透明质酸合成途径在乳酸乳球菌中的建立及微生物合成透明质酸分子量调控机制的初步研究
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摘要
透明质酸(hyaluronic acid,HA)作为一种高分子量的黏多糖,广泛存在于脊椎动物细胞中,发挥着结构、信号传导和信号识别的功能。HA由于其优异的黏弹性、保湿性和支撑作用,被广泛的应用于医药、化妆品、药物传递系统、疫苗和功能健康食品。目前,工业上主要经由革兰氏阳性C型链球菌如兽疫链球菌(Streptococcus zooepidemicus)发酵生产。但作为生产菌株,链球菌生长速度慢、培养基成本高等劣势日益显现出来,而寻找新的优秀野生生产菌株的希望渺茫。所以,利用基因工程构建合成HA的工程菌株不但有利于克服链球菌作为发酵生产HA菌株的劣势,而且对提高HA的品质,扩大HA的应用范围具有理论意义和应用价值。
乳酸乳球菌(Lactoccus lactis)作为革兰氏阳性菌的模式生物,已被广泛应用于食品发酵生产中,且被认定为GRAS(generally regarded as safe)微生物。乳酸乳球菌由于其自身的优良特性,正越来越多地被应用于代谢工程改造过的现代工业中。在过去的25年中,乳酸乳球菌的基因操作方法日益成熟,其中一系列的诱导表达启动子得到了深入的研究,并已开始应用于工业生产。通过改变小肽、单糖的浓度或诱导温度等调控因子,这些启动子能够实现乳酸乳球菌工程菌株中目的蛋白的可控表达。NICE(the nisin controlled gene expression)系统是乳酸乳球菌中研究最深入和最有效的基因工程操作系统之一,已被广泛地应用于该菌株的代谢工程改造。不但诱导剂乳酸链球菌素(nisin)是一种理想的高效安全无毒的食品级诱导分子,而且在NICE系统中可以将染色体上lacF基因缺失的乳酸球菌菌株以及含有lacF基因的质粒结合使用,用食品级筛选标记lacF替代抗生素抗性基因,构建食品级表达系统,而且该方式构建的工程菌株遗传稳定(deRuyter,Kuipers & de Vos,1996b;Mierau & Kleerebezem,2005a;Zhang,Chen,Jia,Chen & Huan,2002)。为了改善现有HA生产菌株的不足,我们计划利用该系统构建既具有合成HA能力的,同时符合公认安全标准的乳酸乳球菌工程菌株。另外,来源于乳糖操纵子PlacR的启动子也是乳酸乳球菌常用的强诱导表达启动子之一。LacR的启动子和NICE系统中nisA启动子已有利用各自诱导剂浓度的变化在乳酸乳球菌工程菌株中成功实现内源或外源蛋白可控表达的先例。这些特点有助于在本论文中实现乳酸乳球菌工程菌株中szHasA和szHasB基因的可控表达,从而在不同诱导条件下改变兽疫链球菌HAS与UDP-葡萄糖脱氢酶之间表达水平比率,为微生物合成HA分子量调控机制的研究提供了可能。
HA在细胞内的合成是一个复杂的过程,有多种酶共同参与。在兽疫链球菌的HA合成途径中,包括透明质酸合酶(szHasA)、UDP-葡萄糖焦磷酸化酶(szHasC)、UDP-葡萄糖脱氢酶(szHasB)、磷酸葡萄糖异构酶、氨基转移酶、乙酰基转移酶、变位酶和UDP-N-乙酰氨基葡糖焦磷酸化酶(szHasD)。其中透明质酸合酶(HA synthase,HAS)是HA合成途径中的关键酶,具有利用UDP-葡萄糖醛酸(UDP-GlcUA)和UDP-N-乙酰-D-氨基葡糖(UDP-GlcNAc)合成HA的催化活性。UDP-葡萄糖脱氢酶(szHasB)、UDP-葡萄糖焦磷酸化酶(szHasC)和UDP-N-乙酰氨基葡糖焦磷酸化酶(szHasD)是参与HA底物合成的酶类,通过一系列催化反应,分别将葡萄糖转化为UDP-GlcUA和UDP-GlcNAc(Blank,Hugenholtz & Nielsen,2008)。在乳酸乳球菌中,也存在着与szHasB和szHasD相同催化活性的UDP-葡萄糖脱氢酶(UDP-GlcDH)和UDP-N-乙酰氨基葡糖焦磷酸化酶(LACR),分别由乳酸乳球菌UDP-葡萄糖脱氢酶基因(ugd)和UDP-N-乙酰氨基葡糖焦磷酸化酶基因(LACR)编码。
本论文分别将基因szHasA、szHasB和szHasC的ORF重组于NICE表达载体pNZ8148的PnisA启动子调控之下,得到含有pSH71来源复制起始位点的表达载体pNZ8148-szHasA、pNZ8148-szHasB和pNZ8148-szHasC。将含有质粒pNZ8148-szHasA、pNZ8148-szHasB、pNZ8148-szHasC和pNZ8148-szHasABC的乳酸乳球菌工程菌株分别命名为N9000-A、N9000-B和N9000-C。通过重组表达载体pNZ8148-szHasABC和pNZ8149-szHasABC将兽疫链球菌来源的HA合成操纵子分别导入乳酸乳球菌NZ9000和NZ3900,得到工程菌株NFHA01和NFHA03。同时,通过融合PCR方法得到一个重组HA合成操纵子,命名为rHAop,在该DNA片断中,原兽疫链球菌HA合成操纵子中的szHasB和szHasC基因的ORF分别被乳酸乳球菌来源的ugd和LACR基因的ORF所取代。重组HA合成操纵子rHAop通过重组表达载体pNZ8149-rHAop导入乳酸乳球菌NZ3900中,得到工程菌株NFHA03。RIA法测定各工程菌株诱导后发酵液中HA的浓度。空白对照菌株NZ9000和分别单独表达szHasB、szHasC的工程菌株NZ9000-B和NZ9000-C的发酵液中没有检测到HA的合成,而工程菌株NZ9000-A发酵液中含有0.22 g/L的HA。证明乳酸乳球菌本身不具有合成HA的能力,而在该菌中单独表达HAS可以使工程菌株获得合成HA的能力,而且该菌本身具有HA底物UDP-GlcA和UDP-GlcNAc的合成途径。工程菌株NFHA01的发酵液中含有0.6 g/L的HA,这一数据约为NZ9000-A发酵液中HA浓度的2.7倍,这一结果证明虽然乳酸乳球菌具有合成UDP-GlcA和UDP-GlcNAc的能力,但自身合成的HA底物的不足限制HAS在工程菌株中合成高浓度的HA,而提高工程菌株HA底物的浓度,有助于HA合成能力的提高。
工程菌株NFHA03和NFHA02发酵液中HA的浓度分别为0.49 g/L和0.38g/L,前者大约高出后者33%。证明在乳酸乳球菌工程菌株中重组表达异源szHasA,同时超表达同源UDP-GlcDH和LACR较同时表达外源szHasA、szHasB和szHasC更利于HA的合成。本论文利用lacF基因作为食品级筛选标记和NICE表达系统,构建得到一株不产生荚膜的具有食品级HA合成能力的工程菌株,而食品级HA在保健食品、医药产品和化妆品的应用中具有明显的优势。
HA的相对分子质量(Mr)一般分布在10~4 Da-10~7 Da范围内。研究表明,在细胞和组织中,HA生物活性与糖链长度相关,不同Mr的HA具有不同甚至相反的生理或药理作用,HA的多种生物活性具有Mr依赖性。通过差异表达HA合成相关酶类,脊椎动物具有在体内调控HA Mr的能力。而目前微生物发酵生产的HA是Mr从大到小的一系列糖链的组合。因此,在活体微生物中确定HA的Mr调控机制并且建立制备特定Mr或特定Mr范围分布的HA的方法,不但具有重要的理论意义,而且对扩展HA的应用范围、改良含HA产品的质量具有应用价值。体外实验已证明,合成能力与底物浓度的比率是影响HA Mr的主要因素之一,我们认为这一理论同样适用于活体微生物内。为实现乳酸乳球菌工程菌株中HA合成能力与底物浓度的比率的可控调节,本论文构建了一个双质粒的调控表达系统,包括表达szHasA的载体pNZ8148-szHasA和表达szHasB的载体pNZ9531-szHasB,而这两个质粒可共存于同一株乳酸乳球菌中。pNZ8148-szHasA包含有乳酸乳球菌常用载体pSH71的复制起始位点,而szHasA基因位于NICE系统启动子nisA的调控之下:而另一个载体pNZ9531-szHasB则含有另一个乳酸乳球菌常用载体pAMb1所携带的复制起始位点,同时szHasB基因的诱导表达受到lacR启动子的调控。蛋白表达结果证明在工程菌株NFHA04中,借助pNZ8148-szHasA和pNZ9531-szHasB组成的双质粒调控表达系统,实现了不同诱导条件下改变szHasA与szHasB之间表达水平比率的实验目的。