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

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 (Bs168) 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 (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-PspoVG-DSdpe-PsrfA-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. Conclusion In this work, the engineered recombinant strain Bs168/pMA5-PspoVG-DSdpe-PsrfA-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.

Engineering of Acid-Resistant d-Allulose 3-Epimerase for Functional Juice Production

“…The nonenzymatic browning reaction is accelerated at a high temperature and under alkaline conditions, which allows the accumulation of additional byproducts and makes downstream processing more challenging. 16 Until now, the molecular modification of KEases has always focused on the improvement of thermostability and catalytic efficiency. Actually, the enhancement of acid resistance of KEases is lacking.…”

“…At these reaction conditions, the hydroxyl group of D-allulose tends to interact with the amino group, leading to an intensification of the nonenzymatic browning reaction. 16 However, KEases are inactivated at low pH, precluding their industrial application under acidic conditions. Accordingly, increasing the acid resistance of KEases is crucial for resolving this issue of industrial D-allulose manufacturing.…”

“…CAG317. − Most KEases exhibit an optimal pH between 7.0 and 8.0 to catalyze d -fructose. At these reaction conditions, the hydroxyl group of d -allulose tends to interact with the amino group, leading to an intensification of the nonenzymatic browning reaction . However, KEases are inactivated at low pH, precluding their industrial application under acidic conditions.…”

Fine-Tuning of Carbon Flux and Artificial Promoters in Bacillus subtilis Enables High-Level Biosynthesis of d-Allulose

“…Thus, the blockage of fructose PTS and fructokinase resulted in a remarkable 7.0-fold increase of the D- subtilis, we screened nine constitutive promoters (P HpaII , P spovG , P luxS , P amyE , P 43 , P gsiB , P srfA , P nprE , and P sigW ), which were reported to exhibit different expression levels of heterologous proteins in B. subtilis. 33,34 For comparative analysis of promoter strength, nine constructed recombinant strains (designated WB-D3P1 to WB-D3P9) were evaluated by RT-qPCR analysis to determine the relative transcription levels of SfDAE. Compared with WB-D3P1, the relative mRNA levels of SfDAE produced by P amyE and P 43 were significantly increased in the recombinant strains WB-D3P4 and WB-D3P5, while the transcriptional activity of P spovG and P srfA was similar in WB-D3P2 and WB-D3P7 (Figure 4A).…”

Enhancement of the d-Allulose 3-Epimerase Expression in Bacillus subtilis through Both Transcriptional and Translational Regulations

“…subtilis for d -allulose production using an autoinducible promoter engineering and a tandem DAEase synergistic expression with Dorea sp. DAEase and Clostridium cellulolyticum DAEase . Liu et al enhanced DAEase expression through protein engineering and catabolite-responsive element box (CRE) engineering .…”