一步单菌发酵生产2-酮基-L-古龙酸工程菌的构建与优化
|
摘要
2-酮基-L-古龙酸(2-keto-L-gulonic acid)是合成维生素C的重要前体。目前国内维生素C生产采用二步发酵法，该过程工艺复杂，影响因素较多，难以精确控制，同时3种微生物生长消耗了大量的原材料和能源。如能以D-山梨醇为碳源进行一步发酵生产维生素C前体2-KLG，对于维生素C产业将是具有重大意义的技术进步。本论文利用基因工程技术将维生素C二步发酵有关基因分别表达于大肠杆菌(Escherichia coli)和氧化葡萄糖酸杆菌(Gluconobacter oxydans)中，构建一步发酵工程菌生产2-KLG，并通过不同连接肽的选择、提高辅酶吡咯喹啉醌(pyrroloquinoline quinine，PQQ)供给以及利用耐热交联蛋白CutA作为蛋白支架等策略，有效提高了G. oxydans一步工程菌生产2-KLG的产量，实现了由D-山梨醇经单菌一步发酵生产2-KLG。主要研究结果如下：
(1)2-KLG合成关键基因的克隆与鉴定
根据Ketogulonicigenium vulgare WSH-001基因组注释，得到7个与2-KLG合成途径相关的关键脱氢酶。为了验证关键酶的酶学性质，将其分别在E. coli中进行了表达与纯化。测定酶活发现7个脱氢酶均为PQQ依赖性的醌酶，KVU_1366、KVU_0203、KVU_2142、KVU_2159及KVU_PA0245同时具有SDH及SNDH的活性，其中KVU_2142具有最高的比酶活为9.68U/mg。而KVU_PB0115及KVU_0095仅具有SNDH活性，后者比酶活为前者的近两倍。体外催化能力的测定表明，将酶液与PQQ孵育5min形成全酶后，5个SDH/SNDH单独及分别与2个SNDH组合催化L-山梨糖均能够生成2-KLG，其中KVU_2142与KVU_0095组合生成2-KLG的浓度最高，为19.7g·L–1。
(2)分子改造大肠杆菌一步发酵生产2-KLG
考察了高浓度的2-KLG对E. coli生长的影响，发现高达100g·L–1的2-KLG对E. coli的生长没有明显的抑制作用。另外，在添加2-KLG的条件下进行E. coli的培养，在发酵120h后未检测到2-KLG的降解，表明E. coli可以作为一步发酵生产2-KLG工程菌构建的宿主。利用具有相容性的两个质粒pETDuet-1及pRSFDuet-1成功构建了一步工程菌株E. coli/pET-k2142-k0095-pRSF-sldh-pqq。通过SDS-PAGE检测及体外催化反应表明，KVU_2142、KVU_0095、sldh及pqqABCDE在E. coli中得到了表达。将一步工程菌经过72h发酵后检测到有4.8g·L–1的山梨糖及0.5g·L–1的2-KLG生成，大部分山梨醇转化成了副产物。经过分析，山梨醇可能是通过山梨醇磷酸转移酶系统进入了糖酵解和磷酸戊糖途径。
(3)氧化葡萄糖酸杆菌全基因组测序及基因操作系统的构建
通过高通量Illumina测序技术得到一个全长为3,364,884bp的G. oxydans WSH-003基因组，共编码3705个ORF，G+C含量为50.42%。G. oxydans WSH-003中涉及碳水化合物代谢的基因共约330个，另外存在300多个膜转运蛋白，使其具有很高的不完全氧化能力和转化效率。考察了高浓度的2-KLG对G. oxydans WSH-003的生长的影响。结果表明，高达100g·L–1的2-KLG对G. oxydans WSH-003的生长没有明显的抑制作用。另外，在添加2-KLG的条件下进行G. oxydans WSH-003的培养，在发酵120h后未检测到2-KLG的降解，表明G. oxydans WSH-003可以作为一步发酵生产2-KLG工程菌构建的宿主。选择G. oxydans621H包含的质粒pGOX3中的复制起始位点序列克隆到E. coli克隆载体pUC18中，得到E. coli-G. oxydans穿梭质粒载体pGUC，该载体具有较高的转化效率并且在没有抗性选择压力的条件下也具有很高的稳定性。
(4)氧化葡萄糖酸杆菌工程菌的构建及利用连接肽工程与辅因子工程对一步工程菌的改造
将来源于K. vulgare WSH-001的5个SDH/SNDH基因和2个SNDH基因分别以不同的组合构建了10株工程菌，经摇瓶发酵均检测到2-KLG的生成，其中G.oxydans/pGUC-k0203-k0095的产量最高，为4.9g·L–1。选择了不同种类及长度的连接肽将k0203和k0095进行融合构建得到10株融合表达工程菌，发酵168h后，2-KLG产量最高达到31.2g·L–1。继续考察了酵母粉浓度对发酵产量的影响，确定酵母粉最佳浓度为10g·L–1，发酵168h后2-KLG产量为32.4g·L–1。在融合表达一步工程菌的基础上进一步表达pqqA和pqqABCDE提高辅酶供给，表达了pqqA和pqqABCDE的两株工程菌PQQ含量较野生菌分别提高了63.2%和136.5%。发酵168h后2-KLG产量分别达到35.1g·L–1和39.2g·L–1，较融合表达最高产量分别提高了8.3%和21.0%。
(5)基于耐热交联蛋白CutA对氧化葡萄糖酸杆菌一步工程菌的改造
首先对k0203-GGGGS-k0095进行了密码子优化，得到工程菌的2-KLG产量为33.2g·L–1。随后对来源于掘越氏热球菌(Pyrococcus horikoshii)的cutA进行密码子优化后在G. oxydans中进行了表达。结果表明CutA在G. oxydans中的表达提高了G. oxydans的耐热性。将优化后的cutA与k0203-GGGGS-k0095在G. oxydans中进行了共表达，得到的一步工程菌生成2-KLG的产量达到40.3g·L–1，较未表达CutA的工程菌提高了24.4%。对基于CutA构建的一步工程菌进行了30℃、35℃及37℃条件下的发酵，结果表明表达了CutA的工程菌生产2-KLG的产量在3个温度条件下均高于对照菌，但是在35℃及37℃条件下，2-KLG的产量均明显下降。最后在表达CutA工程菌的基础上进一步表达了pqqABCDE来提高辅酶供给，发酵168h后2-KLG产量达到42.6g·L–1。
2-Keto-L-gulonic acid is an important precursor of vitamin C. The conventional two-stepfermentation route is the most successful method for L-AA production, and has been used onan industrial scale for several decades. However, unlike most of the common biotechnologicalprocesses, the conventional two-step fermentation of L-AA involves three microorganismsand requires an additional second sterilization process. This significantly increases the cost ofboth raw materials and energy requirement. Furthermore, the mix-culture system composed ofBacillus megaterium and Ketogulonicigenium vulgar makes both strain improvement andprocess optimization difficult. Therefore, a one-step fermentation process is considered to bemore cost-effective and revolutionary in the L-AA industry worldwide. In this paper, thecrtical genes were introduced into Escherichia coli and Gluconobacter oxydans respectivelyto construct engineered strains for one-step fermentation of2-KLG, and the production of2-KLG was effectively improved by the use of connecting peptide and the trimeric proteinCutA，and further by the enhancement of the cofactor PQQ. The main results are listed asfollowing:
(1) Cloning and identification of2-KLG biosynthetic genes
K. vulgare WSH-001is an industrial strain used for vitamin C production. Based ongenome sequencing and pathway analysis of the bacterium, some of dehydrogenases relatedto2-KLG synthesis were predicted, including KVU_PA0245, KVU_2142, KVU_2159,KVU_1366, KVU_0203, KVU_0095and KVU_pmdB_0115. In order to validate theenzymatic properties, these seven putative dehyrogenases were overexpressed in E. coli BL21(DE3) and purified for characterization. It was found that the seven dehydrogenases are PQQdependent enzymes, KVU_1366, KVU_0203, KVU_2142, KVU_2159and KVU_PA0245possess both SDH and SNDH activities, in which KVU_2142has the highest specific activity,9.68U/mg. While KVU_PB0115and KVU_0095only possess SNDH activity, and thespecific activity of the latter is nearly twice that of the former. Furthermore, enzyme kineticparameters were determined, KVU_2142and KVU_0095has the highest substrate bindingability in five SDH/SNDH and two SNDH, respectively. In addition, determination of thecatalytic ability in vitro showed that there was2-KLG generated when L-sorbose wascatalyzed with SDH/SNDH alone or in combination with SNDH, in which the highest2-KLGconcentration was19.7g·L–1with the combination of KVU_2142and KVU_0095.
(2) Construction of E. coli engineered strain for the direct production of2-KLG
The tolerance of E. coli to2-KLG was determined, it showed that even100g·L–1of2-KLG did not significantly affect the cell growth of E. coli. In addition, no obviousdegradation of2-KLG could be detected when E. coli was grown with2-KLG for120h. Theengineered strain E. coli/pET-k2142-k0095-pRSF-sldh-pqq was constructed with twoplasmids pETDuet-1and pRSFDuet-1. It was proved that four enzymes were expressedthrough SDS-PAGE detection. In addition, the crude enzyme was used in catalytic reaction,and2-KLG was generated after sorbitol was catalyzed. After72h fermentation of theengineered strain,4.8g·L–1of sorbose,0.5g·L–1of2-KLG and a great deal of by-products was generated. Sorbitol may be transferred into the glycolysis and the pentose phosphatepathway through sorbitol phosphotransferase system.
(3) The whole genome sequencing and the construction of gene manipulation system ofG. oxydans WSH-003
The whole genome sequence of G. oxydans WSH-003was released by Illuminasequencing technology. The genomic feature was provided in this dissertation. The completeG. oxydans WSH-003genome is3,364,884bp and contains3705protein-encoding genes, theoverall G+C content of the chromosome is50.42%. G. oxydans has a very strong incompleteoxidation ability of carbohydrates, alcohols and polyols. In G. oxydans WSH-003, there areabout330genes involved in the carbohydrate metabolism, and the presence of more than300membrane transport proteins make G. oxydans WSH-003possess a very high conversionefficiency. In addition, the tolerance of G. oxydans WSH-003to2-KLG was determined, itshowed that even100g·L–1of2-KLG did not significantly affect the cell growth of G.oxydans WSH-003. Besides, no obvious degradation of2-KLG could be detected when G.oxydans WSH-003was grown with2-KLG for120h. In order to construct G. oxydansengineered strains, a E. coli-G. oxydans shuttle vector is essential. The par-rep gene from theplasmid pGOX3of G. oxydans621H was ligated into pUC18, resulting in the shuttle vectorpGUC, which has a high conversion efficiency and stability.