szHasA和szHasB mRNA拷贝数之间的比率,表征着HA合成能力与HA底物合成能力之间的相对强弱。不同诱导条件下,分析szHasA/szHasB mRNA比率对乳酸乳球菌工程菌株NFHA04合成HA的重均分子量((?)w)的影响,在微生物体内证明了HA合成能力与底物合成能力之间的相对强弱具有调控HA(?)w的作用。当代表HAS和UDP-GlcDH之间相对强度的szHasA/szHasB mRNA比率高时,发酵液中HA的(?)w相对较小,而当szHasA/szHasB mRNA比率减小时,(?)w则变大。分析认为,合成HA能力与合成HA底物能力之间的相对强弱是影响微生物HA Mr的重要因素之一,szHasA和szHasB mRNA拷贝数之间的比率高时,活细胞体内每一个HAS分子能够分配到的用来合成HA的底物就相对较少,则每一个HAS分子能够合成的HA糖链就相对较短,从而造成合成的HA的(?)w就较小:而当这一比率降低时,每一个HAS分子就有相对较多的底物分子可以用来合成HA糖链,从而造成合成的HA的糖链较长,(?)w也变大。这一理论的确定,为通过调节微生物体内HAS的表达水平和底物浓度来控制微生物生产过程中发酵液中HA Mr特性提供了切实可行的解决方案,具有重大的理论意义和潜在应用价值。
本论文的主要内容:
(1)通过NICE系统,将兽疫链球菌HA合成代谢途径相关基因导入乳酸乳球菌,构建一系列工程菌株。分析各工程菌株合成HA的浓度,证明利用代谢工程手段在乳酸乳球菌中引入编码HAS的基因,可以该菌株获得利用葡萄糖合成HA的能力。但自身合成的HA底物的不足限制HAS在工程菌株中合成高浓度的HA,而提高工程菌株HA底物的浓度,有助于HA合成能力的提高。
(2)通过融合PCR方法得到一个重组HA合成操纵子,利用lacF基因作为食品级筛选标记和NICE表达系统,将该重组HA合成操纵子导入乳酸乳球菌,实现在同一株工程菌株中重组表达异源szHasA,同时超表达同源UDP-GlcDH和LACR,表达这三个合成HA关键酶的乳酸乳球菌工程菌株具备了合成食品级HA的能力。
(3)构建了一个双质粒的调控表达系统,包括表达szHasA的载体pNZ8148-szHasA和表达szHasB的载体pNZ9531-szHasB中。在工程菌株NFHA04中实现了不同诱导条件下改变szHasA与szHasB之间表达水平比率的实验目的。
(4)不同诱导条件下,分析szHasA/szHasB mRNA比率对乳酸乳球菌工程菌株NFHA04合成HA的重均分子量((?)w)的影响。证明合成HA能力与合成HA底物能力之间的相对强弱是影响微生物HA Mr的重要因素之一,szHasA和szHasB mRNA拷贝数之间的比率高时,活细胞体内每一个HAS分子能够分配到的用来合成HA的底物就相对较少,则每一个HAS分子能够合成的HA糖链就相对较短,从而造成合成的HA的(?)w就较小;而当这一比率降低时,每一个HAS分子就有相对较多的底物分子可以用来合成HA糖链,从而造成合成的HA的糖链较长,(?)w也变大。
本论文主要创新点:
(1)首次通过融合PCR方法得到重组HA合成操纵子。并首次在同一乳酸乳球菌工程菌株中实现了重组表达异源szHasA,同时超表达同源UDP-GlcDH和LACR。
(2)首次利用lacF基因作为食品级筛选标记和NICE表达系统,在乳酸乳球菌终建立了透明质酸合成途径。该乳酸乳球菌工程菌株具备合成食品级HA的能力。
(3)首次在乳酸乳球菌工程菌株中实现了兽疫链球菌HAS与UDPGlcDH的同时诱导可控表达。
(4)首次在活体微生物体内证明HA合成能力与底物合成能力之间的相对强弱具有调控HA(?)w的作用。这一理论的确定,为通过调节微生物体内HAS的表达水平和底物浓度来控制微生物生产过程中发酵液中HA Mr特性提供了切实可行的解决方案,具有重大的理论意义和潜在应用价值。
Hyaluronic acid(HA) is one kind of natural high molecular weight polysaccharide and plays structural,recognition and signaling roles in animals.HA has been used in medicines,cosmetics,drug delivery systems,vaccine aids,as well as in health foods. In industry,HA is produced using the gram-positive bacterium Streptococcus zooepidemicus. However,Streptococcus is a less-than-ideal source because of the potential to produce exotoxins,the difficulty in fermentation control or expensive medium.Therefore,it is meaningful and necessary to develop an alternative source of HA that avoids these pitfalls and enlarges the applications.
The relative molecular weight(Mr) of HA is generally ranging from 10~4 to 10~7 Da in vertebrates and bacteria,and the biological roles of HA correlate with the length of the HA chain.HAs of different lengths often appear to have antagonistic or reverse effects in many cellular and tissue systems.The solution of HA with sizes greater than 10 kDa,which has outstanding moisture keeping ability,high viscoelasticity and lubricity,has been widely used in cosmetics and medicines.Recently,several groups have reported that short chain HA(～2 kDa-3.5 kDa) or HA oligosaccharides(～10-20 sugars in length) have interesting effects on cellular behavior.To extend the applications of HA and make better HA-containing biomedical products,it is necessary to obtain specially designed Mr or uniform size-defined HA.Vertebrates may be able to control the length of HA in vivo by differential expression of biosynthetic enzymes,but HA fermented by microbes is a mixture of a wide range of molecular masses.The in vitro study data support the theory that the relative ratio of catalysts to the precursor sugars may be a major factor in HA size control.Therefore, it is meaningful and necessary to investigate the regulation mechanism of HA size in bacteria.
