群体感应信号分子降解酶AiiA的分子进化研究
|
摘要
许多细菌都能够合成并释放一种或多种信号分子,随着菌体密度的增加,信号分子的浓度也随之增加。当信号分子的浓度积累到一定阀值的时候,就会启动菌体中相关基因的表达,调控细菌的某些生物行为。在大部分革兰氏阴性致病菌中,信号分子的积累可以启动下游致病因子的表达。从苏云金芽孢杆菌Bt.4Q7中克隆得到的aiiA基因编码的AiiA蛋白可以降解群体感应信号分子,从而使得致病菌的致病性降低或者消失。AiiA蛋白的活性越高,对致病菌致病性的抑制作用也就越明显。
本文通过PCR扩增,得到了aiiA4Q7、aiiA8010、aiiAWB4、aiiAWB12、aiiAT-HW3、aiiA6A3共6个aiiA同源基因；运用DNA shuffling分子进化技术,通过一步DNaseⅠ(Rnase Free)随机酶切、三轮无引物PCR、一轮有引物PCR过程,成功构建了aiiA基因突变库；以紫色色杆菌突变株CV026为报告菌,从中筛选得到了S29、S47、S21、S4、S36、S52、S7、S14、S63共9株具有较高AiiA蛋白酶活的突变株,并进一步得到了进化后的aiiAS29、aiiAS47、aiiAS21、aiiAS4、aiiAS36、aiiAS52、aiiAS7、aiiAS14、aiiAS63基因；aiiAS29和aiiAS36基因的测序结果完全相同,其余aiiA基因序列各不相同。制备了AiiAT-HW3、AiiA4Q7、AiiA8010、AiiAWB4/WB12、AiiAS29/S36、AiiAS47/S52、AiiAS21、AiiAS4、AiiAS7、AiiAS63共10个AiiA蛋白样品,通过测定各AiiA蛋白样品之间的相对酶活,得到了具有高酶活性的AiiA蛋白即AiiAs29／s36,其进化后的酶活性比原始AiiA4Q7提高了2.2倍,成功实现了对群体感应信号分子降解酶AiiA的分子进化。
aiiA gene cloned from Bt.4Q7 (B. thuringiensis 4Q7) encodes an enzyme which inactivating QS (quorum sensing) signal molecules AHLs (N-acylhomoserine lactones) involved in regulation of divers biological functions especially the expression of pathogenic gene in the Gram negative bacteria. Phenotypic characterization could be abolished or reduced further more when the AHLs inactivated by AiiA with higher enzyme activity which was desired in our molecule evolution. Library of aiiA gene was constructed by DNA shuffling including steps DNase I digestion, three round PCR without primers and one round PCR with primers. Reporter strain CV026(Chromobacterium violaceum 026) was used for the high throughput screening. Nine mutant strains with high enzyme activity were abtained and the evolved aiiA genes were cloned and sequenced. The evolution of AiiA quorum sensing inhabitor was achieved as the activity of AiiAS29/S36 was increased to 2.2 times as the original AiiA4Q7 by the relatively enzyme activity assay of AiiA samples.
