黑曲霉脂肪酶全基因合成及其在毕赤酵母中的表达

The Total Gene Synthesis of Aspergillus Niger Lipase and the Construction Expression in Pcihia Pastoris

黑曲霉脂肪酶是重要的工业用酶,在食品、制药、前体化合物的合成和手性化合物的拆分等方面有广泛的用途。获得高效表达黑曲霉脂肪酶的基因工程菌是该酶工业化生产的前提。根据黑曲霉脂肪酶LIP的氨基酸序列及毕赤酵母密码子的偏爱性,设计合成26条相互重叠20 bp的引物,通过组装PCR分别合成lip基因的4个片段lipA1(300bp)l、ipA2(237 bp)l、ipA3(234 bp)和lipA4(210 bp)。再以基因头lip1和基因尾lip26为引物,以lipA1l、ipA2l、ipA3和lipA4混合物为模板进行第二轮PCR扩增,得到的产物经加A处理后,连接到载体pMD18-T上,挑取重组子测序。通过PCR扩增对人工合成的基因进行校正,得到完全正确的lip基因。将优化后的基因克隆到表达载体pPIC9K上,获得的重组质粒pPIC9K-lip经线性化后转化毕赤酵母GS115菌株,构建分泌型表达LIP的酵母工程菌,G418梯度筛选,得到高拷贝稳定整合菌株。为重组黑曲霉脂肪酶的规模化制备奠定了基础。

Aspergillus niger lipases are important biocatalysts widely used in industries for food processing and pharmaceutical preparation.High-level expression recombinants can lead to cost effective lipase large scale production.Total gene synthesis is an efficient method to enhance the expression level of the gene.According to the mature peptide sequence of Aspergillus niger lipase and codons of preference in Pichia pastoris,26 primers with 20 bp overlaps at both 5′ and 3′ ends between adjacent oligonucleotides were designed and synthesized.Fragments lipA1（300 bp）,lipA2（237 bp）,lipA3（234 bp） and lipA4（210 bp） were separately synthesized by assembly PCR,and then they were used as the template to get the full length gene.The synthetic gene was sequenced and proved to be the same as the designed.The lip gene was cloned into pPIC9K secretory expression vector.The construct was linearized with Sal I and transformed into P.pastoris strain GS115,and stable multicopy integrants were screened in medium containing different concentrations of G418.This offers efficient recombinant P.pastoris strains for mass production of lipase.