以纤维二糖为底物利用重组大肠杆菌合成海藻糖
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摘要
探讨以纤维二糖为底物合成海藻糖的可行性。首先克隆来自施氏假单胞菌A1501的otsA/otsB基因,外源导入Escherichia coli BL21(DE3)构建以葡萄糖为底物合成海藻糖的OtsAB途径。继而通过过表达E. coli本身的galU基因增加海藻糖合成前体物质尿苷二磷酸(uridine diphosphate,UDP)-葡萄糖的含量,使海藻糖产量提高了3倍。通过添加井冈霉素抑制海藻糖的降解,进一步提高海藻糖的产量。在此基础上,过表达来自天然纤维素分解细菌Saccharophagusdegradans的纤维二糖磷酸化酶基因cepA,使重组大肠杆菌具有利用纤维二糖产海藻糖的能力。利用该重组大肠杆菌全细胞催化生产海藻糖,底物为20 g/L纤维二糖,并添加0.05 mmol/L的井冈霉素抑制海藻糖降解时,48 h后可生成1.3 g/L的海藻糖。本研究利用重组大肠杆菌以纤维二糖为底物合成海藻糖,证实了以纤维二糖为底物合成海藻糖的可行性,为纤维二糖来源精细化学品的生产提供了新的思路。
This study aimed to explore the feasibility of synthesizing trehalose using cellobiose as substrate. Firstly, the ots A/ots B gene from Pseudomonas stutzeri A1501 was cloned and exogenously introduced into Escherichia coli BL21(DE3) to construct an OtsAB pathway to synthesize trehalose using glucose as the substrate. Subsequently, the content of uridine diphosphate(UDP)-glucose, the key precursor for biosynthesis of trehalose was increased by overexpressing the galU gene of E. coli itself, resulting in a 4-fold increase in trehalose production. By adding validamycin, the degradation of trehalose was suppressed to increase its yield. Furthermore, by overexpressing the cellobiose phosphorylase gene(cepA) from the naturally occurring cellulolytic bacterium Saccharophagus degradans, the recombinant E. coli was rendered capable of producing trehalose from cellobiose. After 48 h culture with the addition of 0.05 mmol/L validamycin against trehalose degradation, whole recombinant cells catalyzed the transformation of 20 g/L cellobiose to 1.3 g/L trehalose. This study confirms the feasibility of synthesizing trehalose using cellobiose as substrate and provides new ideas for producing fine chemicals from cellobiose.
This study aimed to explore the feasibility of synthesizing trehalose using cellobiose as substrate. Firstly, the ots A/ots B gene from Pseudomonas stutzeri A1501 was cloned and exogenously introduced into Escherichia coli BL21(DE3) to construct an OtsAB pathway to synthesize trehalose using glucose as the substrate. Subsequently, the content of uridine diphosphate(UDP)-glucose, the key precursor for biosynthesis of trehalose was increased by overexpressing the galU gene of E. coli itself, resulting in a 4-fold increase in trehalose production. By adding validamycin, the degradation of trehalose was suppressed to increase its yield. Furthermore, by overexpressing the cellobiose phosphorylase gene(cepA) from the naturally occurring cellulolytic bacterium Saccharophagus degradans, the recombinant E. coli was rendered capable of producing trehalose from cellobiose. After 48 h culture with the addition of 0.05 mmol/L validamycin against trehalose degradation, whole recombinant cells catalyzed the transformation of 20 g/L cellobiose to 1.3 g/L trehalose. This study confirms the feasibility of synthesizing trehalose using cellobiose as substrate and provides new ideas for producing fine chemicals from cellobiose.
引文
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[14]KIM Y C,QUAN F S,COMPANS R W,et al.Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity[J].Journal of Controlled Release,2010,142(2):187-195.DOI:10.1016/j.jconrel.2009.10.013.
[15]PATIST A,ZOERB H.Preservation mechanisms of trehalose in food and biosystems[J].Colloids and Surfaces B:Biointerfaces,2005,40(2):107-113.DOI:10.1016/j.colsurfb.2004.05.003.
