Efficient Ethanol Production from Glucose, Lactose, and Xylose by Recombinant Escherichia colit

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Aug p /89/ $02.00/0 Copyright 1989, American Society for Microbiology Vol. 55 No. 8 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 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 productivities 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 sugars in corn starch or cane syrup by using either Sa(cc(harovmyces cerei'isiae or Zyinomtonas iiobilis (17, 24). However, these are relatively expensive sourses of biomass sugars and have competing value as foods. Starches and sugars represent only a fraction of the total carbohydrates in plants. The dominant forms of plant carbohydrate in stems, leaves, hulls, husks, cobs, etc., are the structural wall polymers, cellulose and hemicellulose (11). Hydrolysis of these polymers releases a mixture of neutral sugars which includes glucose, xylose, mannose, galactose, and arabinose. No organism has been found in nature which can rapidly and efficiently metabolize these different sugars into ethanol or any other single product of value. With the cloning of the genes encoding the enzymes for the ethanol pathway from Z. itnobilis (2-4, 18, 23), it became possible to genetically engineer ethanol production in bacteria which are able to utilize a variety of sugars (8, 9, 30, 31). In most cases, both alcohol dehydrogenase and pyruvate decarboxylase activities were needed for efficient sugar conversion to ethanol (2, 8). Since Z. inobilis pyruvate decarboxylase has a much lower K,,, for pyruvate than competing acidogenic pathways in fermentation, expression of the Z. mobilis enzymes effectively diverted carbon flow in recombinant E. coli (8, 9), Klebsiella planticola (30), and Erw- inia ch-ysanlthemni (31) to ethanol. E. coli has been shown to metabolize all major sugars which are constituents of plant biomass (12), and our studies have focused on this organism (8, 9). Initial studies used strain S17-1 (26) and related strains of E. c oli which contain integrated mobilization elements for plasmid conjugation, recombinants which are unsuitable for commercial application. In this study, we have compared eight strains of E. c oli from the American Type Culture Collection (ATCC) for environmental hardiness and suitability for the further development of commercial ethanol production. * Corresponding author. Florida Agricultural Experiment Station publication MATERIALS AND METHODS Strains and media. E. coli strains were obtained from the ATCC (Table 1). These were grown in a shaking water bath at 30 C in Luria broth (14) containing tryptone (10 g/liter), yeast extract (5 g/liter), sodium chloride (5 g/liter), and a fermentable sugar. Glucose and lactose were added at concentrations of 100 g/liter and xylose was added at a concentration of 80 g/liter unless indicated otherwise. Sugars were autoclaved separately (121 C, 15 min) at double strength in distilled water. Xylose (Sigma Chemical Co., St. Louis, Mo.) solutions were acidic and were neutralized with sodium hydroxide prior to autoclaving; failure to neutralize resulted in extensive browning and decomposition. Similar fermentation results were obtained with sugars which were autoclaved or filter sterilized. Survival in broth and on plates of recombinant strains containing genes encoding the enzymes for the ethanol pathway required the presence of a fermentable sugar. Where indicated, tetracycline was added at a final concentration of 10 mg/liter. Environmental hardiness. Strains were tested for their resistance to sodium chloride, sugars, low ph, and ethanol. Concentrations of sodium chloride and sugars in Table 1 include those in the original medium. The original ph of the medium was 6.8; this was adjusted to lower values with HCI where indicated. Acidified media were sterilized by filtration. Ethanol was added to autoclaved medium after cooling. Sugars were autoclaved separately. Overnight cultures grown in each respective sugar in the absence of test agent were diluted 60-fold into culture tubes (13 by 75 mm) containing 3 ml of test media. Growth was measured as optical density at 550 nm (OD,,() after 48 h. An OD of 1.0 is equivalent to 0.25 mg of cell protein per ml and 0.33 mg of cell dry weight. In tests of environmental hardiness, a final OD below 0.2 reflected less than two doublings and was considered negligible. Sugar utilization. Sugar utilization was tested in two ways. Strains which developed red colonies on MacConkey agar supplemented with 2% carbohydrate were scored positive for sugar utilization (25). Cells were also tested with EC 1943

