Key Laboratory of Industrial Biotechnology of the Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
Microb Cell Fact. 2022 Apr 19;21(1):63. doi: 10.1186/s12934-022-01789-2.
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 (Bs168) for efficient synthesis of D-allulose under the acidic conditions without browning.
First, based on the dicistron expression system, two DPEases with complementary functional characteristics from Dorea sp. CAG:317 (DSdpe) and Clostridium cellulolyticum H10 (RCdpe) were expressed in tandem under the promoter HpaII in one cell. A better potential strain Bs168/pMA5-DSdpe-RCdpe 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 Bs168/pMA5-DSdpe and Bs168/pMA5-RCdpe, 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-DSdpe-P-RCdpe exhibited the highest enzyme activity with 480.1 U/mL on fed-batch fermentation process in a 5 L fermenter at pH 6.5, about 2.1-times higher than the 228.5 U/mL of flask fermentation. Finally, the maximum yield of D-allulose reached as high as 163.5 g/L at the fructose concentration (50% w/v) by whole-cell biocatalyst.
In this work, the engineered recombinant strain Bs168/pMA5-P-DSdpe-P-RCdpe was demonstrated as an effective microbial cell factory for the high-efficient synthesis of D-allulose without browning under acidic conditions. Based on the perspectives from this research, this strategy presented here also made it possible to meet the requirements of the industrial hyper-production of other rare sugars under more acidic conditions in theory.
D-阿洛酮糖是一种低热量、高甜度的己酮糖单糖，通常用作食品和营养中的功能性糖。然而，由于碱性高温下的非酶促褐变，D-果糖制备 D-阿洛酮糖的酶制剂受到严重阻碍，而不必要的副产物进一步增加了工业应用中分离和提取的难度。在这里，为了解决生产过程中的上述问题，在枯草芽孢杆菌 168（Bs168）中利用串联 D-阿洛酮糖 3-差向异构酶（DPEases）异构酶协同表达策略和自动诱导启动子工程，在无褐变的酸性条件下高效合成 D-阿洛酮糖。
首先，基于双顺反子表达系统，来自 Dorea sp. CAG:317（DSdpe）和 Clostridium cellulolyticum H10（RCdpe）的两种具有互补功能特性的 DPEases 在一个细胞中在启动子 HpaII 的控制下串联表达。具有更好潜在性能的菌株 Bs168/pMA5-DSdpe-RCdpe 在酸性条件（pH 6.5）下的酶活提高到 18.9 U/mL，明显高于 Bs168/pMA5-DSdpe 和 Bs168/pMA5-RCdpe 的 17.2 和 16.7 U/mL。随后，在不同表达盒中构建了基于四个组成型启动子的六个重组菌株，以提高蛋白质的表达水平。在这些工程菌株中，在 pH 6.5 下，在 5 L 发酵罐中进行分批补料发酵过程中，Bs168/pMA5-P-DSdpe-P-RCdpe 表现出最高的酶活，达到 480.1 U/mL，约为摇瓶发酵的 228.5 U/mL 的 2.1 倍。最后，通过全细胞生物催化剂，在果糖浓度（50%w/v）下，D-阿洛酮糖的最高产量达到 163.5 g/L。
在这项工作中，工程重组菌株 Bs168/pMA5-P-DSdpe-P-RCdpe 被证明是一种有效的微生物细胞工厂，可在酸性条件下高效合成 D-阿洛酮糖，且无褐变。基于这项研究的观点，从理论上讲，该策略还可以满足更酸性条件下其他稀有糖工业超生产的要求。