本文通过PCR扩增,得到了aiiA4Q7、aiiA8010、aiiAWB4、aiiAWB12、aiiAT-HW3、aiiA6A3共6个aiiA同源基因；运用DNA shuffling分子进化技术,通过一步DNaseⅠ(Rnase Free)随机酶切、三轮无引物PCR、一轮有引物PCR过程,成功构建了aiiA基因突变库；以紫色色杆菌突变株CV026为报告菌,从中筛选得到了S29、S47、S21、S4、S36、S52、S7、S14、S63共9株具有较高AiiA蛋白酶活的突变株,并进一步得到了进化后的aiiAS29、aiiAS47、aiiAS21、aiiAS4、aiiAS36、aiiAS52、aiiAS7、aiiAS14、aiiAS63基因；aiiAS29和aiiAS36基因的测序结果完全相同,其余aiiA基因序列各不相同。制备了AiiAT-HW3、AiiA4Q7、AiiA8010、AiiAWB4/WB12、AiiAS29/S36、AiiAS47/S52、AiiAS21、AiiAS4、AiiAS7、AiiAS63共10个AiiA蛋白样品,通过测定各AiiA蛋白样品之间的相对酶活,得到了具有高酶活性的AiiA蛋白即AiiAs29／s36,其进化后的酶活性比原始AiiA4Q7提高了2.2倍,成功实现了对群体感应信号分子降解酶AiiA的分子进化。
aiiA gene cloned from Bt.4Q7 (B. thuringiensis 4Q7) encodes an enzyme which inactivating QS (quorum sensing) signal molecules AHLs (N-acylhomoserine lactones) involved in regulation of divers biological functions especially the expression of pathogenic gene in the Gram negative bacteria. Phenotypic characterization could be abolished or reduced further more when the AHLs inactivated by AiiA with higher enzyme activity which was desired in our molecule evolution. Library of aiiA gene was constructed by DNA shuffling including steps DNase I digestion, three round PCR without primers and one round PCR with primers. Reporter strain CV026(Chromobacterium violaceum 026) was used for the high throughput screening. Nine mutant strains with high enzyme activity were abtained and the evolved aiiA genes were cloned and sequenced. The evolution of AiiA quorum sensing inhabitor was achieved as the activity of AiiAS29/S36 was increased to 2.2 times as the original AiiA4Q7 by the relatively enzyme activity assay of AiiA samples.
引文
[1]Fuqua, W.C., S.C. Winans, and E.P. Greenberg, Quorum sensing in bacteria:the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol,1994. 176(2):p.269-75.
[2]Nealson, K.H., T. Platt, and J.W. Hastings, Cellular control of the synthesis and activity of the bacterial luminescent system. J Bacteriol,1970.104(1):p.313-22.
[3]Gonzalez, J.E. and N.D. Keshavan, Messing with bacterial quorum sensing. Microbiol Mol Biol Rev,2006.70(4):p.859-75.
[4]Manefield, M. and S.L. Turner, Quorum sensing in context:out of molecular biology and into microbial ecology. Microbiology,2002.148(Pt 12):p.3762-4.
[5]Xavier, K.B. and B.L. Bassler, LuxS quorum sensing:more than just a numbers game. Curr Opin Microbiol,2003.6(2):p.191-7.
[6]Camilli, A. and B.L. Bassler, Bacterial small-molecule signaling pathways. Science, 2006.311(5764):p.1113-6.
[7]Mok, K.C., N.S. Wingreen, and B.L. Bassler, Vibrio harveyi quorum sensing:a coincidence detector for two autoinducers controls gene expression. EMBO J,2003. 22(4):p.870-81.
[8]Winans, S.C., Bacterial esperanto. Nat Struct Biol,2002.9(2):p.83-4.
[9]McClean, K.H., et al., Quorum sensing and Chromobacterium violaceum:exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology,1997.143 (Pt 12):p.3703-11.
[10]Winson, M.K., et al., Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett,1998.163(2):p.185-92.
[11]Swift, S., et al., Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J Bacteriol,1997.179(17):p.5271-81.
[12]Lindsay, A. and B.M. Ahmer, Effect of sdiA on biosensors of N-acylhomoserine lactones. J Bacteriol,2005.187(14):p.5054-8.
[13]Dong, Y.H., et al., The two-component response regulator PprB modulates quorum-sensing signal production and global gene expression in Pseudomonas aeruginosa. Mol Microbiol,2005.56(5):p.1287-301.
[14]Farrand, S.K., Y. Qin, and P. Oger, Quorum-sensing system of Agrobacterium plasmids: analysis and utility. Methods Enzymol,2002.358:p.452-84.
[15]Zhu, J., et al., Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules:detection of autoinducers in Mesorhizobium huakuii. Appl Environ Microbiol,2003.69(11):p.6949-53.
[16]Khan, S.R., et al., Activation of the phz operon of Pseudomonas fluorescens 2-79 requires the LuxR homolog PhzR, N-(3-OH-Hexanoyl)-L-homoserine lactone produced by the LuxI homolog Phzl, and a cis-acting phz box. J Bacteriol,2005.187(18):p. 6517-27.
[17]Llamas, I., N. Keshavan, and J.E. Gonzalez, Use of Sinorhizobium meliloti as an indicator for specific detection of long-chain N-acyl homoserine lactones. Appl Environ Microbiol,2004.70(6):p.3715-23.
