀栀漀欀攀琀漀氀愀猀攀?瀀愀琀栀眀愀礀?攀渀最椀渀攀攀爀椀渀最?昀漀爀?挀愀爀戀漀渀?攀昀昀椀挀椀攀渀琀?戀椀漀挀愀琀愀氀礀猀椀猀嬀?崀???甀爀爀攀渀琀?伀瀀椀渀椀漀渀?椀渀??椀漀琀攀挀栀渀漀氀漀最礀??? ?????????????????戀爀?嬀??崀?匀伀一??刀????刀????匀??唀???????倀?刀??????匀?唀?刀?唀???攀琀愀戀漀氀椀挀?攀渀最椀渀攀攀爀椀渀最?漀昀?愀?瀀栀漀猀瀀栀漀欀攀琀漀氀愀猀攀?瀀愀琀栀眀愀礀?昀漀爀?瀀攀渀琀漀猀攀?挀愀琀愀戀漀氀椀猀洀?椀渀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀?嬀?崀???瀀瀀氀椀攀搀?愀渀搀??渀瘀椀爀漀渀洀攀渀琀愀氀??椀挀爀漀戀椀漀氀漀最礀???  ???? ????????????????戀爀?嬀??崀?匀???????娀??一??夀???吀?一??圀?????娀????猀琀愀戀氀椀猀栀洀攀渀琀?漀昀?愀?挀愀爀戀漀渀?攀昀昀椀挀椀攀渀琀?砀礀氀甀氀漀猀攀?挀氀攀愀瘀愀最攀?瀀愀琀栀眀愀礀?椀渀??椀??猀挀栀攀爀椀挀栀椀愀?挀漀氀椀??椀??琀漀?洀攀琀愀戀漀氀椀稀攀?砀礀氀漀猀攀嬀?崀???椀漀挀栀攀洀椀挀愀氀??渀最椀渀攀攀爀椀渀最??漀甀爀渀愀氀??? ?????????? ??????戀爀?嬀??崀??????????刀???刀唀一儀唀?匀吀????一伀?唀??愀挀甀琀攀??嘀匀????????愀挀甀琀攀?一?????伀刀圀???刀?唀匀?唀一??????匀琀爀攀猀猀?爀攀氀愀琀攀搀?挀栀愀氀氀攀渀最攀猀?椀渀?瀀攀渀琀漀猀攀?昀攀爀洀攀渀琀愀琀椀漀渀?琀漀?攀琀栀愀渀漀氀?戀礀?琀栀攀?礀攀愀猀琀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀?嬀?崀???椀漀琀攀挀栀渀漀氀漀最礀??漀甀爀渀愀氀??? ???????????????????戀爀?嬀??崀???刀?唀??????????一????甀洀氀???刀????????伀刀圀???刀?唀匀?唀一???????渀瘀攀猀琀椀最愀琀椀漀渀?漀昀?氀椀洀椀琀椀渀最?洀攀琀愀戀漀氀椀挀?猀琀攀瀀猀?椀渀?琀栀攀?甀琀椀氀椀稀愀琀椀漀渀?漀昀?砀礀氀漀猀攀?戀礀?爀攀挀漀洀戀椀渀愀渀琀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀??甀猀椀渀最?洀攀琀愀戀漀氀椀挀?攀渀最椀渀攀攀爀椀渀最嬀?崀??夀攀愀猀琀???  ???????????????????戀爀?嬀??崀??伀??夀?匀???夀??匀???刀??吀??伀???夀??匀????????吀??夀???唀???伀刀??????匀礀猀琀攀洀愀琀椀挀?漀瀀琀椀洀椀稀愀琀椀漀渀?漀昀?最攀渀攀?攀砀瀀爀攀猀猀椀漀渀?漀昀?瀀攀渀琀漀猀攀?瀀栀漀猀瀀栀愀琀攀?瀀愀琀栀眀愀礀?攀渀栀愀渀挀攀猀?攀琀栀愀渀漀氀?瀀爀漀搀甀挀琀椀漀渀?昀爀漀洀?愀?最氀甀挀漀猀攀?砀礀氀漀猀攀?洀椀砀攀搀?洀攀搀椀甀洀?椀渀?愀?