Lactoccus lactis,one of the most widely used Gram-positive lactic acid bacteria in food fermentations,is a generally regarded as safe(GRAS) strain and increasingly used in modern biotechnological applications.In virtue of great development of genetic engineering tools and the molecular characterization of this species in the last 25 years,a number of inducible expression promotors have been developed in L. lactis.Through these promotors,gene expression can be controlled by an inducer,a repressor or environmental factors,such as peptide,monosaccharide,or temperature. NICE(nisin controlled gene expression) system has become one of the most successful and widely used tools for regulating gene expression in Gram-positive bacteria.The lacF gene is commonly used as a food-grade selectable marker in the NICE system.In order to overcome the shortcomings of Streptococcus zooepidemicus as a HA production bacterium,we planned to construct some engineering GRAS recombinant strains based on Lactoecus lactis containing HA biosynthesis operon and lacF seletable marker.Another controllable expression system of L.lactis is based on the lacR promoter of the lac operon.The lacR promoter is a strong promoter that is virtually not subjected to catabolite repression and controlled by autoregulated LacR repressor.Both NICE system and the lacR promoter have been used to overexpress homologous or heterologous membrane-located proteins and regulate gene expression by quorum sensing-controlling in L.lactis.In this work,these characters of the lacR and the nisA promoters was used to regulate szHasA and szHasB expression,in investigating regulation mechanism of HA polymer size in recombinant L lactis. A series of enzymes are involved in the biosynthesis of HA in S.zooepidemicus, including hyaluronan synthase(HasA),UDP-glucose-6-dehydrogenase(HasB), UDP-glucose pyrophosphorylase(HasC) and glucosamine-1-phosphate N-acetyltransferase/UDP-N-acetylglucosamine pyrophosphorylase(HasD).HasA,the key enzyme in the production of HA,utilizes two sugar substrates(UDP-GlcA and UDP-GlcNAc) to synthesize HA.HasB,HasC and HasD are the enzymes in the synthesis pathway of the precursor sugars.UDP-glucose-6-dehydrogenase (UDP-GlcDH) and GlcN-1-P acetyl transferase/UDP-GlcNAc pyrophosphorylase (LACR) from L.lactis have the same catalyzing effects as HasB and HasD do. The szHasA,szHasB and szHasC were carried on pSH71-derived replicons and under the control of PnisA promoter in the expression plasmids pNZ8148-szHasA, pNZ8148-szHasB and pNZ8148-szHasC,respectively.The recombinant L.lactis strains with pNZ8148-szHasA,pNZ8148-szHasB and pNZ8148-szHasC were named as N9000-A,N9000-B and N9000-C.The HA biosynthesis operon of S.
zooepidemicus was introduced into L.lactis NZ9000 and NZ3900 with the vectors of pNZ8148-szHasABC and pNZ8149-szHasABC,respectively,producing strain NFHA01 and NFHA03.Meanwhile,a recombinant HA biosynthesis operon was constructed and named as rHaop.In this artificial DNA fragment,the ORFs of szHasB and szHasC were displaced by the ORFs of ugd and LACR from L.lactis respectively.Then rHAop was introduced into L.lactis NZ3900 with the vector pNZ8149-rHAop,producing strain NFHA02.
The concentrations of HA in the culture of these recombinant strains were analyzed and compared.About 0.22 g/L HA was detected in the NZ9000-A culture while HA cannot be detected in the control strain NZ9000,indicating that HA could be synthesized with the expression of HasA alone in L.lactis and the required precursors, UDP-GlcA and UDP-GIcNAc,could also be synthesized in the host strain.However, no HA was detected in the cultures of NZ9000-B and NZ9000-C.Furthermore,a final concentration of HA was about 0.6g/L in the NFHA01 culture,was about 170% higher than that of NZ9000-A,showing that HasA is essential for high heterologous HA production in L.lactis and improving the precursor sugar biosynthesis levels can obviously increase the production of HA.A final concentration of HA was about 0.49g/L in the NFHA03 culture,about 33%higher than that(about 0.38 g/L) in the culture of NFHA02..Therefore,it can be concluded that overexpression of the endogeneous enzymes,directing the steps in the synthesis pathway of the precursor sugars,is a more effective way to increase HA production than expressing exogenous enzymes in recombinant cells.
HA fermented by microbes was a mixture of a wide range of molecular masses,but the biological roles of HA are correlated with the length of the polysaccharide chain, which is one of the limiting-factors to extend the applications of HA and make better HA-containing biomedical products.So,it has theoretical and practical value to obtain specially designed(?)w or uniform size-defined HA by industrial fermentation. The mechanism for size control of HA product has begun to be investigated recently. Jing et al.(2004) proved that HA polymers of a desired size could be obtained by controlling the reaction stoichiometry,such as molar ratio of precursors and acceptor molecules.The theory that the relative strength of the interaction between the catalyst and the precursor sugars may be a major factor in HA size control has been testified by Pummill.et al.(2003) in vitro.We considered that this theory could also be suitable in bacterium cells and should be investigated in vivo.
To adjust the relative strength of the interaction between the catalyst and the precursor sugars,we obtained different ratios of HasA expression level to precursor sugar UDP-GlcA biosynthesis ability by regulating the szHasA and szHasB expression levels in a recombinant L.lactis.Two inducible expression promoters,nisA promoter and lacR promoter,were used in our work and gene expression could be controlled by nisin or lactose through these promoters.Thus,a dual-plasmid controlled expression system,which consisted of szHasA expression vector pNZ8148-szHasA and szHasB expression vector pNZ9531-szHasB,was constructed.The aim to regulate szHasA and szHasB expression levels in recombinant strain NFHA04 was accomplished by using this system.
The ratio of szHasA to szHasB mRNA copies,the surface features of the ratio of catalytic ability of HA synthesis to the precursor sugar biosynthesis level in cells,was regulated strictly by changing the inducing concentration collocations with nisin and lactose.We analyzed the Mw and the concentration of HA under the different ratios, discovering that the theory that the relative strength of the interaction between the catalyst and the precursor sugar may be a major factor in HA size control was also suitable for in vivo HA synthesis of microbes,as we had expected.The average size of HA produced in high HasA/HasB mRNA ratio conditions was smaller than that in low ratio conditions.Altering the ratio of HA synthesizing ability to the concentration of precursor sugars might cause a change in HA size.The reason is probably that the distribution of precursor sugars among the HasA molecules is altered by changing HasA expression level and the precursor sugar biosynthesis ability.The distribution of precursor sugars to each HasA under the inducing condition of high concentration of nisin and low concentration of lactose would be relatively less than under the reverse inducing condition,so each HA polysaccharide chain gets less precursor sugars to lengthen itself catalyzed by HasA in the membrane.In the present study,the ratio of HasA expression level to the precursor sugars biosynthesis ability in bacteria, standing for the relative strength of the interaction between the catalyst and the precursor sugars,was proved to be an important facter for the size control of HA in bacteria.It has theoretical and practical value to determine the regulation mechanism of HA polymer size and this study puts forward a guide for establishing an efficacious way to control the size of HA in fermentation.
