Production Of Human Epidermal Growth Factor (hEGF) Using Immobilized Escherichia Coli

Microorganisms have been widely used in production of recombinant proteins in food and pharmaceutical fields for the simple culture and easy operation.However,efficient expression and secretion of recombinant proteins by Escherichia coli have remained as a challenge.Recombinant Human Epidermal Growth Factor（h EGF）,one of the most popular small molecule protein products for life science and biotechnology research,has received increasing attention for its large-scale industrial production.At present,the production of h EGF in E.coli is mainly based on the traditional free-cell fermentation.The application of biofilm-based immobilized continuous fermentation system in industrial production of chemicals provides a new method for the construction of efficient secretory expression systems for recombinant proteins.In this research,we proposed an immobilized continuous fermentation process for h EGF production taking advantages of biofilm formation of E.coli.Specific studies are as follows:1.E.coli MG1655 and BL21（DE3）were used as the starting strains to explore the influence of genetic modification on biofilm formation in E.coli.We selected four potential genes（moa E,gsh B,yce A and ych J）that inhibit biofilm formation to construct disrupted strains and three genes（bcs B,csg Acsg B and fim H）that have been shown to facilitate biofilm formation to construct plasmid-based strains and integrated strains.Finally,20 recombinant strains were constructed by CRISPR-Cas9 gene editing technology and plasmid expression respectively,which laid the foundation for exploring the effects of these genes on E.coli biofilm formation.2.In order to investigate if the modification of these genes could promote the ability of biofilm formation,crystalline violet and Congo red staining experiments were conducted to verify the biofilm formation of the above strains.It was found that overexpression of bcs B,csg Acsg B and fim H genes could promote the formation of biofilm especially through expression based on plasmids.Disruption of moa E,gsh B,yce A or ych J gene did not show significant effect on biofilm formation in LB medium.However,the gsh B-,yce A-and ych J-disrupted strains showed greatly enhanced biofilm formation after adding 5 g/L glucose in the medium.The overall results showed that most of genetic manipulations could promote biofilm formation to some extent.3.In order to investigate the effect of these modifications on the production of h EGF secretion,we firstly conducted free-cell fermentation.Results showed that compared with the h EGF secretion by wild BL21（DE3）strain,overexpression of bcs B,csg Acsg B and fim H by plasmid increased h EGF secretion by 38%,69%and 10%,respectively.By contrast,genome-integrated expression of these three genes could improve h EGF secretion to a more extent,showing an improvement by 106%,81%and63%,respectively.Genome-integrated expression of these genes was better for cell growth compared to plasmid-based expression.Moreover disruption of moa E,yce A and ych J in E.coli had resulted in an improvement in hEGF secretion by 25%,8%and29%,respectively.In contrast,disruption of gsh B reduced the production of h EGF.4.Based on the modification of biofilm-related genes,we created an immobilized continuous fermentation system taking advantages of E.coli biofilm.The successfully constructed BL21（DE3）chassis cells were used to verify the possibility of continuous production of h EGF by providing cotton fabric in LB medium as biofilm carrier.The best results were observed for the ych J-disrupted strain whose h EGF production was higher than the BL21（DE3）wild strain during all the 8 repeated batches,with an average of 28%higher h EGF production（from around 24 mg/L to 32 mg/L in the shake-flasks）.According to the results of OD₆₀₀ of the fermentation broth and the results of scanning electron microscopy,the expression of bcs B and csg Acsg B genes played a significant role in promoting the adsorption of the cells on the carrier.In addition to increase h EGF production,the growth rates and fermentation rates of cells in biofilm were also improved.The time of fermentation was shortened from 72 h to42 h per batch for plasmid-base strains,and from 48 h to 36 h per batch for for the integrated expression strains and gene disrupted strains.Different from free cells,the biofilm retained cells so that they could be used repeatedly.The existing cells retained in the biofilm at the start of each new batch and their already established production capacity would contribute to the accelerated fermentation rate.In addition,biofilm cells attached on carriers could keep growing throughout the fermentation and reach a cell density much higher than that in liquid culture.In a word,this study systematically investigated the effects of gene modification related to biofilm formation in E.coli on its biofilm forming ability as well as h EGF secretion,showing a potential in the continuous production of recombinant proteins.These genes could potentially be engineering targets for the design of efficient cell factories with improved protein secretory capacity in future.Different from common strategies employed to improve the production efficiency of heterogeneous proteins in E.coli,this study employed biofilm-based systems to develop efficient h EGF production processes,providing an effective new idea and reference for the subsequent creation of a continuous fermentation production process for more proteins.