Construction And Activity Analysis Of Antibacterial Bait-algae And Bait-pastoris

Diseases have been a serious problem in the aquaculture and livestock husbandry industry.The routine method to control the diseases is to use the traditional antibiotics,which may result in the problems of the food safety and the environmental risk.The antimicrobial peptides(AMPs)are considered to be the potential antibiotic alternatives due to their unique antibacterial mechanism to avoid evolution of resistance by microbes..In addition,AMPs do not produce harmful residues in the body because they can be degraded in vivo.It is expensive for chemical synthesis of AMPs.On the other hand,it is difficult to purify the AMPs directly from organisms.Fortunately,the recombinant protein expression technology provides the possibility that AMPs can be produced on an industrial scale.In this study,the AMP pisL9K22 WK and NZ2114 gene sequences were cloned and constructed respectively the expression plasmid regulated by FLD1 promoter.Then the constructs were transformed into the yeast(pichiapink)by electroporation respectively.Gene recombinant yeast strains to express pisL9K22 WK or NZ2114 were obtained.On the other hand,NZ2114 gene sequence was modified according to the codon bias of the chloroplast genome in Chlamydomonas reinhardtii,and was transformed into Chlamydomonas reinhardtii chloroplast.The total proteins from transformants showed very strong antimicrobial activity for the first time.The main work and results are as follows:1.The AOX1 promoter in the pPink-LC vector was replaced by FLD1 promoter.Then pisL9K22 WK and NZ2114 gene were ligated into the vector for constructing the expression vector FPLC and FNLC which express pisL9K22 WK and NZ2114 intracellular respectively.Four repeats of the pisL9K22 WK and NZ2114 gene were fused in tandem and ligated to the ?-factor and inserted into a site after the FLD1 promoter for generating the construct ?FPHLC and ?FNHLC.2.These constructs were transformed into the yeast(pichiapink)by electroporation respectively.A large number of transformants were screened on PAD media.PCR analyses on the transformants proved that the target gene had beenintegrated into the genome of the yeast(pichiapink).Finally some recombinant yeast strains were obtained,PH6 and NZ6 which express intracellular pis L9K22 WK or NZ2114 respectively,and PIS3 and NZ2114-9 which express extracellular pisL9K22 WK or NZ2114 respectively.3.In order to avoid the use of toxic methanol as an inducer to express the recombinant proteins in the yeast.In this study methanol was replaced by the choline chloride as an inducer,which can be used as the food additive.The fermentation conditions for transformants was optimized.The total protein from transformants was tested for the antimicrobial activity.The results indicated that the total proteins from transformants showed the strong antimicrobial activity.And the optimal concentration of choline chloride was 1%(w/v)for PH6 and 2%(w/v)for NZ6 to induce the recombinant AMP expression,the optimal inducing time was 120 h and 72 h for PH6 and NZ6 respectively.4.An AMP NZ2114 gene was modified according to the codon bias of the chloroplast genome in Chlamydomonas reinhardtii.The promoter 16s/psbA was ligated to construct the expression vector p322-NZ which was transformed into Chlamydomonas reinhardtii chloroplast with the vector p228 with spectinomycin resistance gene.After repeated screening,the strains named 16 which expressed NZ2114 with high efficiency was obtained.The total protein from 16 showed a very strong antimicrobial activity within 24 hours.The results of Tris-Tricine SDS-PAGE showed that the total proteins from 16 have a protein bands in 4 kDa.In summary,this study constructed the yeast(pichiapink)strains that can express AMPs,and explored a new way to induce the expression of the recombinant AMP in the yeast(pichiapink)by using choline chloride as an inducer to replace the methanol.The genetically-engineered algae strain was constructed,which can use the chloroplasts genetic system of Chlamydomonas reinhardtii to express AMPs.The NZ2114 was proved to express in the chloroplasts of Chlamydomonas reinhardtii with high efficiency taken together,the present study provided a new approach for production and utilization of antibacterial bait-algae.