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[17]VíLCHEZ J I,GARCíA-FONTANA C,ROMáN-NARANJO D,et al.Plant drought tolerance enhancement by trehalose production of desiccation-tolerant microorganisms[J].Frontiers in Microbiology,2016,7:1577.DOI:10.3389/fmicb.2016.01577.
[18]PADILLA L,KR?MER R,STEPHANOPOULOS G,et al.Overproduction of trehalose:heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum[J].Applied and Environmental Microbiology,2004,70(1):370-376.DOI:10.1128/AEM.70.1.370-376.2004.
[19]WU Y,SUN X,LIN Y,et al.Establishing a synergetic carbon utilization mechanism for non-catabolic use of glucose in microbial synthesis of trehalose[J].Metabolic Engineering,2017,39:1-8.DOI:10.1016/j.ymben.2016.11.001.
[20]CARPINELLI J,KR?MER R,AGOSIN E.Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction:role of the TreYZ trehalose biosynthetic pathway[J].Applied and Environmental Microbiology,2006,72(3):1949-1955.DOI:10.1128/AEM.72.3.1949-1955.2006.
[21]ZHENG Z J,XU Y,SUN Y,et al.Biocatalytic production of trehalose from maltose by using whole cells of permeabilized recombinant Escherichia coli[J].PLoS ONE,2015,10(10):e0140477.DOI:10.1371/journal.pone.0140477.
[22]FREEMAN B C,CHEN C,BEATTIE G A.Identification of the trehalose biosynthetic loci of Pseudomonas syringae and their contribution to fitness in the phyllosphere[J].Environmental Microbiology,2010,12(6):1486-1497.DOI:10.1111/j.1462-2920.2010.02171.x.
[23]DJONOVI?S,URBACH J M,DRENKARD E,et al.Trehalose biosynthesis promotes Pseudomonas aeruginosa pathogenicity in plants[J].PLoS Pathogens,2013,9(3):e1003217.DOI:10.1371/journal.ppat.1003217.
[24]SAMBROOK J,RUSSELL D W,RUSSELL D,et al.Molecular cloning:a laboratory manual[M].UK:Coldspring-Harbour Laboratory Press,2001.
[25]徐颖.海藻糖合酶基因的克隆表达及海藻糖的制备[D].南京:南京林业大学,2015.
[26]STROM A R,KAASEN I.Trehalose metabolism in Escherichia coli:stress protection and stress regulation of gene expression[J].Molecular Microbiology,1993,8(2):205-210.DOI:10.1111/j.1365-2958.1993.tb01564.x.
[27]GIBSON R P,GLOSTER T M,ROBERTS S,et al.Molecular basis for trehalase inhibition revealed by the structure of trehalase in complex with potent inhibitors[J].Angewandte Chemie International Edition,2007,46(22):4115-4119.DOI:10.1002/anie.200604825.
[28]LI H,SU H,KIM S B,et al.Enhanced production of trehalose in Escherichia coli by homologous expression of ots BA in the presence of the trehalase inhibitor,validamycin A,at high osmolarity[J].Journal of Bioscience and Bioengineering,2012,113(2):224-232.DOI:10.1016/j.jbiosc.2011.09.018.
[29]SEKAR R,SHIN H D,CHEN R.Engineering Escherichia coli cells for cellobiose assimilation through a phosphorolytic mechanism[J].Applied and Environmental Microbiology,2012,78(5):1611-1614.DOI:10.1128/AEM.06693-11.
[30]吴秀丽,岳明,丁宏标.海藻糖合酶的研究进展[J].微生物学通报,2009,36(7):1067-1072.DOI:10.13344/j.microbiol.china.2009.07.009.
[31]HABE H,SATO S,MORITA T,et al.Bacterial production of shortchain organic acids and trehalose from levulinic acid:a potential cellulose-derived building block as a feedstock for microbial production[J].Bioresource Technology,2015,177:381-386.DOI:10.1016/j.biortech.2014.11.048.
[32]KAR J R,HALLSWORTH J E,SINGHAL R S.Fermentative production of glycine betaine and trehalose from acid whey using Actinopolyspora halophila(MTCC 263)[J].Environmental Technology&Innovation,2015,3:68-76.DOI:10.1016/j.eti.2015.02.001.
