APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Aug. 1989. p. 1943-1948 Vol. 55 No.

8
0099-2240/89/081943-06$02.00/0
Copyright © 1989, American Society for Microbiology

Efficient Ethanol Production from Glucose, Lactose, and Xylose by

Recombinant Escherichia colit
FLAVIO ALTERTHUM' AND L. 0. INGRAM2*
Department of Microbiology, Inistiti,to de Ciencias Biomnedic as, Univer.sidade de Sao Paiulo, Sao Pal/o, Brazi/ 05508,
and Department of Microbiology anid Cell Science, McCarty Hall, Univer-sity of Florida, Gainesville, Florida 32611-
Received 3 March 1989/Accepted 12 May 1989

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose
and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes
encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid
stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from
glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli
ATCC 9637(pLOI297), ATCC 11303(pLOI297), and ATCC 15224(pLOI297) were selected for further
development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productiv-
ities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter
for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3
g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli
than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose
(8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.

Most fuel ethanol is currently produced from hexose MATERIALS AND METHODS
sugars in corn starch or cane syrup by using either Sa(cc(ha- Strains and media. E. coli strains were obtained from the
rovmyces cerei'isiae or Zyinomtonas iIobilis (17, 24). How- ATCC (Table 1). These were grown in a shaking water bath
ever, these are relatively expensive sourses of biomass
at 30°C in Luria broth (14) containing tryptone (10 g/liter),
sugars and have competing value as foods. Starches and
yeast extract (5 g/liter), sodium chloride (5 g/liter), and a
sugars represent only a fraction of the total carbohydrates in
fermentable sugar. Glucose and lactose were added at con-
plants. The dominant forms of plant carbohydrate in stems, centrations of 100 g/liter and xylose was added at a concen-
leaves, hulls, husks, cobs, etc., are the structural wall tration of 80 g/liter unless indicated otherwise. Sugars were
polymers, cellulose and hemicellulose (11). Hydrolysis of autoclaved separately (121°C, 15 min) at double strength in
these polymers releases a mixture of neutral sugars which distilled water. Xylose (Sigma Chemical Co., St. Louis,
includes glucose, xylose, mannose, galactose, and arabi- Mo.) solutions were acidic and were neutralized with sodium
nose. No organism has been found in nature which can
hydroxide prior to autoclaving; failure to neutralize resulted
rapidly and efficiently metabolize these different sugars into in extensive browning and decomposition. Similar fermen-
ethanol or any other single product of value. tation results were obtained with sugars which were auto-
With the cloning of the genes encoding the enzymes for claved or filter sterilized. Survival in broth and on plates of

Downloaded from https://journals.asm.org/journal/aem on 09 July 2024 by 131.242.30.51.

the ethanol pathway from Z. itnobilis (2-4, 18, 23), it became recombinant strains containing genes encoding the enzymes
possible to genetically engineer ethanol production in bacte- for the ethanol pathway required the presence of a ferment-
ria which are able to utilize a variety of sugars (8, 9, 30, 31). able sugar. Where indicated, tetracycline was added at a
In most cases, both alcohol dehydrogenase and pyruvate final concentration of 10 mg/liter.
decarboxylase activities were needed for efficient sugar Environmental hardiness. Strains were tested for their
conversion to ethanol (2, 8). Since Z. inobilis pyruvate resistance to sodium chloride, sugars, low pH, and ethanol.
decarboxylase has a much lower K,,, for pyruvate than Concentrations of sodium chloride and sugars in Table 1
competing acidogenic pathways in fermentation, expression include those in the original medium. The original pH of the
of the Z. mobilis enzymes effectively diverted carbon flow in medium was 6.8; this was adjusted to lower values with HCI
recombinant E. coli (8, 9), Klebsiella planticola (30), and where indicated. Acidified media were sterilized by filtra-
Erw- inia ch-ysanlthemni (31) to ethanol. tion. Ethanol was added to autoclaved medium after cooling.
E. coli has been shown to metabolize all major sugars Sugars were autoclaved separately. Overnight cultures
which are constituents of plant biomass (12), and our studies grown in each respective sugar in the absence of test agent
have focused on this organism (8, 9). Initial studies used were diluted 60-fold into culture tubes (13 by 75 mm)
strain S17-1 (26) and related strains of E. c oli which contain containing 3 ml of test media. Growth was measured as
integrated mobilization elements for plasmid conjugation, optical density at 550 nm (OD,,() after 48 h. An OD of 1.0 is
recombinants which are unsuitable for commercial applica- equivalent to 0.25 mg of cell protein per ml and 0.33 mg of
tion. In this study, we have compared eight strains of E. c oli cell dry weight. In tests of environmental hardiness, a final
from the American Type Culture Collection (ATCC) for OD below 0.2 reflected less than two doublings and was
environmental hardiness and suitability for the further de- considered negligible.
velopment of commercial ethanol production. Sugar utilization. Sugar utilization was tested in two ways.
Strains which developed red colonies on MacConkey agar
* Corresponding author. supplemented with 2% carbohydrate were scored positive
Florida Agricultural Experiment Station publication 9806. for sugar utilization (25). Cells were also tested with EC
1943
1944 ALTERTHUM AND INGRAM APPL. ENVIRON. MICROBIOL.

