曲霉N1-14′发酵产L－苹果酸代谢的基因调控研究

L-malic acid has been widely used in food industry. In recent years, the advantages of the method of producing L-malic acid by microbial fermentation are gradually emerging and molecular techniques have received increasing attention as a means of improving production of L-malic acid producing strain. Currently, No data has been reported in the study of gene regulation of metabolisn of L－malic acid fermentation production by Aspergillus.Therefore, the L-malic acid producing strain, Aspergillus N1-14′,was chosen as our research object to study effect of the key enzyme gene and oxoglutarate-malate carrier gene (OMC) on regulation of metabolic pathway for L-malic acid accumulation by Aspergillus N1-14′. The temporal and spatial expressions of the pyruvate carboxylase (pyc), malate dehydrogenase (mdh) and OMC gene were observed by the fusion between regulative genes and a fluoreseent gene.Some genetic mutations will been created by gene integration technology. The fermentation kinetics, enzyme expression and activity of these mutants will been analysed quantitatively to study effect of the pyc gene, mdh gene and OMC gene on regulation of metabolic pathway for L-malic acid accumulation by Aspergillus N1-14′. The other genetic mutations will been created by gene knockout technology. The fermentation kinetics, enzyme expression and activity of these mutants will been analysed quantitatively to study effect of the fumarase (fum) gene and succinate dehydrogenase (SDH) gene on regulation of metabolic pathway for L-malic acid accumulation by Aspergillus N1-14′. The aim of this project is to provide important technical support and theoretical basis for acquiring mutants could be used in the industrial production of L-malic acid.

L－苹果酸已广泛应用于食品工业中。近年来微生物发酵法生产L-苹果酸的优势逐渐显现，采用分子技术改良L-苹果酸生产菌日益受到重视，但对曲霉发酵产L-苹果酸代谢的基因调控研究缺乏报道。本项目以发酵产L-苹果酸曲霉N1-14′为研究对象，研究关键酶基因及苹果酸转运蛋白(OMC)基因对苹果酸代谢的调控作用，利用荧光蛋白基因融合技术了解丙酮酸羧化酶(pyc)、苹果酸脱氢酶（mdh）及OMC基因在细胞内表达情况；利用基因整合技术构建基因突变株，对突变株发酵动力学和酶蛋白表达及活性进行研究，定量分析pyc、 mdh及OMC基因对曲霉N1-14′发酵产苹果酸代谢的调控作用；利用基因敲除技术构建基因突变株，对突变株发酵动力学进行研究，定量观察延胡索酸酶（fum）及琥珀酸脱氢酶（sdh）基因对曲霉N1-14′发酵产苹果酸代谢的调控作用；为获得能应用于生产的改良菌株,提供重要技术保障和理论依据。

L-苹果酸是生物体代谢过程中产生的一种重要中间代谢物，参与众多重要的物质和能量代谢。随着对L-苹果酸的需求日益增加、L-苹果酸合成和代谢途径的阐明、分子生物信息学的发展以及基因调控技术的成熟，使得对生产菌株根据代谢通路进行分子水平上定向改造以提升L-苹果酸产量成为可能。.本研究设计同源引物扩增丙酮酸羧化酶基因(pyc)和苹果酸脱氢酶基因(mdh)。通过TA克隆、测序并推导出两种酶的氨基酸序列，Blast比对和3D模拟分析其突变位点与酶活的关系，为后续研究曲霉N-14′的高产LMA机理和酶的特异性奠定基础。本研究通过基因工程手段，过表达PYC、MDH以及异源表达粟酒裂殖酵母的苹果酸转运蛋白(SpMAE1)，一方面增加丙酮酸向LMA的转化量，另一方面优化LMA向细胞外转运从而降低胞内积累的LMA引起的反馈抑制。主要研究结果有：测序结果显示pyc编码区长3582bp,编码1193aa，其中N1-14′在两个保守位点出现突变，分别是A833和F1022；mdh编码区全长1023bp，编码340aa，同样有两个位于保守的氨基酸点突变，分别是K220和R222，这几个突变位点均出现在酶高度保守的功能域，可能与其高产酸活性相关；过表达曲霉自身的丙酮酸羧化酶基因能极大程度上提升细胞内PYC酶活力，释放了曲霉细胞的产LMA潜能，表明丙酮酸羧化酶是曲霉产LMA通路中的一个关键调节酶，通过控制丙酮酸羧化酶的表达能够达到控制N1-14′产LMA量的目的；过表达曲霉自身的苹果酸脱氢酶基因大幅度提升了突变株的MDH酶活力，但其对曲霉菌株产LMA提升空间较小，表明MDH不是曲霉N1-14′产LMA的关键瓶颈，单独过表达曲霉自身的mdh不能达到较大幅度提升突变株产LMA水平的目的；异源表达粟酒裂殖酵母的苹果酸转运蛋白（SpMAE1）对于提升曲霉发酵产LMA水平的效果不明显，但相对于空白载体组能一定程度缓解因空白载体的导入引起的产LMA水平下降，表明Spmae1的表达对提升产LMA水平有一定作用。