2 1944 ALTERTHUM AND INGRAM TABLE 1. Growth of E. coli in Luria broth containing 100 g of glucose per liter under chemical and physical stresses Stress Growth" of E. co/i ATCC str-ain: t) NaCI (g/liter) ± ± Ethanol (% by volume) 3.8 +i t) Acidity (ph) ± ± Temperature ( C) ± 'Growth was scored as 0 (lcss thain two dolublings in ODs,,(). + (two to four doublings), or + + (over four doublings). plates (Biolog, Inc., Hayward, Calif.) according to the directions of the manufacturer. The Biolog plates were rapid and convenient, detecting NADH production (conversion of a tetrazolium salt to the insoluble formazan) as a measure of substrate utilization. Both methods were in complete agreement for the 13 sugars examined. Genetic methods and plasmid constructions. Two new plasmids which contained resistance genes for ampicillin and tetracycline as selectable markers were constructed by standard methods (16). The ethanol production operon (PET operon) containing a cryptic Z. inobilis promoter, pyruvate decarboxylase, alcohol hydrogenase, and transcriptional terminator was removed as a 5.2-kilobase EcoRI fragment from ploi (8) and inserted into the EcoRl site of pbr322 to produce ploi The plasmid ploi297 was constructed by inserting the 2.6-kilobase EcoRI fragment from pcos 2EMBL (22) containing the tetracycline resistance gene into the Sall site of ploi295 (9). Cohesive ends were removed by treatment with the Klenow fragment of E. (oli DNA polymerase (16) before ligation. Plasmids were introduced into the different strains of E. (/li by transformation using the calcium chloride procedure of Mandel and Higa (15). Selections were made on solid medium containing 2% glucose and tetracycline. Plasmid stability is expressed as the percentage of cells retaining antibiotic markers after 25 generations of growth in the absence of antibiotic selection. Pyruvate decarboxylase. Pyru vate decarboxylase activity was measured as previously described (3, 18), except that cells were harvested at an OD of 4.0, approximately half of maximal growth. Fermentation experiments. Luria broth was modified for fermentation experiments by the inclusion of potassium phosphate buffer (ph 7.0) at a final concentration of 0.2 M. Phosphate buffer, complex medium components, and sugars were autoclaved separately and mixed after cooling. Tetracycline was included at a concentration of 10 mg/liter. Inocula were grown from freshly isolated colonies for 24 h, washed in the fermentation medium to be tested, and added to an initial OD,,, of approximately 1.0. Fermentations were carried out at 30 or 37 C in 100-ml volumetric flasks containing 80 ml of broth and fitted with rubber septa and 25-gauge needles to allow gas to escape. Fermentation flasks were immersed in a temperature-controlled water bath and stirred by a magnetic stirrer at 100 rpm. Ethanol concentration was measured by gas chromatography as previously described (5) and is expressed as percentage by volume. The conversion efficiency was calculated on the basis of sugar added, assuming that 100% efficiency results in the production of ml of ethanol (10.2 g) per 20 g of glucose or xylose and 13.5 ml of ethanol (10.8 g) per 20 g of lactose. RESULTS APPL. ENVIRON. MICROBIOL. Environmental hardiness and sugar utilization. Before the introduction of plasmids for ethanol production, the growth of eight different strains of E. (oli was compared for environmental hardiness. Table 1 summarizes the results obtained with medium containing glucose. Similar though not identical results were obtained with media containing lactose and xylose (data not shown). Strains ATCC 8677, ATCC 8739, and ATCC were particularly sensitive to inhibition by sodium chloride. Strains ATCC 8677 and ATCC were inhibited by low concentrations of ethanol. Strains ATCC 9637 and ATCC were the most resistant to low ph, although all strains except ATCC grew for at least two to four doublings at ph 4.0. All strains grew at 45 C. with limited growth at higher temperatures; none could be subcultured above 45 C. All strains grew in media containing 200% glucose, 20%, lactose, or 12% xylose. All strains tested utilized glucose, fructose, galactose, mannose, arabinose, lactose, glucuronic acid, and galacturonic acid. Strain ATCC did not utilize xylose. Maltose and maltotriose were not used by ATCC and ATCC All strains exhibited a weakly positive reaction with cellobiose. Only strain ATCC 9637 utilized sucrose. These results indicated that on the basis of environmental