[18]Andersen, J.B., et al., New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol,1998.64(6):p.2240-6.
[19]Riedel, K., et al., N-acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms. Microbiology, 2001.147(Pt 12):p.3249-62.
[20]Gould, T.A., et al., Specificity of acyl-homoserine lactone synthases examined by mass spectrometry. J Bacteriol,2006.188(2):p.773-83.
[21]Gould, T.A., H.P. Schweizer, and M.E. Churchill, Structure of the Pseudomonas aeruginosa acyl-homoserinelactone synthase LasI. Mol Microbiol,2004.53(4):p. 1135-46.
[22]Kim, M.H., et al., The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase. Proc Natl Acad Sci USA, 2005.102(49):p.17606-11.
[23]Uroz, S., et al., N-Acylhomoserine lactone quorum-sensing molecules are modified and degraded by Rhodococcus erythropolis W2 by both amidolytic and novel oxidoreductase activities. Microbiology,2005.151(Pt 10):p.3313-22.
[24]Park, S.Y., et al., N-acylhomoserine lactonase producing Rhodococcus spp. with different AHL-degrading activities. FEMS Microbiol Lett,2006.261(1):p.102-8.
[25]Lin, Y.H., et al., Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol Microbiol,2003. 47(3):p.849-60.
[26]Sio, C.F., et al., Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1. Infect Immun,2006.74(3):p.1673-82.
[27]Huang, J.J., et al., Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1. Appl Environ Microbiol,2006.72(2):p.1190-7.
[28]Frezza, M., et al., Synthesis and biological evaluation of homoserine lactone derived ureas as antagonists of bacterial quorum sensing. Bioorg Med Chem,2006.14(14):p. 4781-91.
[29]Adonizio, A.L., et al., Anti-quorum sensing activity of medicinal plants in southern Florida. J Ethnopharmacol,2006.105(3):p.427-35.
[30]Thomas, P.W., et al., The quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein. Biochemistry,2005.44(20):p.7559-69.
[31]Dong, Y.H., et al., AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc Natl Acad Sci USA,2000.97(7):p.3526-31.
[32]Dong, Y.H., et al., Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl Environ Microbiol,2002.68(4):p.1754-9.
[33]Draganov, D.I., et al., Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation. J Biol Chem,2000.275(43):p.33435-42.
[34]Lee, S.J., et al., Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Appl Environ Microbiol,2002. 68(8):p.3919-24.
[35]Xu, F., et al., Degradation of N-acylhomoserine lactones, the bacterial quorum-sensing molecules, by acylase. J Biotechnol,2003.101(1):p.89-96.
[36]Zhang, H.B., L.H. Wang, and L.H. Zhang, Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens. Proc Natl Acad Sci USA,2002.99(7):p. 4638-43.
[37]Molina, L., et al., Degradation of pathogen quorum-sensing molecules by soil bacteria:a preventive and curative biological control mechanism. FEMS Microbiol Ecol,2003. 45(1):p.71-81.
[38]Billecke, S., et al., Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters. Drug Metab Dispos,2000.28(11):p.1335-42.
[39]Uroz, S., et al., Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plant-pathogenic bacteria. Microbiology,2003.149(Pt 8):p.1981-9.
[40]Leadbetter, J.R. and E.P. Greenberg, Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus. J Bacteriol,2000.182(24):p.6921-6.
[41]Taga, M.E. and B.L. Bassler, Chemical communication among bacteria. Proc Natl Acad Sci USA,2003.100 Suppl 2:p.14549-54.
[42]Crameri, A., et al., DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature,1998.391(6664):p.288-91.
[43]Aharoni, A., et al., Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization. Proc Natl Acad Sci USA,2004.101(2): p.482-7.
[44]Suen, W.C., et al., Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling. Protein Eng Des Sel,2004.17(2):p.133-40.
[45]Ness, J.E., et al., DNA shuffling of subgenomic sequences of subtilisin. Nat Biotechnol, 1999.17(9):p.893-6.
[46]Baik, S.H., et al., Significantly enhanced stability of glucose dehydrogenase by directed evolution. Appl Microbiol Biotechnol,2003.61(4):p.329-35.
