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Noda Institute for Scientific Research GRANT
2012 Young Investigator Research Grant

Molecular breeding of Clostridia for butanol production fromrice straw
Shunichi Nakayama
Department of Fermentation Science and Technology, Faculty of Applied Bio-science, Tokyo University of Agriculture

Research aims
Biobutanol produced by Clostridium spp. can be used asa biofuel and/or chemical feedstock. For cost-effective andsustainable production of biobutanol, an untapped biomass
such as rice straw could be used as a substrate. Rice straw,
which is composed of cellulose, hemicellulose, and lignin,
is cheap and abundant. However, the utilization of ricestraw is limited because butanol-producing clostridia cannot degrade cellulose, which is the major carbon source inlignocellulose. To achieve butanol production from lignocellulose, cellulose needs to be hydrolyzed to di- or monosaccharides that can then be metabolized by Clostridium
spp.
In this study, we developed a system for butanol production from rice straw using a co-culture of cellulose-degrading Clostridium thermocellum and butanol-producing
Clostridium saccharoperbutylacetonicum. In addition, weshowed that β-glucosidase enhances butanol production
from rice straw, and identified promoters that may be used
for high-level expression of the β-glucosidase gene to increase productivity.
Methods
Growth and co-culture of cellulose-degrading C. thermocellum and butanol-producing C. saccharoperbutylacetonicum were performed using optimized conditions as previously described.1) Briefly, C. thermocellum cells weregrown in National Institute of Technology and Evaluation
Biological Resource Center (NBRC) medium 979 containing cellobiose at 60°C and collected by centrifugation. Cellpellets were washed and suspended in the same mediumwithout any added carbon source and adjusted to an opticaldensity at 600 nm (OD600 nm) of 2. Six microliter of the cellsuspension was used to inoculate 6 mL of NBRC medium
979 containing rice straw treated with 0.1 N NaOH or nontreated rice straw (final OD600 nm=1) and cultured at 60°C.
C. saccharoperbutylacetonicum cells were collected after
24 h by centrifugation, washed, and suspended in clostridial
growth medium (CGM) containing 4% glucose and adjusted to OD600 nm=13. The cell suspension (1 mL) was thenadded to C. thermocellum cultures that had been incubatedat 30°C (final OD600 nm=1). Co-cultures were incubated at30°C. Cellulase and β-glucosidase enzymes were added tothe co-culture medium at the same time as inoculation of C.

saccharoperbutylacetonicum. Fermentation products werequantified by HPLC using an Aminex HPX-87H column(Bio-Rad Laboratories K.K., Japan) with a differential refractive index detector (Shimadzu Corporation, Japan) andan aqueous solution of 0.05 mM H2SO4 as the solvent. Enzyme activities for cellulase were measured according to
the method described by Chundawat et al.2)
Twelve promoter sequences resembling the consensus
promoter sequence in Clostridium spp. (TTGACA-N(17)TATAAT) were screened using in silico molecular cloning
software. Promoters were ligated upstream of the AcGFP
reporter gene in shuttle vector pNAK1. Recombinant plasmids were transfected into C. saccharoperbutylacetonicum
strain N1–4 by electroporation.3) AcGFP expression levels
were compared using western blot analysis using an antiAcGFP antibody.
Results
Butanol production from alkali-treated rice straw
Co-cultures of C. thermocellum and C. saccharoperbutylacetonicum produced very low levels of butanol (1.1±0.1 g/L) from non-pretreated rice straw. The maximum
OD600 nm of co-culture was 2.7 after one day of cultivationand gradually decreased to 1.3 during cultivation, suggesting that C. thermocellum was not metabolizing cellulose torelease usable sugars to sustain growth of C. saccharoperbutylacetonicum. The lack of butanol fermentation fromcellulose in these culture conditions was likely because therice straw contains lignin, which inhibits cellulase accessibility to the substrate.
In order to improve butanol production, the lignin in ricestraw was removed by the alkaline-treatment and the pretreated rice straw was used as a substrate. As expected, butanol production from delignified rice straw—in which thelignin content was decreased to 70%—was 2.3-fold higher
than that of non-pretreated rice straw (2.5±0.1 g/L) (Fig. 1).
This result clearly indicates that delignification improves
butanol production during co-culture of C. thermocellumand C. saccharoperbutylacetonicum. Although there was anincrease in butanol production, the concentration was 67%
lower than that achieved with crystalline cellulose, as previously reported.1) This may be due to the presence of lowlevels of cellulase activity in the co-culture. Therefore, toconfirm whether additional cellulase enhanced butanol production, commercial cellulase from Aspergillus niger was		47

Fig. 1. Fermentation products (g/L) from alkali-treated rice straw C.
thermocellum and C. saccharoperbutylacetonicum co-cultures. Cellulaseor β-glucosidase (10 U and 100 U) were added to the co-culture medium.
Bars indicate amounts of acetate (horizontal lines), butyrate (verticallines), ethanol (solid white), and butanol (solid gray). All samples were assayed three times. Means±standard error are shown with arbitrary units.
Table 1. Comparison of enzyme activity related to lignocellulose degradation (U/mg).

