Enhanced Production Of Poly-γ-glutamic Acid By Overexpression Of The PgsBCA And PgdS In Bacillus Licheniformis

The poly-γ-glutamic acid(γ-PGA),produced mainly by microbes,is a water-soluble polyamino acid compound commonly used in industry.It has broad applications in fields of cosmetics,food,agriculture,medicinal industries and many others.At present,the production of γ-PGA mainly relies on microbial fermentation,but its low yield,high production cost,and inability to meet the needs of industrialized large-scale production are the most important problems.Studies have shown that constructing recombinant strains by overexpression of key enzyme genes in the synthesis pathway of γ-PGA may be one of the methods to increase the yield of γ-PGA.Therefore,in this experiment,the γ-PGA synthetase genes(pgsB,pgsC,pgsA)and degrading enzyme genes(pgdS)were overexpressed in Bacillus licheniformis to construct recombinant strains in order to obtain recombinant strains with high yield of γ-PGA,thereby providing a new strategy for the construction of high yield strains of γ-PGA.The main experimental results are as follows:(1)The γ-PGA production strain provided by Sichuan Food Fermentation Industry Research and Design Institute was identified by morphology,physiology and biochemistry,production performance,and molecular biology.Finally,the strain was named Bacillus licheniformis DY136.Said strain was undergo the processes of fermentation and culture,and the fermentation curve was determined.During the whole fermentation process,the maximum OD600 reached 2.30±0.12,and the γ-PGA yield reached the maximum21.00±4.77 g/L in 54 h.(2)Clone the pgsB,pgsC,pgsA,and pgdS genes in the strain Bacillus licheniformis DY136 and perform bioinformatics analysis on the proteins encoded by them.The results showed that the sizes of genes pgsB,pgsC,pgsA and pgdS were 1182 bp,450 bp,1170 bp and 1245 bp,respectively.The results of bioinformatics analysis showed that the molecular weights of PgsA,PgsB,PgsC and PgdS proteins were 43.89 k Da,43.88 k Da,16.35 k Da,45.61 k Da,respectively,the isoelectric points were 8.74,5.11,9.48,9.20,respectively.PgsA,PgsB,and PgdS proteins were hydrophilic proteins,and PgsC proteins were hydrophobins.PgdS contains signal peptide,which was a secreted protein.(3)Using the vector p HY300 PLK as backbone,the promoter P43,α-amylase gene terminator Pamy L,and GFP reporter gene expression elements were connected to the backbone to construct a new Bacillus licheniformis expression vector PHY300PLK2.0 by means of enzyme digestion.The vector was transformed into Bacillus licheniformis DY136,and the reporter gene GFP expression was detected by SDS-PAGE method,and the target protein band with a size of about 27.00 k Da was obtained.The recombinant strain fermentation broth can see obvious fluorescence under ultraviolet irradiation,indicating the vector can successfully express the target gene in Bacillus licheniformis DY136.At the same time,we verified the stability of the vector PHY300PLK2.0.The results showed that the plasmid carrying rate was 100% after 10 consecutive passages,indicating that the vector is in Bacillus licheniformis DY136 can be maintained stably.In summary,the vector PHY300PLK2.0 is able to efficiently express the target gene in Bacillus licheniformis.(4)Using the vector PHY300PLK2.0,the genes pgsB,pgsC,pgsA,pgdS,and pgsBCA were overexpressed in Bacillus licheniformis DY136 to construct DY136-pgsA,DY136-pgsB,DY136-pgsC,DY136-pgsBCA and DY136-pgdS.The above-mentioned recombinant strain was fermented and cultured,and the results showed that the growth status of the recombinant strain was higher than control strain(DY136-PHY)during the whole fermentation process,and its reducing sugar utilization ability was stronger than control strain after fermenting for 24 h.The γ-PGA yields of DY136-pgsA,DY136-pgsB,DY136-pgsC,DY136-pgsBCA and DY136-pgdS were 25.67±1.04,24.50±0.87,24.00±1.50,21.33±4.04 and 21.67±0.76 g/L,respectively.Compared with the control strains,the increase was 26.27%,20.51%,18.05%,4.92% and 6.59%.The maximum acetoin content was 3.49±0.20,4.13±0.19,3.41±0.09,4.58±0.04 and 3.28±0.12 g/L,respectively,which were 55.87%,31.72%,59.53%,18.78%,65.85% lower than the control strain(5.44±0.13),respectively.In summary,overexpression of pgsB,pgsC,pgsA,pgdS,pgsBCA genes can not only improve the growth of strains,but also increase the production of γ-PGA.(5)The yield curve of γ-PGA was further determined during the fermentation of strain DY136-pgsA.During the whole fermentation process,the yield of γ-PGA was higher than control strain(DY136-PHY).The maximum yield of γ-PGA could reach 29.67±1.61 g/L,an increase of 25.32% compared to control strain,indicating that overexpression of pgsA is beneficial to the synthesis of γ-PGA.