[47]Hao, J. and A. Berry, A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents. Protein Eng Des Sel,2004.17(9):p.689-97.
[48]Kaper, T., et al., DNA family shuffling of hyperthermostable beta-glycosidases. Biochem J,2002.368(Pt2):p.461-70.
[49]Barriault, D., M.M. Plante, and M. Sylvestre, Family shuffling of a targeted bphA region to engineer biphenyl dioxygenase. J Bacteriol,2002.184(14):p.3794-800.
[50]Kagami, O., M. Kikuchi, and S. Harayama, Single-stranded DNA family shuffling. Methods Enzymol,2004.388:p.11-21.
[51]Kikuchi, M., K. Ohnishi, and S. Harayama, An effective family shuffling method using single-stranded DNA. Gene,2000.243(1-2):p.133-7.
[52]Gibbs, M.D., K.M. Nevalainen, and P.L. Bergquist, Degenerate oligonucleotide gene shuffling (DOGS):a method for enhancing the frequency of recombination with family shuffling. Gene,2001.271(1):p.13-20.
[53]Bergquist, P.L., R.A. Reeves, and M.D. Gibbs, Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM):two complementary techniques for enzyme evolution. Biomol Eng,2005.22(1-3):p.63-72.
[54]Zhang, Y.X., et al., Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature,2002.415(6872):p.644-6.
[55]Dai, M. and S.D. Copley, Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. Appl Environ Microbiol,2004.70(4):p.2391-7.
[56]Patnaik, R., et al., Genome shuffling of Lactobacillus for improved acid tolerance. Nat Biotechnol,2002.20(7):p.707-12.
[57]Giudici, P., et al., Strategies and perspectives for genetic improvement of wine yeasts. Appl Microbiol Biotechnol,2005.66(6):p.622-8.
[58]Coco, W.M., et al., DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat Biotechnol,2001.19(4):p.354-9.
[59]Song, J.K., et al., Construction of DNA-shuffled and incrementally truncated libraries by a mutagenic and unidirectional reassembly method:changing from a substrate specificity of phospholipase to that of lipase. Appl Environ Microbiol,2002.68(12):p. 6146-51.
[60]Ghadessy, F.J., J.L. Ong, and P. Holliger, Directed evolution of polymerase function by compartmentalized self-replication. Proc Natl Acad Sci USA,2001.98(8):p.4552-7.
[61]Fujii, R., M. Kitaoka, and K. Hayashi, One-step random mutagenesis by error-prone rolling circle amplification. Nucleic Acids Res,2004.32(19):p. e145.
[62]Osuna, J., et al., Protein evolution by codon-based random deletions. Nucleic Acids Res, 2004.32(17):p. e136.
[2]Nealson, K.H., T. Platt, and J.W. Hastings, Cellular control of the synthesis and activity of the bacterial luminescent system. J Bacteriol,1970.104(1):p.313-22.
[3]Gonzalez, J.E. and N.D. Keshavan, Messing with bacterial quorum sensing. Microbiol Mol Biol Rev,2006.70(4):p.859-75.
[4]Manefield, M. and S.L. Turner, Quorum sensing in context:out of molecular biology and into microbial ecology. Microbiology,2002.148(Pt 12):p.3762-4.
[5]Xavier, K.B. and B.L. Bassler, LuxS quorum sensing:more than just a numbers game. Curr Opin Microbiol,2003.6(2):p.191-7.
[6]Camilli, A. and B.L. Bassler, Bacterial small-molecule signaling pathways. Science, 2006.311(5764):p.1113-6.
[7]Mok, K.C., N.S. Wingreen, and B.L. Bassler, Vibrio harveyi quorum sensing:a coincidence detector for two autoinducers controls gene expression. EMBO J,2003. 22(4):p.870-81.
[8]Winans, S.C., Bacterial esperanto. Nat Struct Biol,2002.9(2):p.83-4.
[9]McClean, K.H., et al., Quorum sensing and Chromobacterium violaceum:exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology,1997.143 (Pt 12):p.3703-11.
[10]Winson, M.K., et al., Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing. FEMS Microbiol Lett,1998.163(2):p.185-92.
[11]Swift, S., et al., Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J Bacteriol,1997.179(17):p.5271-81.