乳酸乳球菌(Lactoccus lactis)作为革兰氏阳性菌的模式生物,已被广泛应用于食品发酵生产中,且被认定为GRAS(generally regarded as safe)微生物。乳酸乳球菌由于其自身的优良特性,正越来越多地被应用于代谢工程改造过的现代工业中。在过去的25年中,乳酸乳球菌的基因操作方法日益成熟,其中一系列的诱导表达启动子得到了深入的研究,并已开始应用于工业生产。通过改变小肽、单糖的浓度或诱导温度等调控因子,这些启动子能够实现乳酸乳球菌工程菌株中目的蛋白的可控表达。NICE(the nisin controlled gene expression)系统是乳酸乳球菌中研究最深入和最有效的基因工程操作系统之一,已被广泛地应用于该菌株的代谢工程改造。不但诱导剂乳酸链球菌素(nisin)是一种理想的高效安全无毒的食品级诱导分子,而且在NICE系统中可以将染色体上lacF基因缺失的乳酸球菌菌株以及含有lacF基因的质粒结合使用,用食品级筛选标记lacF替代抗生素抗性基因,构建食品级表达系统,而且该方式构建的工程菌株遗传稳定(deRuyter,Kuipers & de Vos,1996b;Mierau & Kleerebezem,2005a;Zhang,Chen,Jia,Chen & Huan,2002)。为了改善现有HA生产菌株的不足,我们计划利用该系统构建既具有合成HA能力的,同时符合公认安全标准的乳酸乳球菌工程菌株。另外,来源于乳糖操纵子PlacR的启动子也是乳酸乳球菌常用的强诱导表达启动子之一。LacR的启动子和NICE系统中nisA启动子已有利用各自诱导剂浓度的变化在乳酸乳球菌工程菌株中成功实现内源或外源蛋白可控表达的先例。这些特点有助于在本论文中实现乳酸乳球菌工程菌株中szHasA和szHasB基因的可控表达,从而在不同诱导条件下改变兽疫链球菌HAS与UDP-葡萄糖脱氢酶之间表达水平比率,为微生物合成HA分子量调控机制的研究提供了可能。
HA在细胞内的合成是一个复杂的过程,有多种酶共同参与。在兽疫链球菌的HA合成途径中,包括透明质酸合酶(szHasA)、UDP-葡萄糖焦磷酸化酶(szHasC)、UDP-葡萄糖脱氢酶(szHasB)、磷酸葡萄糖异构酶、氨基转移酶、乙酰基转移酶、变位酶和UDP-N-乙酰氨基葡糖焦磷酸化酶(szHasD)。其中透明质酸合酶(HA synthase,HAS)是HA合成途径中的关键酶,具有利用UDP-葡萄糖醛酸(UDP-GlcUA)和UDP-N-乙酰-D-氨基葡糖(UDP-GlcNAc)合成HA的催化活性。UDP-葡萄糖脱氢酶(szHasB)、UDP-葡萄糖焦磷酸化酶(szHasC)和UDP-N-乙酰氨基葡糖焦磷酸化酶(szHasD)是参与HA底物合成的酶类,通过一系列催化反应,分别将葡萄糖转化为UDP-GlcUA和UDP-GlcNAc(Blank,Hugenholtz & Nielsen,2008)。在乳酸乳球菌中,也存在着与szHasB和szHasD相同催化活性的UDP-葡萄糖脱氢酶(UDP-GlcDH)和UDP-N-乙酰氨基葡糖焦磷酸化酶(LACR),分别由乳酸乳球菌UDP-葡萄糖脱氢酶基因(ugd)和UDP-N-乙酰氨基葡糖焦磷酸化酶基因(LACR)编码。
本论文分别将基因szHasA、szHasB和szHasC的ORF重组于NICE表达载体pNZ8148的PnisA启动子调控之下,得到含有pSH71来源复制起始位点的表达载体pNZ8148-szHasA、pNZ8148-szHasB和pNZ8148-szHasC。将含有质粒pNZ8148-szHasA、pNZ8148-szHasB、pNZ8148-szHasC和pNZ8148-szHasABC的乳酸乳球菌工程菌株分别命名为N9000-A、N9000-B和N9000-C。通过重组表达载体pNZ8148-szHasABC和pNZ8149-szHasABC将兽疫链球菌来源的HA合成操纵子分别导入乳酸乳球菌NZ9000和NZ3900,得到工程菌株NFHA01和NFHA03。同时,通过融合PCR方法得到一个重组HA合成操纵子,命名为rHAop,在该DNA片断中,原兽疫链球菌HA合成操纵子中的szHasB和szHasC基因的ORF分别被乳酸乳球菌来源的ugd和LACR基因的ORF所取代。重组HA合成操纵子rHAop通过重组表达载体pNZ8149-rHAop导入乳酸乳球菌NZ3900中,得到工程菌株NFHA03。RIA法测定各工程菌株诱导后发酵液中HA的浓度。空白对照菌株NZ9000和分别单独表达szHasB、szHasC的工程菌株NZ9000-B和NZ9000-C的发酵液中没有检测到HA的合成,而工程菌株NZ9000-A发酵液中含有0.22 g/L的HA。证明乳酸乳球菌本身不具有合成HA的能力,而在该菌中单独表达HAS可以使工程菌株获得合成HA的能力,而且该菌本身具有HA底物UDP-GlcA和UDP-GlcNAc的合成途径。工程菌株NFHA01的发酵液中含有0.6 g/L的HA,这一数据约为NZ9000-A发酵液中HA浓度的2.7倍,这一结果证明虽然乳酸乳球菌具有合成UDP-GlcA和UDP-GlcNAc的能力,但自身合成的HA底物的不足限制HAS在工程菌株中合成高浓度的HA,而提高工程菌株HA底物的浓度,有助于HA合成能力的提高。
工程菌株NFHA03和NFHA02发酵液中HA的浓度分别为0.49 g/L和0.38g/L,前者大约高出后者33%。证明在乳酸乳球菌工程菌株中重组表达异源szHasA,同时超表达同源UDP-GlcDH和LACR较同时表达外源szHasA、szHasB和szHasC更利于HA的合成。本论文利用lacF基因作为食品级筛选标记和NICE表达系统,构建得到一株不产生荚膜的具有食品级HA合成能力的工程菌株,而食品级HA在保健食品、医药产品和化妆品的应用中具有明显的优势。
HA的相对分子质量(Mr)一般分布在10~4 Da-10~7 Da范围内。研究表明,在细胞和组织中,HA生物活性与糖链长度相关,不同Mr的HA具有不同甚至相反的生理或药理作用,HA的多种生物活性具有Mr依赖性。通过差异表达HA合成相关酶类,脊椎动物具有在体内调控HA Mr的能力。而目前微生物发酵生产的HA是Mr从大到小的一系列糖链的组合。因此,在活体微生物中确定HA的Mr调控机制并且建立制备特定Mr或特定Mr范围分布的HA的方法,不但具有重要的理论意义,而且对扩展HA的应用范围、改良含HA产品的质量具有应用价值。体外实验已证明,合成能力与底物浓度的比率是影响HA Mr的主要因素之一,我们认为这一理论同样适用于活体微生物内。为实现乳酸乳球菌工程菌株中HA合成能力与底物浓度的比率的可控调节,本论文构建了一个双质粒的调控表达系统,包括表达szHasA的载体pNZ8148-szHasA和表达szHasB的载体pNZ9531-szHasB,而这两个质粒可共存于同一株乳酸乳球菌中。pNZ8148-szHasA包含有乳酸乳球菌常用载体pSH71的复制起始位点,而szHasA基因位于NICE系统启动子nisA的调控之下:而另一个载体pNZ9531-szHasB则含有另一个乳酸乳球菌常用载体pAMb1所携带的复制起始位点,同时szHasB基因的诱导表达受到lacR启动子的调控。蛋白表达结果证明在工程菌株NFHA04中,借助pNZ8148-szHasA和pNZ9531-szHasB组成的双质粒调控表达系统,实现了不同诱导条件下改变szHasA与szHasB之间表达水平比率的实验目的。szHasA和szHasB mRNA拷贝数之间的比率,表征着HA合成能力与HA底物合成能力之间的相对强弱。不同诱导条件下,分析szHasA/szHasB mRNA比率对乳酸乳球菌工程菌株NFHA04合成HA的重均分子量((?)w)的影响,在微生物体内证明了HA合成能力与底物合成能力之间的相对强弱具有调控HA(?)w的作用。当代表HAS和UDP-GlcDH之间相对强度的szHasA/szHasB mRNA比率高时,发酵液中HA的(?)w相对较小,而当szHasA/szHasB mRNA比率减小时,(?)w则变大。分析认为,合成HA能力与合成HA底物能力之间的相对强弱是影响微生物HA Mr的重要因素之一,szHasA和szHasB mRNA拷贝数之间的比率高时,活细胞体内每一个HAS分子能够分配到的用来合成HA的底物就相对较少,则每一个HAS分子能够合成的HA糖链就相对较短,从而造成合成的HA的(?)w就较小:而当这一比率降低时,每一个HAS分子就有相对较多的底物分子可以用来合成HA糖链,从而造成合成的HA的糖链较长,(?)w也变大。这一理论的确定,为通过调节微生物体内HAS的表达水平和底物浓度来控制微生物生产过程中发酵液中HA Mr特性提供了切实可行的解决方案,具有重大的理论意义和潜在应用价值。
本论文的主要内容:
(1)通过NICE系统,将兽疫链球菌HA合成代谢途径相关基因导入乳酸乳球菌,构建一系列工程菌株。分析各工程菌株合成HA的浓度,证明利用代谢工程手段在乳酸乳球菌中引入编码HAS的基因,可以该菌株获得利用葡萄糖合成HA的能力。但自身合成的HA底物的不足限制HAS在工程菌株中合成高浓度的HA,而提高工程菌株HA底物的浓度,有助于HA合成能力的提高。
(2)通过融合PCR方法得到一个重组HA合成操纵子,利用lacF基因作为食品级筛选标记和NICE表达系统,将该重组HA合成操纵子导入乳酸乳球菌,实现在同一株工程菌株中重组表达异源szHasA,同时超表达同源UDP-GlcDH和LACR,表达这三个合成HA关键酶的乳酸乳球菌工程菌株具备了合成食品级HA的能力。
(3)构建了一个双质粒的调控表达系统,包括表达szHasA的载体pNZ8148-szHasA和表达szHasB的载体pNZ9531-szHasB中。在工程菌株NFHA04中实现了不同诱导条件下改变szHasA与szHasB之间表达水平比率的实验目的。
(4)不同诱导条件下,分析szHasA/szHasB mRNA比率对乳酸乳球菌工程菌株NFHA04合成HA的重均分子量((?)w)的影响。证明合成HA能力与合成HA底物能力之间的相对强弱是影响微生物HA Mr的重要因素之一,szHasA和szHasB mRNA拷贝数之间的比率高时,活细胞体内每一个HAS分子能够分配到的用来合成HA的底物就相对较少,则每一个HAS分子能够合成的HA糖链就相对较短,从而造成合成的HA的(?)w就较小;而当这一比率降低时,每一个HAS分子就有相对较多的底物分子可以用来合成HA糖链,从而造成合成的HA的糖链较长,(?)w也变大。
本论文主要创新点:
(1)首次通过融合PCR方法得到重组HA合成操纵子。并首次在同一乳酸乳球菌工程菌株中实现了重组表达异源szHasA,同时超表达同源UDP-GlcDH和LACR。
(2)首次利用lacF基因作为食品级筛选标记和NICE表达系统,在乳酸乳球菌终建立了透明质酸合成途径。该乳酸乳球菌工程菌株具备合成食品级HA的能力。
(3)首次在乳酸乳球菌工程菌株中实现了兽疫链球菌HAS与UDPGlcDH的同时诱导可控表达。