TABLE 1. Growth of E. coli in Luria broth containing 100 g of cycline was included at a concentration of 10 mg/liter.
glucose per liter under chemical and physical stresses Inocula were grown from freshly isolated colonies for 24 h,
Growth" of E. co/i ATCC str-ain: washed in the fermentation medium to be tested, and added
Stress to an initial OD,,, of approximately 1.0. Fermentations were
8677 8739 9637 113t)3 11775 14948 15224 23227 carried out at 30 or 37°C in 100-ml volumetric flasks contain-
NaCI (g/liter) ing 80 ml of broth and fitted with rubber septa and 25-gauge
50 + ++ ++ +-+ +±+ ++ ++ needles to allow gas to escape. Fermentation flasks were
60 0 + ++ ±+ + ++ + ++ immersed in a temperature-controlled water bath and stirred
70 0 0 + + 0 + + + by a magnetic stirrer at 100 rpm.
Ethanol concentration was measured by gas chromatogra-
Ethanol (% by phy as previously described (5) and is expressed as percent-
volume) age by volume. The conversion efficiency was calculated on
3.8 +i+- ++ ++ ++ ++ +-+ ++ ++ the basis of sugar added, assuming that 100% efficiency
5.0 ++ ++ + + + + + +
results in the production of 12.75 ml of ethanol (10.2 g) per 20
6.3 0 ++ + + + + + t)
7.5 0 + + 0 0 + 0 0 g of glucose or xylose and 13.5 ml of ethanol (10.8 g) per 20
8.8 0 0 0 0 0 0 0 0 g of lactose.
Acidity (pH) RESULTS
4.50 ++ ++ ++ ++ +±+ ++ ++ ++
4.25 +++ + + ++ ± ++ ++ ++ + Environmental hardiness and sugar utilization. Before the
4.00 + + ++ + ++ + + 0 introduction of plasmids for ethanol production, the growth
3.75 0 0 + 0 + 0 0 0 of eight different strains of E. (oli was compared for envi-
Temperature (°C) ronmental hardiness. Table 1 summarizes the results ob-
45 ++ ++ ±+ ++ + ++ ++ ++ tained with medium containing glucose. Similar though not
47 + + + + + + + + identical results were obtained with media containing lactose
49 0 0 + + 0 0 + + and xylose (data not shown). Strains ATCC 8677, ATCC
8739, and ATCC 11775 were particularly sensitive to inhibi-
'Growth was scored as 0 (lcss thain two doLublings in ODs,,(). + (two to four tion by sodium chloride. Strains ATCC 8677 and ATCC
doublings), or + + (over four doublings).
23227 were inhibited by low concentrations of ethanol.
Strains ATCC 9637 and ATCC 11775 were the most resistant
plates (Biolog, Inc., Hayward, Calif.) according to the to low pH, although all strains except ATCC 23227 grew for
directions of the manufacturer. The Biolog plates were rapid at least two to four doublings at pH 4.0. All strains grew at
and convenient, detecting NADH production (conversion of 45°C. with limited growth at higher temperatures; none could
a tetrazolium salt to the insoluble formazan) as a measure of be subcultured above 45°C. All strains grew in media con-
substrate utilization. Both methods were in complete agree- taining 200% glucose, 20%, lactose, or 12% xylose.
ment for the 13 sugars examined. All strains tested utilized glucose, fructose, galactose,
Genetic methods and plasmid constructions. Two new mannose, arabinose, lactose, glucuronic acid, and galactu-
plasmids which contained resistance genes for ampicillin and ronic acid. Strain ATCC 11775 did not utilize xylose. Malt-
tetracycline as selectable markers were constructed by stan- ose and maltotriose were not used by ATCC 11303 and
dard methods (16). The ethanol production operon (PET ATCC 23227. All strains exhibited a weakly positive reaction
operon) containing a cryptic Z. inobilis promoter, pyruvate with cellobiose. Only strain ATCC 9637 utilized sucrose.
decarboxylase, alcohol hydrogenase, and transcriptional ter- These results indicated that on the basis of environmental

