黑曲霉SH-2基因组中注释的7个α-葡萄糖苷酶基因的鉴定与表达分析

Identification and Expression Analysis of the Seven Predicted α-glucosidase Genes in Aspergillus niger SH-2 Genome

摘要:

已公布的黑曲霉CBS513.88基因组中注释了7个α-葡萄糖苷酶基因信息，但其中主要高活性基因一直未报道。本文通过同源重组构建了黑曲霉SH-2菌株7个α-葡萄糖苷酶基因的系列多重敲除株。α-葡萄糖苷酶活性测定结果显示仅agdB敲除株的酶活相较于野生株SH-2降低36%，而其余多重敲除株菌株酶活影响较小。以敲除糖化酶、淀粉酶背景的黑曲霉SH-2-3菌株为宿主，分别敲除和过量表达基因agdB及与已报道的黑曲霉α-葡萄糖苷酶基因序列相似度最高的基因agdA，结果显示agdB敲除株生长速度在不含葡萄糖的培养条件下最慢，并随着葡萄糖添加量增加；agdB过量表达株酶活提高到野生型的10倍。本研究结果说明目前黑曲霉SH-2基因组中所注释的7个α-葡萄糖苷酶基因中仅agdB为主要高活性α-葡萄糖苷酶基因（占总体酶活36%），黑曲霉SH-2中α-葡萄糖苷酶基因仍需进一步探索。

Abstract:

Seven α-glucosidase genes have been annotated in the genome of Aspergillus niger CBS513.88, but the gene with predominant α-glucosidase activity has not been reported. A set of seven α-glucosidase-gene-knockout mutant strains of A. niger SH-2 were constructed by homologous recombination, with deletion of α-glucosidase genes B, A, E, G, C, D, and F. Measurement of α-glucosidase activity showed that in the agdB disruption mutant strain (ΔagdB) activity was decreased by 36% compared with that of the control strain, but there was no significant decrease with the other deletion mutants. Using the A. niger SH-2-3 strain with glucoamylase and amylase gene deletions as the host, and the agdA gene, that shows the highest identity with the reported α-glucosidase gene sequence from A. niger, and agdB were knocked out and overexpressed, respectively. The results showed that the growth rate of agdB disruption mutant strains was the slowest in culture medium without glucose, and increased with increasing glucose concentrations. The α-glucosidase activity in the agdB overexpressing strain (3-agdB) was ten times as much as that of the SH-2 wild-type strain. Therefore, the results of this study suggest that agdB is the gene with predominant α-glucosidase activity (36% of all α-glucosidase activity) among the seven predicted α-glucosidase genes in the A. niger SH-2 genome, but further investigation is required.