红色亚栖热菌海藻糖合成酶相关基因的克隆、表达以及海藻糖代谢途径的研究
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摘要
海藻糖因其具有保护生物分子活性的功能,被广泛应用于食品加工业、农业、医药业等诸多领域。在海藻糖的酶转化法工业生产中,海藻糖合酶(trehalosesynthase)只需一步反应即可将麦芽糖转化为海藻糖,生产原料可来源于淀粉的水解产物,更为廉价,因此备受关注。而筛选稳定的、转化率高的、适合工业生产的海藻糖合酶,并构建高表达的工程菌株,成为降低海藻糖生产成本的关键因素。
在本实验的前期工作中,从地热水中筛选得到一株能够生产海藻糖合酶的耐热菌株CBS-01。对其海藻糖合酶的初步分析显示,该酶的最适反应温度为50℃,最适pH为6.5,在50℃以下和pH 5.0-9.0的范围内该酶能够保持80%以上的酶活。鉴于该海藻糖合酶具有较强的稳定性,有必要克隆得到该酶的基因并构建工程菌株,为海藻糖的工业生产奠定基础;另一方面,也有必要对该酶的结构、作用机制以及耐热机制进行研究,为进一步提高该酶的热稳定性和转化率提供充分的理论依据。
首先,对这株能够生产海藻糖合酶的菌株进行了鉴定。该菌为革兰氏阴性的杆菌,多个细胞相连接可形成丝状,能够产生暗红色色素,好氧,生长的最适温度和pH分别为55℃～60℃和7.5～8.5。在生理生化试验中,该菌与标准菌株红色亚栖热菌(Meiothermus ruber ATCC 35948)的测试结果完全一致。该菌基因组的G+C含量为62.1%,与红色亚栖热菌标准菌株的DNA-DNA杂交率为63.9%。该菌的16srDNA基因与Genbank中红色亚栖热菌呈现最大的相似性。综合以上实验结果,该菌被鉴定为亚栖热菌属红色亚栖热菌(Meiothermus ruber CBS-01)。
第二,克隆得到了海藻糖合酶的基因。根据已报道的海藻糖合酶同源序列中保守的氨基酸序列设计简并引物进行PCR,结果得到一长度为762bp,编码254个氨基酸的基因片段。通过对其编码的氨基酸序列的相似性搜索表明,所扩增片段为新的潜在的海藻糖合酶基因的部分DNA序列。在此基础上,采用接头PCR、TAIL-PCR以及混合质粒PCR筛选基因组DNA文库的方法共得到全长为6200 bp的核苷酸序列。序列分析发现,该序列中共含有三个ORF区:第一个ORF区的长度为1350bp、编码449个氨基酸;第二个ORF区的长度为690 bp、编码229个氨基酸;第三个ORF区的长度为2889 bp、编码962个氨基酸;通过比对发现这三个ORF区分别是潜在的6-磷酸海藻糖合成酶(TPS)、6-磷酸海藻糖磷酸酶(TPP)和海藻糖合酶(TreS)基因。即在红色亚栖热菌CBS-01中存在两条海藻糖的合成途径,而且这三个基因很可能位于同一个多顺反子中。通过对所得到的潜在的海藻糖合酶氨基酸序列的分析,发现红色亚栖热菌的海藻糖合酶与栖热菌属的海藻糖合酶的同源性最高,且蛋白的分子量远远大于其他种属的海藻糖合酶,预示所发现的海藻糖合酶可能具有较好的热稳定性。同时还发现,克隆得到的潜在的海藻糖合酶属于α-淀粉酶家族,在其氨基酸序列中具有四个α-淀粉酶家族的保守区。这些结果也进一步证实所得到的序列为潜在的海藻糖合酶基因。
第三,构建了生产海藻糖合酶的工程菌株,并优化了表达条件。将潜在的编码海藻糖合酶的基因序列转化到大肠杆菌中进行表达,通过高效液相色谱(HPLC)检测了粗酶液的酶活。结果显示,该序列所编码的蛋白具有海藻糖合酶的活性,能够实现麦芽糖和海藻糖之间的相互转化反应,并有少量副产物葡萄糖生成。该基因序列已经注册到NCBI的GenBank中(序列接收号为EU443098)。为了增加海藻糖合酶在大肠杆菌中的表达量,利用PCR定点突变技术,将海藻糖合酶基因5'末端的核苷酸进行了优化,大幅度提高了该酶的表达量。同时通过对诱导表达条件的优化,确定了发酵生产海藻糖合酶的最适条件:采用pET-21a载体在胞质中表达目的蛋白,选用E.coli Rosetta-gami(DE3)作为宿主菌,20℃、低转速过夜诱导,以降低包涵体的生成,获得更多的可溶且有活性的目的蛋白。利用所构建的大肠杆菌工程菌株发酵生产海藻糖合酶的比活为0.60U/ml发酵液,比发酵红色亚栖热菌的0.08U/ml提高了大约8倍。
