2. 690
sucrose) and confirmed by lactate production in anaer-
obic tubes filled with modified LB medium containing
sucrose.
Genetic methods
Standard methods were used for plasmid construc-
tion (Sambrook & Russell 2001). Genomic DNA from
E. coli KO11 was partially digested with Sau3A1.
Fragments (4–6 kbp) were purified, ligated into the
BamHI site of pUC18, and transformed into DH5α.
Colonies were pooled and used to prepare a plas-
mid library. DNA sequencing was provided by the
University of Florida Interdisciplinary Center for Bio-
technology Research. Terminal regions of the sucrose-
positive inserts were sequenced using M13 forward
and reverse primers. Primer-walking was used to se-
quence the sucrose genes from KO11 in both direc-
tions (GenBank database Accession No. AY314757).
Fermentation
Seed cultures were prepared by inoculating fresh
colonies into modified LB medium containing 2%
sugar (150 ml in 500 ml flask). After incubation (15 h,
37 ◦C, 200 rpm), cells were harvested by centrifuga-
tion (5000 × g, 10 min) and used as an inoculum
(165 mg l−1; OD550 0.5). Fermentations were car-
ried out in 500 ml vessels (37 ◦C, 150 rpm agitation)
containing 350 ml modified LB broth with either gluc-
ose, sucrose, molasses, or artificial molasses (mix-
ture of 7.3 g glucose l−1, 7.3 g fructose l−1, and
35.4 g sucrose l−1). Broth was maintained at pH 7 by
the automatic addition of 6 M KOH. Ampicillin was
included in seed cultures and fermentations.
Analyses
Cell mass was estimated by measuring optical density
at 550 nm and converted to cell dry wt by an appropri-
ate calibration curve. Organic acid production (primar-
ily lactate) was estimated by measuring the amount
of base consumed to maintain pH 7 during ferment-
ation. At the end of fermentation, individual organic
acids were analyzed by HPLC as described previ-
ously (Zhou et al. 2003a). Sugar mixtures (molasses;
sucrose, fructose and glucose) were analyzed using a
Biorad Aminex HPX-87C column (7.8 × 300 mm)
at 85 ◦C with distilled water as the eluent. Prior to
sugar analysis, organic acids were removed by batch
treatment with the carbonate form of Dowex 1-X8.
Fig. 1. Plasmid pLOI3501 containing genes for sucrose utilization.
Arrows indicate direction of transcription.
Results and discussion
Cloning sucrose-utilization genes from E. coli KO11
We previously demonstrated that Escherichia coli
KO11, an ethanologenic derivative of the B strain,
has the native ability to ferment sucrose (Moniruzza-
man et al. 1997). Genomic DNA from this organism
was used to construct a gene library containing ap-
prox. 8000 clones in pUC18. Transformants of DH5α
were selected for ampicillin resistance and screened
for sucrose fermentation using MacConkey plates con-
taining 1% sucrose. Of the 7000 colonies screened,
five were red indicating sucrose fermentation. Sucrose
fermentation was confirmed by HPLC analysis of
lactate produced during growth in anaerobic tubes
containing LB and sucrose. Two clones were found
unstable during transfers and discarded.
Plasmids from the three stable clones were di-
gested with a series of restriction enzymes and par-
tially sequenced (ends) using M13 primers. Both
the patterns of DNA fragments and DNA sequences
confirmed that all were siblings. One, designated
pLOI3501, was sequenced fully in both directions
(Accession No. AY314757). This fragment contained
four open reading frames, one of which was truncated
at the N-terminus (Figure 1). Genes in this fragment
shared 98% identity to the chromosomal cluster of
sucrose genes (Accession No. AF473544) from E.
coli strain EC3132 (Jahreis et al. 2002). The sucrose
gene cluster consists of an operon encoding a repressor
protein (cscR), an operon encoding invertase (cscA),
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3. 691
Fig. 2. Fermentation of sugar substrates (5% w/v). (A) Glucose;
(B) sucrose; (C) mixed sugars; (D) diluted molasses. , Lactate
production by SZ63(pLOI3501); , SZ63(pLOI3501) cell mass; ,
lactate production by SZ63; , SZ63 cell mass.
and a bicistronic operon (cscKB) encoding fructok-
inase and an anion symport for sucrose, respectively.
Native promoters were retained with the latter two op-
erons. The N-terminus, ribosomal-binding site, and
promoter region of cscR are absent in pLOI3501.
Production of lactate from sucrose
Plasmid pLOI3501 was readily transformed into the
strain engineered for D(−)-lactate production, SZ63,
using CaCl2 treatment or electroporation. Transform-
ation efficiency with SZ85 was more than 1000-fold
lower. Although ampicillin selection was sufficient to
maintain this plasmid in SZ63, all transformants of
SZ85 rapidly lost the sucrose-utilization phenotype
during subculture. This instability precluded further
investigation of L(+)-lactate production from sucrose
with SZ85(pLOI3501). Since SZ85 is a near isogenic
derivative of SZ63, the instability of SZ85(pLOI3501)
is presumed to result from the cryptic mutations in
SZ85 that improved expression of P. acidilactici ldhL
(Zhou et al. 2003b).
Lactate fermentations with SZ63 and
SZ63(pLOI3501) were initially compared with 5%
(w/v) glucose in LB medium to evaluate the effect
of plasmid burden on growth and metabolism (Fig-
ure 2A and Table 1). Both were similar and rapidly
converted the glucose into lactate (about 510 mM) in
15–18 h with a yield of 92%. Small amounts of acetate
(9–10 mM) were also present, higher than previously
reported (1–2 mM acetate) for SZ63 in minimal salts
medium (Zhou et al. 2003a). This additional acetate
may be produced from the metabolism of complex
nutrients in the medium.