Endoglucanase*
Exoglucanase*β-glucosidase
xylanase

C. thermocellum

N1-4

Cellulase (10U)

101.15±3.00  4.51±0.40  0.07±0.00   2.85±0.03

1.00±0.28
0.47±0.08
0.35±0.02
0.38±0.02

25.42±2.48
10.79±0.24
17.38±1.94
0.17±0.01*Enzyme activity is expressed as milliunits/mg

added to the co-culture system. As expected, butanol production in the co-culture increased significantly with theaddition of cellulase (Fig. 1). This result indicated that thelower yields of butanol in the co-culture system werecaused by low activities of cellulase.
Delignified rice straw consists mainly of cellulose andhemicellulose, and the cellulase complex that degrades
cellulose and hemicellulose contains endo-glucanase, exoglucanase, β-glucosidase, and xylanase. The identificationand expression of enzymes that contribute to high butanol
production during co-culture is required for cost–effective
butanol production. Therefore, cellulase activity in supernatants from single-cultures of C. thermocellum and C.
saccharoperbutylacetonicum was compared to that of commercial cellulase (Table 1). Endo-glucanase and xylanase
activities were the highest in C. thermocellum. By contrast,
exo-glucanase and β-glucosidase activities were highest in
the commercial enzyme (Table 1).
Interestingly, cellulase showed the highest β-glucosidase
activity among the enzyme activities tested. Considering
this result, we tested the effect of β-glucosidase in the coculture and found that the addition of this enzyme significantly enhanced butanol production (Fig. 1). Butanol
concentrations with 10 U and 100 U of β-glucosidase added
per gram of biomass were 4.4 and 4.7 g/L, respectively.
This result clearly indicates that the supplementation ofβ-glucosidase activity is important for butanol production
from delignified rice straw during co-culture of C. thermocellum and C. saccharoperbutylacetonicum.

Fig. 2. Comparison of expression levels of AcGFP and Chloramphenicol acetyltransferase (CAT) by western blot analysis using anti-AcGFP
and anti-CAT antibodies. Crude extracts from C. saccharoperbutylacetonicum strain N1–4 transformants harboring expression plasmids grown for
24 h were separated using SDS-PAGE. Lanes: 1, pNAK1/phosphotransferase systemIIC component promoter; 2, pNAK1/thiolase promoter; 3,pNAK1/hsp90 promoter; 4, pNAK1/rubrerythrin promoter; 5, pNAK1/ribosomal protein L13 promoter; 6, pNAK1/uncharacterized protein promoter; 7, pNAK1/butanol dehydrogenase promoter; 8, pNAK1/phosphatebutyryltransferase promoter; 9, pNAK1/superfamilyII DNA and RNA helicases promoter; 10, pNAK1/adenine deaminase promoter; 11, pNAK1/
prolyl-tRNA synthetase promoter; 12, pNAK1/ Transcriptional antiterminater promoter 13, pNAK1 (control vector). Since the shuttle vector,
pNAK1, contains CAT as a selective marker, maintenance of this vector
was confirmed using an anti-CAT antibody (lower panel).

Screening of promoter sequences for β-glucosidase
expression
Our data suggested that the expression of β-glucosidase
by Clostridium spp. in co-cultures would be necessary to increase butanol production from rice straw. Therefore,
promoter sequences that were similar to the consensus
promoter sequence in Clostridium spp. were screened for activity in C. saccharoperbutylacetonicum using the reporter
gene AcGFP. Among the promoter sequences selected fromthe whole genome sequence of strain N1–4, the strong expression of AcGFP was confirmed with promoters for phosphotransferase systemIIC component and thiolase genes.
Such an expression of β-glucosidase using the promoters is
required for high butanol production from rice straw.
Conclusion
We demonstrated that butanol production from rice straw
by using a co-culture of cellulose-degrading C. thermocellum and butanol-producing C. saccharoperbutylacetonicum
is increased by the addition of β-glucosidase. This result indicates that the molecular breeding of butanol-producing
strains for secretion of β-glucosidase should increase butanol yield. In addition, we identified promoter sequencesthat upregulate the expression of β-glucosidase in C. saccharoperbutylacetonicum. In the future, we intend to develop an effective biobutanol production system involving
clostridia by increasing β-glucosidase activity using promoters that we have identified.
References
1)2)3)Nakayama S., Kiyoshi K., Kadokura T., Nakazato A. (2011) Butanol
production from crystalline cellulose by co-cultured Clostridium
thermocellum and Clostridium saccharoperbutylacetonicum N1–4.
Appl. Environ. Microbiol. 77: 6470–6475.
Chundawat S. P. et al. (2011) Proteomics-based compositional analysis of complex cellulase-hemicellulase mixtures. J. Proteome. Res.
10: 4365–4372.
Nakayama S., Irie R., Kosaka T., Matsuura K., Yoshino S., Furukawa K. (2007) New host-vector system in solvent-producing Clostridium saccharoperbutylacetonicum strain N1–4. J. Gen. Appl. Microbiol. 53: 53–56.