An efficient ABC transporter signal peptide directs heterologous protein secretion in food-grade hosts.

An efficient expression-secretion system for heterologous protein production in food-grade hosts, Lactobacillus plantarum and Bacillus subtilis, is still required to broaden their applications. The optimal signal peptide compatible with both the desired protein and the target host is important for the system. Here, we constructed new expression-secretion vectors to be used in both bacteria. A natural plasmid originating from food-grade L. plantarum BCC9546 was used as a core vector combined with a strong constitutive promoter, L-ldh promoter, and various signal peptides from several types of L. plantarum proteins: ABC transporter, cell wall-associated and extracellular proteins. A gene encoding 88-kDa amylase isolated from starch-related L. plantarum TBRC470 was used as a gene model to evaluate the systems. By comparing the amounts of secreted amylase from the recombinant strains to that of wild type, all signal peptides gave higher yields of secreted amylase in recombinant B. subtilis. Interestingly, two ABC transporter signal peptides from glutamine and mannose ABC transporters provided noticeably high levels of secreted amylase in recombinant L. plantarum. Moreover, these signal peptides also gave high yields of secreted amylase in recombinant B. subtilis. From the results, the signal peptide of glutamine ABC transporter, which functions in essential amino acid transportation that is a precursor for synthesis of nitrogen-containing compounds and nitrogen homeostasis, has a potential use in development of an efficient expression-secretion system for heterologous protein production in both food-grade hosts.

A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis.

Bacillus subtilis is commonly used as a host for heterologous protein production via plasmid-based expression system. In order to improve product safety, avoid carbon catabolite repression and lower production cost, a novel salt-inducible vector, pSaltExSePR5, was developed based on a natural plasmid of Lactobacillus plantarum BCC9546. Salt-inducible promoter opuAA and a DNA fragment encoding a signal peptide of subtilisin E (SubE) were sequentially added to the core shuttle vector to facilitate expression and secretion of a target protein in B. subtilis. To evaluate the effectiveness of this system under salt induction, a protease gene from Halobacillus sp. without its native signal sequence was inserted in the pSaltExSePR5 plasmid downstream of SubE signal sequence and transformed into B. subtilis WB800. Protease activities from cell-free supernatants of the recombinant bacteria cultures induced with 0.5-6% NaCl were analyzed. The highest protease activity of 9.1 U/ml was obtained after induction with 4% NaCl, while the non-induced culture exhibited activity of 0.128 U/ml. The results demonstrated that pSaltExSePR5 provides an alternative vector for efficient and simple production of heterologous proteins in B. subtilis with a safer and more economic inducer.

Monitoring Lactobacillus plantarum BCC 9546 starter culture during fermentation of Nham, a traditional Thai pork sausage.

The use of Lactobacillus plantarum BCC 9546 (LpBCC9546) as a starter culture for Nham, a traditional Thai fermented pork sausage ensures product quality and consistency. However, no direct evidence has confirmed the growth of this starter during Nham fermentation. In order to investigate its role during Nham fermentation, LpBCC9546 was genetically modified to distinguish it from the natural microflora in Nham. LpBCC9546 was transformed with a recombinant plasmid pRV85 to produce the recombinant strain LpG11, which is resistant to erythromycin and emits green fluorescence. LpG11 was used as a starter culture for Nham fermentation, and its growth was monitored by plating on a selective medium and assay of fluorescent activity. During Nham fermentation the numbers of LpG11 increased ten fold during the first 12 h of fermentation, reaching maximum numbers of between 10(7) and 10(8) cfu g(-1) after 24 h, and then declining after 60 h to 10(5) cfu g(-1) at 168 h. The growth of LpG11 starter culture during Nham fermentation was very similar to that of the untransformed LpBCC9546, although after a prolonged period of fermentation the recombinant LpG11 bacteria appeared to lose the plasmid, or were outgrown by naturally present L. plantarum. The acidity, texture and color of fermented Nham inoculated with recombinant LpG11 or untransformed LpBCC9546 were similar. These results indicated that the recombinant L. plantarum strain LpG11 is a suitable starter culture for Nham fermentation, and that the ability to monitor its growth directly during Nham fermentation could be exploited to further improve Nham production.