Downloaded from https://journals.asm.org/journal/aem on 09 July 2024 by 131.242.30.51.

minator was removed as a 5.2-kilobase EcoRI fragment from hardiness, ATCC 8677, ATCC 11775, and ATCC 23227 were
pLOI308-10 (8) and inserted into the EcoRl site of pBR322 to less suitable than the other four strains for further develop-
produce pLOI308-11. The plasmid pLOI297 was constructed ment. Strain ATCC 11775 also lacked the ability to metabo-
by inserting the 2.6-kilobase EcoRI fragment from pCOS lize xylose, one of the most abundant sugars in biomass.
2EMBL (22) containing the tetracycline resistance gene into Growth, plasmid stability, and expression of pyruvate de-
the Sall site of pLOI295 (9). Cohesive ends were removed by carboxylase. Recombinant strains harboring plasmids with
treatment with the Klenow fragment of E. (oli DNA poly- the genes for ethanol production grew as unusually large
merase (16) before ligation. colonies which became yellow after 24 to 48 h on solid
Plasmids were introduced into the different strains of E. medium containing a fermentable sugar. In liquid medium,
(/li by transformation using the calcium chloride procedure the final cell densities of these recombinants were two to
of Mandel and Higa (15). Selections were made on solid three times higher than that of the control lacking plasmid
medium containing 2% glucose and tetracycline. Plasmid (Table 2). No transformants were obtained after several
stability is expressed as the percentage of cells retaining attempts from ATCC 14948(pLOI297) or from ATCC
antibiotic markers after 25 generations of growth in the 11775(pLOI308-11). Strain ATCC 11775 did not utilize xy-
absence of antibiotic selection. lose, and recombinants of this strain did not grow to higher
Pyruvate decarboxylase. Pyru vate decarboxylase activity densities than the control with xylose as the fermentable
was measured as previously described (3, 18), except that sugar, although increased growth was observed with lactose
cells were harvested at an OD of 4.0, approximately half of and glucose.
maximal growth. Plasmid stability was examined after growth in medium
Fermentation experiments. Luria broth was modified for containing glucose for 25 generations (Table 3). Both plas-
fermentation experiments by the inclusion of potassium mids contained the same replicons and were maintained well
phosphate buffer (pH 7.0) at a final concentration of 0.2 M. in all strains except ATCC 8677 and ATCC 8739.
Phosphate buffer, complex medium components, and sugars The expression of Z. Imobilis pyruvate decarboxylase
were autoclaved separately and mixed after cooling. Tetra- activity was examined after growth in the presence of
VOL. 55, 1989 ETHANOL PRODUCTION BY E. COLI 1945