爀攀挀漀洀戀椀渀愀渀琀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀?嬀?崀???????砀瀀爀攀猀猀??? ???????????????戀爀?嬀??崀?倀?刀??匀????????????一???圀?????一??匀???攀琀愀戀漀氀椀挀?攀渀最椀渀攀攀爀椀渀最?漀昀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀??昀漀爀?瀀爀漀搀甀挀琀椀漀渀?漀昀?猀栀椀渀漀爀椀渀攀??愀?猀甀渀猀挀爀攀攀渀?洀愀琀攀爀椀愀氀??昀爀漀洀?砀礀氀漀猀攀嬀?崀????匀?匀礀渀琀栀攀琀椀挀??椀漀氀漀最礀??? ???????????????????戀爀?嬀??崀?夀唀?一?堀匀????伀?夀???吀唀?匀????一??倀??匀??一?????夀?一???刀??圀唀??????渀挀爀攀愀猀椀渀最?一??倀??愀瘀愀椀氀愀戀椀氀椀琀礀?昀漀爀?砀礀氀椀琀漀氀?瀀爀漀搀甀挀琀椀漀渀?瘀椀愀?瀀攀渀琀漀猀攀?瀀栀漀猀瀀栀愀琀攀?瀀愀琀栀眀愀礀?最攀渀攀?漀瘀攀爀攀砀瀀爀攀猀猀椀漀渀?愀渀搀?攀洀戀搀攀渀?洀攀礀攀爀栀漀昀?瀀愀爀渀愀猀??瀀愀琀栀眀愀礀?最攀渀攀?搀攀氀攀琀椀漀渀?椀渀??椀??猀挀栀攀爀椀挀栀椀愀?挀漀氀椀??椀?嬀?崀???漀甀爀渀愀氀?漀昀??最爀椀挀甀氀琀甀爀愀氀?愀渀搀??漀漀搀??栀攀洀椀猀琀爀礀??? ???????????????????????戀爀?嬀??崀?刀伀????????????????伀?匀匀伀一?????渀最椀渀攀攀爀椀渀最?漀昀?挀愀爀戀漀渀?挀愀琀愀戀漀氀椀琀攀?爀攀瀀爀攀猀猀椀漀渀?椀渀?爀攀挀漀洀戀椀渀愀渀琀?砀礀氀漀猀攀?昀攀爀洀攀渀琀椀渀最??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀?嬀?崀???瀀瀀氀椀攀搀??椀挀爀漀戀椀漀氀漀最礀?愀渀搀??椀漀琀攀挀栀渀漀氀漀最礀???  ???????????????????戀爀?嬀?  崀?娀??一?????刀?一?????娀??伀?娀倀??娀??一??匀夀??堀唀??夀??娀?一??堀?????????椀最栀?琀攀洀瀀攀爀愀琀甀爀攀?砀礀氀椀琀漀氀?瀀爀漀搀甀挀琀椀漀渀?琀栀爀漀甀最栀?猀椀洀甀氀琀愀渀攀漀甀猀?挀漀?甀琀椀氀椀稀愀琀椀漀渀?漀昀?最氀甀挀漀猀攀?愀渀搀?砀礀氀漀猀攀?戀礀?攀渀最椀渀攀攀爀攀搀??椀??氀甀礀瘀攀爀漀洀礀挀攀猀?洀愀爀砀椀愀渀甀猀??椀?嬀?崀???椀漀挀栀攀洀椀挀愀氀??渀最椀渀攀攀爀椀渀最??漀甀爀渀愀氀??? ?????????? ??? ??戀爀?嬀? ?崀?吀伀倀??伀???????唀?????匀?一??伀甀洀氀???吀唀刀?一???? ???夀??? ??????? ??娀?????刀匀??一??????挀攀搀椀氀????刀?娀倀???爀漀洀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀??琀漀?攀琀栀愀渀漀氀??甀渀氀漀挀欀椀渀最?琀栀攀?瀀漀眀攀爀?