Effects Of Over-expression Of Psd Gene And Addition Of Surfactant On Cytidine Fermentation

Cytidine is one of the raw materials of nucleotide health food and functional food.It is also the in-termediate of synthesizing many kinds of antiviral and antineoplastic drugs.It is mainly obtained by microbial direct fermentation.Cell membrane plays an important role in the secretion of cytidine during fermentation.In this paper,the genes psd related to cell membrane permeability and different surfac-tants were studied,and the effects of overexpression of psd gene on cytidine fermentation were ana-lyzed.The role of psd gene in the process of cytidine secretion was discussed.Four different surface activities were added during the fermentation of Escherichia coli BG-12.The effect of the agent on cyt-idine fermentation and cell membrane permeability was studied.The main results are as follows:1.Construction of psd gene overexpression vector.Through pathway analysis,it was found that phosphatidylserine decarboxylase(EC:4.1.1.65,coded by psd gene)in Bacillus amyloliquefaciens was closely related to phospholipid synthesis in cell membrane.Using genomic DNA of Bacillus amyloliq-uefaciens BG-09 as template,psd gene was amplified and linked to plasmid pHT43.The recombinant expression vector pHT43-psd was successfully constructed by restriction enzyme digestion and electro-phoresis analysis.2.The effect of overexpression of psd gene on the cytidine fermentation of Bacillus amyloliquefa-ciens BG-09.The recombinant expression vector pHT43-psd was electrochemically transformed into Bacillus amyloliquefaciens BG-09,and the recombinant strain B.amyloliquefaciens BG-09-psd was obtained for enzyme gene expression and fermentation.The results showed that the psd gene could be expressed in B.amyloliquefaciens BG-09 and the correct band appeared in SDS-PAGE electrophoresis.Fermentation results showed that the cytidine content in fermentation broth was 0.882 g/L after 50 h fermentation at 37? and 200 rpm.Compared with the original strain B.amyloliquefaciens BG-09,the cytidine yield increased by 18.07%.It indicated that the overexpression of psd gene promoted the fer-mentation of cytidine and could change the permeability of cell membrane.The effect makes the bacte-ria enter the logarithmic growth earlier and enter the stable growth later.3.The effect of overexpression of psd gene on the cytidine fermentation of Escherichia coli BG-12.The recombinant expression vector pHT43-psd was transfected into Escherichia coli BG-12 by chemi-cal transformation method,and the recombinant strain E.coli BG-12-psd was obtained for enzyme gene expression and fermentation.The results showed that the gene psd could be correctly expressed in E.coli BG-12 and the target band appeared in SDS-PAGE electrophoresis.Fermentation results showed that the yield of cytidine in fermentation broth was 0.532 g/L in shaking flask fermentation at 37? and 200 rpm for 40 h.Although the glucose consumption rate of recombinant bacteria increased compared with the control strain,the concentration of cytidine in fermentation broth did not increase,only 89.11%of the control strain,indicating that the over-expression of psd gene in E.coli BG-12 did not produce cytidine fermentation.The reasons for the promotion need to be further studied.4.The effects of different surfactants on the fermentation and cell membrane permeability of Esche-richia coli BG-12 cytidine.Cytidine-producing bacteria during the fermentation of E.coli BG-12,sur-factants Tween-80,Triton X-100,CTAB and EMB with different concentrations were added to analyze the growth of bacteria,the rate of sugar consumption and the change of cytidine concentration in fer-mentation broth,and their effects on cytidine fermentation were studied.The results showed that the addition of non-ionic surfactant Tween-80 had a significant effect on the cytidine fermentation.When the amount of Tween-80 was less than 5 g/L,it had little effect on the growth of bacteria and the sugar consumption rate showed a downward trend.However,the concentration of cytidine in fermentation broth increased.At 3 g/L and 40 h after fermentation,the concentration of cytidine was 1.696 g/L,2.42 times as that of the control group,and the mass concentration was 0.4-2.0 g/L Triton X-100,respective-ly.Cytidine production was inhibited when the concentration was higher than 0.8 g/L,and the growth of the strain would be inhibited when the concentration was higher than 0.8 g/L.When the concentration was 1.6 g/L for 40 h,the yield of cytidine was 0.362 g/L,which was 0.52 times higher than that of the control group.Adding 0.4-2 mg/L CTAB had little effect on the growth and sugar consumption rate of the strain.The inhibition of cytidine was strengthened with the increase of CTAB concentration.When the concentration of CTAB was 0.4 mg/L for 40 h,the yield of cytidine was 0.229 g/L,which was 0.33 times higher than that of the control group.When 5-25 mg/L EMB was added,the growth rate and sug-ar consumption rate of the strain were not significantly affected.Cytidine production was inhibited to varying degrees.When 5 mg/L was added,the inhibition of cytidine production was the strongest.Cyti-dine production was 0.028 g/L at 40 h fermentation,which was 0.04 times of that of the control group.Therefore,the over-expression of psd gene in Bacillus amyloliquefaciens BG-09 can promote the se-cretion of cytidine,affect the permeability of cell membrane and increase the concentration of cytidine in fermentation broth,while the over-expression of psd gene in Escherichia coli BG-12 has no positive effect on the fermentation of cytidine.Adding different surfactants to the fermentation process of Esch-erichia coli BG-12 produced different effects.Tween-80 was beneficial to the secretion of cytidine,while Triton X-100,CTAB and EMB did not promote the secretion of cytidine.