Overproduction of Rummeliibacillus pycnus arginase with multi-copy insertion of the arg R.pyc cassette into the Bacillus subtilis chromosome

Abstract

A plasmid-less and marker-less strain with multi-copy integration of the arginase gene from Rummeliibacillus pycnus was constructed using Bacillus subtilis 168 as a host. A total of nine copies of the arg cassettes, in which the R. pycnus arginase gene was fused with the strong promoter P43, were inserted into the recipient chromosome. These multiple insertions were completed via step-by-step integrations into designed (2 copies) and random (9 copies) sites, respectively. A strategy for random site integration was developed based on the construction of the arg cassette sandwiched between "front" and "back" homologous arms which were randomly restricted from chromosomal DNA. An antibiotic resistance marker was applied in transformant selection and was eliminated via the Cre/lox system. Performance showed that the highest enzyme activity (14.5 U/mL) was obtained after culture in flasks, and this segregation stable strain could efficiently hydrolyze L-arginine with a 97.2% molar yield, showing potential application in the food industry.

Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

“…Several methods are available for multi-copy insertions of the target gene. Among them, integration at two and more different chromosomal sites may be the most stable [ 28 ], so the expression cassette was inserted into both the amyE and the lacA loci to increase the magnitude of expression of the target gene. The vector was first transformed into competent B. subtilis cells and the integration occurred at the amyE locus in the presence of spectinomycin.…”

“…In another study, nine copies of a arg R.pyc cassette containing the Rummeliibacillus pycnus arginase gene regulated by the strong promoter P43 were inserted into the recipient’s genome. Tests showed that the highest arginase activity (14.5 U/mL) was obtained from flask cultures, and this segregation-stable strain could efficiently hydrolyze l -arginine with a 97.2 % molar yield, suggesting a potential application for the food industry [ 28 ]. A strong promoter was engineered that allowed synthesis of BgaB and sfGFP to levels of 43 % and 30 % of intracellular proteins, respectively.…”

“…In addition, it has a long history of industrial use because of its excellent growth on cheap carbon sources, and robustness under industrial conditions [ 4 ]. Another advantage is that B. subtilis is regarded as a Generally Recognized as Safe (GRAS) organism that lacks endotoxins and is non-pathogenic [ 5 , 6 ]. Furthermore, it has no significant bias in codon usage [ 3 ].…”

“…Recently, several new vectors have been developed resulting in more efficient integration. Some of them allow production of recombinant proteins by incorporating many copies of the recombinant gene at different sites in the bacterial genome [ 5 , 13 ]. For example, an integrated vector engineered with the strong inducer-free promoter NBP3510 exhibited β-galactosidase (BgaB) and GFP (green fluorescent protein) production of 43 % and 30 % of the total cellular proteins, respectively [ 14 ].…”

Draft Genome Sequence of Rummeliibacillus sp. Strain TYF005, a Physiologically Recalcitrant Bacterium with High Ethanol and Salt Tolerance Isolated from Spoilage Vinegar

“…For instance, it can convert palm oil mill effluent into terpolymer polyhydroxyalkanoate and biodiesel (3), and it has potential for biomineralization (4). Furthermore, a thermally stable arginase from R. pycnus is used in the production of l -ornithine (5, 6). In this study, we report the draft genome sequence of Rummeliibacillus sp.…”