[2]KAWAGUCHI H,HASUNUMA T,OGINO C,et al.Bioprocessing of bio-based chemicals produced from lignocellulosic feedstocks[J].Current Opinion in Biotechnology,2016,42:30-39.DOI:10.1016/j.copbio.2016.02.031.
[3]WHEELDON I,CHRISTOPHER P,BLANCH H.Integration of heterogeneous and biochemical catalysis for production of fuels and chemicals from biomass[J].Current Opinion in Biotechnology,2017,45:127-135.DOI:10.1016/j.copbio.2017.02.019.
[4]ALVIRA P,TOMáS-PEJóE,BALLESTEROS M J,et al.Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis:a review[J].Bioresource Technology,2010,101(13):4851-4861.DOI:10.1016/j.biortech.2009.11.093.
[5]KUMAR P,BARRETT D M,DELWICHE M J,et al.Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production[J].Industrial&Engineering Chemistry Research,2009,48(8):3713-3729.DOI:10.1021/ie801542g.
[6]BEZERRA R M F,DIAS A A.Enzymatic kinetic of cellulose hydrolysis[J].Applied Biochemistry and Biotechnology,2005,126(1):49-59.DOI:10.1007/s12010-005-0005-5.
[7]GRUNO M,V?LJAM?E P,PETTERSSON G,et al.Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate[J].Biotechnology and Bioengineering,2004,86(5):503-511.DOI:10.1002/bit.10838.
[8]ZHAO Y,WU B,YAN B,et al.Mechanism of cellobiose inhibition in cellulose hydrolysis by cellobiohydrolase[J].Science in China Series C:Life Sciences,2004,47(1):18-24.DOI:10.1360/02yc0163.
[9]ADACHI N,TAKAHASHI C,ONO-MUROTA N,et al.Direct L-lysine production from cellobiose by Corynebacterium glutamicum displayingβ-glucosidase on its cell surface[J].Applied Microbiology and Biotechnology,2013,97(16):7165-7172.DOI:10.1007/s00253-013-5009-4.
[10]MU?OZ-GUTIéRREZ I,OROPEZA R,GOSSET G,et al.Cell surface display of aβ-glucosidase employing the type V secretion system on ethanologenic Escherichia coli for the fermentation of cellobiose to ethanol[J].Journal of Industrial Microbiology&Biotechnology,2012,39(8):1141-1152.DOI:10.1007/s10295-012-1122-0.
[11]SOMA Y,INOKUMA K,TANAKA T,et al.Direct isopropanol production from cellobiose by engineered Escherichia coli using a synthetic pathway and a cell surface display system[J].Journal of Bioscience and Bioengineering,2012,114(1):80-85.DOI:10.1016/j.jbiosc.2012.02.019.
[12]OHTAKE S,WANG Y J.Trehalose:current use and future applications[J].Journal of Pharmaceutical Sciences,2011,100(6):2020-2053.DOI:10.1002/jps.22458.
[13]SCHIRALDI C,DI LERNIA I,DE ROSA M.Trehalose production:exploiting novel approaches[J].Trends in Biotechnology,2002,20(10):420-425.DOI:10.1016/S0167-7799(02)02041-3.
[14]KIM Y C,QUAN F S,COMPANS R W,et al.Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity[J].Journal of Controlled Release,2010,142(2):187-195.DOI:10.1016/j.jconrel.2009.10.013.
[15]PATIST A,ZOERB H.Preservation mechanisms of trehalose in food and biosystems[J].Colloids and Surfaces B:Biointerfaces,2005,40(2):107-113.DOI:10.1016/j.colsurfb.2004.05.003.
[16]MARTINETTI D,COLAROSSI C,BUCCHERI S,et al.Effect of trehalose on cryopreservation of pure peripheral blood stem cells[J].Biomedical Reports,2017,6(3):314-318.DOI:10.3892/br.2017.859.
[17]VíLCHEZ J I,GARCíA-FONTANA C,ROMáN-NARANJO D,et al.Plant drought tolerance enhancement by trehalose production of desiccation-tolerant microorganisms[J].Frontiers in Microbiology,2016,7:1577.DOI:10.3389/fmicb.2016.01577.
