State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
Microb Cell Fact. 2022 May 28;21(1):95. doi: 10.1186/s12934-022-01833-1.
Glucoamylase is an important industrial enzyme for the saccharification of starch during sugar production, but the production cost of glucoamylase is a major limiting factor for the growth of the starch-based sugar market. Therefore, seeking strategies for high-level expression of glucoamylase in heterologous hosts are considered as the main way to reduce the enzyme cost.
ReGa15A from Rasamsonia emersonii and TlGa15B-GA2 from Talaromyces leycettanus have similar properties. However, the secretion level of ReGa15A was significantly higher than TlGa15B-GA2 in Pichia pastoris. To explore the underlying mechanisms affecting the differential expression levels of glucoamylase in P. pastoris, the amino acid sequences and three-dimensional structures of them were compared and analyzed. First, the CBM region was identified by fragment replacement as the key region affecting the expression levels of ReGa15A and TlGa15B-GA2. Then, through the substitution and site-directed mutation of the motifs in the CBM region, three mutants with significantly increased expression levels were obtained. The eight-point mutant TlGA-M4 (S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q), the three-point mutant TlGA-M6 (Q599A/G600Y/V603Q) and the five-point mutant TlGA-M7 (S589D/T607I/V608L/N609D/R613Q) have the same specific activity with the wild-type, and the enzyme activity and secretion level have increased by 4-5 times, respectively. At the same time, the expression levels were 5.8-, 2.0- and 2.4-fold higher than that of wild type, respectively. Meanwhile, the expression of genes related to the unfolded protein responses (UPR) in the endoplasmic reticulum (ER) did not differ significantly between the mutants and wild type. In addition, the most highly expressed mutant, TlGA-M7 exhibits rapidly and effectively hydrolyze raw corn starch.
Our results constitute the first demonstration of improved expression and secretion of a glucoamylase in P. pastoris by introducing mutations within the non-catalytic CBM. This provides a novel and effective strategy for improving the expression of recombinant proteins in heterologous host expression systems.
糖化酶是制糖过程中淀粉糖化的重要工业酶，但糖化酶的生产成本是淀粉糖市场发展的主要限制因素。因此，寻求在异源宿主中高水平表达糖化酶的策略被认为是降低酶成本的主要途径。
来自拉沙姆逊毛壳菌的 ReGa15A 和来自塔宾曲霉的 TlGa15B-GA2 具有相似的性质。然而，ReGa15A 在毕赤酵母中的分泌水平明显高于 TlGa15B-GA2。为了探讨影响毕赤酵母中糖化酶差异表达水平的潜在机制，对它们的氨基酸序列和三维结构进行了比较和分析。首先，通过片段替换鉴定出 CBM 区域是影响 ReGa15A 和 TlGa15B-GA2 表达水平的关键区域。然后，通过替换 CBM 区域中的基序和定点突变，获得了三个表达水平显著提高的突变体。八个点突变体 TlGA-M4（S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q）、三个点突变体 TlGA-M6（Q599A/G600Y/V603Q）和五个点突变体 TlGA-M7（S589D/T607I/V608L/N609D/R613Q）的比活性与野生型相同，酶活性和分泌水平分别提高了 4-5 倍。同时，表达水平分别比野生型高 5.8、2.0-和 2.4 倍。同时，突变体和野生型之间内质网（ER）中未折叠蛋白反应（UPR）相关基因的表达没有显著差异。此外，表达水平最高的突变体 TlGA-M7 对生玉米淀粉具有快速有效的水解作用。
本研究首次证明了通过在非催化 CBM 内引入突变可以提高毕赤酵母中糖化酶的表达和分泌。这为提高异源宿主表达系统中重组蛋白的表达提供了一种新颖有效的策略。