(4)首次在活体微生物体内证明HA合成能力与底物合成能力之间的相对强弱具有调控HA(?)w的作用。这一理论的确定,为通过调节微生物体内HAS的表达水平和底物浓度来控制微生物生产过程中发酵液中HA Mr特性提供了切实可行的解决方案,具有重大的理论意义和潜在应用价值。
Hyaluronic acid(HA) is one kind of natural high molecular weight polysaccharide and plays structural,recognition and signaling roles in animals.HA has been used in medicines,cosmetics,drug delivery systems,vaccine aids,as well as in health foods. In industry,HA is produced using the gram-positive bacterium Streptococcus zooepidemicus. However,Streptococcus is a less-than-ideal source because of the potential to produce exotoxins,the difficulty in fermentation control or expensive medium.Therefore,it is meaningful and necessary to develop an alternative source of HA that avoids these pitfalls and enlarges the applications.
The relative molecular weight(Mr) of HA is generally ranging from 10~4 to 10~7 Da in vertebrates and bacteria,and the biological roles of HA correlate with the length of the HA chain.HAs of different lengths often appear to have antagonistic or reverse effects in many cellular and tissue systems.The solution of HA with sizes greater than 10 kDa,which has outstanding moisture keeping ability,high viscoelasticity and lubricity,has been widely used in cosmetics and medicines.Recently,several groups have reported that short chain HA(～2 kDa-3.5 kDa) or HA oligosaccharides(～10-20 sugars in length) have interesting effects on cellular behavior.To extend the applications of HA and make better HA-containing biomedical products,it is necessary to obtain specially designed Mr or uniform size-defined HA.Vertebrates may be able to control the length of HA in vivo by differential expression of biosynthetic enzymes,but HA fermented by microbes is a mixture of a wide range of molecular masses.The in vitro study data support the theory that the relative ratio of catalysts to the precursor sugars may be a major factor in HA size control.Therefore, it is meaningful and necessary to investigate the regulation mechanism of HA size in bacteria.
Lactoccus lactis,one of the most widely used Gram-positive lactic acid bacteria in food fermentations,is a generally regarded as safe(GRAS) strain and increasingly used in modern biotechnological applications.In virtue of great development of genetic engineering tools and the molecular characterization of this species in the last 25 years,a number of inducible expression promotors have been developed in L. lactis.Through these promotors,gene expression can be controlled by an inducer,a repressor or environmental factors,such as peptide,monosaccharide,or temperature. NICE(nisin controlled gene expression) system has become one of the most successful and widely used tools for regulating gene expression in Gram-positive bacteria.The lacF gene is commonly used as a food-grade selectable marker in the NICE system.In order to overcome the shortcomings of Streptococcus zooepidemicus as a HA production bacterium,we planned to construct some engineering GRAS recombinant strains based on Lactoecus lactis containing HA biosynthesis operon and lacF seletable marker.Another controllable expression system of L.lactis is based on the lacR promoter of the lac operon.The lacR promoter is a strong promoter that is virtually not subjected to catabolite repression and controlled by autoregulated LacR repressor.Both NICE system and the lacR promoter have been used to overexpress homologous or heterologous membrane-located proteins and regulate gene expression by quorum sensing-controlling in L.lactis.In this work,these characters of the lacR and the nisA promoters was used to regulate szHasA and szHasB expression,in investigating regulation mechanism of HA polymer size in recombinant L lactis. A series of enzymes are involved in the biosynthesis of HA in S.zooepidemicus, including hyaluronan synthase(HasA),UDP-glucose-6-dehydrogenase(HasB), UDP-glucose pyrophosphorylase(HasC) and glucosamine-1-phosphate N-acetyltransferase/UDP-N-acetylglucosamine pyrophosphorylase(HasD).HasA,the key enzyme in the production of HA,utilizes two sugar substrates(UDP-GlcA and UDP-GlcNAc) to synthesize HA.HasB,HasC and HasD are the enzymes in the synthesis pathway of the precursor sugars.UDP-glucose-6-dehydrogenase (UDP-GlcDH) and GlcN-1-P acetyl transferase/UDP-GlcNAc pyrophosphorylase (LACR) from L.lactis have the same catalyzing effects as HasB and HasD do. The szHasA,szHasB and szHasC were carried on pSH71-derived replicons and under the control of PnisA promoter in the expression plasmids pNZ8148-szHasA, pNZ8148-szHasB and pNZ8148-szHasC,respectively.The recombinant L.lactis strains with pNZ8148-szHasA,pNZ8148-szHasB and pNZ8148-szHasC were named as N9000-A,N9000-B and N9000-C.The HA biosynthesis operon of S.
