Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
RSC Advances
PAPER
1-Butanol production from glycerol by engineered
Klebsiella pneumoniae
Cite this: RSC Adv., 2014, 4, 57791
*
Miaomiao Wang, Lihai Fan and Tianwei Tan
To utilize the by-product of biodiesel production, Klebsiella pneumoniae, a well-known glycerol-
fermenting microorganism, was engineered to produce 1-butanol. The modified CoA-dependent and 2-
keto acid pathways were established by expressing the genes ter-bdhB-bdhA and kivd, respectively. The
1-butanol titer and specific BuOH yield were 15.03 mg Lꢀ1 and 27.79 mg-BuOH per g cell in KpTBB (K.
pneumoniae overexpressing the genes ter-bdhB-bdhA), and 28.7 mg Lꢀ1 and 51.58 mg-BuOH per g cell
in Kp-kivd (K. pneumoniae overexpressing the gene kivd), respectively. Moreover, the native products in
K. pneumoniae fermentation were down regulated using the antisense RNA strategy. The resulting yield
of 1,3-propanediol and 2,3-butanediol was reduced by 81% and 15%, respectively. This work reports a
new strain, K. pneumoniae, for 1-butanol production and the application of an antisense RNA strategy as
an effective method for reducing the main by-products.
Received 27th August 2014
Accepted 20th October 2014
DOI: 10.1039/c4ra09016k
tolerance to ethanol and potentially to 1-butanol than Clos-
tridium species,12 was engineered with some substituted Clos-
tridium enzymes for 1-butanol production.13 Clostridium
1. Introduction
Alternative energy sources have gained increasing popularity
due to the shortage of fuel and the increasing global environ-
mental concerns. Over the past few decades, ethanol has been
regarded as a major renewable substitute of fuel.1,2 Ethanol,
however, is not ideally suited because of its lower energy density
and hygroscopicity-resulting storage and distribution prob-
lems.3–5 In addition, due to the limits set by regulations and the
requirements for engine modication, the percentage of
ethanol blended with gasoline is generally limited to 10%.
Compared to ethanol, 1-butanol has higher energy density and
lower vapor pressure, and can be blended with gasoline up to
85% and used to fuel cars without engine modication.6
Therefore, 1-butanol, especially biobutanol, is considered a
highly promising alternative fuel.6–8
The microbial production of 1-butanol using lower cost
substrates has received worldwide attention, and Clostridium
species, a solventogenic bacteria, can produce 1-butanol by
anaerobic fermentation.9,10 On the other hand, the industrial
production of 1-butanol by Clostridium species has been
restricted due to its complex physiology and difficulty for
genetic modication.9–11 Many other engineered strains have
been used for 1-butanol production. E. coli, which has a higher
growth rate than Clostridium species,12 was engineered to
produce 1-butanol by transferring the CoA-dependent 1-butanol
production pathway9 or by introducing the synthetic 2-keto acid
pathway.3,10 Saccharomyces cerevisiae, which has a higher
tyrobutyricum,14 a native butyric acid-producing bacterium, was
introduced the aldehyde/alcohol dehydrogenase of Clostridium
acetobutylicum to produce 1-butanol. Lactobacillus brevis,15
which has a higher tolerance to 1-butanol, was engineered to
utilize C5 and C6 substrates to produce 1-butanol. Synecho-
coccus elongatus,16 a kind of autotrophic photosynthetic micro-
organism, was engineered by introducing a CoA-dependent
pathway to produce 1-butanol utilizing CO2. The byproducts10,17
in host fermentation, however, are normally reduced by RED
system or homologous recombination. Although they are suit-
able for knocking out a single gene, they are not practical for
more advanced metabolic engineering strategies according to
the introduced multi FRT sites or the multi selection markers.
The asRNA strategy18 could be an alternative and possibly a
more exible and empowering method to down regulate the
enzyme level and product titer.
Glycerol, as
a by-product of biodiesel production, is
produced abundantly every year.19,20 Utilizing the crude glycerol
and converting it to value-added products have become
increasingly attractive.21,22 Klebsiella pneumoniae, which can
grow on glycerol as the sole carbon source, has been studied
extensively since the 1980s. Although K. pneumoniae is a path-
ogenic microorganism, a great deal of research on chemicals
(1,3-propanediol,17,19 3-hydroxypropionic acid,23,24 2-butanol25)
production has been conducted. This is due mainly to its
capacity to metabolize glycerol and having a similar genetic
background to Escherichia coli, together with rapid cell prolif-
eration.23 To our knowledge, K. pneumoniae has not been engi-
neered to produce 1-butanol.
National Energy R&D Center for Biorenery, Beijing Key Laboratory of Bioprocess,
Beijing University of Chemical Technology, Beijing 100029, China. E-mail: twtan@
mail.buct.edu.cn; Fax: +86 10 64715443; Tel: +86 10 64416691
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 57791–57798 | 57791
Wang, Miaomiao