第四,纯化了重组海藻糖合酶,并对其酶学性质做了详细的研究。利用NTA-Ni柱亲和层析法纯化得到了重组海藻糖合酶。测定了重组海藻糖合酶的各项动力学常数,并发现葡萄糖是该反应的竞争性抑制剂。测定海藻糖合酶的酶活为80U/mg,最适反应温度为50℃,最适pH为6.0-7.0。并且发现该酶在较宽的温度(4-60℃)和pH值(pH 4.0-8.5)范围内都极其稳定;更为突出的是,该酶在60℃中保温5h后,残余酶活仍然能够达到90%以上,而且4℃冷藏保存2个月对该酶的活性几乎不会产生影响。从而证明该酶非常适合工业化生产海藻糖。同时,实验还确定在低温下反应更利于降低副产物葡萄糖的形成,提高海藻糖的产量。以麦芽糖作为底物,20℃下反应,最终可以得到大约64%的海藻糖,而副产物葡萄糖的含量不到2%。
第五,构建了缺失C端结构域的海藻糖合酶蛋白,发现该蛋白并不具有海藻糖合酶的活性。根据Native-PAGE的结果,TreS以四聚体形式存在,而TreSΔC蛋白形成二聚体结构。从而,我们推测来源于红色亚栖热菌的海藻糖合酶是以四聚体的形式行使功能,而C端结构域对四聚体的形成具有十分重要的作用,该结构域的缺失导致酶不能以正确的构象存在,故而活性丧失。
第六,对红色亚栖热菌的海藻糖代谢途径做了初步研究。将潜在的编码TPS和TPP的基因序列转化到大肠杆菌中进行表达,利用NTA-Ni柱纯化得到了两个蛋白,并通过薄层层析(TLC)检测到了其酶活。基因序列已经注册到NCBI的GenBank中(序列接收号分别为FJ360766和FJ360767)。同时,研究发现红色亚栖热菌在高盐环境压力下胞内积累的内溶物为6-磷酸海藻糖。我们推测6-磷酸海藻糖磷酸酶在表达方面可能具有一定缺陷,不能将生成的6-磷酸海藻糖转化为海藻糖。
Trehalose is widely used in food processing industry, agriculture, pharmaceutical industry and many other fields, because of its ability to protect the biological molecules. In industrial production of trehalose, trehalose synthase (TreS) has been getting so much concern. That was because TreS can convert maltose into trehalose in one step reaction, and the cheaper raw material can be derived from starch hydrolysates. Screening of trehalose synthase gene and building a high-expression strain become key factors in decreasing the cost of trehalose industrial production.
At the early study of our laboratory, a thermophilic strain CBS-01 producing trehalose synthase was isolated from geothermal water. The preliminary analysis of the TreS showed that the optimal reaction temperature is 50℃, and the optimum reaction pH is 6.5. The enzyme can maintain more than 80% of its activity below 50℃and at pH range of 5.0-9.0. In view of the trehalose synthase with strong stability, it is necessary to clone the TreS gene and build engineering strains producing TreS for trehalose industrial production. On the other hand, it is necessary to study the structure, reaction mechanism and thermoadapatation mechanism of the enzyme, so that modified TreS with stronger thermostability and a favorable conversion rate could be obtained by molecular biological technology.
