Development of a lipase gene expression and secretion system for the protein over-production in Bacillus licheniformis
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Date
2006-01
Authors
Ramagoma, Faranani
Journal Title
Journal ISSN
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Publisher
University of the Free State
Abstract
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.
Description
Keywords
Dissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2006, Bacillus (Bacteria) -- Genetics, Lipase, Gene expression