[18]PADILLA L,KR?MER R,STEPHANOPOULOS G,et al.Overproduction of trehalose:heterologous expression of Escherichia coli trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in Corynebacterium glutamicum[J].Applied and Environmental Microbiology,2004,70(1):370-376.DOI:10.1128/AEM.70.1.370-376.2004.
[19]WU Y,SUN X,LIN Y,et al.Establishing a synergetic carbon utilization mechanism for non-catabolic use of glucose in microbial synthesis of trehalose[J].Metabolic Engineering,2017,39:1-8.DOI:10.1016/j.ymben.2016.11.001.
[20]CARPINELLI J,KR?MER R,AGOSIN E.Metabolic engineering of Corynebacterium glutamicum for trehalose overproduction:role of the TreYZ trehalose biosynthetic pathway[J].Applied and Environmental Microbiology,2006,72(3):1949-1955.DOI:10.1128/AEM.72.3.1949-1955.2006.
[21]ZHENG Z J,XU Y,SUN Y,et al.Biocatalytic production of trehalose from maltose by using whole cells of permeabilized recombinant Escherichia coli[J].PLoS ONE,2015,10(10):e0140477.DOI:10.1371/journal.pone.0140477.
[22]FREEMAN B C,CHEN C,BEATTIE G A.Identification of the trehalose biosynthetic loci of Pseudomonas syringae and their contribution to fitness in the phyllosphere[J].Environmental Microbiology,2010,12(6):1486-1497.DOI:10.1111/j.1462-2920.2010.02171.x.
[23]DJONOVI?S,URBACH J M,DRENKARD E,et al.Trehalose biosynthesis promotes Pseudomonas aeruginosa pathogenicity in plants[J].PLoS Pathogens,2013,9(3):e1003217.DOI:10.1371/journal.ppat.1003217.
[24]SAMBROOK J,RUSSELL D W,RUSSELL D,et al.Molecular cloning:a laboratory manual[M].UK:Coldspring-Harbour Laboratory Press,2001.
[25]徐颖.海藻糖合酶基因的克隆表达及海藻糖的制备[D].南京:南京林业大学,2015.
[26]STROM A R,KAASEN I.Trehalose metabolism in Escherichia coli:stress protection and stress regulation of gene expression[J].Molecular Microbiology,1993,8(2):205-210.DOI:10.1111/j.1365-2958.1993.tb01564.x.
[27]GIBSON R P,GLOSTER T M,ROBERTS S,et al.Molecular basis for trehalase inhibition revealed by the structure of trehalase in complex with potent inhibitors[J].Angewandte Chemie International Edition,2007,46(22):4115-4119.DOI:10.1002/anie.200604825.
[28]LI H,SU H,KIM S B,et al.Enhanced production of trehalose in Escherichia coli by homologous expression of ots BA in the presence of the trehalase inhibitor,validamycin A,at high osmolarity[J].Journal of Bioscience and Bioengineering,2012,113(2):224-232.DOI:10.1016/j.jbiosc.2011.09.018.
[29]SEKAR R,SHIN H D,CHEN R.Engineering Escherichia coli cells for cellobiose assimilation through a phosphorolytic mechanism[J].Applied and Environmental Microbiology,2012,78(5):1611-1614.DOI:10.1128/AEM.06693-11.
[30]吴秀丽,岳明,丁宏标.海藻糖合酶的研究进展[J].微生物学通报,2009,36(7):1067-1072.DOI:10.13344/j.microbiol.china.2009.07.009.
[31]HABE H,SATO S,MORITA T,et al.Bacterial production of shortchain organic acids and trehalose from levulinic acid:a potential cellulose-derived building block as a feedstock for microbial production[J].Bioresource Technology,2015,177:381-386.DOI:10.1016/j.biortech.2014.11.048.
[32]KAR J R,HALLSWORTH J E,SINGHAL R S.Fermentative production of glycine betaine and trehalose from acid whey using Actinopolyspora halophila(MTCC 263)[J].Environmental Technology&Innovation,2015,3:68-76.DOI:10.1016/j.eti.2015.02.001.