zooepidemicus was introduced into L.lactis NZ9000 and NZ3900 with the vectors of pNZ8148-szHasABC and pNZ8149-szHasABC,respectively,producing strain NFHA01 and NFHA03.Meanwhile,a recombinant HA biosynthesis operon was constructed and named as rHaop.In this artificial DNA fragment,the ORFs of szHasB and szHasC were displaced by the ORFs of ugd and LACR from L.lactis respectively.Then rHAop was introduced into L.lactis NZ3900 with the vector pNZ8149-rHAop,producing strain NFHA02.
The concentrations of HA in the culture of these recombinant strains were analyzed and compared.About 0.22 g/L HA was detected in the NZ9000-A culture while HA cannot be detected in the control strain NZ9000,indicating that HA could be synthesized with the expression of HasA alone in L.lactis and the required precursors, UDP-GlcA and UDP-GIcNAc,could also be synthesized in the host strain.However, no HA was detected in the cultures of NZ9000-B and NZ9000-C.Furthermore,a final concentration of HA was about 0.6g/L in the NFHA01 culture,was about 170% higher than that of NZ9000-A,showing that HasA is essential for high heterologous HA production in L.lactis and improving the precursor sugar biosynthesis levels can obviously increase the production of HA.A final concentration of HA was about 0.49g/L in the NFHA03 culture,about 33%higher than that(about 0.38 g/L) in the culture of NFHA02..Therefore,it can be concluded that overexpression of the endogeneous enzymes,directing the steps in the synthesis pathway of the precursor sugars,is a more effective way to increase HA production than expressing exogenous enzymes in recombinant cells.
HA fermented by microbes was a mixture of a wide range of molecular masses,but the biological roles of HA are correlated with the length of the polysaccharide chain, which is one of the limiting-factors to extend the applications of HA and make better HA-containing biomedical products.So,it has theoretical and practical value to obtain specially designed(?)w or uniform size-defined HA by industrial fermentation. The mechanism for size control of HA product has begun to be investigated recently. Jing et al.(2004) proved that HA polymers of a desired size could be obtained by controlling the reaction stoichiometry,such as molar ratio of precursors and acceptor molecules.The theory that the relative strength of the interaction between the catalyst and the precursor sugars may be a major factor in HA size control has been testified by Pummill.et al.(2003) in vitro.We considered that this theory could also be suitable in bacterium cells and should be investigated in vivo.
To adjust the relative strength of the interaction between the catalyst and the precursor sugars,we obtained different ratios of HasA expression level to precursor sugar UDP-GlcA biosynthesis ability by regulating the szHasA and szHasB expression levels in a recombinant L.lactis.Two inducible expression promoters,nisA promoter and lacR promoter,were used in our work and gene expression could be controlled by nisin or lactose through these promoters.Thus,a dual-plasmid controlled expression system,which consisted of szHasA expression vector pNZ8148-szHasA and szHasB expression vector pNZ9531-szHasB,was constructed.The aim to regulate szHasA and szHasB expression levels in recombinant strain NFHA04 was accomplished by using this system.
The ratio of szHasA to szHasB mRNA copies,the surface features of the ratio of catalytic ability of HA synthesis to the precursor sugar biosynthesis level in cells,was regulated strictly by changing the inducing concentration collocations with nisin and lactose.We analyzed the Mw and the concentration of HA under the different ratios, discovering that the theory that the relative strength of the interaction between the catalyst and the precursor sugar may be a major factor in HA size control was also suitable for in vivo HA synthesis of microbes,as we had expected.The average size of HA produced in high HasA/HasB mRNA ratio conditions was smaller than that in low ratio conditions.Altering the ratio of HA synthesizing ability to the concentration of precursor sugars might cause a change in HA size.The reason is probably that the distribution of precursor sugars among the HasA molecules is altered by changing HasA expression level and the precursor sugar biosynthesis ability.The distribution of precursor sugars to each HasA under the inducing condition of high concentration of nisin and low concentration of lactose would be relatively less than under the reverse inducing condition,so each HA polysaccharide chain gets less precursor sugars to lengthen itself catalyzed by HasA in the membrane.In the present study,the ratio of HasA expression level to the precursor sugars biosynthesis ability in bacteria, standing for the relative strength of the interaction between the catalyst and the precursor sugars,was proved to be an important facter for the size control of HA in bacteria.It has theoretical and practical value to determine the regulation mechanism of HA polymer size and this study puts forward a guide for establishing an efficacious way to control the size of HA in fermentation.
引文
Albrecht, C, Schlegel, W., Eckl, P., Jagersberger, T., Sadeghi, K., Berger, A., Vecsei,V., & Marlovits, S. (2009). Alterations in CD44 isoforms and HAS expression in human articular chondrocytes during the de- and re-differentiation processes. Int J Mol Med, 23(2), 253-259.
Armstrong, D. C, & Johns, M. R. (1997). Culture Conditions Affect the Molecular Weight Properties of Hyaluronic Acid Produced by Streptococcus zooepidemicus. Appl Environ Microbiol, 63(7), 2759-2764.
Bermudez-Humaran, L. G. (2009). Lactococcus lactis as a live vector for mucosal delivery of therapeutic proteins. Hum Vaccin, 5(4).
Blank, L. M., Hugenholtz, P., & Nielsen, L. K. (2008). Evolution of the Hyaluronic Acid Synthesis (has) Operon in Streptococcus zooepidemicus and Other Pathogenic Streptococci. J Mol Evol, 67(1), 13-22.
Bodevin-Authelet, S., Kusche-Gullberg, M., Pummill, P. E., DeAngelis, P. L., & Lindahl, U. (2005). Biosynthesis of hyaluronan: direction of chain elongation. J Biol Chem, 280(10), 8813-8818.
Boels, I. C, Kleerebezem, M, & de Vos, W. M. (2003). Engineering of carbon distribution between glycolysis and sugar nucleotide biosynthesis in Lactococcus lactis. Appl Environ Microbiol, 69(2), 1129-1135.
Brown, S. H., & Pummill, P. E. (2008). Recombinant production of hyaluronic acid. Curr Pharm Biotechnol, 9(4), 239-241.
Bustin, S. A. (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol, 25(2), 169-193.
Casadaban, M. J., & Cohen, S. N. (1980). Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol, 138(2), 179-207.
Chien, L. J., & Lee, C. K. (2007). Hyaluronic acid production by recombinant Lactococcus lactis. Appl Microbiol Biotechnol, 77(2), 339-346.
Chong, B. F., Blank, L. M., McLaughlin, R., & Nielsen, L. K. (2005). Microbial hyaluronic acid production. Appl Microbiol Biotechnol, 66(4), 341-351.