漀昀?攀瘀漀氀甀琀椀漀渀愀爀礀?攀渀最椀渀攀攀爀椀渀最?椀渀?洀攀琀愀戀漀氀椀挀?攀渀最椀渀攀攀爀椀渀最?愀瀀瀀氀椀挀愀琀椀漀渀猀嬀?崀???漀甀爀渀愀氀?漀昀??甀渀最椀??? ??????? ????????戀爀?嬀? ?崀???刀?匀?圀??吀????????吀???搀愀瀀琀椀瘀攀?氀愀戀漀爀愀琀漀爀礀?攀瘀漀氀甀琀椀漀渀?漀昀?洀椀挀爀漀漀爀最愀渀椀猀洀猀??洀攀琀栀漀搀漀氀漀最礀?愀渀搀?愀瀀瀀氀椀挀愀琀椀漀渀?昀漀爀?戀椀漀瀀爀漀搀甀挀琀椀漀渀嬀?崀???椀挀爀漀漀爀最愀渀椀猀洀猀??? ???????????????戀爀?嬀? ?崀???嘀????????一??匀???夀?娀??一??匀匀???搀愀瀀琀愀琀椀漀渀?漀渀?砀礀氀漀猀攀?椀洀瀀爀漀瘀攀猀?最氀甀挀漀猀攀?砀礀氀漀猀攀?挀漀?甀琀椀氀椀稀愀琀椀漀渀?愀渀搀?攀琀栀愀渀漀氀?瀀爀漀搀甀挀琀椀漀渀?椀渀?愀?挀愀爀戀漀渀?挀愀琀愀戀漀氀椀琀攀?爀攀瀀爀攀猀猀椀漀渀????刀??挀漀洀瀀爀漀洀椀猀攀搀?攀琀栀愀渀漀氀漀最攀渀椀挀?猀琀爀愀椀渀嬀?崀???椀挀爀漀戀椀愀氀??攀氀氀??愀挀琀漀爀椀攀猀??? ????????????????戀爀?嬀? ?崀??伀唀??夀??圀?一???圀??夀?一??夀???夀?一??儀?????刀堀???唀????????儀一???唀????圀?一??堀????????夀?一??匀????攀瘀攀氀漀瀀洀攀渀琀?愀渀搀?挀栀愀爀愀挀琀攀爀椀稀愀琀椀漀渀?漀昀?攀昀昀椀挀椀攀渀琀?砀礀氀漀猀攀?甀琀椀氀椀稀愀琀椀漀渀?猀琀爀愀椀渀猀?漀昀??椀?娀礀洀漀洀漀渀愀猀?洀漀戀椀氀椀猀??椀?嬀?崀???椀漀琀攀挀栀渀漀氀漀最礀?昀漀爀??椀漀昀甀攀氀猀??? ????????????????戀爀?嬀? ?崀?倀刀伀??伀一?伀夀?倀????唀?一吀伀一??圀??????倀刀????嘀??吀?一?倀伀一?倀?倀?吀?匀??刀唀一?唀倀??一?圀???洀瀀爀漀瘀攀洀攀渀琀?椀渀?搀?砀礀氀漀猀攀?甀琀椀氀椀稀愀琀椀漀渀?愀渀搀?椀猀漀戀甀琀愀渀漀氀?瀀爀漀搀甀挀琀椀漀渀?椀渀?椀??匀??椀???椀??挀攀爀攀瘀椀猀椀愀攀??椀??戀礀?愀搀愀瀀琀椀瘀攀?氀愀戀漀爀愀琀漀爀礀?攀瘀漀氀甀琀椀漀渀?愀渀搀?爀愀琀椀漀渀愀氀?攀渀最椀渀攀攀爀椀渀最嬀?崀???漀甀爀渀愀氀?漀昀??渀搀甀猀琀爀椀愀氀??椀挀爀漀戀椀漀氀漀最礀??愀洀瀀???椀漀琀攀挀栀渀漀氀漀最礀??? ? ????????????????? ??戀爀?嬀? ?崀????????吀刀????一??????刀?匀匀?????倀唀刀?夀???????一?????刀??????夀?倀???刀????????匀礀猀琀攀洀猀?洀攀琀愀戀漀氀椀挀?攀渀最椀渀攀攀爀椀渀最?漀昀??椀??猀挀栀攀爀椀挀栀椀愀?挀漀氀椀??椀??椀洀瀀爀漀瘀攀猀?挀漀挀漀渀瘀攀爀猀椀漀渀?漀昀?氀椀最渀漀挀攀氀氀甀氀漀猀攀?搀攀爀椀瘀攀搀?猀甀最愀爀猀嬀?崀???椀漀琀攀挀栀渀漀氀漀最礀??漀甀爀渀愀氀??? ???????????攀??  ?????戀爀?嬀? ?崀?匀?刀??刀?