First of all, this strain producing trehalose synthase was identified. It was rod, Gram-negative, aerobic, and could produce dark red pigment. The optimum temperature and pH of its growth are 55℃～60℃and 7.5～8.5, respectively. At physiological and biochemical experiments, the strain CBS-01 showed the same results as the standard strains of Meiothermus ruber ATCC 35948. The G+C content of its genome was 62.1%, and the DNA-DNA hybridization rate with M. ruber ATCC 35948 was 63.9%. The sequence of 16s rDNA gene of CBS-01 showed the greatest identity to M. ruber in Genbank. Based on the above experimental results, the strain producing TreS was identified as Meiothermus ruber.
Secondly, the trehalose synthase gene was cloned. According to the conserved amino acid sequences of trehalose synthase reported, degenerate primers were designed for PCR. A fragment of 762 bp was obtained. The deduced amino acid sequence of the DNA product had high identity to other trehalose synthase, indicating that a partial putative
TreS gene was isolated from M. ruber. On this basis, A 6200 bp DNA sequence containing three open reading frames was isolated, by using linker mediated PCR, TAIL-PCR, and PCR-based method for screening genomic DNA library. The length of the three ORFs were 1350, 690, and 2889 bp, encoding 449, 229, and 962 amino acids, respectively. Sequence alignment analysis revealed that the deduced amino acid sequences of these ORFs were potential trehalose 6-phosphate synthase (TPS), trehalose 6-phosphate phosphatase (TPP) and trehalose synthase (TreS), respectively. Hence, there were two trehalose synthetic pathways in M. ruber CBS-01 and these three genes are likely to exist in the same operon-like structure. The potential TreS from M.ruber showed the highest homology to those of Thermus genus, and the molecular weight was much larger than those from mesophilic bacteria, indicating the TreS was likely to have better thermal stability. At the same time, it was found that the potential trehalose synthase belonged to theα-amylase family. These results indicated a putative TreS gene was isolated.
Third, an engineering strain producing TreS was built, and protein expression conditions were optimized. The potential trehalose synthase gene was transformed into E. coli and induced to express. The activity of crude enzyme solution was detected by using high-performance liquid chromatography (HPLC). The result revealed that the recombinant protein showed the activity of trehalose synthase, and could catalyze the conversion reaction of maltose into trehalose with glucose as the byproduct. This novel gene has been assigned in GenBank, the accession No. is EU443098. In order to increase the yield of trehalose synthase in E. coli, the 5' terminal nucleotides of the TreS were optimized by using PCR-based site-directed mutagenesis technology. At the same time, conditions of protein expression were also optimized, and the optimum conditions were below: using pET-21a as expression vector and E. coli Rosetta-gami (DE3) as host bacterium, the induction was carried out at 20℃, low speed, and overnight. The results suggested the formation of inclusion bodies was decreased, and the yield of soluble and active protein was increased. The TreS activity was 0.60 U/ml broth by fermentation of E. coli engineering strain, and it was about 8-fold than that of M. ruber.
Fourth, the recombinant TreS was purified and its enzymatic properties were studied in detail. The TreS was purified by using NTA-Ni column affinity chromatography. The kinetic values of the enzyme were determined, and glucose was found a competitive inhibitor of the reaction. The activity of the recombinant TreS was 80 U/mg, the optimum reaction temperature was 50℃, the optimum pH was 6.0-7.0. It was found that the enzyme could maintain 90% of its activity at a wide temperature (4-60℃) and pH range (pH 4.0-8.5). Because of its stability, the TreS proved very suitable for industrial production of trehalose. Meanwhile, it was found that low reaction temperature could decrease the formation of glucose, and increased the yield of trehalose.
Fifth, the vector for expressing the truncated TreS without C-terminal domain was constructed. It was found that the TreSΔC protein did not have the TreS activity. According to the Native-PAGE results, TreS was a tetramer, but TreSΔC was a dimer. The results suggested that the C-terminal domain of TreS played an important role in the formation of the tetramer, which had the TreS activity. However, TreSΔC without C-terminal domain could not form a tetramer, so its activity lost.
Sixth, the trehalose biosynthetic pathway of M. ruber was studied. The potential TPS and TPP gene were transformed into E. coli, and the recombinant proteins were purified by using NTA-Ni column. The results of thin layer chromatography (TLC) revealed that the two proteins showed the activity of TPS and TPP, respectively. The novel genes have been assigned in GenBank, the accession No. were FJ360766 and FJ360767. At the same time, it was found that M. ruber could accumulate 6-phosphate trehalose as the compatible solute in response to water stress imposed by salt. The results suggested that the TPP expression of M. ruber was defective, so the 6-trehalose phosphate producted by TPS could not be converted into trehalose.