TABLE 2. Growth of E. coli strains harboring pLO1297 and pLO1308-11

Final OD,5, of E. co/i ATCC str-ain:
Sugar Plasmid
8677 8739 9637 11303 11775 14948 15224 23227
Glucose None 4.0 3.7 6.1 6.0 4.7 5.6 7.0 6.6
pLO1297 10.0 10.5 10.5 10.0 9.5 " 9.5 10.2
pLO1308-11 9.8 9.5 11.4 11.2 9.3 10.8 11.4
Lactose None 4.3 3.8 7.5 6.0 4.5 6.1 7.0 6.4
pLO1297 13.0 6.8 11.6 10.8 7.6 10.5 7.0
pLO1308-11 10.0 10.0 11.5 11.0 7.3 10.( 10.0
Xylose None 4.1 3.7 7.7 7.3 4.9 5.9 7.2 7.0
pLO1297 8.1 10.6 10.8 10.6 4.7 11.0 11.0
pLO1308-11 10.0 6.8 11.4 8.5 11.4 10.6 12.0
No data available.

tetracycline (Table 4). With pLOI297, Z. inobilis genes are the best constructs for ethanol production. The time courses
expressed under the control of the E. c(li kac promoter; of growth and ethanol production were examined with both
pLOI308-11 utilizes the cryptic Z. inobilis promoter for strains in 12% glucose, 12% lactose, and 8% xylose (Fig. 1).
expression of the PET operon. Strains ATCC 11303 Cell mass increased approximately 10-fold, reaching a final
(pLOI297), ATCC 11775(pLOI297), and ATCC 15224 density of 3.6 g of dry weight per liter. With xylose, cell mass
(pLOI297) contained the highest levels of activity. increased at half the rate observed with glucose or lactose.
Comparison of ethanol production during batch fermenta- Ethanol production and growth were approximately linear
tion. All genetically engineered strains of E. c(li produced for the three sugars until the concentration of ethanol
significant amounts of ethanol from sugars (Table 5). Prelim- reached 5%.
inary experiments with strain ATCC 15224(pLOI297) indi- To compute the conversion efficiency of sugar to ethanol,
cated that higher levels of ethanol were produced in medium final ethanol concentrations after 120 h were averaged from
containing 0.2 M potassium phosphate buffer (pH 7.0). With three sets of experiments (Table 6). The final concentration
15% glucose, higher ethanol levels were produced at 30°C of ethanol in cultures grown with 12% glucose was 7.2% (by
than at 37°C after 48 h. The fermentation of lactose and volume), representing 94% of theoretical yield from glucose.
xylose was examined only at the lower temperature, 30°C. In With 12% lactose, the final ethanol concentration was 6.5%,
general, higher levels of ethanol were produced by strains 80% of the theoretical yield from lactose. With 8% xylose,
harboring pLOI297 than by those with pLOI308-11. Strains we consistently obtained yields of 100% and higher. These
ATCC 11303(pLOI297), ATCC 11775(pLOI297), and ATCC high yields during slower growth with xylose may reflect the
15224(pLOI297) produced the highest levels of ethanol after conversion of pyruvate from the catabolism of complex
48 h from 15% glucose, 5.8 to 6.9% by volume. Most strains nutrients into ethanol, in addition to conversion of pyruvate
were less tolerant of xylose in initial experiments, and from glucose.
comparisons of fermentation were carried out with 8% The rate of ethanol production was computed from the
xylose. Strains ATCC 9637(pLOI297), ATCC 11303 graphs in Fig. 1 and are summarized in Table 6. Volumetric
(pLOI297), and ATCC 15224(pLOI297) produced the highest productivity of ethanol ranged from 1.4 g/liter per h for

Downloaded from https://journals.asm.org/journal/aem on 09 July 2024 by 131.242.30.51.