倀???唀???刀????????伀匀圀?????????匀?????搀愀瀀琀椀瘀攀?氀愀戀漀爀愀琀漀爀礀?攀瘀漀氀甀琀椀漀渀?椀渀搀甀挀攀搀?渀漀瘀攀氀?洀甀琀愀琀椀漀渀猀?椀渀??椀?娀礀洀漀洀漀渀愀猀?洀漀戀椀氀椀猀??椀???吀???娀圀?????愀?瀀漀琀攀渀琀椀愀氀?瀀氀愀琀昀漀爀洀?昀漀爀?挀漀?甀琀椀氀椀稀愀琀椀漀渀?漀昀?最氀甀挀漀猀攀?愀渀搀?砀礀氀漀猀攀嬀?崀???漀甀爀渀愀氀?漀昀??渀搀甀猀琀爀椀愀氀??椀挀爀漀戀椀漀氀漀最礀??愀洀瀀???椀漀琀攀挀栀渀漀氀漀最礀??? ? ??????????????????戀爀?嬀? ?崀???一?匀????匀??伀刀????匀礀渀琀栀攀琀椀挀?洀椀挀爀漀戀椀愀氀?攀挀漀氀漀最礀??攀渀最椀渀攀攀爀椀渀最?栀愀戀椀琀愀琀猀?昀漀爀?洀漀搀甀氀愀爀?挀漀渀猀漀爀琀椀愀嬀?崀???爀漀渀琀椀攀爀猀?椀渀??椀挀爀漀戀椀漀氀漀最礀??? ?????????????戀爀?嬀? ?崀?一伀匀刀?吀????伀?匀?一????刀刀?匀伀一?匀吀????匀???????????吀???匀????琀栀愀渀漀氀?昀爀漀洀?戀椀漀洀愀猀猀?栀礀搀爀漀氀礀猀愀琀攀猀?戀礀?攀昀昀椀挀椀攀渀琀?昀攀爀洀攀渀琀愀琀椀漀渀?漀昀?最氀甀挀漀猀攀?愀渀搀?砀礀氀漀猀攀??愀?爀攀瘀椀攀眀嬀?崀???栀攀洀?椀漀?渀最?刀攀瘀椀攀眀猀??? ???????????????????戀爀?嬀?? 崀?匀??一???????伀?匀儀???唀?一夀匀??嘀?一??堀?一?夀倀??????????唀?吀????一?夀匀???漀洀瀀漀猀椀琀椀漀渀愀氀?愀渀搀?琀攀洀瀀漀爀愀氀?搀椀瘀椀猀椀漀渀?漀昀?氀愀戀漀爀?洀漀搀甀氀愀琀攀猀?洀椀砀攀搀?猀甀最愀爀?昀攀爀洀攀渀琀愀琀椀漀渀?戀礀?愀渀?攀渀最椀渀攀攀爀攀搀?礀攀愀猀琀?挀漀渀猀漀爀琀椀甀洀嬀?崀??一愀琀甀爀攀??漀洀洀甀渀椀挀愀琀椀漀渀猀??? ?????????????戀爀?嬀???崀???伀刀?匀??????夀????娀??一???匀?一??刀??圀?一??堀???渀最椀渀攀攀爀椀渀最?愀?猀礀渀琀栀攀琀椀挀??挀愀琀愀戀漀氀椀挀愀氀氀礀?漀爀琀栀漀最漀渀愀氀?挀漀挀甀氀琀甀爀攀?猀礀猀琀攀洀?昀漀爀?攀渀栀愀渀挀攀搀?挀漀渀瘀攀爀猀椀漀渀?漀昀?氀椀最渀漀挀攀氀氀甀氀漀猀攀?搀攀爀椀瘀攀搀?猀甀最愀爀猀?琀漀?攀琀栀愀渀漀氀嬀?崀????匀?匀礀渀琀栀攀琀椀挀??椀漀氀漀最礀??? ??????????? ???? ????戀爀?嬀???崀???刀刀??刀?????匀?一吀伀匀?嘀?儀???刀唀娀???????琀栀愀渀漀氀?瀀爀漀搀甀挀琀椀漀渀?戀礀?挀漀?挀甀氀琀甀爀攀?漀昀??椀?娀礀洀漀洀漀渀愀猀?洀漀戀椀氀椀猀??椀??愀渀搀??椀?倀愀挀栀礀猀漀氀攀渀?琀愀渀渀漀瀀栀椀氀甀猀??椀??甀猀椀渀最?戀愀渀愀渀愀?瀀攀攀氀猀?栀礀搀爀漀氀礀猀愀琀攀?愀猀?猀甀戀猀琀爀愀琀攀嬀?崀???挀琀愀?匀挀椀攀渀琀椀愀爀甀洀?吀攀挀栀渀漀氀漀最礀??? ????? ????????????戀爀?嬀???崀?娀伀唀???????一?