(4) Construction of G. oxydans engineered strain for the direct production of2-KLG andthe modification by connecting peptide engineering and cofactor engineering
Different combinations of five SDH/SNDH and two SNDH from K. vulgare WSH-001were introduced into G. oxydans WSH-003, an industrial strain used for the conversion ofD-sorbitol to L-sorbose. The optimum combination produced4.9g·L–1of2-KLG. In addition,10different linker peptides were used for the fusion expression of SDH/SNDH and SNDH inG. oxydans. The best recombinant strain produced31.2g·L–1of2-KLG after168h. After theoptimization of yeast extract concentration, the production of2-KLG reached32.4g·L–1.Furthermore, overexpression of pqqA and pqqABCDE gene clusters under the control of tufBpromoter enhanced PQQ concentration by126.1%and273.6%, respectively, compared withthat noted in the wild-type strain. Overexpression of PQQ biosynthesis gene(s) significantlyenhanced cell growth, and the2-KLG production by G.oxydans/pGUC-k0203-GS-k0095-pqqA and G. oxydans/pGUC-k0203-GS-k0095-pqqABCDEreached35.1g·L–1and39.2g·L–1, which was8.3%and21.0%higher than that by G.oxydans/pGUC-k0203-GS-k0095, respectively.
(5) Efficient optimization of G. oxydans based on trimeric protein CutA for the directproduction of2-KLG
The codon optimization was conducted to further improve the expression of SDH andSNDH in G. oxydans, the production of2-KLG was33.2g·L–1after the codon optimization.In order to verify whether the expression of cutA could take effect in G. oxydans, after thecodon optimization of cutA, G. oxydans strain harboring pGUC-tufB-cutA was constructed.The growth curve of G. oxydans/pGUC-tufB-cutA and the wild strain were determined, whichindicated that CutA was correctly expressed in G. oxydans, and the heat resistance of G.oxydans was improved. Furthermore, based on the adaptor protein SH3and its ligand SH3lig, the codon-optimized cutA and sdh-GGGGS-sndh were expressed in G. oxydans. Theengineered strain G. oxydans/pGUC-tufB-k0203-GS-k0095-cutAproduced40.3g·L–1of2-KLG. In order to investigate the fermentation performance at different temperatures, theengineered strain G. oxydans/pGUC-tufB-k0203-GS-k0095-cutA was fermented at30℃,35℃and37℃, respectively. G. oxydans/pGUC-tufB-k0203-GS-k0095-cutA grew better at35℃and37℃than the control strain. The production of2-KLG of G.oxydans/pGUC-tufB-k0203-GS-k0095-cutA were higher than the control at differenttemperatures, which revealed that the expression of CutA improved the catalytic efficiency ofthe dehydrogenases. However, the strain G. oxydans/pGUC-tufB-k0203-GS-k0095-cutAproduced less2-KLG at35℃and37℃than30℃. Utimately, pqqABCDE gene clusterswere overexpressed and the production of2-KLG reached to42.6g·L–1.
(1)2-KLG合成关键基因的克隆与鉴定
根据Ketogulonicigenium vulgare WSH-001基因组注释，得到7个与2-KLG合成途径相关的关键脱氢酶。为了验证关键酶的酶学性质，将其分别在E. coli中进行了表达与纯化。测定酶活发现7个脱氢酶均为PQQ依赖性的醌酶，KVU_1366、KVU_0203、KVU_2142、KVU_2159及KVU_PA0245同时具有SDH及SNDH的活性，其中KVU_2142具有最高的比酶活为9.68U/mg。而KVU_PB0115及KVU_0095仅具有SNDH活性，后者比酶活为前者的近两倍。体外催化能力的测定表明，将酶液与PQQ孵育5min形成全酶后，5个SDH/SNDH单独及分别与2个SNDH组合催化L-山梨糖均能够生成2-KLG，其中KVU_2142与KVU_0095组合生成2-KLG的浓度最高，为19.7g·L–1。
(2)分子改造大肠杆菌一步发酵生产2-KLG
考察了高浓度的2-KLG对E. coli生长的影响，发现高达100g·L–1的2-KLG对E. coli的生长没有明显的抑制作用。另外，在添加2-KLG的条件下进行E. coli的培养，在发酵120h后未检测到2-KLG的降解，表明E. coli可以作为一步发酵生产2-KLG工程菌构建的宿主。利用具有相容性的两个质粒pETDuet-1及pRSFDuet-1成功构建了一步工程菌株E. coli/pET-k2142-k0095-pRSF-sldh-pqq。通过SDS-PAGE检测及体外催化反应表明，KVU_2142、KVU_0095、sldh及pqqABCDE在E. coli中得到了表达。将一步工程菌经过72h发酵后检测到有4.8g·L–1的山梨糖及0.5g·L–1的2-KLG生成，大部分山梨醇转化成了副产物。经过分析，山梨醇可能是通过山梨醇磷酸转移酶系统进入了糖酵解和磷酸戊糖途径。
(3)氧化葡萄糖酸杆菌全基因组测序及基因操作系统的构建
通过高通量Illumina测序技术得到一个全长为3,364,884bp的G. oxydans WSH-003基因组，共编码3705个ORF，G+C含量为50.42%。G. oxydans WSH-003中涉及碳水化合物代谢的基因共约330个，另外存在300多个膜转运蛋白，使其具有很高的不完全氧化能力和转化效率。考察了高浓度的2-KLG对G. oxydans WSH-003的生长的影响。结果表明，高达100g·L–1的2-KLG对G. oxydans WSH-003的生长没有明显的抑制作用。另外，在添加2-KLG的条件下进行G. oxydans WSH-003的培养，在发酵120h后未检测到2-KLG的降解，表明G. oxydans WSH-003可以作为一步发酵生产2-KLG工程菌构建的宿主。选择G. oxydans621H包含的质粒pGOX3中的复制起始位点序列克隆到E. coli克隆载体pUC18中，得到E. coli-G. oxydans穿梭质粒载体pGUC，该载体具有较高的转化效率并且在没有抗性选择压力的条件下也具有很高的稳定性。
(4)氧化葡萄糖酸杆菌工程菌的构建及利用连接肽工程与辅因子工程对一步工程菌的改造
将来源于K. vulgare WSH-001的5个SDH/SNDH基因和2个SNDH基因分别以不同的组合构建了10株工程菌，经摇瓶发酵均检测到2-KLG的生成，其中G.oxydans/pGUC-k0203-k0095的产量最高，为4.9g·L–1。选择了不同种类及长度的连接肽将k0203和k0095进行融合构建得到10株融合表达工程菌，发酵168h后，2-KLG产量最高达到31.2g·L–1。继续考察了酵母粉浓度对发酵产量的影响，确定酵母粉最佳浓度为10g·L–1，发酵168h后2-KLG产量为32.4g·L–1。在融合表达一步工程菌的基础上进一步表达pqqA和pqqABCDE提高辅酶供给，表达了pqqA和pqqABCDE的两株工程菌PQQ含量较野生菌分别提高了63.2%和136.5%。发酵168h后2-KLG产量分别达到35.1g·L–1和39.2g·L–1，较融合表达最高产量分别提高了8.3%和21.0%。
(5)基于耐热交联蛋白CutA对氧化葡萄糖酸杆菌一步工程菌的改造
首先对k0203-GGGGS-k0095进行了密码子优化，得到工程菌的2-KLG产量为33.2g·L–1。随后对来源于掘越氏热球菌(Pyrococcus horikoshii)的cutA进行密码子优化后在G. oxydans中进行了表达。结果表明CutA在G. oxydans中的表达提高了G. oxydans的耐热性。将优化后的cutA与k0203-GGGGS-k0095在G. oxydans中进行了共表达，得到的一步工程菌生成2-KLG的产量达到40.3g·L–1，较未表达CutA的工程菌提高了24.4%。对基于CutA构建的一步工程菌进行了30℃、35℃及37℃条件下的发酵，结果表明表达了CutA的工程菌生产2-KLG的产量在3个温度条件下均高于对照菌，但是在35℃及37℃条件下，2-KLG的产量均明显下降。最后在表达CutA工程菌的基础上进一步表达了pqqABCDE来提高辅酶供给，发酵168h后2-KLG产量达到42.6g·L–1。
2-Keto-L-gulonic acid is an important precursor of vitamin C. The conventional two-stepfermentation route is the most successful method for L-AA production, and has been used onan industrial scale for several decades. However, unlike most of the common biotechnologicalprocesses, the conventional two-step fermentation of L-AA involves three microorganismsand requires an additional second sterilization process. This significantly increases the cost ofboth raw materials and energy requirement. Furthermore, the mix-culture system composed ofBacillus megaterium and Ketogulonicigenium vulgar makes both strain improvement andprocess optimization difficult. Therefore, a one-step fermentation process is considered to bemore cost-effective and revolutionary in the L-AA industry worldwide. In this paper, thecrtical genes were introduced into Escherichia coli and Gluconobacter oxydans respectivelyto construct engineered strains for one-step fermentation of2-KLG, and the production of2-KLG was effectively improved by the use of connecting peptide and the trimeric proteinCutA，and further by the enhancement of the cofactor PQQ. The main results are listed asfollowing:
(1) Cloning and identification of2-KLG biosynthetic genes
K. vulgare WSH-001is an industrial strain used for vitamin C production. Based ongenome sequencing and pathway analysis of the bacterium, some of dehydrogenases relatedto2-KLG synthesis were predicted, including KVU_PA0245, KVU_2142, KVU_2159,KVU_1366, KVU_0203, KVU_0095and KVU_pmdB_0115. In order to validate theenzymatic properties, these seven putative dehyrogenases were overexpressed in E. coli BL21(DE3) and purified for characterization. It was found that the seven dehydrogenases are PQQdependent enzymes, KVU_1366, KVU_0203, KVU_2142, KVU_2159and KVU_PA0245possess both SDH and SNDH activities, in which KVU_2142has the highest specific activity,9.68U/mg. While KVU_PB0115and KVU_0095only possess SNDH activity, and thespecific activity of the latter is nearly twice that of the former. Furthermore, enzyme kineticparameters were determined, KVU_2142and KVU_0095has the highest substrate bindingability in five SDH/SNDH and two SNDH, respectively. In addition, determination of thecatalytic ability in vitro showed that there was2-KLG generated when L-sorbose wascatalyzed with SDH/SNDH alone or in combination with SNDH, in which the highest2-KLGconcentration was19.7g·L–1with the combination of KVU_2142and KVU_0095.
