Heterologous Expression Of Antimicrobial Peptides And Phytase In Yeast

Antimicrobial peptides are a kind of small-molecule proteins that are originated by various organisms.These compounds have the effects of stable anti-bacterial,anti-fungal,and anti-viral without any drug resistance,as well as the unique mechanism and broad spectrum.Antimicrobial peptides have been widely used as animal feed additives to prevent feed deterioration during producing and transportation,and occurrence of disease after animal consumption.Therefore,antibacterial peptides are urgent need feed additives in animal husbandry industry.Phytate is the main storage form of phosphorus and inositol,which is widely present in plant tissues and grain products.However,non-ruminant animals cannot use phosphorus in phytate directly due to lack of phytase.Phytase can catalyze phytate to produce inositol and phosphate.The addition of phytase in animal foods has been shown to effectively improve the utilization of phosphorus in foods and reduce environment pollution caused by phosphorus in animal excrements.Antimicrobial peptides and phytase have been used as two kinds of high-efficiency feed additive proteins for preventing feed deterioration and promoting digestion and absorption of phosphorus in foods.The natural antimicrobial peptides have a low yield and are difficult to separate and purify.In present study,the synthesized gene of antimicrobial peptide and phytase gene coloned from Penicillium were recombined into expression vectors and then introduced in yeast cell to construct the genetic engineering yeast stranins.Our aim is to realize the heterologous expression of antimicrobial peptides and phytase in recombined yeast strains.Furthermore,high density fermentation of antimicrobial peptides in 100 L fermentor was also studied.The main results of this study are as follows.1.Odoranagin is an antimicrobial peptide that originated from Odorrana species.The odoranagin gene was synthesized and recombined into an expression vector to construct expression vector p PICOG.After transformation of p PICOG into cells of Pichia pastoris,we selected a recombined strain Pichia pastoris OG9 with broad-spectrum antibacterial activity.The highest activity of antibacterial peptide odoranagin reaches up to 2.47×109 U/L after 96 h of fermentation in the shake flasks.Further studies showed that after 96 h of cultivation,the activity of odoranagin expressed by yeast OG9 began to decline.The SDS-PAGE result showed that the antibacterial peptide odoranagin was expressed by the recombinant yeast strain OG9 correctly.The optimum conditions for the production of odoranagin by strain OG9 were: p H 6.5,50 g/L of inoculum size,and the BMMY medium.2.The high density fermentation technology for the production of antimicrobial peptide PSI in a 100 L fermentor using a recombinant Kluyveromyces lactis strain has been studied.The effects of mediums,cell density,lactose,as well as the fed-batch models of inducer lactose into the fermentor,on the production of PSI were investigated.Results obtained showed that the inducer lactose should be added when the cell density was at 190 g/L during the fermentation using BSM medium,the addition of lactose was 5 kg with feeding velocity of 1 L/h.When the induction culture time was 25 h,the cell wet weight,the protein yield,and the titer of PSI reached to 280 g/L,27.8 mg/L,and 2.36×109 U/L,respectively.The separation and purification of the PSI in the fermentation broth were also investigated.The purification efficiency of anion exchange chromatography,ultrafiltration,precipitation of ammonium sulphate or acetone were 8.83×109,6.35×109,4.56×109 and 3.28×109 U/L,respectively,indicating that the most efficient technique for the purification of PSI was anion exchange chromatography.3.The phytase gene of Penicillium glabrum NMH 2-3-1 was amplified by PCR using specific primers and cloned into the yeast expression vector p KLAC1.The recombinant plasmid p KLAC1-phy was introduced into Kluyveromyces lactis after linearization using Sac II.The recombinant strain which contain the phytase gene in the chromosome was obtained after screening.The SDS-PAGE identification indicated the exactly expression of the phytase protein,which is a 22 KD protein.The protein yield and the titer of phytase reached to 2.3 mg/L and 1.37×104 U/L,respectively,when the induction culture time was 120 h.The expression of phytase in Kluyveromyces lactis was successfully achieved.4.The enzymatic properties of heterogenous expressed phytase were investigated.The phytase retain its activity in range of p H 3.0 to 7.0 and its highest activity at p H 5.0.The optimum reaction temperature is 50℃.The enzyme activity remained by 58.3%,47.5%,and 25.6%,respectively,after incubation in water bath at 70℃ for 10,20,and 40 min,indicating its good thermal stability.The enzyme activity was increased by high concentrations of Ca2+ and Mg2+,but suppressed by high concentrations of Cu2+,Mn2+,and Al3+.The enzyme also exhibits strong anti-hydrolysis of pepsin and trypsin.