堀娀??吀?伀?夀???娀??一??娀???伀唀夀?一?????刀攀挀攀渀琀?愀搀瘀愀渀挀攀猀?椀渀?琀栀攀?攀砀瀀氀漀椀琀愀琀椀漀渀?愀渀搀?愀瀀瀀氀椀挀愀琀椀漀渀?漀昀?挀漀挀甀氀琀甀爀攀?猀礀猀琀攀洀猀?昀漀爀?琀栀攀?戀椀漀挀漀渀瘀攀爀猀椀漀渀?漀昀?氀椀最渀漀挀攀氀氀甀氀漀猀椀挀?昀攀攀搀猀琀漀挀欀猀嬀?崀???渀搀甀猀琀爀椀愀氀??爀漀瀀猀?愀渀搀?倀爀漀搀甀挀琀猀??? ????? ???????????戀爀?嬀???崀???唀?夀刀??夀?一??匀夀??????堀儀???漀渀猀琀爀甀挀琀椀漀渀?漀昀?愀??渠甀琀爀椀琀椀漀渀?猀甀瀀瀀氀礀?搀攀琀漀砀椀昀椀挀愀琀椀漀渀??挀漀挀甀氀琀甀爀攀?挀漀渀猀漀爀琀椀甀洀?昀漀爀?洀攀搀椀甀洀?挀栀愀椀渀?氀攀渀最琀栀?瀀漀氀礀栀礀搀爀漀砀礀愀氀欀愀渀漀愀琀攀?瀀爀漀搀甀挀琀椀漀渀?眀椀琀栀?愀?最氀甀挀漀猀攀?砀礀氀漀猀攀?洀椀砀琀甀爀攀嬀?崀???漀甀爀渀愀氀?漀昀??渀搀甀猀琀爀椀愀氀??椀挀爀漀戀椀漀氀漀最礀??愀洀瀀???椀漀琀攀挀栀渀漀氀漀最礀??? ? ??????????????????戀爀?嬀???崀???吀???一?????????匀?????吀??一?刀????吀??一??????猀甀戀猀琀爀愀琀攀?猀攀氀攀挀琀椀瘀攀?挀漀?昀攀爀洀攀渀琀愀琀椀漀渀?猀琀爀愀琀攀最礀?眀椀琀栀??椀??猀挀栀攀爀椀挀栀椀愀?挀漀氀椀??椀??瀀爀漀搀甀挀攀猀?氀愀挀琀愀琀攀?戀礀?猀椀洀甀氀琀愀渀攀漀甀猀氀礀?挀漀渀猀甀洀椀渀最?砀礀氀漀猀攀?愀渀搀?最氀甀挀漀猀攀嬀?崀???椀漀琀攀挀栀渀漀氀漀最礀?愀渀搀??椀漀攀渀最椀渀攀攀爀椀渀最???  ???? ???????????????戀爀?嬀???崀?娀??一???刀??倀?刀??刀????????娀???匀吀?倀??一伀倀伀唀?伀匀?????渀最椀渀攀攀爀椀渀最??椀??猀挀栀攀爀椀挀栀椀愀?挀漀氀椀??椀??挀漀挀甀氀琀甀爀攀?猀礀猀琀攀洀猀?昀漀爀?琀栀攀?瀀爀漀搀甀挀琀椀漀渀?漀昀?戀椀漀挀栀攀洀椀挀愀氀?瀀爀漀搀甀挀琀猀嬀?崀??倀爀漀挀攀攀搀椀渀最猀?漀昀?琀栀攀?一愀琀椀漀渀愀氀??挀愀搀攀洀礀?漀昀?匀挀椀攀渀挀攀猀?漀昀?琀栀攀?唀渀椀琀攀搀?匀琀愀琀攀猀?漀昀??洀攀爀椀挀愀??? ????????????????????????戀爀?嬀???崀?娀??伀?????匀?一唀?嘀?夀伀??倀??娀??一??夀倀?????夀???攀猀椀最渀?愀渀搀?搀攀瘀攀氀漀瀀洀攀渀琀?漀昀?愀??夠?猀栀愀瀀攀搀??洀椀挀爀漀戀椀愀氀?挀漀渀猀漀爀琀椀甀洀?挀愀瀀愀戀氀攀?漀昀?猀椀洀甀氀琀愀渀攀漀甀猀氀礀?甀琀椀氀椀稀椀渀最?戀椀漀洀愀猀猀?猀甀最愀爀猀?昀漀爀?攀昀昀椀挀椀攀渀琀?瀀爀漀搀甀挀琀椀漀渀?漀昀?戀甀琀愀渀漀氀嬀?崀???攀琀愀戀漀氀椀挀??渀最椀渀攀攀爀椀渀最??? ?????????????????戀爀?嬀???崀?圀?一?倀??娀????????圀?一??娀??夀?一??堀匀??吀??一?匀???琀栀愀渀漀氀?瀀爀漀搀甀挀琀椀漀渀?昀爀漀洀?渀漀渀搀攀琀漀砀椀昀椀攀搀?