[12]Lindsay, A. and B.M. Ahmer, Effect of sdiA on biosensors of N-acylhomoserine lactones. J Bacteriol,2005.187(14):p.5054-8.
[13]Dong, Y.H., et al., The two-component response regulator PprB modulates quorum-sensing signal production and global gene expression in Pseudomonas aeruginosa. Mol Microbiol,2005.56(5):p.1287-301.
[14]Farrand, S.K., Y. Qin, and P. Oger, Quorum-sensing system of Agrobacterium plasmids: analysis and utility. Methods Enzymol,2002.358:p.452-84.
[15]Zhu, J., et al., Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules:detection of autoinducers in Mesorhizobium huakuii. Appl Environ Microbiol,2003.69(11):p.6949-53.
[16]Khan, S.R., et al., Activation of the phz operon of Pseudomonas fluorescens 2-79 requires the LuxR homolog PhzR, N-(3-OH-Hexanoyl)-L-homoserine lactone produced by the LuxI homolog Phzl, and a cis-acting phz box. J Bacteriol,2005.187(18):p. 6517-27.
[17]Llamas, I., N. Keshavan, and J.E. Gonzalez, Use of Sinorhizobium meliloti as an indicator for specific detection of long-chain N-acyl homoserine lactones. Appl Environ Microbiol,2004.70(6):p.3715-23.
[18]Andersen, J.B., et al., New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol,1998.64(6):p.2240-6.
[19]Riedel, K., et al., N-acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms. Microbiology, 2001.147(Pt 12):p.3249-62.
[20]Gould, T.A., et al., Specificity of acyl-homoserine lactone synthases examined by mass spectrometry. J Bacteriol,2006.188(2):p.773-83.
[21]Gould, T.A., H.P. Schweizer, and M.E. Churchill, Structure of the Pseudomonas aeruginosa acyl-homoserinelactone synthase LasI. Mol Microbiol,2004.53(4):p. 1135-46.
[22]Kim, M.H., et al., The molecular structure and catalytic mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase. Proc Natl Acad Sci USA, 2005.102(49):p.17606-11.
[23]Uroz, S., et al., N-Acylhomoserine lactone quorum-sensing molecules are modified and degraded by Rhodococcus erythropolis W2 by both amidolytic and novel oxidoreductase activities. Microbiology,2005.151(Pt 10):p.3313-22.
[24]Park, S.Y., et al., N-acylhomoserine lactonase producing Rhodococcus spp. with different AHL-degrading activities. FEMS Microbiol Lett,2006.261(1):p.102-8.
[25]Lin, Y.H., et al., Acyl-homoserine lactone acylase from Ralstonia strain XJ12B represents a novel and potent class of quorum-quenching enzymes. Mol Microbiol,2003. 47(3):p.849-60.
[26]Sio, C.F., et al., Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1. Infect Immun,2006.74(3):p.1673-82.
[27]Huang, J.J., et al., Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1. Appl Environ Microbiol,2006.72(2):p.1190-7.
[28]Frezza, M., et al., Synthesis and biological evaluation of homoserine lactone derived ureas as antagonists of bacterial quorum sensing. Bioorg Med Chem,2006.14(14):p. 4781-91.
[29]Adonizio, A.L., et al., Anti-quorum sensing activity of medicinal plants in southern Florida. J Ethnopharmacol,2006.105(3):p.427-35.
[30]Thomas, P.W., et al., The quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein. Biochemistry,2005.44(20):p.7559-69.
[31]Dong, Y.H., et al., AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc Natl Acad Sci USA,2000.97(7):p.3526-31.
[32]Dong, Y.H., et al., Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl Environ Microbiol,2002.68(4):p.1754-9.
[33]Draganov, D.I., et al., Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation. J Biol Chem,2000.275(43):p.33435-42.
[34]Lee, S.J., et al., Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Appl Environ Microbiol,2002. 68(8):p.3919-24.
[35]Xu, F., et al., Degradation of N-acylhomoserine lactones, the bacterial quorum-sensing molecules, by acylase. J Biotechnol,2003.101(1):p.89-96.
