Development of a lipase gene expression and secretion system for the protein over-production in Bacillus licheniformis

[摘要] Bacillus represents a genus of Gram-positive bacteria, which are ubiquitous in naturebeing found in soil, water, and airborne dust. They are distinguished from otherGram- positive bacteria by their ability to produce endospores when environmentalconditions become unfavourable. Certain Bacilli species have been implicated ascausative agents of diseases such as food poisoning and anthrax while others havebeen applied in the homologous production of products such as riboflavin, ribose,poly-g-glutamic acid and industrially important enzymes like alpha-amylases, lipasesand 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 speciesincluding Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens;Bacillus species have been employed as industrial workhorses in the production ofhomologous proteins. This has attracted Bacilli for development as expressionsystems for expression of heterologous proteins. The promoters that have been used todrive heterologous expression of proteins are predominantly of genes expressingextracellular proteins. These promoters are even more attractive due to the fact thattheir genes also contain sequences encoding signal peptides that direct thetranslocation of proteins to the extracellular, which make the downstream processingof the expressed proteins easy. A physiological study on the production ofextracellular lipase from Bacillus licheniformis revealed that the protein is expressedat relatively high levels particularly when grown on nutrient broth in the presence ofthe 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 andthe signal peptide region of the Bacillus licheniformis MBB01 extracellular lipase andto evaluate its potential as a tool for expression and secretion of endogenous andheterologous proteins.The study described the improved method for genome walking based on the cassetteligation-mediated PCR principle which was used to clone the promoter and signalpeptide region of the lipase gene from Bacillus licheniformis MBB01. A 200 bp DNAcassette flanked by various restriction enzymes was introduced within the multiplecloning site of pUC18 plasmid to yield the pLigCas plasmid. Excision of the cassettewith restriction enzymes located at opposite ends of the cassette resulted in the releaseof an efficiently annealed cassette with ends that are ligatable to a compatiblyenzyme-restricted genomic DNA sample. Treatment of the excised cassette withalkaline phosphatase prevented self ligation between cassette DNA molecules andensured preferential ligation with targeted enzyme restricted genomic DNAfragments. The PCR technique referred to as Single-Strand Amplification PCR whichinvolves an initial amplification using a lone primer designed based on the knownregion of the target region and the cassette-target DNA ligation mixture as thetemplate was employed. The single stranded DNA product obtained during the initialSSA-PCR is used as a template in the second PCR by employing a nested locusspecific primer paired with a cassette specific primer in a conventional PCR whichresults in increased selectivity and specificity of the PCR product. The SSA-PCRtechnique was used to amplify DNA fragments of 800 and 2200 base pairsrespectively corresponding to the regions upstream and downstream to the maturelipase gene fragment of Bacillus licheniformis MBB01. Nucleotide sequence analysis revealed the presence of the open reading frame encoding the isochorismatase genelocated downstream to the lipase gene. Isochorismatase is a family of hydrolaseenzymes, that is also referred to as dihydro-2,3 dihydroxybenzoate synthase. Someenzymes belonging to this family have been found to be capable of catalyzing thebioconversion of isochorismate in the presence of water to produce dihydro-2,3-dihydroxybenzoate and pyruvate, which are used as important chiral starting materialsin the manufacture of bioactive substances such as the siderophore enterobactin andcarbasugars. The other ORF encoding a hypothetical conserved protein of unknownfunction in Bacillus was located on the complementary strand upstream to thepromoter region of the lipase gene.The promoter and signal sequence of the extracellular lipase from Bacilluslicheniformis was incorporated into an Escherichia coli / Bacillus shuttle vectorcomprising of replicative elements from pUB110 and pUC18. The shuttle vectordenoted pSV6 contained the multiple cloning site, a sequence encoding 6 His tag andthe rrnB T1T2 terminator from Escherichia coli. The genes encoding thecarboxylesterase from Bacillus pumilus, Taq DNA polymerase from Thermusaquaticus and the mature lipase from Bacillus licheniformis were subcloned into theshuttle and the enzymes were expressed as C-terminally His tagged proteins inBacillus licheniformis MBB01 strain. The endogenous mature lipase gene could notbe expressed while the Taq DNA polymerase and the carboxylesterase genes weresuccessfully expressed and processed to the extracellular medium. The level ofexpression was however different, with the carboxylesterase being expressed at levelsthat according to visual estimations on SDS-PAGE, were 50 % more than that of thebackground proteins. The fact that the activities of the carboxylesterase and the Taq DNA polymerase couldbe detected in the supernatant is a preliminary indication that the host strain is capableof translocating the otherwise intracellular proteins to the extracellular medium.Further studies, are however required to confirm the nature and status of the Nterminalsequences of the secreted proteins. A provision has been made to introducewithin the pSV6 vector at a later stage the gene encoding type 1 Signal peptidases thatcould be co-expressed to facilitate cleavage of the signal peptide and prevent theprobable bottleneck of signal peptide processing. The signal peptidase gene togetherwith a functional promoter could be introduced by subcloning using theCla1/Nhe1/AscI polycloning sites within the pSV6 expression vector. The Nhe1 site iscompatible with Spe1, Xba1 and AvrII, and the Asc1 is a rare cutter (as it is an 8nucleotide recognizing restriction enzyme) which is also compatible with sticky endsgenerated by Mlu1. The size of the pSV6 plasmid is also large and this could restrictthe cloning of genes containing large open reading frames, and the bigger the size thehigher the instability of the recombinant plasmid. The size could be effectivelyreduced by the replacing the kanamycin and ampicillin resistance genes in pSV6 by asingle gene encoding the chloramphenical acetyltransferase gene from pNW33Nplasmid (available from Bacillus Genetic Stock Center, Ohio State University, Ohio,USA), which is reportedly capable of serving as a selection marker in bothEscherichia coli and Bacillus hosts.

[发布机构] University of the Free State