(2) Construction of E. coli engineered strain for the direct production of2-KLG
The tolerance of E. coli to2-KLG was determined, it showed that even100g·L–1of2-KLG did not significantly affect the cell growth of E. coli. In addition, no obviousdegradation of2-KLG could be detected when E. coli was grown with2-KLG for120h. Theengineered strain E. coli/pET-k2142-k0095-pRSF-sldh-pqq was constructed with twoplasmids pETDuet-1and pRSFDuet-1. It was proved that four enzymes were expressedthrough SDS-PAGE detection. In addition, the crude enzyme was used in catalytic reaction,and2-KLG was generated after sorbitol was catalyzed. After72h fermentation of theengineered strain,4.8g·L–1of sorbose,0.5g·L–1of2-KLG and a great deal of by-products was generated. Sorbitol may be transferred into the glycolysis and the pentose phosphatepathway through sorbitol phosphotransferase system.
(3) The whole genome sequencing and the construction of gene manipulation system ofG. oxydans WSH-003
The whole genome sequence of G. oxydans WSH-003was released by Illuminasequencing technology. The genomic feature was provided in this dissertation. The completeG. oxydans WSH-003genome is3,364,884bp and contains3705protein-encoding genes, theoverall G+C content of the chromosome is50.42%. G. oxydans has a very strong incompleteoxidation ability of carbohydrates, alcohols and polyols. In G. oxydans WSH-003, there areabout330genes involved in the carbohydrate metabolism, and the presence of more than300membrane transport proteins make G. oxydans WSH-003possess a very high conversionefficiency. In addition, the tolerance of G. oxydans WSH-003to2-KLG was determined, itshowed that even100g·L–1of2-KLG did not significantly affect the cell growth of G.oxydans WSH-003. Besides, no obvious degradation of2-KLG could be detected when G.oxydans WSH-003was grown with2-KLG for120h. In order to construct G. oxydansengineered strains, a E. coli-G. oxydans shuttle vector is essential. The par-rep gene from theplasmid pGOX3of G. oxydans621H was ligated into pUC18, resulting in the shuttle vectorpGUC, which has a high conversion efficiency and stability.
(4) Construction of G. oxydans engineered strain for the direct production of2-KLG andthe modification by connecting peptide engineering and cofactor engineering
Different combinations of five SDH/SNDH and two SNDH from K. vulgare WSH-001were introduced into G. oxydans WSH-003, an industrial strain used for the conversion ofD-sorbitol to L-sorbose. The optimum combination produced4.9g·L–1of2-KLG. In addition,10different linker peptides were used for the fusion expression of SDH/SNDH and SNDH inG. oxydans. The best recombinant strain produced31.2g·L–1of2-KLG after168h. After theoptimization of yeast extract concentration, the production of2-KLG reached32.4g·L–1.Furthermore, overexpression of pqqA and pqqABCDE gene clusters under the control of tufBpromoter enhanced PQQ concentration by126.1%and273.6%, respectively, compared withthat noted in the wild-type strain. Overexpression of PQQ biosynthesis gene(s) significantlyenhanced cell growth, and the2-KLG production by G.oxydans/pGUC-k0203-GS-k0095-pqqA and G. oxydans/pGUC-k0203-GS-k0095-pqqABCDEreached35.1g·L–1and39.2g·L–1, which was8.3%and21.0%higher than that by G.oxydans/pGUC-k0203-GS-k0095, respectively.
(5) Efficient optimization of G. oxydans based on trimeric protein CutA for the directproduction of2-KLG
The codon optimization was conducted to further improve the expression of SDH andSNDH in G. oxydans, the production of2-KLG was33.2g·L–1after the codon optimization.In order to verify whether the expression of cutA could take effect in G. oxydans, after thecodon optimization of cutA, G. oxydans strain harboring pGUC-tufB-cutA was constructed.The growth curve of G. oxydans/pGUC-tufB-cutA and the wild strain were determined, whichindicated that CutA was correctly expressed in G. oxydans, and the heat resistance of G.oxydans was improved. Furthermore, based on the adaptor protein SH3and its ligand SH3lig, the codon-optimized cutA and sdh-GGGGS-sndh were expressed in G. oxydans. Theengineered strain G. oxydans/pGUC-tufB-k0203-GS-k0095-cutAproduced40.3g·L–1of2-KLG. In order to investigate the fermentation performance at different temperatures, theengineered strain G. oxydans/pGUC-tufB-k0203-GS-k0095-cutA was fermented at30℃,35℃and37℃, respectively. G. oxydans/pGUC-tufB-k0203-GS-k0095-cutA grew better at35℃and37℃than the control strain. The production of2-KLG of G.oxydans/pGUC-tufB-k0203-GS-k0095-cutA were higher than the control at differenttemperatures, which revealed that the expression of CutA improved the catalytic efficiency ofthe dehydrogenases. However, the strain G. oxydans/pGUC-tufB-k0203-GS-k0095-cutAproduced less2-KLG at35℃and37℃than30℃. Utimately, pqqABCDE gene clusterswere overexpressed and the production of2-KLG reached to42.6g·L–1.
引文
1. Stone I. The natural history of ascorbic acid in the evolution of the mammals andprimates and its significance for present-day man [J]. J Orthomol Psychiat,1972,1(2-3):82-89
2. Wenzel U, Nickel A, Kuntz S, et al. Ascorbic acid suppresses drug-induced apoptosis inhuman colon cancer cells by scavenging mitochondrial superoxide anions [J].Carcinogenesis,2004,25(5):703-712
3. May J M, Qu Z. Transport and intracellular accumulation of vitamin C in endothelialcells: relevance to collagen synthesis [J]. Arch Biochem Biophys,2005,434(1):178-186
4. De Tullio M, Arrigoni O. Hopes, disillusions and more hopes from vitamin C [J]. CellMol Life Sci,2004,61(2):209-219
5. Chatterjee I. Evolution and the biosynthesis of ascorbic acid [J]. Science,1973,182(4118):1271-1272
6. Reichstein T, Grüssner A, Oppenauer R. Die Synthese der D-Ascorbins ure (D-Form desC-Vitamins)[J]. Helv Chim Acta,1933,16(1):561-565
7. Boudrant J. Microbial processes for ascorbic acid biosynthesis: a review [J]. EnzymeMicrob Technol,1990,12(5):322-329
8. Hancock R D, Viola R. Biotechnological approaches for L-ascorbic acid production [J].Trends Biotechnol,2002,20(7):299-305
9. Bremus C, Herrmann U, Bringer-Meyer S, et al. The use of microorganisms inL-ascorbic acid production [J]. J Biotechnol,2006,124(1):196-205
10. Sonoyama T, Tani H, Matsuda K, et al. Production of2-keto-L-gulonic acid fromD-glucose by two-stage fermentation [J]. Appl Environ Microbiol,1982,43(5):1064-1069
11. Sugisawa T, Miyazaki T, Hoshino T. Microbial production of L-ascorbic acid fromD-sorbitol, L-sorbose, L-gulose, and L-sorbosone by Ketogulonicigenium vulgare DSM4025[J]. Biosci Biotechnol Biochem,2005,69(3):659-662
12. Zhou B, Li Y, Liu Y, et al. Microbiological eco-regulation in VC two-step fermentation[J]. Chin J Appl Ecol,2002,13(11):1452-1454
13. Zhang J, Liu J, Shi Z, et al. Manipulation of B. megaterium growth for efficient2-KLGproduction by K. vulgare [J]. Process Biochem,2010,45(4):602-606
14. Takagi Y, Sugisawa T, Hoshino T. Continuous2-keto-L-gulonic acid fermentation bymixed culture of Ketogulonicigenium vulgare DSM4025and Bacillus megaterium orXanthomonas maltophilia [J]. Appl Microbiol Biotechnol,2010,86(2):469-480
15. Takagi Y, Sugisawa T, Hoshino T. Continuous2-keto-L-gulonic acid fermentation froml-sorbose by Ketogulonigenium vulgare DSM4025[J]. Appl Microbio Biotechnol,2009,82(6):1049-1056
16. Leduc S, Troostembergh J C, Lebeault J M. Folate requirements of the2-keto-L-gulonicacid-producing strain Ketogulonigenium vulgare LMP P-20356in L-sorbose/CSLmedium [J]. Appl Microbiol Biotechnol,2004,65(2):163-167
17. Giridhar R, Srivastava A. Productivity enhancement in L-sorbose fermentation usingoxygen vector [J]. Enzyme Microb Technol,2000,27(7):537-541
18. Yang F C, Lim Y H. Kinetic study of the bioconversion of D-sorbitol to L-sorbose byAcetobacter pasteurianus [J]. Process Biochem,1997,32(3):233-236
19. Anderson S, Marks C B, Lazarus R, et al. Production of2-keto-L-gulonate, anintermediate in L-ascorbate synthesis, by a genetically modffied erwinia herbicola [J].Science,1985,230(4722):144-149
20. Rao Y M, Sureshkumar G. Direct biosynthesis of ascorbic acid from glucose byXanthomonas campestris through induced free-radicals [J]. Biotechnol Lett,2000,22(5):407-411
21. Hori K, Kaneko M, Tanji Y, et al. Construction of self-disruptive Bacillus megaterium inresponse to substrate exhaustion for polyhydroxybutyrate production [J]. Appl MicrobiolBiotechnol,2002,59(2-3):211-216
22. Sugisawa T, Hoshino T, Nomura S, et al. Isolation and characterization ofmembrane-bound L-sorbose dehydrogenase from Gluconobacter melanogenus UV10[J].Agric Biol Chem,1991,55(2):363-370
23. Hoshino T, Sugisawa T, Fujiwara A. Isolation and characterization of NAD(P)-dependentL-sorbosone dehydrogenase from Gluconobacter melanogenus UV10[J]. Agric BiolChem,1991,55(3):665-670
24. Shinjoh M, Tomiyama N, Asakura A, et al. Cloning and nucleotide sequencing of themembrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO12258and its expression in Gluconobacter oxydans [J]. Appl Environ Microbio,1995,61(2):413-420
25. Xiong X, Han S, Wang J, et al. Complete genome sequence of the bacteriumKetogulonicigenium vulgare Y25[J]. J Bacteriol,2011,193(1):315-316
26. Liu L, Li Y, Zhang J, et al. Complete Genome Sequence of the Industrial StrainKetogulonicigenium vulgare WSH-001[J]. J Bacteriol,2011,193(21):6108-6109