levels of ethanol (4.8 to 5.2%) from 8% xylose after 72 h. All glucose to 0.64 g/liter per h for xylose. Specific productivity
strains grew well in 15% lactose. Strains ATCC of ethanol was highest during the initial growth period for
11303(pLOI297) and ATCC 15224(pLOI297) produced the each of the three sugars. The highest productivity was
highest levels of ethanol from lactose after 48 h (6.1 and obtained with glucose, 2.1 g of ethanol per g of cell dry
5.6%, respectively). weight per h. The highest yield of eth-anot per g of sugar was
On the basis of these comparative studies, strains ATCC obtained with xylose, exceeding the maximal theoretical
11303(pLOI297) and ATCC 15224(pLOI297) appeared to be yield for sugar alone.
TABLE 4. Expression of Z. mnobilis pyruvate decarboxylase in
TABLE 3. Stability of pLOI297 and pLOI308-11 after E. coli strains harboring pLO1297 and pLO1308-11
25 generations of growth with glucose in the during growth with glucose
absence of antibiotic selection
Pyruvate decarboxylase activity"
% of cells retaining plasmid ATCC strain
ATCC strain pLO1297 pLO1308-11
pLO1297 pLO1308-11
8677 5.7 6.0
8677 75 83 8739 0.8 1.4
8739 44 47 9637 1.1 1.4
9637 100 9() 11303 16.7 2.1
11303 98 98 11775 17.1 -h
11775 100 " 14948 2.5
14948 97 15224 16.3 1.8
15224 99 100 23227 2.3 1.7
23227 91 100
International units per milligram of cell protein.
-. No data available. " No data available.
1946 ALTERTHUM AND INGRAM APPL. ENVIRON. MICROBIOL.

TABLE 5. Ethanol production in batch fermentations from glucose (48 h), xylose (72 h), and lactose (48 h)
by E. coli strains harboring pLO1297 and pLO1308-11
Carbohydrate a % Ethanol (vol/vol) produced by E. coli ATCC strain:
(%) Plasmid 8677 8739 9637 11303 11775 14948 15224 23227
Glucose (15)" pLOI297 2.4 4.7 4.2 4.3 4.8 C
4.8 0.9
pLO1308-11 3.6 1.4 2.1 1.3 3.4 2.8 1.3
Glucose (15)" pLOI297 3.2 4.7 4.1 5.8 6.9 6.1 3.1
pLOI308-11 5.8 5.0 3.5 1.5 3.8 3.0 3.2
Lactose (15)" pLOI297 2.3 4.4 5.3 6.1 4.5 5.6 3.7
pLO1308-11 2.3 2.1 3.4 0.9 2.9 2.7 3.0
Xylose (8)"' pLO1297 0.9 4.1 4.8 5.2 4.8 1.2
pLO1308-11 2.0 2.8 2.8 1.2 - 2.0 3.5 1.0
'Incubation with plasmids was at 37'C.
"Incubation with plasmids was at 30'C.
-, No data available.

Experiments were conducted with ATCC 11303(pLOI297) 115

to examine ethanol production from arabinose, galactose, CD

and mannose. Ethanol concentrations of 3 to 4% were 12 3

obtained after 48 h at 30°C but were not investigated further. a)
cn
These sugars are metabolized by pathways similar to those 9
for glucose and xylose and would be expected to produce O
C)
analogous yields (12). LJD 6*a-n
a_n
3 ,3
DISCUSSION 'UL
Brau and Sahm (2) first demonstrated that ethanol produc-
tion could be increased in recombinant E. coli by the
overexpression of Z. mobilis pyruvate decarboxylase, al- Time (hours)
though very low ethanol concentrations were produced.
Subsequent studies by Tolan and Finn extended this work by 15
using two other enteric bacteria (E. chrysanthemi [301 and K. C-,)
planticola [31]) and achieved much higher levels of ethanol
from hexoses, pentoses, and sugar mixtures. Alcohol dehy- 6 11 ,
:::a-~-~---------- 12
tn
drogenase is also essential for this conversion, and ethanol Jo 5 Al 93 cn

production in these previous recombinant systems was de- s

pendent on native activities in the host organisms. Mutant E.
1~~~~~~~~~~~~~~~
3I3

Downloaded from https://journals.asm.org/journal/aem on 09 July 2024 by 131.242.30.51.

