Development of a lipase gene expression and secretion system for the protein over-production in Bacillus licheniformis
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Bacillus represents a genus of Gram-positive bacteria, which are ubiquitous in nature being found in soil, water, and airborne dust. They are distinguished from other Gram- positive bacteria by their ability to produce endospores when environmental conditions become unfavourable. Certain Bacilli species have been implicated as causative agents of diseases such as food poisoning and anthrax while others have been applied in the homologous production of products such as riboflavin, ribose, poly-g-glutamic acid and industrially important enzymes like alpha-amylases, lipases and proteases. Due to their ability to express and secrete large quantities of proteins, coupled with the Generally Regarded As Safe (GRAS) status of some of the species including Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens; Bacillus species have been employed as industrial workhorses in the production of homologous proteins. This has attracted Bacilli for development as expression systems for expression of heterologous proteins. The promoters that have been used to drive heterologous expression of proteins are predominantly of genes expressing extracellular proteins. These promoters are even more attractive due to the fact that their genes also contain sequences encoding signal peptides that direct the translocation of proteins to the extracellular, which make the downstream processing of the expressed proteins easy. A physiological study on the production of extracellular lipase from Bacillus licheniformis revealed that the protein is expressed at relatively high levels particularly when grown on nutrient broth in the presence of the detergent Tween 80. The DNA fragment encoding mature lipase from this strain has been cloned previously. The objective of the study was to clone the promoter and the signal peptide region of the Bacillus licheniformis MBB01 extracellular lipase and to evaluate its potential as a tool for expression and secretion of endogenous and heterologous proteins. The study described the improved method for genome walking based on the cassette ligation-mediated PCR principle which was used to clone the promoter and signal peptide region of the lipase gene from Bacillus licheniformis MBB01. A 200 bp DNA cassette flanked by various restriction enzymes was introduced within the multiple cloning site of pUC18 plasmid to yield the pLigCas plasmid. Excision of the cassette with restriction enzymes located at opposite ends of the cassette resulted in the release of an efficiently annealed cassette with ends that are ligatable to a compatibly enzyme-restricted genomic DNA sample. Treatment of the excised cassette with alkaline phosphatase prevented self ligation between cassette DNA molecules and ensured preferential ligation with targeted enzyme restricted genomic DNA fragments. The PCR technique referred to as Single-Strand Amplification PCR which involves an initial amplification using a lone primer designed based on the known region of the target region and the cassette-target DNA ligation mixture as the template was employed. The single stranded DNA product obtained during the initial SSA-PCR is used as a template in the second PCR by employing a nested locus specific primer paired with a cassette specific primer in a conventional PCR which results in increased selectivity and specificity of the PCR product. The SSA-PCR technique was used to amplify DNA fragments of 800 and 2200 base pairs respectively corresponding to the regions upstream and downstream to the mature lipase gene fragment of Bacillus licheniformis MBB01. Nucleotide sequence analysis revealed the presence of the open reading frame encoding the isochorismatase gene located downstream to the lipase gene. Isochorismatase is a family of hydrolase enzymes, that is also referred to as dihydro-2,3 dihydroxybenzoate synthase. Some enzymes belonging to this family have been found to be capable of catalyzing the bioconversion of isochorismate in the presence of water to produce dihydro-2,3- dihydroxybenzoate and pyruvate, which are used as important chiral starting materials in the manufacture of bioactive substances such as the siderophore enterobactin and carbasugars. The other ORF encoding a hypothetical conserved protein of unknown function in Bacillus was located on the complementary strand upstream to the promoter region of the lipase gene. The promoter and signal sequence of the extracellular lipase from Bacillus licheniformis was incorporated into an Escherichia coli / Bacillus shuttle vector comprising of replicative elements from pUB110 and pUC18. The shuttle vector denoted pSV6 contained the multiple cloning site, a sequence encoding 6 His tag and the rrnB T1T2 terminator from Escherichia coli. The genes encoding the carboxylesterase from Bacillus pumilus, Taq DNA polymerase from Thermus aquaticus and the mature lipase from Bacillus licheniformis were subcloned into the shuttle and the enzymes were expressed as C-terminally His tagged proteins in Bacillus licheniformis MBB01 strain. The endogenous mature lipase gene could not be expressed while the Taq DNA polymerase and the carboxylesterase genes were successfully expressed and processed to the extracellular medium. The level of expression was however different, with the carboxylesterase being expressed at levels that according to visual estimations on SDS-PAGE, were 50 % more than that of the background proteins. The fact that the activities of the carboxylesterase and the Taq DNA polymerase could be detected in the supernatant is a preliminary indication that the host strain is capable of translocating the otherwise intracellular proteins to the extracellular medium. Further studies, are however required to confirm the nature and status of the Nterminal sequences of the secreted proteins. A provision has been made to introduce within the pSV6 vector at a later stage the gene encoding type 1 Signal peptidases that could be co-expressed to facilitate cleavage of the signal peptide and prevent the probable bottleneck of signal peptide processing. The signal peptidase gene together with a functional promoter could be introduced by subcloning using the Cla1/Nhe1/AscI polycloning sites within the pSV6 expression vector. The Nhe1 site is compatible with Spe1, Xba1 and AvrII, and the Asc1 is a rare cutter (as it is an 8 nucleotide recognizing restriction enzyme) which is also compatible with sticky ends generated by Mlu1. The size of the pSV6 plasmid is also large and this could restrict the cloning of genes containing large open reading frames, and the bigger the size the higher the instability of the recombinant plasmid. The size could be effectively reduced by the replacing the kanamycin and ampicillin resistance genes in pSV6 by a single gene encoding the chloramphenical acetyltransferase gene from pNW33N plasmid (available from Bacillus Genetic Stock Center, Ohio State University, Ohio, USA), which is reportedly capable of serving as a selection marker in both Escherichia coli and Bacillus hosts.