27. Yang F, Jia Q, Xiong Z, et al. Complete genome analysis of Ketogulonigenium sp.WB0104[J]. Chin Sci Bull,2006,51(8):941-945
28. Liu L, Li Y, Zhang J, et al. Complete Genome Sequence of the Industrial Strain Bacillusmegaterium WSH-002[J]. J Bacteriol,2011,193(22):6389-6390
29. Saito Y, Ishii Y, Hayashi H, et al. Cloning of genes coding for L-sorbose andL-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain [J].Appl Environ Microbiol,1997,63(2):454-460
30. Saito Y, Ishii Y, Hayashi H, et al. Direct fermentation of2-keto-gulonic acid inrecombinant Gluconobacter oxydans [J]. Biotechnol Bioeng,1998,58:309-315
31.林红雨,陈尹.欧文氏菌和棒杆菌的属间融合研究[J].微生物学通报,1999,26(1):3-6
32. Leifson E. Determination of carbohydrate metabolism of marine bacteria [J]. J Bacteriol,1963,85(5):1183
33. Jain A K, Nessler C L. Metabolic engineering of an alternative pathway for ascorbic acidbiosynthesis in plants [J]. Mol Breed,2000,6(1):73-78
34. Smirnoff N. Ascorbic acid: metabolism and functions of a multi-facetted molecule [J].Curr Opin Plant Biol,2000,3(3):229-235
35. Running J, Severson D, Schneider K. Extracellular production of L-ascorbic acid byChlorella protothecoides, Prototheca species, and mutants of P. moriformis duringaerobic culturing at low pH [J]. J Ind Microbiol Biotechnol,2002,29(2):93-98
36. Conklin P L, Williams E H, Last R L. Environmental stress sensitivity of an ascorbicacid-deficient Arabidopsis mutant [J]. Proc Nat Acad Sci,1996,93(18):9970-9974
37. Wheeler G L, Jones M A, Smirnoff N. The biosynthetic pathway of vitamin C in higherplants [J]. Nature,1998,393(6683):365-369
38. Valpuesta V, Botella M A. Biosynthesis of L-ascorbic acid in plants: new pathways for anold antioxidant [J]. Trends Plant Sci,2004,9(12):573-577
39. Debolt S, Melino V, Ford C M. Ascorbate as a biosynthetic precursor in plants [J]. AnnBot,2007,99(1):3-8
40. DeBolt S, Gutierrez R, Ehrhardt D W, et al. Nonmotile cellulose synthase subunitsrepeatedly accumulate within localized regions at the plasma membrane in Arabidopsishypocotyl cells following2,6-dichlorobenzonitrile treatment [J]. Plant Physiol,2007,145(2):334-338
41. Kumano Y, Sakamoto T, Egawa M, et al. In vitro and in vivo prolonged biologicalactivities of novel vitamin C derivative,2-O-alpha-D-glucopyranosyl-L-ascorbic acid(AA-2G), in cosmetic fields [J]. J Nutr Sci Vitaminol,1998,44(3):345-359
42. Asakura A, Hoshino T. Isolation and characterization of a new quinoproteindehydrogenase, L-sorbose/L-sorbosone dehydrogenase [J]. Biosci Biotechnol Biochem,1999,63(1):46-53
43. Miyazaki T, Sugisawa T, Hoshino T. Pyrroloquinoline quinone-dependentdehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion ofL-sorbosone to L-ascorbic acid [J]. Appl Environ Microbio,2006,72(2):1487-1495
44. Lazarus R A, Seymour J L. Determination of2-keto-L-gulonic and other ketoaldonic andaldonic acids produced by ketogenic bacterial fermentation [J]. Anal Biochem,1986,157(2):360-366
45. Grindley J, Payton M, Van De Pol H, et al. Conversion of glucose to2-keto-L-gulonate,an intermediate in L-ascorbate synthesis, by a recombinant strain of Erwinia citreus [J].Appl Environ Microbiol,1988,54(7):1770-1775
46. Hancock R, Viola R. The use of micro-organisms for L-ascorbic acid production: currentstatus and future perspectives [J]. Appl Microbiol Biotechnol,2001,56(5-6):567-576
47. Shinjoh M, Tomiyama N, Asakura A, et al. Cloning and nucleotide sequencing of themembrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO12258and its expression in Gluconobacter oxydans [J]. Appl Environ Microbiol,1995,61(2):413-420
48. Hoshino T, Sugisawa T, Tazoe M, et al. Metabolic pathway for2-keto-L-gulonic acidformation in Gluconobacter melanogenus IFO3293[J]. Agri Biol Chem,1990,54(5):1211-1218
49. Chen F, Chen C, Li Y, et al. Studies on gene knocking out of2-keto aldose reductasesfrom Erwinia sp. SCB125[J]. Acta microbiologica Sinica,2000,40(5):475-481
50. Chowanadisai W, Bauerly K A, Tchaparian E, et al. Pyrroloquinoline quinone stimulatesmitochondrial biogenesis through cAMP response element-binding proteinphosphorylation and increased PGC-1α expression [J]. J Biol Chem,2010,285(1):142-152
51. Misra H, Rajpurohit Y, Khairnar N. Pyrroloquinoline-quinone and its versatile roles inbiological processes [J]. J Biosci (Bangalore),2012,37(2):313-325
52. Razumiene J, Vilkanauskyte A, Gureviciene V, et al. New bioorganometallic ferrocenederivatives as efficient mediators for glucose and ethanol biosensors based onPQQ-dependent dehydrogenases [J]. J Organometal Chem,2003,668(1):83-90
53. Kim Y P, Park S J, Lee D, et al. Electrochemical glucose biosensor by electrostaticbinding of PQQ-glucose dehydrogenase onto self-assembled monolayers on gold [J]. JAppl Electrochem,2012,42(6):383-390
54. Durand F, Stines-Chaumeil C, Flexer V, et al. Designing a highly active solublePQQ–glucose dehydrogenase for efficient glucose biosensors and biofuel cells [J].Biochem Biophys Res Commun,2010,402(4):750-754
55. Malinauskas A, Me kys R, Ramanavi ius A. Bioelectrochemical sensor based onPQQ-dependent glucose dehydrogenase [J]. Sensors Actuators B: Chem,2004,100(3):387-394
56. Razumiene J, Vilkanauskyte A, Gureviciene V, et al. Direct electron transfer betweenPQQ dependent glucose dehydrogenases and carbon electrodes: an approach forelectrochemical biosensors [J]. Electrochim Acta,2006,51(24):5150-5156
57. Matsushita K, Toyama H, Yamada M, et al. Quinoproteins: structure, function, andbiotechnological applications [J]. Appl Microbiol Biotechnol,2002,58(1):13-22
58. Gvozdev A, Tukhvatullin I, Gvozdev R. Quinone-dependent alcohol dehydrogenases andfad-dependent alcohol oxidases [J]. Biochemistry (Moscow),2012,77(8):843-856
59. Sugisawa T, Hoshino T. Purification and properties of membrane-bound D-sorbitoldehydrogenase from Gluconobacter suboxydans IFO3255[J]. Biosci BiotechnolBiochem,2002,66(1):57-64
60. Dokter P, Frank J, Duine J. Purification and characterization of quinoprotein glucosedehydrogenase from Acinetobacter calcoaceticus L.M.D.79.41[J]. Biochem J,1986,239(1):163-167
61. Xia Z, Dai W, Xiong J, et al. The three-dimensional structures of methanoldehydrogenase from two methylotrophic bacteria at2.6-A resolution [J]. J Biol Chem,1992,267(31):22289-22297
62. Davidson V L. Generation of protein-derived redox cofactors by posttranslationalmodification [J]. Mol BioSyst,2011,7(1):29-37
63. Murakawa T, Hayashi H, Taki M, et al. Structural insights into the substrate specificity ofbacterial copper amine oxidase obtained by using irreversible inhibitors [J]. J Biochem,2012,151(2):167-178
64. Anthony C. Pyrroloquinoline quinone (PQQ) and quinoprotein enzymes [J]. AntioxidRedox Signal,2001,3(5):757-774
65. Nosjean O, Ferro M, Cogé F, et al. Identification of the Melatonin-binding SiteMT3asthe Quinone Reductase2[J]. J Biol Chem,2000,275(40):31311-31317
66. Laurinavicius V, Razumiene J, Kurtinaitiene B, et al. Bioelectrochemical application ofsome PQQ-dependent enzymes [J]. Bioelectrochemistry,2002,55(1):29-32
67. Oubrie A. Structure and mechanism of soluble glucose dehydrogenase and otherPQQ-dependent enzymes [J]. BBA-Proteins Proteomics,2003,1647(1):143-151
68. Lapenaite I, Kurtinaitiene B, Razumiene J, et al. Properties and analytical application ofPQQ-dependent glycerol dehydrogenase from Gluconobacter sp.33[J]. Anal Chim Acta,2005,549(1):140-150
69. Adachi O, Fujii Y, Ghaly M F, et al. Membrane-bound quinoprotein D-arabitoldehydrogenase of Gluconobacter suboxydans IFO3257: a versatile enzyme for theoxidative fermentation of various ketoses [J]. Biosci Biotechnol Biochem,2001,65(12):2755-2762
70. Kay C W M, Mennenga B, G risch H, et al. Structure of the pyrroloquinoline quinoneradical in quinoprotein ethanol dehydrogenase [J]. J Biol Chem,2006,281(3):1470-1476
71. Yamada M, Sumi K, Matsushita K, et al. Topological analysis of quinoprotein glucosedehydrogenase in Escherichia coli and its ubiquinone-binding site [J]. J Biol Chem,1993,268(17):12812-12817
72. Magnusson O T, Toyama H, Saeki M, et al. The structure of a biosynthetic intermediateof pyrroloquinoline quinone (PQQ) and elucidation of the final step of PQQ biosynthesis[J]. J Am Chem Soc,2004,126(17):5342-5343
73. Puehringer S, Metlitzky M, Schwarzenbacher R. The pyrroloquinoline quinonebiosynthesis pathway revisited: a structural approach [J]. BMC Biochem,2008,9(1):8
74. Goldstein A, Lester T, Brown J. Research on the metabolic engineering of the directoxidation pathway for extraction of phosphate from ore has generated preliminaryevidence for PQQ biosynthesis in Escherichia coli as well as a possible role for thehighly conserved region of quinoprotein dehydrogenases [J]. BBA-Proteins Proteomics,2003,1647(1):266-271
75. Xia Z, Dai W, He Y, et al. X-ray structure of methanol dehydrogenase from Paracoccusdenitrificans and molecular modeling of its interactions with cytochrome c-551i [J]. JBiological Inorg Chem,2003,8(8):843-854
76. Banta S, Swanson B A, Wu S, et al. Optimizing an artificial metabolic pathway:engineering the cofactor specificity of Corynebacterium2,5-diketo-D-gluconic acidreductase for use in vitamin C biosynthesis [J]. Biochem,2002,41(20):6226-6236