9--
coli which overproduced native alcohol dehydrogenase pro- cn
Ln
duced much higher levels of ethanol with Z. mobilis pyru- 3
vate decarboxylase than E. coli recombinants with the native LLJ a~~~~~~~~~~~~~
2 =3

activity did (9). 0

The dependence upon host alcohol dehydrogenase activity 0 20 40 60 80 100 120
was eliminated by combining Z. mobilis genes encoding Time (hours) C-,

alcohol dehydrogenase II and pyruvate decarboxylase to

form a portable, plasmid-borne operon for ethanol produc-
tion, the PET operon (8, 9). Considering the vast amount of CD
information available concerning the physiology, genetics,
and metabolism of E. coli, this organism is an obvious choice C)

for further exploration of ethanol production. E. coli has an 'n

W

extremely wide substrate range which includes hexoses, >1

_^

pentoses, dissacharides, hexuronic acids, sugar alcohols,

purines, pyrimidines, and amino acids, among others. Many Ln
nT
potentially useful genes have been cloned from other organ- -- C>

isms and expressed in E. coli, the most widely used host for =:

molecular genetics.
A variety of factors need to be considered in selecting E. 0 20 40 60 80 100 120
coli strains suitable for ethanol production, including sub- Time (hours)
strate range and environmental hardiness (sugar tolerance, FIG. 1. Growth and ethanol production from 12% glucose (A),
salt tolerance, ethanol tolerance, tolerance to low pH, and 12% lactose (B), and 8% xylose (C). Symbols: , ethanol; - - -,
thermal tolerance). Strain ATCC 9637 (Waksman strain W) cell mass; 0, strain ATCC 11303(pLO1297); A, strain ATCC
appeared superior in terms of environmental hardiness, 15224(pLO1297).
VOL. 55, 1989 ETHANOL PRODUCTION BY E. COLI 1947

