High-efficiency expression and secretion of human FGF21 in Bacillus subtilis by intercalation of a mini-cistron cassette and combinatorial optimization of cell regulatory components

Abstract

BackgroundRecombinant human Fibroblast growth factor 21 (rhFGF21) is an endocrine hormone that has profound effects on treatment of metabolic diseases. However, rhFGF21 is prone to form inclusion body when expressed in bacteria, which results in, the downstream process of purification of bioactive rhFGF21 is time-consuming and labor intensive. The aim of this work is to explore a new method for improving the soluble expression and secretion level of rhFGF21 in B. subtilis.ResultsA codon optimized rhFGF21 gene was expressed under the control of a strong inducible promoter PmalA in B. subtilis. A mini-cistron cassette (from gsiB) was located upstream of rhFGF21 in expression vector (pMATEFc5), which could reduce the locally stabilized mRNA secondary structure of transcripts and enhance the efficiency of translation initiation. Then various chaperones were further overexpressed to improve the expression efficiency of rhFGF21. Results showed that overexpression of the chaperone DnaK contributed to the increase of solubility of rhFGF21. Moreover, an extracellular proteases deficient strain B. subtilis Kno6cf was used to accumulate the secreted rhFGF21 solidly. In addition, eleven signal peptides from B. subtilis were evaluated and the SPdacB appeared the highest secretion yield of rhFGF21 in B. subtilis. Finally, the combinatorial optimized strain achieved an about ninefold increase of the soluble rhFGF21 production after 24 h of flask fermentation in comparison with the initial production strain.ConclusionThis work provided a comprehensive strategy for secretory expressing the heterologous protein rhFGF21 in B. subtilis. To our knowledge, this is the first report of the highly efficient production of rhFGF21 in B. subtilis and this approach may provide some suggestions for heterologous proteins production in B. subtilis.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1066-4) contains supplementary material, which is available to authorized users.

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

“…Several studies provide guidelines to utilize the existing techniques to their best efficiency and compatibility. One such report describes a combinatorial optimization scheme involving fine-tuning of promoter strength, translation, and folding efficiency in a protease-deficient B. subtilis, which altogether led to a ninefold increase in the production of human fibroblast growth factor 21, whose soluble expression is difficult due to protein aggregation [14]. Other recent technical aspects of recombinant protein expression in Bacillus have been reviewed elsewhere [92].…”

“…It was reported that proteins with large molecular weight or those that harbor several membrane domains pose a far greater tendency to form insoluble aggregates and are prone to proteolysis [10]. Moreover, additional challenges include mimicking eukaryotic post-translational modifications (such as glycosylation) [11], production of harmful endotoxins (E. coli) [12], reduced cell viability resulting from unwanted by-products [13], and low protein yields [14]. To tackle these obstacles, different recombinant DNA technologies, including manipulation of gene expression control [15][16][17], directing proper bond formation and protein folding [18,19], interfering host metabolic pathways [20,21], random or directed evolutions of bacterial strains or enzymes [22], and series of other methods have been systematically employed (summarized in [23]).…”

Efficient production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

“…It is especially important when a mammalian protein of interest is produced in a heterologous, such as bacterial, host. GFs are a protein type produced in a heterologous host [1,[15][16][17][18][19][20]. To meet increasing demand, highly productive systems, such as E. coli or yeast, are used for GF production.…”

“…With an increasing interest in functional cosmetics, the demand for GFs has also increased. Various expression systems, including Escherichia coli [1,[15][16][17][18][19], Bacillus subtilis [20], mammalian cell [21], baculovirus [22], silkworm [23,24], and plant [11,12] systems, are used for cost-effective and e cient production of recombinant GFs. Among them, the E. coli system is frequently used for the production of recombinant proteins because of facile genetic modi cation, rapid protein expression, and high growth rate.…”

Enhanced production of d‐psicose 3‐epimerase in Bacillus subtilis by regulation of segmented fermentation

“…Compared with Gramnegative bacterium Escherichia coli, Gram-positive B. subtilis has obvious advantages such as GRAS organism (Generally Recognized As Safe), lacking obvious codon bias, simplified fermentation, and the ability of secreting recombinant proteins. Among them, secretion of exogenous protein is the main advantage of B. subtilis for its application in industrial enzyme production [5][6][7].…”