77. Yang X, Zhong G, Lin J, et al. Pyrroloquinoline quinone biosynthesis in Escherichia colithrough expression of the Gluconobacter oxydans pqqABCDE gene cluster [J]. J IndMicrobiol Biotechnol,2010,37(6):575-580
78. Alterovitz G, Muso T, Ramoni M F. The challenges of informatics in synthetic biology:from biomolecular networks to artificial organisms [J]. Brief Bioinform,2010,11(1):80-95
79. Keasling J D. Synthetic biology for synthetic chemistry [J]. ACS Chem Biol,2008,3(1):64-76
80. Zhou H, Liao X, Wang T, et al. Enhanced L-phenylalanine biosynthesis by co-expressionof pheAfbrand aroFwt[J]. Bioresour Technol,2010,101(11):4151-4156
81. Eppinger M, Bunk B, Johns M A, et al. Genome sequences of the biotechnologicallyimportant Bacillus megaterium strains QM B1551and DSM319[J]. J Bacteriol,2011,193(16):4199-4213
82. Prust C, Hoffmeister M, Liesegang H, et al. Complete genome sequence of the aceticacid bacterium Gluconobacter oxydans [J]. Nat Biotechnol,2005,23(2):195-200
83. Gao L, Zhou J, Liu J, et al. Draft genome sequence of Gluconobacter oxydans WSH-003,a strain that is extremely tolerant of saccharides and alditols [J]. J Bacteriol,2012,194(16):4455-4456
84. Sebaihia M, Bocsanczy A, Biehl B, et al. Complete genome sequence of the plantpathogen Erwinia amylovora strain ATCC49946[J]. J Bacteriol,2010,192(7):2020-2021
85. Kalinowski J, Bathe B, Bartels D, et al. The complete Corynebacterium glutamicumATCC13032genome sequence and its impact on the production of L-aspartate-derivedamino acids and vitamins [J]. J Biotechnol,2003,104(1):5-25
86. Zhang L, Wang Z, Xia Y, et al. Metabolic engineering of plant L-ascorbic acidbiosynthesis: recent trends and applications [J]. Crit. Rev. Biotechnol,2007,27(3):173-182
87. Domingues A, Vinga S, Lemos J M. Optimization strategies for metabolic networks [J].BMC Syst Biol,2010,4(1):113
88. Christian N, May P, Kempa S, et al. An integrative approach towards completinggenome-scale metabolic networks [J]. Mol BioSyst,2009,5(12):1889-1903
89. Park J M, Kim T Y, Lee S Y. Constraints-based genome-scale metabolic simulation forsystems metabolic engineering [J]. Biotechnol Adv,2009,27(6):979-988
90. Price N D, Reed J L, Palsson B. Genome-scale models of microbial cells: evaluating theconsequences of constraints [J]. Nat Rev Microbiol,2004,2(11):886-897
91. Lee S Y, Lee D Y, Kim T Y. Systems biotechnology for strain improvement [J]. TrendsBiotechnol,2005,23(7):349-358
92. Zou W, Liu L, Zhang J, et al. Reconstruction and analysis of a genome-scale metabolicmodel of the vitamin C producing industrial strain Ketogulonicigenium vulgareWSH-001[J]. J Biotechnol,2012,161(1):42-48
93. Liu L, Agren R, Bordel S, et al. Use of genome-scale metabolic models forunderstanding microbial physiology [J]. FEBS Lett,2010,584(12):2556-2564
94. Curran K A, Alper H S. Expanding the chemical palate of cells by combining systemsbiology and metabolic engineering [J]. Metab Eng,2012,14(4):289-297
95. Bradford M M. A rapid and sensitive method for the quantification of microgramquantities of protein utilizing the principle of protein-dye binding [J]. Anal Biochem,1976,72:248-254
96. Salvatore C A, Mallee J J, Bell I M, et al. Identification and pharmacologicalcharacterization of domains involved in binding of CGRP receptor antagonists to thecalcitonin-like receptor [J]. Biochem,2006,45(6):1881-1887
97. Pinter R Y, Rokhlenko O, Yeger-Lotem E, et al. Alignment of metabolic pathways [J].Bioinformatics,2005,21(16):3401-3408
98. Pósfai G, Plunkett G, Fehér T, et al. Emergent properties of reduced-genome Escherichiacoli [J]. Science,2006,312(5776):1044-1046
99. Levskaya A, Chevalier A A, Tabor J J, et al. Synthetic biology: engineering Escherichiacoli to see light [J]. Nature,2005,438(7067):441-442
100. Sorensen H P, Mortensen K K. Advanced genetic strategies for recombinant proteinexpression in Escherichia coli [J]. J Biotechnol,2005,115(2):113-128
101. Quail M A, Kozarewa I, Smith F, et al. A large genome center's improvements to theIllumina sequencing system [J]. Nat Methods,2008,5(12):1005-1010
102. Li R, Zhu H, Ruan J, et al. De novo assembly of human genomes with massively parallelshort read sequencing [J]. Genome Res,2010,20(2):265-272
103. Delcher A L, Bratke K A, Powers E C, et al. Identifying bacterial genes andendosymbiont DNA with Glimmer [J]. Bioinformatics,2007,23(6):673-679
104. Tatusov R L, Natale D A, Garkavtsev I V, et al. The COG database: new developments inphylogenetic classification of proteins from complete genomes [J]. Nucleic Acids Res,2001,29(1):22-28
105. Apweiler R, Attwood T K, Bairoch A, et al. The InterPro database, an integrateddocumentation resource for protein families, domains and functional sites [J]. NucleicAcids Res,2001,29(1):37-40
106. Griffiths-Jones S, Bateman A, Marshall M, et al. Rfam: an RNA family database [J].Nucleic Acids Res,2003,31(1):439-441
107. Lowe T M, Eddy S R. tRNAscan-SE: a program for improved detection of transfer RNAgenes in genomic sequence [J]. Nucleic Acids Res,1997,25(5):955-964
108. Panda A K, Khan R, Appa Rao K, et al. Kinetics of inclusion body production in batchand high cell density fed-batch culture of Escherichia coli expressing ovine growthhormone [J]. J Biotechnol,1999,75(2):161-172
109. Deppenmeier U, Hoffmeister M, Prust C. Biochemistry and biotechnologicalapplications of Gluconobacter strains [J]. Appl Microbiol Biotechnol,2002,60(3):233-242
110. Tkac J, Svitel J, Vostiar I, et al. Membrane-bound dehydrogenases from Gluconobactersp.: Interfacial electrochemistry and direct bioelectrocatalysis [J]. Bioelectrochemistry,2009,76(1):53-62
111. Odaci D, Timur S, Telefoncu A. A microbial biosensor based on bacterial cellsimmobilized on chitosan matrix [J]. Bioelectrochemistry,2009,75(1):77-82
112. Kallnik V, Meyer M, Deppenmeier U, et al. Construction of expression vectors forprotein production in Gluconobacter oxydans [J]. J Biotechnol.,2010,150(4):460-465
113. Schleyer U, Bringer-Meyer S, Sahm H. An easy cloning and expression vector systemfor Gluconobacter oxydans [J]. Int J Food Microbiol,2008,125(1):91-95
114. Shinjoh M, Hoshino T. Development of a stable shuttle vector and a conjugative transfersystem for Gluconobacter oxydans [J]. J Ferment Bioeng,1995,79(2):95-99
115. Cha J S, Pujol C, Ducusin A, et al. Studies on Pantoea citrea, the causal agent of pinkdisease of pineapple [J]. J Phytopathol,1997,145(7):313-319
116. Mostafa H E, Heller K J, Geis A. Cloning of Escherichia coli lacZ and lacY genes andtheir expression in Gluconobacter oxydans and Acetobacter liquefaciens [J]. ApplEnviron Microbiol,2002,68(5):2619-2623
117. Albertsen L, Chen Y, Bach L S, et al. Diversion of flux toward sesquiterpene productionin Saccharomyces cerevisiae by fusion of host and heterologous enzymes [J]. ApplEnviron Microbiol,2011,77(3):1033-1040
118. Sanchez A M, Bennett G N, San K Y. Effect of different levels of NADH availability onmetabolic fluxes of Escherichia coli chemostat cultures in defined medium [J]. JBiotechnol,2005,117(4):395-405
119. Fluckiger R, Paz M, Mah J, et al. Characterization of the glycine-dependentredox-cycling activity in animal fluids and tissues using specific inhibitors and activators:evidence for presence of PQQ [J]. Biochem Biophys Res Commun,1993,196(1):61-68
120. Jorgensen K, Rasmussen A V, Morant M, et al. Metabolon formation and metabolicchanneling in the biosynthesis of plant natural products [J]. Curr Opin Plant Biol,2005,8(3):280-291
121. Yan Y, Li Z, Koffas M A. High-yield anthocyanin biosynthesis in engineered Escherichiacoli [J]. Biotechnol Bioeng,2008,100(1):126-140
122. Conrado R J, Varner J D, DeLisa M P. Engineering the spatial organization of metabolicenzymes: mimicking nature's synergy [J]. Curr Opin Biotechnol,2008,19(5):492-499
123. Wang J, Tsai M, Lee G, et al. Construction of a recombinant thermostableβ-amylase-trehalose synthase bifunctional enzyme for facilitating the conversion ofstarch to trehalose [J]. J Agric Food Chem,2007,55(4):1256-1263
124. Hong S Y, Lee J S, Cho K M, et al. Assembling a novel bifunctional cellulase–xylanasefrom Thermotoga maritima by end-to-end fusion [J]. Biotechnol Lett,2006,28(22):1857-1862
125. Orita I, Sakamoto N, Kato N, et al. Bifunctional enzyme fusion of3-hexulose-6-phosphate synthase and6-phospho-3-hexuloisomerase [J]. Appl MicrobiolBiotechnol,2007,76(2):439-445
126. Karp, Matti, Oker-Blom, et al. A streptavidin-luciferase fusion protein: comparisons andapplications [J]. Biomol Eng,1999,16(1):101-104
127. Blazyk J L, Lippard S J. Domain engineering of the reductase component of solublemethane monooxygenase from Methylococcus capsulatus (Bath)[J]. J Biol Chem,2004,279(7):5630-5640
128. Holsch K, Weuster Botz D. Enantioselective reduction of prochiral ketones byengineered bifunctional fusion proteins [J]. Biotechnol Appl Biochem,2010,56(4):131-140
129. Kavoosi M, Creagh A L, Kilburn D G, et al. Strategy for selecting and characterizinglinker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli [J].Biotechnol Bioeng,2007,98(3):599-610
130. Wriggers W, Chakravarty S, Jennings P A. Control of protein functional dynamics bypeptide linkers [J]. Peptide Science,2005,80(6):736-746
131. Lu P, Feng M. Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme byoptimization of peptide linkers [J]. Appl Microbiol Biotechnol,2008,79(4):579-587