Colon, G. E., Jetten, M. S., Nguyen, T. T., Gubler, M. E., Follettie, M. T., Sinskey, A. J., & Stephanopoulos, G (1995). Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799. Appl Environ Micmbiol, 67(1), 74-78.
Crescenzi, V. (1995). Microbial polysaccharides of applied interest: ongoing research activities in Europe. Biotechnol Prog, 11(3), 251-259.
de Ruyter, P. G, Kuipers, O. P., Beerthuyzen, M. M., van Alen-Boerrigter, I., & de Vos,W. M. (1996a). Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis. J Bacteriol, 178(12), 3434-3439.
de Ruyter, P. G, Kuipers, O. P., & de Vos, W. M. (1996b). Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol, 62(10), 3662-3667.
de Vos, W. M. (1999). Gene expression systems for lactic acid bacteria. Curr Opin Microbiol, 2(3), 289-295.
de Vos, W. M. (2001). Advances in genomics for microbial food fermentations and safety. Curr Opin Biotechnol, 12(5), 493-498.
de Vos, W. M., Boerrigter, I., van Rooyen, R. J., Reiche, B., & Hengstenberg, W.(1990). Characterization of the lactose-specific enzymes of the phosphotransferase system in Lactococcus lactis. J Biol Chem, 265(36), 22554-22560.
DeAngelis, P. L. (1996). Enzymological characterization of the Pasteurella multocida hyaluronic acid synthase. Biochemistry, 35(30), 9768-9771.
DeAngelis, P. L. (1999a). Hyaluronan synthases: fascinating glycosyltransferases from vertebrates, bacterial pathogens, and algal viruses. Cell Mol Life Sci, 56(7-8),670-682.
DeAngelis, P. L. (1999b). Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase. J Biol Chem, 274(37),26557-26562.
DeAngelis, P. L. (2002a). Evolution of glycosaminoglycans and their glycosyltransferases: Implications for the extracellular matrices of animals and the capsules of pathogenic bacteria. Anat Rec, 268(3), 317-326.
DeAngelis, P. L. (2002b). Microbial glycosaminoglycan glycosyltransferases.Glycobiology, 72(1), 9R-16R.
DeAngelis,P.L.,Jing,W.,Graves,M.V.,Burbank,D.E.,& Van Etten,J.L.(1997).Hyaluronan synthase of chlorella virus PBCV-1.Science,278(5344),1800-1803.
DeAngelis,P.L.,Oatman,L.C.,& Gay,D.F.(2003).Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors.J Biol Chem,278(37),35199-35203.
DeAngelis,P.L.,Papaconstantinou,J.,& Weigel,P.H.(1993a).Isolation of a Streptococcus pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria.J Biol Chem,268(20),14568-14571.
DeAngelis,P.L.,Papaconstantinou,J.,& Weigel,P.H.(1993b).Molecular cloning,identification,and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes.J Biol Chem,268(26),19181-19184.
Deangelis,P.L.,& White,C.L.(2004).Identification of a distinct,cryptic heparosan synthase from Pasteurella multocida types A,D,and F.J Bacteriol,186(24),8529-8532.
Deed,R.,Rooney,P.,Kumar,P.,Norton,J.D.,Smith,J.,Freemont,A.J.,& Kumar,S.(1997).Early-response gene signalling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells.Inhibition by non-angiogenic,high-molecular-weight hyaluronan.Int J Cancer,71(2),251-256.
Dieye,Y.,Usai,S.,Clier,F.,Gruss,A.,& Piard,J.C.(2001).Design of a protein-targeting system for lactic acid bacteria.J Bacteriol,183(14),4157-4166.
Eichenbaum,Z.,Federle,M.J.,Marra,D.,de Vos,W.M.,Kuipers,O.P.,Kleerebezem,M.,& Scott,J.R.(1998).Use of the lactococcal nisA promoter to regulate gene expression in gram-positive bacteria:comparison of induction level and promoter strength.App! Environ Microbiol,64(8),2763-2769.
Golshani,R.,Lopez,L.,Estrella,V.,Kramer,M.,Iida,N.,& Lokeshwar,V.B.(2008).Hyaluronic acid synthase-1 expression regulates bladder cancer growth,invasion,and angiogenesis through CD44.Cancer Res,68(2),483-491.
郭学平,凌沛学,&张天民(2002).低分子量及寡聚玻璃酸,山东省药学会第六次生化药物学术研讨会论文集,日照。
Hoshi,H.,Nakagawa,H.,Nishiguchi,S.,Iwata,K.,Niikura,K.,Monde,K.,&Nishimura,S.(2004).An engineered hyaluronan synthase:characterization for recombinant human hyaluronan synthase 2 Escherichia coli. J Biol Chem, 279(4),2341-2349.
Huang, S. L., Ling, P. X., & Zhang, T. M. (2007). Oral absorption of hyaluronic acid and phospholipids complexes in rats. World J Gastroenterol, 13(6), 945-949.
Jing, W., & DeAngelis, P. L. (2003). Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida. Glycobiology, 13(10),661-671.
Jing, W., & DeAngelis, P. L. (2004). Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers. J Biol Chem, 279(40), 42345-42349.
Kleerebezem, M., Beerthuyzen, M. M., Vaughan, E. E., de Vos, W. M., & Kuipers, O.P. (1997a). Controlled gene expression systems for lactic acid bacteria: transferablenisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl Environ Microbiol, 65(11), 4581-4584.
Kleerebezem, M., Quadri, L. E., Kuipers, O. P., & de Vos, W. M. (1997b). Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria. Mol Microbiol, 24(5), 895-904.
Kuipers, O. P., de Ruyter, P. G., Kleerebezem, M., & de Vos, W. M. (1997). Controlled overproduction of proteins by lactic acid bacteria. Trends Biotechnol, 15(4), 135-140.
Kumari, K., Tlapak-Simmons, V. L., Baggenstoss, B. A., & Weigel, P. H. (2002). The streptococcal hyaluronan synthases are inhibited by sulfhydryl-modifying reagents,but conserved cysteine residues are not essential for enzyme function. J Biol Chem,277(16), 13943-13951.
Kumari, K., & Weigel, P. H. (1997). Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis. J Biol Chem, 272(51), 32539-32546.
Lee, C. Y., Bagdasarian, M., Meng, M. H., & Zeikus, J. G. (1990). Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes.凌沛学 (2007), 透明质酸,中国轻工业出版社。
Characterization of the structural gene and function of active site histidine. J Biol Chem, 265(31), 19082-19090.
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Armstrong, D. C, & Johns, M. R. (1997). Culture Conditions Affect the Molecular Weight Properties of Hyaluronic Acid Produced by Streptococcus zooepidemicus. Appl Environ Microbiol, 63(7), 2759-2764.
Bermudez-Humaran, L. G. (2009). Lactococcus lactis as a live vector for mucosal delivery of therapeutic proteins. Hum Vaccin, 5(4).
Blank, L. M., Hugenholtz, P., & Nielsen, L. K. (2008). Evolution of the Hyaluronic Acid Synthesis (has) Operon in Streptococcus zooepidemicus and Other Pathogenic Streptococci. J Mol Evol, 67(1), 13-22.
Bodevin-Authelet, S., Kusche-Gullberg, M., Pummill, P. E., DeAngelis, P. L., & Lindahl, U. (2005). Biosynthesis of hyaluronan: direction of chain elongation. J Biol Chem, 280(10), 8813-8818.
Boels, I. C, Kleerebezem, M, & de Vos, W. M. (2003). Engineering of carbon distribution between glycolysis and sugar nucleotide biosynthesis in Lactococcus lactis. Appl Environ Microbiol, 69(2), 1129-1135.