在本实验的前期工作中,从地热水中筛选得到一株能够生产海藻糖合酶的耐热菌株CBS-01。对其海藻糖合酶的初步分析显示,该酶的最适反应温度为50℃,最适pH为6.5,在50℃以下和pH 5.0-9.0的范围内该酶能够保持80%以上的酶活。鉴于该海藻糖合酶具有较强的稳定性,有必要克隆得到该酶的基因并构建工程菌株,为海藻糖的工业生产奠定基础;另一方面,也有必要对该酶的结构、作用机制以及耐热机制进行研究,为进一步提高该酶的热稳定性和转化率提供充分的理论依据。
首先,对这株能够生产海藻糖合酶的菌株进行了鉴定。该菌为革兰氏阴性的杆菌,多个细胞相连接可形成丝状,能够产生暗红色色素,好氧,生长的最适温度和pH分别为55℃～60℃和7.5～8.5。在生理生化试验中,该菌与标准菌株红色亚栖热菌(Meiothermus ruber ATCC 35948)的测试结果完全一致。该菌基因组的G+C含量为62.1%,与红色亚栖热菌标准菌株的DNA-DNA杂交率为63.9%。该菌的16srDNA基因与Genbank中红色亚栖热菌呈现最大的相似性。综合以上实验结果,该菌被鉴定为亚栖热菌属红色亚栖热菌(Meiothermus ruber CBS-01)。
第二,克隆得到了海藻糖合酶的基因。根据已报道的海藻糖合酶同源序列中保守的氨基酸序列设计简并引物进行PCR,结果得到一长度为762bp,编码254个氨基酸的基因片段。通过对其编码的氨基酸序列的相似性搜索表明,所扩增片段为新的潜在的海藻糖合酶基因的部分DNA序列。在此基础上,采用接头PCR、TAIL-PCR以及混合质粒PCR筛选基因组DNA文库的方法共得到全长为6200 bp的核苷酸序列。序列分析发现,该序列中共含有三个ORF区:第一个ORF区的长度为1350bp、编码449个氨基酸;第二个ORF区的长度为690 bp、编码229个氨基酸;第三个ORF区的长度为2889 bp、编码962个氨基酸;通过比对发现这三个ORF区分别是潜在的6-磷酸海藻糖合成酶(TPS)、6-磷酸海藻糖磷酸酶(TPP)和海藻糖合酶(TreS)基因。即在红色亚栖热菌CBS-01中存在两条海藻糖的合成途径,而且这三个基因很可能位于同一个多顺反子中。通过对所得到的潜在的海藻糖合酶氨基酸序列的分析,发现红色亚栖热菌的海藻糖合酶与栖热菌属的海藻糖合酶的同源性最高,且蛋白的分子量远远大于其他种属的海藻糖合酶,预示所发现的海藻糖合酶可能具有较好的热稳定性。同时还发现,克隆得到的潜在的海藻糖合酶属于α-淀粉酶家族,在其氨基酸序列中具有四个α-淀粉酶家族的保守区。这些结果也进一步证实所得到的序列为潜在的海藻糖合酶基因。
第三,构建了生产海藻糖合酶的工程菌株,并优化了表达条件。将潜在的编码海藻糖合酶的基因序列转化到大肠杆菌中进行表达,通过高效液相色谱(HPLC)检测了粗酶液的酶活。结果显示,该序列所编码的蛋白具有海藻糖合酶的活性,能够实现麦芽糖和海藻糖之间的相互转化反应,并有少量副产物葡萄糖生成。该基因序列已经注册到NCBI的GenBank中(序列接收号为EU443098)。为了增加海藻糖合酶在大肠杆菌中的表达量,利用PCR定点突变技术,将海藻糖合酶基因5'末端的核苷酸进行了优化,大幅度提高了该酶的表达量。同时通过对诱导表达条件的优化,确定了发酵生产海藻糖合酶的最适条件:采用pET-21a载体在胞质中表达目的蛋白,选用E.coli Rosetta-gami(DE3)作为宿主菌,20℃、低转速过夜诱导,以降低包涵体的生成,获得更多的可溶且有活性的目的蛋白。利用所构建的大肠杆菌工程菌株发酵生产海藻糖合酶的比活为0.60U/ml发酵液,比发酵红色亚栖热菌的0.08U/ml提高了大约8倍。
第四,纯化了重组海藻糖合酶,并对其酶学性质做了详细的研究。利用NTA-Ni柱亲和层析法纯化得到了重组海藻糖合酶。测定了重组海藻糖合酶的各项动力学常数,并发现葡萄糖是该反应的竞争性抑制剂。测定海藻糖合酶的酶活为80U/mg,最适反应温度为50℃,最适pH为6.0-7.0。并且发现该酶在较宽的温度(4-60℃)和pH值(pH 4.0-8.5)范围内都极其稳定;更为突出的是,该酶在60℃中保温5h后,残余酶活仍然能够达到90%以上,而且4℃冷藏保存2个月对该酶的活性几乎不会产生影响。从而证明该酶非常适合工业化生产海藻糖。同时,实验还确定在低温下反应更利于降低副产物葡萄糖的形成,提高海藻糖的产量。以麦芽糖作为底物,20℃下反应,最终可以得到大约64%的海藻糖,而副产物葡萄糖的含量不到2%。
第五,构建了缺失C端结构域的海藻糖合酶蛋白,发现该蛋白并不具有海藻糖合酶的活性。根据Native-PAGE的结果,TreS以四聚体形式存在,而TreSΔC蛋白形成二聚体结构。从而,我们推测来源于红色亚栖热菌的海藻糖合酶是以四聚体的形式行使功能,而C端结构域对四聚体的形成具有十分重要的作用,该结构域的缺失导致酶不能以正确的构象存在,故而活性丧失。