132. Arai R, Wriggers W, Nishikawa Y, et al. Conformations of variably linked chimericproteins evaluated by synchrotron X-ray small-angle scattering [J]. Proteins: Struct FuncBioinformatics,2004,57(4):829-838
133. Gao L, Du G, Zhou J, et al. Characterization of a group of pyrroloquinolinequinone-dependent dehydrogenases that are involved in the conversion of L-sorbose to2-keto-L-gulonic acid in Ketogulonicigenium vulgare WSH-001[J]. Biotechnol Prog,2013,29(6):1398-1404
134. Du J, Bai W, Song H, et al. Combinational expression of sorbose/sorbosonedehydrogenases and cofactor pyrroloquinoline quinone increases2-keto-L-gulonic acidproduction in Ketogulonigenium vulgare-Bacillus cereus consortium [J]. Metab Eng,2013,19:50-56
135. Choi O, Kim J, Kim J G, et al. Pyrroloquinoline quinone is a plant growth promotionfactor produced by Pseudomonas fluorescens B16[J]. Plant Physiol,2008,146(2):657-668
136. Shrivastava M, Rajpurohit Y S, Misra H S, et al. Survival of phosphate-solubilizingbacteria against DNA damaging agents [J]. Can J Microbiol,2010,56(10):822-830
137. Sawano M, Yamamoto H, Ogasahara K, et al. Thermodynamic basis for the stabilities ofthree CutA1s from Pyrococcus horikoshii, Thermus thermophilus, and Oryza sativa, withunusually high denaturation temperatures [J]. Biochemistry,2008,47(2):721-730
138. Ramirez M, Guan D, Ugaz V, et al. Intein-triggered artificial protein hydrogels thatsupport the immobilization of bioactive proteins [J]. J Am Chem Soc,2013,135(14):5290-5293
139. Lithwick G, Margalit H. Hierarchy of sequence-dependent features associated withprokaryotic translation [J]. Genome Res,2003,13(12):2665-2673
140. Zehentgruber D, Lundemo P, Svensson D, et al. Substrate complexation and aggregationinfluence the cyclodextrin glycosyltransferase (CGTase) catalyzed synthesis of alkylglycosides [J]. J Biotechnol,2011,155(2):232-235
141. De Muynck C, Pereira C S, Naessens M, et al. The genus Gluconobacter oxydans:comprehensive overview of biochemistry and biotechnological applications [J]. Crit RevBiotechnol,2007,27(3):147-171
142. Tanaka Y, Tsumoto K, Nakanishi T, et al. Structural implications for heavy metal-inducedreversible assembly and aggregation of a protein: the case of Pyrococcus horikoshii CutA[J]. FEBS Lett,2004,556(1):167-174
143. Olijve W, Kok J. An analysis of the growth of Gluconobacter oxydans in chemostatcultures [J]. Arch Microbiol,1979,121(3):291-297
2. Wenzel U, Nickel A, Kuntz S, et al. Ascorbic acid suppresses drug-induced apoptosis inhuman colon cancer cells by scavenging mitochondrial superoxide anions [J].Carcinogenesis,2004,25(5):703-712
3. May J M, Qu Z. Transport and intracellular accumulation of vitamin C in endothelialcells: relevance to collagen synthesis [J]. Arch Biochem Biophys,2005,434(1):178-186
4. De Tullio M, Arrigoni O. Hopes, disillusions and more hopes from vitamin C [J]. CellMol Life Sci,2004,61(2):209-219
5. Chatterjee I. Evolution and the biosynthesis of ascorbic acid [J]. Science,1973,182(4118):1271-1272
6. Reichstein T, Grüssner A, Oppenauer R. Die Synthese der D-Ascorbins ure (D-Form desC-Vitamins)[J]. Helv Chim Acta,1933,16(1):561-565
7. Boudrant J. Microbial processes for ascorbic acid biosynthesis: a review [J]. EnzymeMicrob Technol,1990,12(5):322-329
8. Hancock R D, Viola R. Biotechnological approaches for L-ascorbic acid production [J].Trends Biotechnol,2002,20(7):299-305
9. Bremus C, Herrmann U, Bringer-Meyer S, et al. The use of microorganisms inL-ascorbic acid production [J]. J Biotechnol,2006,124(1):196-205
10. Sonoyama T, Tani H, Matsuda K, et al. Production of2-keto-L-gulonic acid fromD-glucose by two-stage fermentation [J]. Appl Environ Microbiol,1982,43(5):1064-1069
11. Sugisawa T, Miyazaki T, Hoshino T. Microbial production of L-ascorbic acid fromD-sorbitol, L-sorbose, L-gulose, and L-sorbosone by Ketogulonicigenium vulgare DSM4025[J]. Biosci Biotechnol Biochem,2005,69(3):659-662
12. Zhou B, Li Y, Liu Y, et al. Microbiological eco-regulation in VC two-step fermentation[J]. Chin J Appl Ecol,2002,13(11):1452-1454
13. Zhang J, Liu J, Shi Z, et al. Manipulation of B. megaterium growth for efficient2-KLGproduction by K. vulgare [J]. Process Biochem,2010,45(4):602-606
14. Takagi Y, Sugisawa T, Hoshino T. Continuous2-keto-L-gulonic acid fermentation bymixed culture of Ketogulonicigenium vulgare DSM4025and Bacillus megaterium orXanthomonas maltophilia [J]. Appl Microbiol Biotechnol,2010,86(2):469-480
15. Takagi Y, Sugisawa T, Hoshino T. Continuous2-keto-L-gulonic acid fermentation froml-sorbose by Ketogulonigenium vulgare DSM4025[J]. Appl Microbio Biotechnol,2009,82(6):1049-1056
16. Leduc S, Troostembergh J C, Lebeault J M. Folate requirements of the2-keto-L-gulonicacid-producing strain Ketogulonigenium vulgare LMP P-20356in L-sorbose/CSLmedium [J]. Appl Microbiol Biotechnol,2004,65(2):163-167
17. Giridhar R, Srivastava A. Productivity enhancement in L-sorbose fermentation usingoxygen vector [J]. Enzyme Microb Technol,2000,27(7):537-541
18. Yang F C, Lim Y H. Kinetic study of the bioconversion of D-sorbitol to L-sorbose byAcetobacter pasteurianus [J]. Process Biochem,1997,32(3):233-236
19. Anderson S, Marks C B, Lazarus R, et al. Production of2-keto-L-gulonate, anintermediate in L-ascorbate synthesis, by a genetically modffied erwinia herbicola [J].Science,1985,230(4722):144-149
20. Rao Y M, Sureshkumar G. Direct biosynthesis of ascorbic acid from glucose byXanthomonas campestris through induced free-radicals [J]. Biotechnol Lett,2000,22(5):407-411
21. Hori K, Kaneko M, Tanji Y, et al. Construction of self-disruptive Bacillus megaterium inresponse to substrate exhaustion for polyhydroxybutyrate production [J]. Appl MicrobiolBiotechnol,2002,59(2-3):211-216
22. Sugisawa T, Hoshino T, Nomura S, et al. Isolation and characterization ofmembrane-bound L-sorbose dehydrogenase from Gluconobacter melanogenus UV10[J].Agric Biol Chem,1991,55(2):363-370
23. Hoshino T, Sugisawa T, Fujiwara A. Isolation and characterization of NAD(P)-dependentL-sorbosone dehydrogenase from Gluconobacter melanogenus UV10[J]. Agric BiolChem,1991,55(3):665-670
24. Shinjoh M, Tomiyama N, Asakura A, et al. Cloning and nucleotide sequencing of themembrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO12258and its expression in Gluconobacter oxydans [J]. Appl Environ Microbio,1995,61(2):413-420
25. Xiong X, Han S, Wang J, et al. Complete genome sequence of the bacteriumKetogulonicigenium vulgare Y25[J]. J Bacteriol,2011,193(1):315-316
26. Liu L, Li Y, Zhang J, et al. Complete Genome Sequence of the Industrial StrainKetogulonicigenium vulgare WSH-001[J]. J Bacteriol,2011,193(21):6108-6109
27. Yang F, Jia Q, Xiong Z, et al. Complete genome analysis of Ketogulonigenium sp.WB0104[J]. Chin Sci Bull,2006,51(8):941-945
28. Liu L, Li Y, Zhang J, et al. Complete Genome Sequence of the Industrial Strain Bacillusmegaterium WSH-002[J]. J Bacteriol,2011,193(22):6389-6390
29. Saito Y, Ishii Y, Hayashi H, et al. Cloning of genes coding for L-sorbose andL-sorbosone dehydrogenases from Gluconobacter oxydans and microbial production of2-keto-L-gulonate, a precursor of L-ascorbic acid, in a recombinant G. oxydans strain [J].Appl Environ Microbiol,1997,63(2):454-460
30. Saito Y, Ishii Y, Hayashi H, et al. Direct fermentation of2-keto-gulonic acid inrecombinant Gluconobacter oxydans [J]. Biotechnol Bioeng,1998,58:309-315
31.林红雨,陈尹.欧文氏菌和棒杆菌的属间融合研究[J].微生物学通报,1999,26(1):3-6
32. Leifson E. Determination of carbohydrate metabolism of marine bacteria [J]. J Bacteriol,1963,85(5):1183
33. Jain A K, Nessler C L. Metabolic engineering of an alternative pathway for ascorbic acidbiosynthesis in plants [J]. Mol Breed,2000,6(1):73-78
34. Smirnoff N. Ascorbic acid: metabolism and functions of a multi-facetted molecule [J].Curr Opin Plant Biol,2000,3(3):229-235
35. Running J, Severson D, Schneider K. Extracellular production of L-ascorbic acid byChlorella protothecoides, Prototheca species, and mutants of P. moriformis duringaerobic culturing at low pH [J]. J Ind Microbiol Biotechnol,2002,29(2):93-98
36. Conklin P L, Williams E H, Last R L. Environmental stress sensitivity of an ascorbicacid-deficient Arabidopsis mutant [J]. Proc Nat Acad Sci,1996,93(18):9970-9974
37. Wheeler G L, Jones M A, Smirnoff N. The biosynthetic pathway of vitamin C in higherplants [J]. Nature,1998,393(6683):365-369
38. Valpuesta V, Botella M A. Biosynthesis of L-ascorbic acid in plants: new pathways for anold antioxidant [J]. Trends Plant Sci,2004,9(12):573-577
39. Debolt S, Melino V, Ford C M. Ascorbate as a biosynthetic precursor in plants [J]. AnnBot,2007,99(1):3-8
40. DeBolt S, Gutierrez R, Ehrhardt D W, et al. Nonmotile cellulose synthase subunitsrepeatedly accumulate within localized regions at the plasma membrane in Arabidopsishypocotyl cells following2,6-dichlorobenzonitrile treatment [J]. Plant Physiol,2007,145(2):334-338
41. Kumano Y, Sakamoto T, Egawa M, et al. In vitro and in vivo prolonged biologicalactivities of novel vitamin C derivative,2-O-alpha-D-glucopyranosyl-L-ascorbic acid(AA-2G), in cosmetic fields [J]. J Nutr Sci Vitaminol,1998,44(3):345-359