搀椀氀甀琀攀?愀挀椀搀?氀椀最渀漀挀攀氀氀甀氀漀猀椀挀?栀礀搀爀漀氀礀猀愀琀攀?戀礀?挀漀挀甀氀琀甀爀攀猀?漀昀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀??夀??愀渀搀??椀?倀椀挀栀椀愀?猀琀椀瀀椀琀椀猀??椀????匀? ??嬀?崀???椀漀琀攀挀栀渀漀氀漀最礀?刀攀猀攀愀爀挀栀??渀琀攀爀渀愀琀椀漀渀愀氀??? ????? ????????????戀爀?嬀???崀???????愀挀甀琀攀?一???伀匀吀??????伀匀伀嘀?一唀?????????一琀椀氀搀攀??刀伀匀???伀愀挀甀琀攀?倀?娀?倀??吀?伀?匀?一?匀吀????刀一??夀??嘀????一??????刀?夀伀??刀???漀?挀甀氀琀椀瘀愀琀椀漀渀?漀昀?愀?渀漀瘀攀氀??椀??甀猀愀爀椀甀洀??椀???椀?猀琀爀椀愀琀甀洀??椀??猀琀爀愀椀渀?愀渀搀?愀?砀礀氀漀猀攀?挀漀渀猀甀洀椀渀最??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀??礀椀攀氀搀猀?愀渀?攀昀昀椀挀椀攀渀琀?瀀爀漀挀攀猀猀?昀漀爀?猀椀洀甀氀琀愀渀攀漀甀猀?搀攀琀漀砀椀昀椀挀愀琀椀漀渀?愀渀搀?昀攀爀洀攀渀琀愀琀椀漀渀?漀昀?氀椀最渀漀挀攀氀氀甀氀漀猀椀挀?栀礀搀爀漀氀礀猀愀琀攀猀嬀?崀???栀攀洀椀挀愀氀??渀最椀渀攀攀爀椀渀最??漀甀爀渀愀氀??? ?????????????????戀爀?嬀?? 崀???圀??????圀?一?????圀唀?夀???刀????一?伀??匀伀一??夀堀??堀唀?刀??娀??一??儀????伀??倀??堀唀??????椀最渀漀挀攀氀氀甀氀漀猀椀挀?攀琀栀愀渀漀氀?愀渀搀?戀甀琀愀渀漀氀?瀀爀漀搀甀挀琀椀漀渀?戀礀??椀?匀愀挀挀栀愀爀漀洀礀挀攀猀?挀攀爀攀瘀椀猀椀愀攀??椀??愀渀搀??椀??氀漀猀琀爀椀搀椀甀洀?戀攀椀樀攀爀椀渀挀欀椀椀??椀??挀漀?挀甀氀琀甀爀攀?甀猀椀渀最?渀漀渀?搀攀琀漀砀椀昀椀攀搀?挀漀爀渀?猀琀漀瘀攀爀?栀礀搀爀漀氀礀猀愀琀攀嬀?崀???漀甀爀渀愀氀?漀昀??椀漀琀攀挀栀渀漀氀漀最礀??? ?????????????
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

王倩,高教琪,周雍进. 葡萄糖和木糖高效共利用代谢工程研究进展[J]. 生物工程学报, 2024, 40(8): 2710-2730

复制
分享
文章指标
  • 点击次数:383
  • 下载次数: 911
  • HTML阅读次数: 403
  • 引用次数: 0
历史
  • 收稿日期:2024-03-07
  • 在线发布日期: 2024-08-08
  • 出版日期: 2024-08-25
文章二维码
您是第5984799位访问者
生物工程学报 ® 2025 版权所有

通信地址:中国科学院微生物研究所    邮编:100101

电话:010-64807509   E-mail:cjb@im.ac.cn

技术支持:北京勤云科技发展有限公司