Synthesis Of α-arbutin By Biotransformation Of Bacillus Subtilis

α-arbutin is a glycosylated product of hydroquinone,which is widely used in whitening cosmetics due to its excellent performance in preventing melanin formation.α-arbutin can be synthesized by whole cell catalysis,which has great value in industrial application.In this study,the gene encoding sucrose phosphorylase was integrated into the genome of Bacillus subtilis,which improved the genetic stability of the engineered strain and realized the stable and efficient synthesis ofα-arbutin.Then,the sucrose metabolism pathway was engineered by means of metabolic engineering to reduce the sucrose consumption in the whole cell catalytic process.Finally,the genes related to the transformation of by-product into substrate were overexpressed,and protein degradation tags were fused to the key protein in the downstream competitive pathway to control its expression and tunable degradation,thus improving the carbon utilization efficiency of sucrose.The main research results of this study are as follows:（1）The encoding gene smut of sucrose phosphorylase derived from Streptococcus mutans was integrated at multiple sites using P43 promoter in B.subtilis WB800,and the recombinant strain BS8S4 was obtained by optimazing the RBS sequence.The production ofα-arbutin was87.6 g·L^-1,and the conversion rate of HQ was 70.8%.（2）The whole cell catalytic time,dissolved oxygen level and catalytic temperature of BS8S4 strain were optimized.It was found that cells were cultured in a 250 m L shake flask with 50 m L medium at a rotational speed of 220 r·min^-1 and then collected.After 48 h of catalysis at 30°C,the titer ofα-arbutin was 116.0 g·L^-1,and the HQ conversion rate was 93.8%.（3）The results showed that the yield ofα-arbutin of BS8S4 strain was 93.5%,78.8%,67.7%and 43.9%of that of the first time after the second to fifth repeated use,respectively.Scanning electron microscopy showed that with the increase of catalytic times,the damage degree of cell surface was aggravated,and the aggregation of cells became more obvious.（4）BS8S4 strain was treated with whole-cell catalysis at the fermenter level.BS8S4 was first cultured in a 3 L tank at 37°C for 12 h at 500 r·min^-1 and a ventilation rate of 3 vvm.Then the cells were collected for whole cell catalysis in a 1 L tank,which was catalyzed at 30°C for36 h under dark conditions.The stirring speed was 200 r·min^-1,and the ventilation rate was 2vvm.The production ofα-arbutin was 123.3 g·L^-1,and the HQ conversion rate was 99.7%.（5）The recombinant strain BS8S404 was obtained by knockout of sac B（levansucrase encoding gene）and sac A（sucrose 6-phosphate hydrolase encoding gene）.Compared with the original strain BS8S4,the sucrose consumption of BS8S404 reduced by 23%-66%.（6）The xylose-inducible protein degradation system was constructed to degrade Pfk A under the premise of the normal cell growth.The enhanced expression of pgi（glucose 6-phosphate isomerase encoding gene）and pgc A（glucose phosphate isomerase encoding gene）in the pathway from fructose 6-phosphate to glucose 1-phosphate makes fructose,the by-product produced in the catalytic process,also used in the synthesis ofα-arbutin.The sucrose addition decreased by 35.6%and the molar conversion rate of sucrose increased by 15.4%.