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首页> 外文期刊>Microbial Cell Factories >Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid
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Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid

机译：使用双重表达盒质粒增强大肠杆菌中伴侣蛋白依赖性脂肪酶的功能性生产

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Background The lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA) and its chaperone (LipB) from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems. Results To produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB), six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a) were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein) and only a small fraction of the overexpressed lipase was folded in vivo into the functional lipase in soluble form whereas the main fraction was still inactive in the form of inclusion bodies. Another controversial finding was that the dual expression cassette plasmid systems E. coli BL21/pELipAB-LipB1a and E. coli/pELipAB-LipB3a secreted the active lipase into the culture medium of 51 and 29 times as high as the single expression cassette plasmid system E. coli pELipABa did, respectively, which has never been reported before. Another interesting finding was that the lipase form LipA6xHis (mature lipase fused with 6× histidine tag) expressed in the dual expression cassette plasmid systems (BL21/pELipA-LipB1a and BL21/pELipA-LipB3a) showed no lipase activity although the expression level of the lipase and two chaperone forms LipB1 and LipB3 in these systems remained as high as that in E. coli BL21/pELipABa + pELipB1k, BL21/pELipABa + pELipB3k, BL21/pELipAB-LipB1a, and BL21/pELipAB-LipB3a. The addition of Neptune oil or detergents into the LB medium increased the lipase production and secretion by up to 94%. Conclusions Our findings demonstrated that a dual expression cassette plasmid system E. coli could overproduce and secrete the active chaperone-dependent lipase (subfamilies I.1 and I.2) in vivo and an improved dual expression cassette plasmid system E. coli could be potentially applied for industrial-scale production of subfamily I.1 and I.2 lipases.
机译：背景脂肪酶亚家族I.1和I.2在氨基酸序列中显示超过33％的同源性，并且大多数成员具有另一个共同的特性，即它们的基因与次级基因聚类，次级基因的蛋白质产物是将脂肪酶折叠成活性蛋白所需的构象和分泌到培养基中。在以前的研究中，来自Ralstonia sp。的脂肪酶（LipA）及其伴侣（LipB）。 M1在大肠杆菌中过表达，并且脂肪酶在体外成功折叠。这项研究的目的是提高来自Ralstonia sp。的活性脂肪酶LipA的产生。使用两个质粒共表达系统和双重表达盒质粒系统，无需体外重折叠过程即可在异源宿主大肠杆菌中产生M1。结果从Ralstonia sp。产生更多活性的脂肪酶。大肠杆菌中的M1，无需体外重折叠过程，但借助伴侣蛋白的过表达（LipB1和LipB3分别对应于56-aa截短的伴侣和26-aa截短的伴侣LipB），六个不同的表达系统，包括2个两质粒共表达系统（大肠杆菌BL21 / pELipAB a + pELipB1 k 和BL21 / pELipAB a + pELipB3 k ）和4种双表达盒质粒系统（BL21 / pELipAB-LipB1 a ，BL21 / pELipAB-LipB3 a ，BL21 / pELipA-LipB1 a 和构建了BL21 / pELipA-LipB3 a 。两质粒共表达系统（大肠杆菌BL21 / pELipAB a + pELipB1 k 和BL21 / pELipAB a + pELipB3 k ）产生的活性脂肪酶的水平是单表达盒质粒系统大肠杆菌BL21 / pELipAB a did的4倍。双重表达盒质粒系统BL21 / pELipAB-LipB1 a 和BL21 / pELipAB-LipB3 a 首次产生了29倍和19倍的活性脂肪酶与单表达盒质粒系统E. coli BL21 / pELipAB a 分别比较。尽管脂肪酶量在所有这些表达系统中均等表达（占总细胞蛋白的40％），并且过表达的脂肪酶中只有一小部分在体内折叠成可溶形式的功能性脂肪酶，而主要部分在形式上仍然没有活性包涵体。另一个有争议的发现是双表达盒质粒系统大肠杆菌BL21 / pELipAB-LipB1 a 和大肠杆菌/ pELipAB-LipB3 a 将活性脂肪酶分泌到培养物中培养基分别是单表达盒质粒系统大肠杆菌pELipAB a did的51倍和29倍，以前从未报道过。另一个有趣的发现是在双表达盒质粒系统（BL21 / pELipA-LipB1 a 和BL21 / pELipA-LipB3 ）中表达的脂肪酶形式LipA6xHis（与6x组氨酸标签融合的成熟脂肪酶） a ）没有脂肪酶活性，尽管在这些系统中脂肪酶和两个伴侣形式的LipB1和LipB3的表达水平仍然与大肠杆菌BL21 / pELipAB a + pELipB1 < sup> k ，BL21 / pELipAB a + pELipB3 k ，BL21 / pELipAB-LipB1 a 和BL21 / pELipAB-LipB3 a 。向LB培养基中添加海王星油或去污剂可使脂肪酶的产生和分泌增加多达94％。结论我们的发现表明，双表达盒质粒系统大肠杆菌可以在体内过量生产和分泌活性伴侣依赖性脂肪酶（亚家族I.1和I.2），改进的双表达盒质粒系统大肠杆菌可能应用于工业化生产亚家族I.1和I.2脂肪酶。

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