42. Asakura A, Hoshino T. Isolation and characterization of a new quinoproteindehydrogenase, L-sorbose/L-sorbosone dehydrogenase [J]. Biosci Biotechnol Biochem,1999,63(1):46-53
43. Miyazaki T, Sugisawa T, Hoshino T. Pyrroloquinoline quinone-dependentdehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion ofL-sorbosone to L-ascorbic acid [J]. Appl Environ Microbio,2006,72(2):1487-1495
44. Lazarus R A, Seymour J L. Determination of2-keto-L-gulonic and other ketoaldonic andaldonic acids produced by ketogenic bacterial fermentation [J]. Anal Biochem,1986,157(2):360-366
45. Grindley J, Payton M, Van De Pol H, et al. Conversion of glucose to2-keto-L-gulonate,an intermediate in L-ascorbate synthesis, by a recombinant strain of Erwinia citreus [J].Appl Environ Microbiol,1988,54(7):1770-1775
46. Hancock R, Viola R. The use of micro-organisms for L-ascorbic acid production: currentstatus and future perspectives [J]. Appl Microbiol Biotechnol,2001,56(5-6):567-576
47. Shinjoh M, Tomiyama N, Asakura A, et al. Cloning and nucleotide sequencing of themembrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO12258and its expression in Gluconobacter oxydans [J]. Appl Environ Microbiol,1995,61(2):413-420
48. Hoshino T, Sugisawa T, Tazoe M, et al. Metabolic pathway for2-keto-L-gulonic acidformation in Gluconobacter melanogenus IFO3293[J]. Agri Biol Chem,1990,54(5):1211-1218
49. Chen F, Chen C, Li Y, et al. Studies on gene knocking out of2-keto aldose reductasesfrom Erwinia sp. SCB125[J]. Acta microbiologica Sinica,2000,40(5):475-481
50. Chowanadisai W, Bauerly K A, Tchaparian E, et al. Pyrroloquinoline quinone stimulatesmitochondrial biogenesis through cAMP response element-binding proteinphosphorylation and increased PGC-1α expression [J]. J Biol Chem,2010,285(1):142-152
51. Misra H, Rajpurohit Y, Khairnar N. Pyrroloquinoline-quinone and its versatile roles inbiological processes [J]. J Biosci (Bangalore),2012,37(2):313-325
52. Razumiene J, Vilkanauskyte A, Gureviciene V, et al. New bioorganometallic ferrocenederivatives as efficient mediators for glucose and ethanol biosensors based onPQQ-dependent dehydrogenases [J]. J Organometal Chem,2003,668(1):83-90
53. Kim Y P, Park S J, Lee D, et al. Electrochemical glucose biosensor by electrostaticbinding of PQQ-glucose dehydrogenase onto self-assembled monolayers on gold [J]. JAppl Electrochem,2012,42(6):383-390
54. Durand F, Stines-Chaumeil C, Flexer V, et al. Designing a highly active solublePQQ–glucose dehydrogenase for efficient glucose biosensors and biofuel cells [J].Biochem Biophys Res Commun,2010,402(4):750-754
55. Malinauskas A, Me kys R, Ramanavi ius A. Bioelectrochemical sensor based onPQQ-dependent glucose dehydrogenase [J]. Sensors Actuators B: Chem,2004,100(3):387-394
56. Razumiene J, Vilkanauskyte A, Gureviciene V, et al. Direct electron transfer betweenPQQ dependent glucose dehydrogenases and carbon electrodes: an approach forelectrochemical biosensors [J]. Electrochim Acta,2006,51(24):5150-5156
57. Matsushita K, Toyama H, Yamada M, et al. Quinoproteins: structure, function, andbiotechnological applications [J]. Appl Microbiol Biotechnol,2002,58(1):13-22
58. Gvozdev A, Tukhvatullin I, Gvozdev R. Quinone-dependent alcohol dehydrogenases andfad-dependent alcohol oxidases [J]. Biochemistry (Moscow),2012,77(8):843-856
59. Sugisawa T, Hoshino T. Purification and properties of membrane-bound D-sorbitoldehydrogenase from Gluconobacter suboxydans IFO3255[J]. Biosci BiotechnolBiochem,2002,66(1):57-64
60. Dokter P, Frank J, Duine J. Purification and characterization of quinoprotein glucosedehydrogenase from Acinetobacter calcoaceticus L.M.D.79.41[J]. Biochem J,1986,239(1):163-167
61. Xia Z, Dai W, Xiong J, et al. The three-dimensional structures of methanoldehydrogenase from two methylotrophic bacteria at2.6-A resolution [J]. J Biol Chem,1992,267(31):22289-22297
62. Davidson V L. Generation of protein-derived redox cofactors by posttranslationalmodification [J]. Mol BioSyst,2011,7(1):29-37
63. Murakawa T, Hayashi H, Taki M, et al. Structural insights into the substrate specificity ofbacterial copper amine oxidase obtained by using irreversible inhibitors [J]. J Biochem,2012,151(2):167-178
64. Anthony C. Pyrroloquinoline quinone (PQQ) and quinoprotein enzymes [J]. AntioxidRedox Signal,2001,3(5):757-774
65. Nosjean O, Ferro M, Cogé F, et al. Identification of the Melatonin-binding SiteMT3asthe Quinone Reductase2[J]. J Biol Chem,2000,275(40):31311-31317
66. Laurinavicius V, Razumiene J, Kurtinaitiene B, et al. Bioelectrochemical application ofsome PQQ-dependent enzymes [J]. Bioelectrochemistry,2002,55(1):29-32
67. Oubrie A. Structure and mechanism of soluble glucose dehydrogenase and otherPQQ-dependent enzymes [J]. BBA-Proteins Proteomics,2003,1647(1):143-151
68. Lapenaite I, Kurtinaitiene B, Razumiene J, et al. Properties and analytical application ofPQQ-dependent glycerol dehydrogenase from Gluconobacter sp.33[J]. Anal Chim Acta,2005,549(1):140-150
69. Adachi O, Fujii Y, Ghaly M F, et al. Membrane-bound quinoprotein D-arabitoldehydrogenase of Gluconobacter suboxydans IFO3257: a versatile enzyme for theoxidative fermentation of various ketoses [J]. Biosci Biotechnol Biochem,2001,65(12):2755-2762
70. Kay C W M, Mennenga B, G risch H, et al. Structure of the pyrroloquinoline quinoneradical in quinoprotein ethanol dehydrogenase [J]. J Biol Chem,2006,281(3):1470-1476
71. Yamada M, Sumi K, Matsushita K, et al. Topological analysis of quinoprotein glucosedehydrogenase in Escherichia coli and its ubiquinone-binding site [J]. J Biol Chem,1993,268(17):12812-12817
72. Magnusson O T, Toyama H, Saeki M, et al. The structure of a biosynthetic intermediateof pyrroloquinoline quinone (PQQ) and elucidation of the final step of PQQ biosynthesis[J]. J Am Chem Soc,2004,126(17):5342-5343
73. Puehringer S, Metlitzky M, Schwarzenbacher R. The pyrroloquinoline quinonebiosynthesis pathway revisited: a structural approach [J]. BMC Biochem,2008,9(1):8
74. Goldstein A, Lester T, Brown J. Research on the metabolic engineering of the directoxidation pathway for extraction of phosphate from ore has generated preliminaryevidence for PQQ biosynthesis in Escherichia coli as well as a possible role for thehighly conserved region of quinoprotein dehydrogenases [J]. BBA-Proteins Proteomics,2003,1647(1):266-271
75. Xia Z, Dai W, He Y, et al. X-ray structure of methanol dehydrogenase from Paracoccusdenitrificans and molecular modeling of its interactions with cytochrome c-551i [J]. JBiological Inorg Chem,2003,8(8):843-854
76. Banta S, Swanson B A, Wu S, et al. Optimizing an artificial metabolic pathway:engineering the cofactor specificity of Corynebacterium2,5-diketo-D-gluconic acidreductase for use in vitamin C biosynthesis [J]. Biochem,2002,41(20):6226-6236
77. Yang X, Zhong G, Lin J, et al. Pyrroloquinoline quinone biosynthesis in Escherichia colithrough expression of the Gluconobacter oxydans pqqABCDE gene cluster [J]. J IndMicrobiol Biotechnol,2010,37(6):575-580
78. Alterovitz G, Muso T, Ramoni M F. The challenges of informatics in synthetic biology:from biomolecular networks to artificial organisms [J]. Brief Bioinform,2010,11(1):80-95
79. Keasling J D. Synthetic biology for synthetic chemistry [J]. ACS Chem Biol,2008,3(1):64-76
80. Zhou H, Liao X, Wang T, et al. Enhanced L-phenylalanine biosynthesis by co-expressionof pheAfbrand aroFwt[J]. Bioresour Technol,2010,101(11):4151-4156
81. Eppinger M, Bunk B, Johns M A, et al. Genome sequences of the biotechnologicallyimportant Bacillus megaterium strains QM B1551and DSM319[J]. J Bacteriol,2011,193(16):4199-4213
82. Prust C, Hoffmeister M, Liesegang H, et al. Complete genome sequence of the aceticacid bacterium Gluconobacter oxydans [J]. Nat Biotechnol,2005,23(2):195-200
83. Gao L, Zhou J, Liu J, et al. Draft genome sequence of Gluconobacter oxydans WSH-003,a strain that is extremely tolerant of saccharides and alditols [J]. J Bacteriol,2012,194(16):4455-4456
84. Sebaihia M, Bocsanczy A, Biehl B, et al. Complete genome sequence of the plantpathogen Erwinia amylovora strain ATCC49946[J]. J Bacteriol,2010,192(7):2020-2021
85. Kalinowski J, Bathe B, Bartels D, et al. The complete Corynebacterium glutamicumATCC13032genome sequence and its impact on the production of L-aspartate-derivedamino acids and vitamins [J]. J Biotechnol,2003,104(1):5-25
86. Zhang L, Wang Z, Xia Y, et al. Metabolic engineering of plant L-ascorbic acidbiosynthesis: recent trends and applications [J]. Crit. Rev. Biotechnol,2007,27(3):173-182
87. Domingues A, Vinga S, Lemos J M. Optimization strategies for metabolic networks [J].BMC Syst Biol,2010,4(1):113
88. Christian N, May P, Kempa S, et al. An integrative approach towards completinggenome-scale metabolic networks [J]. Mol BioSyst,2009,5(12):1889-1903
89. Park J M, Kim T Y, Lee S Y. Constraints-based genome-scale metabolic simulation forsystems metabolic engineering [J]. Biotechnol Adv,2009,27(6):979-988
90. Price N D, Reed J L, Palsson B. Genome-scale models of microbial cells: evaluating theconsequences of constraints [J]. Nat Rev Microbiol,2004,2(11):886-897
91. Lee S Y, Lee D Y, Kim T Y. Systems biotechnology for strain improvement [J]. TrendsBiotechnol,2005,23(7):349-358
