Promoter engineering for efficient production of sucrose phosphorylase in Bacillus subtilis and its application in enzymatic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid

L-ascorbic acid (L-AA) is known as vitamin C and is one of the essential nutrients. It displays various physiological activities to maintain and promote human health[1]. It contains four hydroxyl groups in positions 2, 3, 5 and 6 and is widely used as acidifier, reducing agent, antioxidant, bleaching agent and stabilizer in cosmetics, food and medicine. However, the 2-OH group is extremely unstable upon exposure to oxygen, light, heat, alkaline, and metal ion. In addition, its excess absorption will cause cytotoxicity[2]. For example, 0.05-0.25 mM of L-AA can inhibit the growth of monolayer cultured fibroblasts from chick embryos, human and mouse skin[3]. In contrast, the glycosylated derivative of L-AA, 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) displays much more stable in aqueous solution and less cytotoxicity. It also readily releases L-AA by in vivo hydrolysis and exerts similar physiological activities[2], [4], [5], making it an excellent substitute for L-AA.

At present, the synthesis of AA-2G mainly depends upon enzyme-mediated biocatalysis. These enzymes used for producing AA-2G include α-glucosidases[6], cyclodextrin glycosyltransferase (CGTase)[7] and sucrose phosphorylase (SPase)[8], [9]. Among them, the SPases from Bifidobacterium longum (BloSPase) and B. breve (BbrSPase) have been reported to efficiently and exclusively produce AA-2G via a one-step glycosylation process using the inexpensive substrate sucrose[8], [9]. The SPase-based biotransformation method shows great potential for the substitute of the existing CGTase-based two-step method for industrial production of AA-2G, which is first developed by the Hayashibara Biochemical Laboratories of Japan in the 1990s[7]. The CGTase and glucoamylase used in the two-step method are both produced by the generally regarded as safe (GRAS) microorganism. However, the high-level production of BloSPase and BbrSPase were both just reported in the easy-to-operate Escherichia coli. Therefore, the one-step method based on the GRAS strain for the production of AA-2G need to be evaluated for the purpose of its nutraceutical and pharmaceutical application.

Bacillus subtilis, a generally considered to be a GRAS Gram-positive bacteria, has its various advantages for the production of industrially enzymes[10]. It does not produce pathogenic potential like endotoxin[11], [12], and has excellent protein secretion capacity to concentration in excess of 20 g/L[13]. With low nutritional requirements, its fermentation is easy to scale up[14]. Moreover, it has no significant codon usage preference[15]. Nonetheless, the secretory expression of heterologous proteins in B. subtilis still has many limitations and often cannot meet the needs of industrial applications[16]. In view of the problems with secretion of heterogenous protein B. subtilis, a large amount of researches has been reported on its secretion pathway[17], transcription elements[18] and host strains[19]. However, a specific expression element/host may be optimal for the secretion of one particular target protein, it is often found not to be optimal for another. Therefore, systematic optimization on the key gene expression elements is generally necessary to maximize the secretion of heterologous proteins in B. subtilis. Among them, constructing highly active promoters to drive the expression of exogenous genes is an effective strategy[20], which can be achieved by mining new promoters, optimizing and modifying core elements of promoters, and constructing tandem promoters. Meng et al. identified four new strong promoters (PsodA, Phag, PtufA, and PfusA) from B. subtilis genome based on transcriptome and bioinformatic data[21]. An optimal dual-promoter expression structure is also a commonly used strategy, for example, PamyL–PspovG is created to achieve a high extracellular pullulanase activity of 625.5 U/mL[22]. In addition, some new promoters were engineered by interlocking special binding motifs (-35 and -10 regions) in response to high salinity or low pH to increase transcriptional activities [23].

In this study, the SPases, which have L-AA glycosylation activity, were produced in B. subtilis for one-step production of AA-2G. The expression of various SPase genes proved that its secretion did not require signal peptide. Interestingly, BloSPase and BbrSPase with 95.5% of identity in amino acid sequence displayed quite different secretion level. A series of dual-promoters based on the effective and strong P43 promoter were screened and further optimized by fine-tuning the distance between two promoters. The optimal recombinant B. subtilis strain was demonstrated to secret a high extracellular SPase activity in 5-L fermenter for the one-step effective production of AA-2G based the GRAS strain.

留言 (0)

沒有登入
gif