Efficient D-allulose synthesis under acidic conditions by auto-inducing expression of the tandem D-allulose 3-epimerase genes in Bacillus subtilis

作者：Mengkai Hu, Yuxia Wei, Rongzhen Zhang, Minglong Shao, Taowei Yang, Meijuan Xu, Xian Zhang, Zhiming Rao · 发表于：Microbial Cell Factories · 年份：2022 · DOI：10.1186/s12934-022-01789-2 · 被引用次数：22 · 研究领域：Diet, Metabolism, and Disease、GABA and Rice Research、Freezing and Crystallization Processes

Abstract Background D-allulose, a hexulose monosaccharide with low calorie content and high sweetness, is commonly used as a functional sugar in food and nutrition. However, enzyme preparation of D-allulose from D-frutose was severely hindered by the non-enzymatic browning under alkaline and high-temperature, and the unnecessary by-products further increased the difficulties in separation and extraction for industrial applications. Here, to address the above issue during the production process, a tandem D-allulose 3-epimerase (DPEases) isomerase synergistic expression strategy and an auto-inducible promoter engineering were levered in Bacillus subtilis 168 ( Bs 168) for efficient synthesis of D-allulose under the acidic conditions without browning. Results First, based on the dicistron expression system, two DPEases with complementary functional characteristics from Dorea sp. CAG:317 ( DS dpe) and Clostridium cellulolyticum H10 ( RC dpe) were expressed in tandem under the promoter HpaII in one cell. A better potential strain Bs 168/pMA5- DS dpe- RC dpe increases enzyme activity to 18.9 U/mL at acidic conditions (pH 6.5), much higher than 17.2 and 16.7 U/mL of Bs 168/pMA5- DS dpe and Bs 168/pMA5- RC dpe, respectively. Subsequently, six recombinant strains based on four constitutive promoters were constructed in variable expression cassettes for improving the expression level of protein. Among those engineered strains, Bs168 /pMA5-P spoVG - DS dpe-P srfA - RC dpe exhibited the ...