92. Zou W, Liu L, Zhang J, et al. Reconstruction and analysis of a genome-scale metabolicmodel of the vitamin C producing industrial strain Ketogulonicigenium vulgareWSH-001[J]. J Biotechnol,2012,161(1):42-48
93. Liu L, Agren R, Bordel S, et al. Use of genome-scale metabolic models forunderstanding microbial physiology [J]. FEBS Lett,2010,584(12):2556-2564
94. Curran K A, Alper H S. Expanding the chemical palate of cells by combining systemsbiology and metabolic engineering [J]. Metab Eng,2012,14(4):289-297
95. Bradford M M. A rapid and sensitive method for the quantification of microgramquantities of protein utilizing the principle of protein-dye binding [J]. Anal Biochem,1976,72:248-254
96. Salvatore C A, Mallee J J, Bell I M, et al. Identification and pharmacologicalcharacterization of domains involved in binding of CGRP receptor antagonists to thecalcitonin-like receptor [J]. Biochem,2006,45(6):1881-1887
97. Pinter R Y, Rokhlenko O, Yeger-Lotem E, et al. Alignment of metabolic pathways [J].Bioinformatics,2005,21(16):3401-3408
98. Pósfai G, Plunkett G, Fehér T, et al. Emergent properties of reduced-genome Escherichiacoli [J]. Science,2006,312(5776):1044-1046
99. Levskaya A, Chevalier A A, Tabor J J, et al. Synthetic biology: engineering Escherichiacoli to see light [J]. Nature,2005,438(7067):441-442
100. Sorensen H P, Mortensen K K. Advanced genetic strategies for recombinant proteinexpression in Escherichia coli [J]. J Biotechnol,2005,115(2):113-128
101. Quail M A, Kozarewa I, Smith F, et al. A large genome center's improvements to theIllumina sequencing system [J]. Nat Methods,2008,5(12):1005-1010
102. Li R, Zhu H, Ruan J, et al. De novo assembly of human genomes with massively parallelshort read sequencing [J]. Genome Res,2010,20(2):265-272
103. Delcher A L, Bratke K A, Powers E C, et al. Identifying bacterial genes andendosymbiont DNA with Glimmer [J]. Bioinformatics,2007,23(6):673-679
104. Tatusov R L, Natale D A, Garkavtsev I V, et al. The COG database: new developments inphylogenetic classification of proteins from complete genomes [J]. Nucleic Acids Res,2001,29(1):22-28
105. Apweiler R, Attwood T K, Bairoch A, et al. The InterPro database, an integrateddocumentation resource for protein families, domains and functional sites [J]. NucleicAcids Res,2001,29(1):37-40
106. Griffiths-Jones S, Bateman A, Marshall M, et al. Rfam: an RNA family database [J].Nucleic Acids Res,2003,31(1):439-441
107. Lowe T M, Eddy S R. tRNAscan-SE: a program for improved detection of transfer RNAgenes in genomic sequence [J]. Nucleic Acids Res,1997,25(5):955-964
108. Panda A K, Khan R, Appa Rao K, et al. Kinetics of inclusion body production in batchand high cell density fed-batch culture of Escherichia coli expressing ovine growthhormone [J]. J Biotechnol,1999,75(2):161-172
109. Deppenmeier U, Hoffmeister M, Prust C. Biochemistry and biotechnologicalapplications of Gluconobacter strains [J]. Appl Microbiol Biotechnol,2002,60(3):233-242
110. Tkac J, Svitel J, Vostiar I, et al. Membrane-bound dehydrogenases from Gluconobactersp.: Interfacial electrochemistry and direct bioelectrocatalysis [J]. Bioelectrochemistry,2009,76(1):53-62
111. Odaci D, Timur S, Telefoncu A. A microbial biosensor based on bacterial cellsimmobilized on chitosan matrix [J]. Bioelectrochemistry,2009,75(1):77-82
112. Kallnik V, Meyer M, Deppenmeier U, et al. Construction of expression vectors forprotein production in Gluconobacter oxydans [J]. J Biotechnol.,2010,150(4):460-465
113. Schleyer U, Bringer-Meyer S, Sahm H. An easy cloning and expression vector systemfor Gluconobacter oxydans [J]. Int J Food Microbiol,2008,125(1):91-95
114. Shinjoh M, Hoshino T. Development of a stable shuttle vector and a conjugative transfersystem for Gluconobacter oxydans [J]. J Ferment Bioeng,1995,79(2):95-99
115. Cha J S, Pujol C, Ducusin A, et al. Studies on Pantoea citrea, the causal agent of pinkdisease of pineapple [J]. J Phytopathol,1997,145(7):313-319
116. Mostafa H E, Heller K J, Geis A. Cloning of Escherichia coli lacZ and lacY genes andtheir expression in Gluconobacter oxydans and Acetobacter liquefaciens [J]. ApplEnviron Microbiol,2002,68(5):2619-2623
117. Albertsen L, Chen Y, Bach L S, et al. Diversion of flux toward sesquiterpene productionin Saccharomyces cerevisiae by fusion of host and heterologous enzymes [J]. ApplEnviron Microbiol,2011,77(3):1033-1040
118. Sanchez A M, Bennett G N, San K Y. Effect of different levels of NADH availability onmetabolic fluxes of Escherichia coli chemostat cultures in defined medium [J]. JBiotechnol,2005,117(4):395-405
119. Fluckiger R, Paz M, Mah J, et al. Characterization of the glycine-dependentredox-cycling activity in animal fluids and tissues using specific inhibitors and activators:evidence for presence of PQQ [J]. Biochem Biophys Res Commun,1993,196(1):61-68
120. Jorgensen K, Rasmussen A V, Morant M, et al. Metabolon formation and metabolicchanneling in the biosynthesis of plant natural products [J]. Curr Opin Plant Biol,2005,8(3):280-291
121. Yan Y, Li Z, Koffas M A. High-yield anthocyanin biosynthesis in engineered Escherichiacoli [J]. Biotechnol Bioeng,2008,100(1):126-140
122. Conrado R J, Varner J D, DeLisa M P. Engineering the spatial organization of metabolicenzymes: mimicking nature's synergy [J]. Curr Opin Biotechnol,2008,19(5):492-499
123. Wang J, Tsai M, Lee G, et al. Construction of a recombinant thermostableβ-amylase-trehalose synthase bifunctional enzyme for facilitating the conversion ofstarch to trehalose [J]. J Agric Food Chem,2007,55(4):1256-1263
124. Hong S Y, Lee J S, Cho K M, et al. Assembling a novel bifunctional cellulase–xylanasefrom Thermotoga maritima by end-to-end fusion [J]. Biotechnol Lett,2006,28(22):1857-1862
125. Orita I, Sakamoto N, Kato N, et al. Bifunctional enzyme fusion of3-hexulose-6-phosphate synthase and6-phospho-3-hexuloisomerase [J]. Appl MicrobiolBiotechnol,2007,76(2):439-445
126. Karp, Matti, Oker-Blom, et al. A streptavidin-luciferase fusion protein: comparisons andapplications [J]. Biomol Eng,1999,16(1):101-104
127. Blazyk J L, Lippard S J. Domain engineering of the reductase component of solublemethane monooxygenase from Methylococcus capsulatus (Bath)[J]. J Biol Chem,2004,279(7):5630-5640
128. Holsch K, Weuster Botz D. Enantioselective reduction of prochiral ketones byengineered bifunctional fusion proteins [J]. Biotechnol Appl Biochem,2010,56(4):131-140
129. Kavoosi M, Creagh A L, Kilburn D G, et al. Strategy for selecting and characterizinglinker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli [J].Biotechnol Bioeng,2007,98(3):599-610
130. Wriggers W, Chakravarty S, Jennings P A. Control of protein functional dynamics bypeptide linkers [J]. Peptide Science,2005,80(6):736-746
131. Lu P, Feng M. Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme byoptimization of peptide linkers [J]. Appl Microbiol Biotechnol,2008,79(4):579-587
132. Arai R, Wriggers W, Nishikawa Y, et al. Conformations of variably linked chimericproteins evaluated by synchrotron X-ray small-angle scattering [J]. Proteins: Struct FuncBioinformatics,2004,57(4):829-838
133. Gao L, Du G, Zhou J, et al. Characterization of a group of pyrroloquinolinequinone-dependent dehydrogenases that are involved in the conversion of L-sorbose to2-keto-L-gulonic acid in Ketogulonicigenium vulgare WSH-001[J]. Biotechnol Prog,2013,29(6):1398-1404
134. Du J, Bai W, Song H, et al. Combinational expression of sorbose/sorbosonedehydrogenases and cofactor pyrroloquinoline quinone increases2-keto-L-gulonic acidproduction in Ketogulonigenium vulgare-Bacillus cereus consortium [J]. Metab Eng,2013,19:50-56
135. Choi O, Kim J, Kim J G, et al. Pyrroloquinoline quinone is a plant growth promotionfactor produced by Pseudomonas fluorescens B16[J]. Plant Physiol,2008,146(2):657-668
136. Shrivastava M, Rajpurohit Y S, Misra H S, et al. Survival of phosphate-solubilizingbacteria against DNA damaging agents [J]. Can J Microbiol,2010,56(10):822-830
137. Sawano M, Yamamoto H, Ogasahara K, et al. Thermodynamic basis for the stabilities ofthree CutA1s from Pyrococcus horikoshii, Thermus thermophilus, and Oryza sativa, withunusually high denaturation temperatures [J]. Biochemistry,2008,47(2):721-730
138. Ramirez M, Guan D, Ugaz V, et al. Intein-triggered artificial protein hydrogels thatsupport the immobilization of bioactive proteins [J]. J Am Chem Soc,2013,135(14):5290-5293
139. Lithwick G, Margalit H. Hierarchy of sequence-dependent features associated withprokaryotic translation [J]. Genome Res,2003,13(12):2665-2673
140. Zehentgruber D, Lundemo P, Svensson D, et al. Substrate complexation and aggregationinfluence the cyclodextrin glycosyltransferase (CGTase) catalyzed synthesis of alkylglycosides [J]. J Biotechnol,2011,155(2):232-235
141. De Muynck C, Pereira C S, Naessens M, et al. The genus Gluconobacter oxydans:comprehensive overview of biochemistry and biotechnological applications [J]. Crit RevBiotechnol,2007,27(3):147-171
142. Tanaka Y, Tsumoto K, Nakanishi T, et al. Structural implications for heavy metal-inducedreversible assembly and aggregation of a protein: the case of Pyrococcus horikoshii CutA[J]. FEBS Lett,2004,556(1):167-174
143. Olijve W, Kok J. An analysis of the growth of Gluconobacter oxydans in chemostatcultures [J]. Arch Microbiol,1979,121(3):291-297