第六,对红色亚栖热菌的海藻糖代谢途径做了初步研究。将潜在的编码TPS和TPP的基因序列转化到大肠杆菌中进行表达,利用NTA-Ni柱纯化得到了两个蛋白,并通过薄层层析(TLC)检测到了其酶活。基因序列已经注册到NCBI的GenBank中(序列接收号分别为FJ360766和FJ360767)。同时,研究发现红色亚栖热菌在高盐环境压力下胞内积累的内溶物为6-磷酸海藻糖。我们推测6-磷酸海藻糖磷酸酶在表达方面可能具有一定缺陷,不能将生成的6-磷酸海藻糖转化为海藻糖。
Trehalose is widely used in food processing industry, agriculture, pharmaceutical industry and many other fields, because of its ability to protect the biological molecules. In industrial production of trehalose, trehalose synthase (TreS) has been getting so much concern. That was because TreS can convert maltose into trehalose in one step reaction, and the cheaper raw material can be derived from starch hydrolysates. Screening of trehalose synthase gene and building a high-expression strain become key factors in decreasing the cost of trehalose industrial production.
At the early study of our laboratory, a thermophilic strain CBS-01 producing trehalose synthase was isolated from geothermal water. The preliminary analysis of the TreS showed that the optimal reaction temperature is 50℃, and the optimum reaction pH is 6.5. The enzyme can maintain more than 80% of its activity below 50℃and at pH range of 5.0-9.0. In view of the trehalose synthase with strong stability, it is necessary to clone the TreS gene and build engineering strains producing TreS for trehalose industrial production. On the other hand, it is necessary to study the structure, reaction mechanism and thermoadapatation mechanism of the enzyme, so that modified TreS with stronger thermostability and a favorable conversion rate could be obtained by molecular biological technology.
First of all, this strain producing trehalose synthase was identified. It was rod, Gram-negative, aerobic, and could produce dark red pigment. The optimum temperature and pH of its growth are 55℃～60℃and 7.5～8.5, respectively. At physiological and biochemical experiments, the strain CBS-01 showed the same results as the standard strains of Meiothermus ruber ATCC 35948. The G+C content of its genome was 62.1%, and the DNA-DNA hybridization rate with M. ruber ATCC 35948 was 63.9%. The sequence of 16s rDNA gene of CBS-01 showed the greatest identity to M. ruber in Genbank. Based on the above experimental results, the strain producing TreS was identified as Meiothermus ruber.