TABLE 6. Average kinetic parameters for ethanol production by ture. Alcohol Fuels Program (88-37233-3987) and from the Depart-
ATCC 11303(pLO1297) and ATCC 15224(pLOI297) ment of Energy. Office of Basic Energy Research (FG-
05-86ER3574). We ar-e graiteful to the Conselho Nacional de
Productivity E Desenvolvimento Cientifico e Teconologico. Brazil (Processo
Sugar (%) Yield" Efficicncy Ethinol'
Volumetric' Specific" (Ce) 20.0915/87) for providing support for Flavio Alterthum.
Glucose (12) 1.4 2.1 0.48 95 58 LITERATURE CITED
Lactose (12) 1.3 2.0 0.43 80 52 1. Beck, M. J. 1989. Faictor-s affecting efficiency of biomass fer-
Xylose (8) 0.6 1.3 0.52 102 42 mentation to ethanol. Biotechnol. Bioeng. Symp. 17:617-627.
Grams of ethanol per gram of sugar. 2. Brau, B., and H. Sahm. 1986. Cloning and expression of the
"Calculated as: (ethanol produced/theoretical maximum from sugair sub- structural gene for pyruvate decarboxylase of Zyoinoonas
strate) x 100. iniobilis in Escherichia (oli. Arch. Microbiol. 144:296-301.
Final ethanol concentration in grams per liter. 3. Conway, T., Y. A. Osman, J. I. Konnan, E. M. Hoffmann, and
' Grams of ethanol per liter pei- hour. L. 0. Ingram. 1987. Promoter and nucleotide sequences of the
Grams of ethanol per g of cell dry weight per hour. Zv10nomonas mzob/ilius pyruvate decarboxylase. J. Bacteriol. 169:
949-954.
although ethanol production from glucose was lower than 4. Conway, T., G. W. Sewell, Y. A. Osman, and L. 0. Ingram.
with other strains. ATCC 11303 (Luria strain B) and ATCC 1987. Cloning and sequencing of the alcohol dehydrogenase 11
15224 (Kepes strain ML308) containing pLOI297 produced gene from Zvinomona)ns miobdilis. J. Bacteriol. 169:2591-2597.
5. Dombek, K. M., and L. 0. Ingram. 1985. Determination of
the highest levels of ethanol and exhibited acceptable levels intracellular concentrations of ethanol in Saccharon'vccs (cere-
of environmental hardiness. Plasmids were quite stable in viisima during fermentation. Appl. Environ. Microbiol. 51:197-
these two constructs, and they were selected as the best 2)0(.
candidates for further development of ethanol production. 6. Ingram, L. 0. 1986. Microbial tolerance to alcohols: role of the
Both constructs expressed the high levels of Z. iniobilis cell membrane. Trends Biotechnol. 4:40-44.
pyruvate decarboxylase which are required for efficient 7. Ingram, L. 0. 1988. Effects of ethanol on E.sclherichia coli. p.
ethanol production (9). 227-237. In N. van Uden (ed.). Alcohol toxicity in yeasts and
All major sugar components of plant biomass were con- bacteria. CRC Press. Inc.. Boca Raton. Fla.
8. Ingram, L. O., and T. Conway. 1988. Expression of different
verted to ethanol by recombinant E. coli containing the levels of the ethanologenic enzymes from ZYnlononas ,tnohili.s
ethanol pathway from Z. inobilis. The conversion efficiency in recombinant strains of Lscherichia (o/i. AppI. Environ.
of glucose and xylose into ethanol exceeded that for S. Microbiol. 54:397-404.
erev'isiae (13) and pentose-fermenting yeasts systems (1. 10. 9. Ingram, L. O., T. Conway, D. P. Clark, G. W. Sewell, and J. F.
27, 28). Xylose was converted to ethanol by recombinant E. Preston. 1987. Genetic engineering of ethanol production in
coli with a higher efficiency than glucose by S. (-ei-(eil,sia(e Escher/chia co/i. Appl. Environ. Microbiol. 53:2420-2425.
(13). The unusually high ethanol yields with xylose (over 10. Jeffries, T. W., and H. K. Sreenath. 1988. Fermentation of
100%7 of theoretical values) may include ethanol derived hemicellulose sugars and sugar mixtures by Cantcdidai slilioitw(e.
from the catabolism of complex nutrients. Many amino acids Biotechnol. Bioeng. 31:502-506.
11. Krull, L. H., and G. 1. Inglet. 1980. Analysis of neutral carbo-
and complex-medium components are catabolized to glyco- hydrates in agricultural residues by gas-liquid chromatography.
lytic intermediates which are converted to pyruvate. This J. Agric. Food Chem. 28:917-919.
pyruvate could then be converted to ethanol. 12. Lin, E. C. C. 1987. Dissimilatory pathways for sugars, polyols,
The ethanol tolerance of E. c oli (6, 7) is lower than that of and carboxylates. p. 244-284. In F. C. Neidhardt, J. L. In-
S. (erevisiae (6, 19) or Z. mobilis (20, 21). However, it is graham. K. B. Low. B. Magasanik. and M. Schaechter (ed.).
unlikely that this will be a limitation for ethanol production E.scherichia coli and Salmontella typlhimumrium, vol. 1. American
from biomass. The hydrolysis of biomass by a combination Society for Microbiology. Washington. D.C.

Downloaded from https://journals.asm.org/journal/aem on 09 July 2024 by 131.242.30.51.

