Construction Of Rhizopus Nigricans G6PDH Engineered Strains And Its Application

11?-hydroxy-16a,17a-epoxy-progesterone(HEP)is one of important pharmaceutical intermediates,which can be widely used in producing steroid hormone drugs,such as hydrocortisone and prednisone.In industry,generally,16a,17a-epoxy-progesterone(EP)is catalyzed by the fungus Rhizopus nigricans to get HER NAPDH is quite important in the process of biocatalysis,as it participates in hydroxylation of EP.In this study,glucose-6-phosphate dehydrogenase(G6PDH)from R.oryzae was cloned and expressed successfully in R.nigricans in order to increase NADPH supply for strengthening HEP production.Firstly,the cDNA sequence of G6PDH was obtained from R.oryzae by reversing transcription and the sequences of promoter PgpdA and terminator TtrpC were amplified from pAN7-1 by PCR.After analyzed by restriction enzymes,the fragments of G6PDH,PgpdA and TtrpC were cloned into expression vector pCB 1004 to get the pCB1004-G6PDH.Secondly,conditions of enzymatic hydrolysis to prepare R.nigricans protoplasts were optimized as follows:75 ?g/mL of Yatalase and 50?g/mL of Lywallzyme.The best osmotic stabilizer was 0.6 mol/L MgSO4·7H2O.The experiments of hygromycin B sensitivity showed that the minimum inhibitory concentration of hygromycin B was 250?g/mL.After the further screening,more than 120 transformants were successfully selected from the hygromycin B-resistant plates and 5 of them had ability of improving the HEP production.According to fermentation experiments,the biotransformation rate of EP increased by 10.08%and the NADPH content in engineered strain was 1.90-fold compared to that in original strain in vivo.Furthermore,the fermentation time of engineered strain was shortened to 52 h,which was 4 h less than the schedule.These results indicated that the strategy of strengthening NADPH regeneration was effective in improving the biosynthesis of HEP.Thirdly,the Response Surface Method(RSM)was employed to optimize fermentation conditions of engineered strain in 50 mL fermentation system.Optimized fermentation conditions were(g/L):36.00 of glucose,14.00 of silkworm pupa powder,22.60 of corn steep liquor,0.80 of(NH4)2SO4,3.00 of KH2PO4,200 rpm,and pH 4.60.After fermentation,the bioconversion rate of EP increased by 3.58%compared to that before optimization.Finally,the glucose consumption in engineered strain was studied in fed-batch fermentation.According to fermentation profile,30.00 g/L of glucose was obtained as the initial fermentation medium.But it did not meet the consumption of cell growth.Thus,12 g/L of extra glucose was added in 20 h and 36 h,respectively.As a result,the highest bioconversion rate of EP in engineered strain was obtained,which was 17.28%higher than that of the original strain.