Secondly, the trehalose synthase gene was cloned. According to the conserved amino acid sequences of trehalose synthase reported, degenerate primers were designed for PCR. A fragment of 762 bp was obtained. The deduced amino acid sequence of the DNA product had high identity to other trehalose synthase, indicating that a partial putative
TreS gene was isolated from M. ruber. On this basis, A 6200 bp DNA sequence containing three open reading frames was isolated, by using linker mediated PCR, TAIL-PCR, and PCR-based method for screening genomic DNA library. The length of the three ORFs were 1350, 690, and 2889 bp, encoding 449, 229, and 962 amino acids, respectively. Sequence alignment analysis revealed that the deduced amino acid sequences of these ORFs were potential trehalose 6-phosphate synthase (TPS), trehalose 6-phosphate phosphatase (TPP) and trehalose synthase (TreS), respectively. Hence, there were two trehalose synthetic pathways in M. ruber CBS-01 and these three genes are likely to exist in the same operon-like structure. The potential TreS from M.ruber showed the highest homology to those of Thermus genus, and the molecular weight was much larger than those from mesophilic bacteria, indicating the TreS was likely to have better thermal stability. At the same time, it was found that the potential trehalose synthase belonged to theα-amylase family. These results indicated a putative TreS gene was isolated.
Third, an engineering strain producing TreS was built, and protein expression conditions were optimized. The potential trehalose synthase gene was transformed into E. coli and induced to express. The activity of crude enzyme solution was detected by using high-performance liquid chromatography (HPLC). The result revealed that the recombinant protein showed the activity of trehalose synthase, and could catalyze the conversion reaction of maltose into trehalose with glucose as the byproduct. This novel gene has been assigned in GenBank, the accession No. is EU443098. In order to increase the yield of trehalose synthase in E. coli, the 5' terminal nucleotides of the TreS were optimized by using PCR-based site-directed mutagenesis technology. At the same time, conditions of protein expression were also optimized, and the optimum conditions were below: using pET-21a as expression vector and E. coli Rosetta-gami (DE3) as host bacterium, the induction was carried out at 20℃, low speed, and overnight. The results suggested the formation of inclusion bodies was decreased, and the yield of soluble and active protein was increased. The TreS activity was 0.60 U/ml broth by fermentation of E. coli engineering strain, and it was about 8-fold than that of M. ruber.
Fourth, the recombinant TreS was purified and its enzymatic properties were studied in detail. The TreS was purified by using NTA-Ni column affinity chromatography. The kinetic values of the enzyme were determined, and glucose was found a competitive inhibitor of the reaction. The activity of the recombinant TreS was 80 U/mg, the optimum reaction temperature was 50℃, the optimum pH was 6.0-7.0. It was found that the enzyme could maintain 90% of its activity at a wide temperature (4-60℃) and pH range (pH 4.0-8.5). Because of its stability, the TreS proved very suitable for industrial production of trehalose. Meanwhile, it was found that low reaction temperature could decrease the formation of glucose, and increased the yield of trehalose.
Fifth, the vector for expressing the truncated TreS without C-terminal domain was constructed. It was found that the TreSΔC protein did not have the TreS activity. According to the Native-PAGE results, TreS was a tetramer, but TreSΔC was a dimer. The results suggested that the C-terminal domain of TreS played an important role in the formation of the tetramer, which had the TreS activity. However, TreSΔC without C-terminal domain could not form a tetramer, so its activity lost.
Sixth, the trehalose biosynthetic pathway of M. ruber was studied. The potential TPS and TPP gene were transformed into E. coli, and the recombinant proteins were purified by using NTA-Ni column. The results of thin layer chromatography (TLC) revealed that the two proteins showed the activity of TPS and TPP, respectively. The novel genes have been assigned in GenBank, the accession No. were FJ360766 and FJ360767. At the same time, it was found that M. ruber could accumulate 6-phosphate trehalose as the compatible solute in response to water stress imposed by salt. The results suggested that the TPP expression of M. ruber was defective, so the 6-trehalose phosphate producted by TPS could not be converted into trehalose.
引文
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