of chemical and enzymatic methods typically yields sugar 13. Lovitt, R. W., B. H. Kim, G.-J. Shen, and J. G. Zeikus. 1988.
concentrations well below 12% (1). Recombinant E. (oli- Solvent production by microorganisms. Crit. Rev. Biotechnol.
7:107-186.
based ethanol production would result in a small increase in 14. Luria, S. E., and M. Delbruck. 1943. Mutations of bacteria from
the costs of product recovery. This would be offset by virus sensitivity to virus resistance. Genetics 28:491-511.
increased ethanol yield and decreased costs of biomass 15. Mandel, M., and A. Higa. 1970. Calcium dependent bacterio-
feedstocks. phage DNA infection. J. Mol. Biol. 53:159-162.
Although further investigations are needed to optimize 16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
ethanol production by recombinant E. coli, the conversion cloning: a laboratory manual. Cold Spring Harbor Laboratory.
rates observed in batch culture for glucose and lactose Cold Spring Harbor-. N.Y.
fermentation compared favorably with those observed for 17. Murtagh, J. E. 1986. Fuel ethanol production-the U.S. expe-
rience. Process Biochem. 21:6145.
yeasts (13, 29). The conversion rate of xylose to ethanol 18. Neale, A. D., R. K. Scopes, R. E. H. Wettenhall, and N. J.
equaled or exceeded previous rates by yeasts even in expen- Hoogenraad. 1987. Nucleotide sequence of the pyruvate decair-
sive systems using added xylose isomerase (1. 10. 26. 28). boxylase gene from Zxvnolnonas It10bili.s. Nucleic Acids Res.
The ability of recombinant E. (coli to convert the diversity of 15:1753-1761.
sugars present in biomass to ethanol offers potential for the 19. Ohta, K., and S. Havashida. 1983. Role of Tween 80 and
commercial expansion of fuel ethanol production by using monolein in a lipid-sterol-protein complex which enhances
alternative feedstocks which do not have competing value as ethanol tolerance of sake yeasts. Appl. Environ. Microbiol.
food. Such expansion could involve a blend of current 46:821-825.
feedstocks with cellulosic biomass from undervalued agri- 20. Ohta, K., K. Supanwong, and S. Hayashida. 1981. Environmen-
cultural products. tatl effects on ethanol tolerance of Zymnoinona.s minobilis'. J. Ferm.
Technol. 59:435-439.
21. Osman, Y. A., and L. 0. Ingram. 1987. Zv,ynolnona.s iiobilis
ACKNOWLEDGMENTS mutants with an increatsed rate of alcohol pr-oduction. Appl.
This work was supported in part by the Florida Agriculturtal Environ. Microbiol. 53:1425-1432.
Experiment Station and by grants from the Department of Agricul- 22. Poustka, A., H. R. Rackwitz, A.-M. Firschauf, and H. Lehrach.
1948 ALTERTHUM AND INGRAM AppI. ENVIRON. MICROBIOL.

1984. Selective isolation of cosmid clones by homologous re- Enzyme Microbiol. Technol. 10:66-79.
combination in Escherichia coli. Proc. Natl. Acad. Sci. USA 28. Slininger, P. J., R. J. Bothast, M. R. Okos, and M. R. Ladisch.
81:4129-4133. 1985. Comparative evaluation of ethanol production by xylose-
23. Reynen, M., and H. Sahm. 1988. Comparison of the structural fermenting yeasts presented high xylose concentrations. Bio-
genes for pyruvate decarboxylase in different Zymomonas mo- technol. Lett. 7:431-436.
bilis strains. J. Bacteriol. 170:3310-3313. 29. Terrell, S. L., A. Bernard, and R. B. Bailey. 1984. Ethanol from
24. Rosillo-Calle, F., and D. 0. Hall. 1987. Brazilian alcohol: food whey: continuous fermentation with a catabolite repression-
versus fuel? Biomass 12:97-128. resistant Saccharotnvces cerev'isiae mutant. Appl. Environ.
25. Silhavy, T. J., and J. R. Beckwith. 1985. Uses of lac fusions for Microbiol. 48:577-580.
the study of biological problems. Microbiol. Rev. 49:398-418. 30. Tolan, J. S., and R. K. Finn. 1987. Fermentation of D-xylose and
26. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range L-arabinose to ethanol by Erwinia Chrysanthemni. Appl. Envi-
mobilization system for in i'iio genetic engineering: transposon ron. Microbiol. 53:2033-2038.
mutagenesis in gram negative bacteria. Biotechnology 1:784- 31. Tolan, J. S., and R. K. Finn. 1987. Fermentation of D-xylose to
791. ethanol by genetically modified Klebsiella planticola. Appl.
27. Skoog, K., and B. Hahn-Hagerdal. 1988. Xylose fermentation. Environ. Microbiol. 53:2039-2044.

Downloaded from https://journals.asm.org/journal/aem on 09 July 2024 by 131.242.30.51.