[36]Zhang, H.B., L.H. Wang, and L.H. Zhang, Genetic control of quorum-sensing signal turnover in Agrobacterium tumefaciens. Proc Natl Acad Sci USA,2002.99(7):p. 4638-43.
[37]Molina, L., et al., Degradation of pathogen quorum-sensing molecules by soil bacteria:a preventive and curative biological control mechanism. FEMS Microbiol Ecol,2003. 45(1):p.71-81.
[38]Billecke, S., et al., Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters. Drug Metab Dispos,2000.28(11):p.1335-42.
[39]Uroz, S., et al., Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plant-pathogenic bacteria. Microbiology,2003.149(Pt 8):p.1981-9.
[40]Leadbetter, J.R. and E.P. Greenberg, Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus. J Bacteriol,2000.182(24):p.6921-6.
[41]Taga, M.E. and B.L. Bassler, Chemical communication among bacteria. Proc Natl Acad Sci USA,2003.100 Suppl 2:p.14549-54.
[42]Crameri, A., et al., DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature,1998.391(6664):p.288-91.
[43]Aharoni, A., et al., Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization. Proc Natl Acad Sci USA,2004.101(2): p.482-7.
[44]Suen, W.C., et al., Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling. Protein Eng Des Sel,2004.17(2):p.133-40.
[45]Ness, J.E., et al., DNA shuffling of subgenomic sequences of subtilisin. Nat Biotechnol, 1999.17(9):p.893-6.
[46]Baik, S.H., et al., Significantly enhanced stability of glucose dehydrogenase by directed evolution. Appl Microbiol Biotechnol,2003.61(4):p.329-35.
[47]Hao, J. and A. Berry, A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents. Protein Eng Des Sel,2004.17(9):p.689-97.
[48]Kaper, T., et al., DNA family shuffling of hyperthermostable beta-glycosidases. Biochem J,2002.368(Pt2):p.461-70.
[49]Barriault, D., M.M. Plante, and M. Sylvestre, Family shuffling of a targeted bphA region to engineer biphenyl dioxygenase. J Bacteriol,2002.184(14):p.3794-800.
[50]Kagami, O., M. Kikuchi, and S. Harayama, Single-stranded DNA family shuffling. Methods Enzymol,2004.388:p.11-21.
[51]Kikuchi, M., K. Ohnishi, and S. Harayama, An effective family shuffling method using single-stranded DNA. Gene,2000.243(1-2):p.133-7.
[52]Gibbs, M.D., K.M. Nevalainen, and P.L. Bergquist, Degenerate oligonucleotide gene shuffling (DOGS):a method for enhancing the frequency of recombination with family shuffling. Gene,2001.271(1):p.13-20.
[53]Bergquist, P.L., R.A. Reeves, and M.D. Gibbs, Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM):two complementary techniques for enzyme evolution. Biomol Eng,2005.22(1-3):p.63-72.
[54]Zhang, Y.X., et al., Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature,2002.415(6872):p.644-6.
[55]Dai, M. and S.D. Copley, Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723. Appl Environ Microbiol,2004.70(4):p.2391-7.
[56]Patnaik, R., et al., Genome shuffling of Lactobacillus for improved acid tolerance. Nat Biotechnol,2002.20(7):p.707-12.
[57]Giudici, P., et al., Strategies and perspectives for genetic improvement of wine yeasts. Appl Microbiol Biotechnol,2005.66(6):p.622-8.
[58]Coco, W.M., et al., DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat Biotechnol,2001.19(4):p.354-9.
[59]Song, J.K., et al., Construction of DNA-shuffled and incrementally truncated libraries by a mutagenic and unidirectional reassembly method:changing from a substrate specificity of phospholipase to that of lipase. Appl Environ Microbiol,2002.68(12):p. 6146-51.
[60]Ghadessy, F.J., J.L. Ong, and P. Holliger, Directed evolution of polymerase function by compartmentalized self-replication. Proc Natl Acad Sci USA,2001.98(8):p.4552-7.
[61]Fujii, R., M. Kitaoka, and K. Hayashi, One-step random mutagenesis by error-prone rolling circle amplification. Nucleic Acids Res,2004.32(19):p. e145.
[62]Osuna, J., et al., Protein evolution by codon-based random deletions. Nucleic Acids Res, 2004.32(17):p. e136.