红球菌Rhodococcus sp.DS-3 dszABC基因和dszD基因在大肠杆菌中的共表达

二苯并噻吩(DBT)及其衍生物微生物脱硫的4S途径需要4个酶(DszA,DszB,DszC and DszD)参与催化。其中DBT单加氧酶(DszC or DBT-MO)和DBT-砜单加氧酶(DszA or DBTO2-MO)都是黄素依赖型氧化酶,它们的催化反应需要菌体中还原型的黄素单核苷酸(FMNH2),FMNH2由辅酶黄素还原酶(DszD)再生。因此,共表达DszA,DszB,DszC和DszD可以提高整个脱硫途径的速率。构建了两个不相容性表达载体pBADD和paN2并在大肠杆菌中实现了4个脱硫酶基因的共表达。DszA,DszB,DszC和DszD的可溶性蛋白表达量分别占菌体总蛋白质的7.6%,3.5%,3.1%和18%。共表达时的脱硫活性是单独用paN2表达时的5.4倍,并对工程菌休止细胞脱除模拟柴油中DBT的活性进行了研究。

Co-expression of Rhodococcus sp. DS-3 dszABC and dszD gene with incompatible plasmids in Escherichia coli

Biological dusulfurizaion of petroleum feedstocks and products may offer an attractive alternative to reduce sulfur oxide emissions that cause serious environmental pollution. Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, can be microbially desulfurized without degradation of the organic structure by 4S pathway. Three desulfurization enzymes (DszA, DszB and DszC) and flavin reductase (DszD) are involved in sulfur-specific DBT desulfurization. DszA and DszC are FMNH2-dependent monoxygenases, FMNH2 is provided from the freely diffusible FMNH2 pool in the cell, and is replenished by DszD. So, co-expression of the desulfurization enzymes and flavin reductase can enhance the rate of sulfur removal. In the present work two incompatible plasmids: pBADD and paN2 were constructed. The paN2 allows Escherichia coli to liberate the sulfur of DBT and DBTs and pBADD produces a flavin reductase. They were co-expressed in Escherichia coli B121 (DE3). The soluble products of DszA, DszB, DszC and DszD accounted for 7.6%, 3.5%, 3.1% and 18% of the total proteins in co-expressed system. The desulfurization rate of lysate of E. coli BL21- pBADD + paN2 is 12.03 micromol/(h x mg) Dsz protein and about 5.4-fold of that of E. coli BL2-paN2. Experiment were also conducted using resting cell with the 0.6 wt% DBT in n-hexadecane as model diesel oil. After 24 hours reaction, 0.42 mmol/L (about 84%) DBT was converted to 2-HBP by E. coli BL21- pBADD + paN2, however, there was only 0.08 mmol/L (about 16%) DBT was desulfurized by E. coli BL2-paN2. The maximum desulfurization rate of E.coli BL21-pBADD + paN2 is about 67 micromol/h. The result shows that DszD enhances the rate of 2-HBP production when co-expressed in vivo with the desulfurization enzymes.