Plasmid pLOI3501 contains all essential genes
for sucrose utilization as evidenced by effective pro-
duction of D(−)-lactate from sucrose (Figure 2B).
Lactate yields were 11% higher with sucrose than
with glucose although minor product were very
similar. SZ63(pLOI3501) fermented 5% sucrose to
568 mM lactate, 97% of the maximum theoretical
yield (Table 1). With sucrose, cells grew more slowly
than with glucose but reached a higher final density.
SZ63 was unable to produce lactate from sucrose
in LB without plasmid pLOI3501. Growth was very
limited despite the presence of complex nutrients.
Production of lactate from molasses
The sugars in cane molasses represent an inexpensive
carbon source for bioconversion, consisting primar-
ily of sucrose, glucose and fructose. A mixture of
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4. 692
Table 1. Summary of fermentation results.
Substrate Strain Cell mass KOH useda Lactate Co-products produced (mM)
(g l−1) (mmol) Produced Yieldb Succinate Formate Acetate Ethanol
(mM) (%)
Glucose SZ63 1.6 ± 0.06 507 ± 4 512 ± 5 92 2.7 ± 0.8 ≤ 0.5 9.5 ± 1.5 ≤ 1
SZ63(pLOI3501) 1.76 ± 0.05 506 ± 5 510 ± 8 92 2.1 ± 0.3 ≤ 0.5 8.7 ± 2.5 ≤ 1
Sucrose SZ63 0.31 ± 0.02 10.6 ± 0.5 0 0 ≤1 ≤ 0.5 ≤0.5 ≤ 1
SZ63(pLOI3501) 2.23 ± 0.07 596 ± 11 568 ± 6 97 1.7 ± 0.3 ≤ 0.5 6.4 ± 0.9 ≤ 1
Sugar mixturec SZ63 1.59 ± 0.14 164 ± 1 163 ± 1 28 2.6 ± 0.5 ≤ 0.5 5.5 ± 0.8 ≤ 1
SZ63(pLOI3501) 2.33 ± 0.04 549 ± 5 540 ± 6 94 3.4 ± 1 ≤ 0.5 8.7 ± 2.5 ≤ 1
Diluted SZ63 2.12 ± 0.29 155 ± 6 163 ± 14 26 13.5 ± 1.3 ≤ 0.5 38.2 ± 5 ≤ 1
molassesd SZ63(pLOI3501) 2.54 ± 0.15 542 ± 14 541 ± 3 90 13.3 ± 1 ≤ 0.5 21.9 ± 0.1 ≤ 1
aKOH (mmol) added per liter of fermentation broth to maintain pH 7.
bMaximum lactate yield is 2 mol per mol of hexose (glucose or fructose) and 4 mol lactate per mol sucrose. Yield values presented are based
on total sugar added to fermentation broth.
cSugar mixture: 7.3 g glucose l−1, 7.3 g fructose l−1, and 35.4 g sucrose l−1.
dTotal sugars = 5.4% (w/v).
Fig. 3. Sugar utilization by SZ63(pLOI3501). (A) Mixed sugars
(5%); (B) diluted molasses (5.4% w/v total sugar). , Glucose; ,
fructose; , sucrose.
these three sugars approximating the ratio in molasses
was fermented efficiently by SZ63 (pLOI3501) to pro-
duce 540 mM of D(−)-lactate (yield of 94%) in 24 h
(Figure 2C, Table 1). Co-product levels were low,
similar to those for glucose and for sucrose. Without
pLOI3501, SZ63 fermented only glucose and fructose.
No evidence of diauxie was observed for the
growth of SZ63(pLOI3501) in this sugar mixture.
Sugar analyses during fermentation (Figure 3A) indic-
ated that all were fermented at different rates. Unlike
growth, however, the rate of fructose and sucrose
metabolism increased after exhaustion of glucose con-
sistent with partial glucose repression. In both the
cscKB and cscA operons, putative cAMP-CrpA bind-
ing sites are present in the −35 regions that could serve
as regulatory sites for glucose-repression.
Diluted cane molasses (5% total sugars) were fer-
mented more slowly than a mixture of purified sugars
(Figure 2D). Again, rates of fructose and sucrose
metabolism increased after exhaustion of glucose
(Figure 3B). Incubation for 96 h was required to com-
plete sugar utilization with a yield of 90%, 540 mM
D(−)-lactate (Table 1). This slower fermentation in
diluted molasses as compared to mixed laboratory sug-
ars can be attributed to inhibitors present in molasses
as well as additional inhibitors that may be produced
during sterilization. Sterilization by membrane filtra-
tion was impractical for fermentation studies due to
rapid clogging (even after centrifugation). In small
broth experiments, filtered molasses supported more
vigorous growth than autoclaved molasses.
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Acetate levels with diluted molasses were over
twice that produced from laboratory sugars (Table 1).
Other product levels were similar in all fermentations
with SZ63(pLOI3501).
Conclusions
These results demonstrate that strain SZ63 harbor-
ing pLOI3501 can be used to efficiently metabolize
sucrose, diluted molasses (5% total sugar), and sugar
mixtures resembling molasses to D(−)-lactate. Addi-
tional components of steam sterilized molasses slowed
but did not prevent fermentation. Opportunities remain
for process improvements that reduce the toxicity
of this inexpensive substrate and decrease the time
required for complete sugar utilization.
Acknowledgements
This research was supported by grants from the U.S.
Department of Agriculture (01-35504-10669 and 00-
52104-9704), the U.S. Department of Energy (FG02-
96ER20222), and the Florida Agricultural Experiment
Station (Journal Series No. R-10032).
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