Brown, S. H., & Pummill, P. E. (2008). Recombinant production of hyaluronic acid. Curr Pharm Biotechnol, 9(4), 239-241.
Bustin, S. A. (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol, 25(2), 169-193.
Casadaban, M. J., & Cohen, S. N. (1980). Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol, 138(2), 179-207.
Chien, L. J., & Lee, C. K. (2007). Hyaluronic acid production by recombinant Lactococcus lactis. Appl Microbiol Biotechnol, 77(2), 339-346.
Chong, B. F., Blank, L. M., McLaughlin, R., & Nielsen, L. K. (2005). Microbial hyaluronic acid production. Appl Microbiol Biotechnol, 66(4), 341-351.
Colon, G. E., Jetten, M. S., Nguyen, T. T., Gubler, M. E., Follettie, M. T., Sinskey, A. J., & Stephanopoulos, G (1995). Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799. Appl Environ Micmbiol, 67(1), 74-78.
Crescenzi, V. (1995). Microbial polysaccharides of applied interest: ongoing research activities in Europe. Biotechnol Prog, 11(3), 251-259.
de Ruyter, P. G, Kuipers, O. P., Beerthuyzen, M. M., van Alen-Boerrigter, I., & de Vos,W. M. (1996a). Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis. J Bacteriol, 178(12), 3434-3439.
de Ruyter, P. G, Kuipers, O. P., & de Vos, W. M. (1996b). Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol, 62(10), 3662-3667.
de Vos, W. M. (1999). Gene expression systems for lactic acid bacteria. Curr Opin Microbiol, 2(3), 289-295.
de Vos, W. M. (2001). Advances in genomics for microbial food fermentations and safety. Curr Opin Biotechnol, 12(5), 493-498.
de Vos, W. M., Boerrigter, I., van Rooyen, R. J., Reiche, B., & Hengstenberg, W.(1990). Characterization of the lactose-specific enzymes of the phosphotransferase system in Lactococcus lactis. J Biol Chem, 265(36), 22554-22560.
DeAngelis, P. L. (1996). Enzymological characterization of the Pasteurella multocida hyaluronic acid synthase. Biochemistry, 35(30), 9768-9771.
DeAngelis, P. L. (1999a). Hyaluronan synthases: fascinating glycosyltransferases from vertebrates, bacterial pathogens, and algal viruses. Cell Mol Life Sci, 56(7-8),670-682.
DeAngelis, P. L. (1999b). Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase. J Biol Chem, 274(37),26557-26562.
DeAngelis, P. L. (2002a). Evolution of glycosaminoglycans and their glycosyltransferases: Implications for the extracellular matrices of animals and the capsules of pathogenic bacteria. Anat Rec, 268(3), 317-326.
DeAngelis, P. L. (2002b). Microbial glycosaminoglycan glycosyltransferases.Glycobiology, 72(1), 9R-16R.
DeAngelis,P.L.,Jing,W.,Graves,M.V.,Burbank,D.E.,& Van Etten,J.L.(1997).Hyaluronan synthase of chlorella virus PBCV-1.Science,278(5344),1800-1803.
DeAngelis,P.L.,Oatman,L.C.,& Gay,D.F.(2003).Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors.J Biol Chem,278(37),35199-35203.
DeAngelis,P.L.,Papaconstantinou,J.,& Weigel,P.H.(1993a).Isolation of a Streptococcus pyogenes gene locus that directs hyaluronan biosynthesis in acapsular mutants and in heterologous bacteria.J Biol Chem,268(20),14568-14571.
DeAngelis,P.L.,Papaconstantinou,J.,& Weigel,P.H.(1993b).Molecular cloning,identification,and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes.J Biol Chem,268(26),19181-19184.
Deangelis,P.L.,& White,C.L.(2004).Identification of a distinct,cryptic heparosan synthase from Pasteurella multocida types A,D,and F.J Bacteriol,186(24),8529-8532.
Deed,R.,Rooney,P.,Kumar,P.,Norton,J.D.,Smith,J.,Freemont,A.J.,& Kumar,S.(1997).Early-response gene signalling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells.Inhibition by non-angiogenic,high-molecular-weight hyaluronan.Int J Cancer,71(2),251-256.
Dieye,Y.,Usai,S.,Clier,F.,Gruss,A.,& Piard,J.C.(2001).Design of a protein-targeting system for lactic acid bacteria.J Bacteriol,183(14),4157-4166.
Eichenbaum,Z.,Federle,M.J.,Marra,D.,de Vos,W.M.,Kuipers,O.P.,Kleerebezem,M.,& Scott,J.R.(1998).Use of the lactococcal nisA promoter to regulate gene expression in gram-positive bacteria:comparison of induction level and promoter strength.App! Environ Microbiol,64(8),2763-2769.
Golshani,R.,Lopez,L.,Estrella,V.,Kramer,M.,Iida,N.,& Lokeshwar,V.B.(2008).Hyaluronic acid synthase-1 expression regulates bladder cancer growth,invasion,and angiogenesis through CD44.Cancer Res,68(2),483-491.
郭学平,凌沛学,&张天民(2002).低分子量及寡聚玻璃酸,山东省药学会第六次生化药物学术研讨会论文集,日照。
Hoshi,H.,Nakagawa,H.,Nishiguchi,S.,Iwata,K.,Niikura,K.,Monde,K.,&Nishimura,S.(2004).An engineered hyaluronan synthase:characterization for recombinant human hyaluronan synthase 2 Escherichia coli. J Biol Chem, 279(4),2341-2349.
Huang, S. L., Ling, P. X., & Zhang, T. M. (2007). Oral absorption of hyaluronic acid and phospholipids complexes in rats. World J Gastroenterol, 13(6), 945-949.
Jing, W., & DeAngelis, P. L. (2003). Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida. Glycobiology, 13(10),661-671.
Jing, W., & DeAngelis, P. L. (2004). Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers. J Biol Chem, 279(40), 42345-42349.
Kleerebezem, M., Beerthuyzen, M. M., Vaughan, E. E., de Vos, W. M., & Kuipers, O.P. (1997a). Controlled gene expression systems for lactic acid bacteria: transferablenisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl Environ Microbiol, 65(11), 4581-4584.
Kleerebezem, M., Quadri, L. E., Kuipers, O. P., & de Vos, W. M. (1997b). Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria. Mol Microbiol, 24(5), 895-904.
Kuipers, O. P., de Ruyter, P. G., Kleerebezem, M., & de Vos, W. M. (1997). Controlled overproduction of proteins by lactic acid bacteria. Trends Biotechnol, 15(4), 135-140.
Kumari, K., Tlapak-Simmons, V. L., Baggenstoss, B. A., & Weigel, P. H. (2002). The streptococcal hyaluronan synthases are inhibited by sulfhydryl-modifying reagents,but conserved cysteine residues are not essential for enzyme function. J Biol Chem,277(16), 13943-13951.
Kumari, K., & Weigel, P. H. (1997). Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis. J Biol Chem, 272(51), 32539-32546.
Lee, C. Y., Bagdasarian, M., Meng, M. H., & Zeikus, J. G. (1990). Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes.凌沛学 (2007), 透明质酸,中国轻工业出版社。
Characterization of the structural gene and function of active site histidine. J Biol Chem, 265(31), 19082-19090.
Maguin, E., Prevost, H., Ehrlich, S. D., & Grass, A. (1996). Efficient insertional mutagenesis in lactococci and other gram-positive bacteria. J Bacteriol, 178(3),931-935.
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