Biobutanol - Biotechnology in China: Biofuels and Bioenergy - page 89

which self-excised from the RNA, can form a ribonucleoprotein complex with a
conserved IEP (intron-encoded protein), which can recognize the DNA target site
and make an invasion event [ 36 ]. Further research revealed that the target-site
recognition rules were determined by two sites located on the intron RNA
(EBS1d and EBS2). Therefore, the modification of the two sites according to a
statistical mathematical model will lead to artificial insertion of the intron into
target sites. A commercial gene-knockout system, Targetron (Sigma-Aldrich, St.
Louis, MO, USA), has been developed based on these rules. Minton's group in
the University of Nottingham, UK [ 19 ] and Jiang's group (Shanghai Institute of
Plant Physiology and Ecology, Chinese Academy of Sciences) [ 45 ] have
almost simultaneously reported the application of such a gene-knockout system
in C. acetobutylicum. Following the establishment of this method, the genetic
manipulation of C. acetobutylicum has become feasible in many laboratories.
Green [ 13 ] believes that this method based on the group II intron system for
gene knockout of C. acetobutylicum has made significant advances and that it is
now possible to construct multi-step biosynthetic pathways paving the way for
new synthetic clostridia.
2.2.2 Intron-Anchored Targeted Gene Deletion in C. acetobutylicum
In 2011, Li's group in the Institute of Microbiology, CAS, proposed an intron-
anchored gene deletion approach for C. acetobutylicum, which combines the
advantage of the group II intron ''ClosTron'' system and homologous recombi-
nation [ 23 ]. In this approach, an allele homologous to the upstream or downstream
of the intron target site was constructed into the intron. Upon introducing this
construct into the target microorganism, an intron retrotransposition might occur
initially. The deletion of the target genes could then be achieved by homologous
recombination. Using this method, the ctfA/B and CAC1493/1494 operons
were successfully deleted. To date, the group II intron system has been used to
inactivate genes in at least ten different bacterial species [ 44 ], for most of which
targeted gene deletion is impossible. Therefore, the approach developed in this
study has the potential to be applied for gene deletion in those species where
first-step insertion via intron retrotransposition has been established.
2.2.3 Engineering C. acetobutylicum to Accept Unmethylated DNA
It is difficult to genetically manipulate the important genus Clostridium, due to the
existence of the restriction and modification (RM) systems. Most RM systems
comprise a DNA methyltransferase (MTase) and a restriction endonuclease
(REase). The MTase enables recognition of 'self' DNA by methylation of
specific nucleotides within particular DNA sequences, whereas the REase
enzymatically cleaves the 'foreign' unmodified DNA. Li's Group in the Institute
of Microbiology, CAS, reported the construction of a CAC1502 disrupted mutant