hardiness, ATCC 8677, ATCC 11775, and ATCC were less suitable than the other four strains for further development. Strain ATCC also lacked the ability to metabolize xylose, one of the most abundant sugars in biomass. Growth, plasmid stability, and expression of pyruvate decarboxylase. Recombinant strains harboring plasmids with the genes for ethanol production grew as unusually large colonies which became yellow after 24 to 48 h on solid medium containing a fermentable sugar. In liquid medium, the final cell densities of these recombinants were two to three times higher than that of the control lacking plasmid (Table 2). No transformants were obtained after several attempts from ATCC 14948(pLOI297) or from ATCC 11775(pLOI308-11). Strain ATCC did not utilize xylose, and recombinants of this strain did not grow to higher densities than the control with xylose as the fermentable sugar, although increased growth was observed with lactose and glucose. Plasmid stability was examined after growth in medium containing glucose for 25 generations (Table 3). Both plasmids contained the same replicons and were maintained well in all strains except ATCC 8677 and ATCC The expression of Z. Imobilis pyruvate decarboxylase activity was examined after growth in the presence of

3 VOL. 55, 1989 ETHANOL PRODUCTION BY E. COLI 1945 Sugar Plasmid TABLE 2. Growth of E. coli strains harboring plo1297 and plo Final OD,5, of E. co/i ATCC str-ain: Glucose None plo " plo Lactose None plo plo ( 10.0 Xylose None plo plo No data available. tetracycline (Table 4). With ploi297, Z. inobilis genes are expressed under the control of the E. c(li kac promoter; ploi utilizes the cryptic Z. inobilis promoter for expression of the PET operon. Strains ATCC (ploi297), ATCC 11775(pLOI297), and ATCC (ploi297) contained the highest levels of activity. Comparison of ethanol production during batch fermentation. All genetically engineered strains of E. c(li produced significant amounts of ethanol from sugars (Table 5). Preliminary experiments with strain ATCC 15224(pLOI297) indicated that higher levels of ethanol were produced in medium containing 0.2 M potassium phosphate buffer (ph 7.0). With 15% glucose, higher ethanol levels were produced at 30 C than at 37 C after 48 h. The fermentation of lactose and xylose was examined only at the lower temperature, 30 C. In general, higher levels of ethanol were produced by strains harboring ploi297 than by those with ploi Strains ATCC 11303(pLOI297), ATCC 11775(pLOI297), and ATCC 15224(pLOI297) produced the highest levels of ethanol after 48 h from 15% glucose, 5.8 to 6.9% by volume. Most strains were less tolerant of xylose in initial experiments, and comparisons of fermentation were carried out with 8% xylose. Strains ATCC 9637(pLOI297), ATCC (ploi297), and ATCC 15224(pLOI297) produced the highest levels of ethanol (4.8 to 5.2%) from 8% xylose after 72 h. All strains grew well in 15% lactose. Strains ATCC 11303(pLOI297) and ATCC 15224(pLOI297) produced the highest levels of ethanol from lactose after 48 h (6.1 and 5.6%, respectively). On the basis of these comparative studies, strains ATCC 11303(pLOI297) and ATCC 15224(pLOI297) appeared to be TABLE 3. Stability of ploi297 and ploi after 25 generations of growth with glucose in the absence of antibiotic selection ATCC strain plo1297 % of cells retaining plasmid plo () " No data available. the best constructs for ethanol production. The time courses of growth and ethanol production were examined with both strains in 12% glucose, 12% lactose, and 8% xylose (Fig. 1). Cell mass increased approximately 10-fold, reaching a final density of 3.6 g of dry weight per liter. With xylose, cell mass increased at half the rate observed with glucose or lactose. Ethanol production and growth were approximately linear for the three sugars until the concentration of ethanol reached 5%. To compute the conversion efficiency of sugar to ethanol, final ethanol concentrations after 120 h were averaged from three sets of experiments (Table 6). The final concentration of ethanol in cultures grown with 12% glucose was 7.2% (by volume), representing 94% of theoretical yield from glucose. With 12% lactose, the final ethanol concentration was 6.5%, 80% of the theoretical yield from lactose. With 8% xylose, we consistently obtained yields of 100% and higher. These high yields during slower growth with xylose may reflect the conversion of pyruvate from the catabolism of complex nutrients into ethanol, in addition to conversion of pyruvate from glucose. The rate of ethanol production was computed from the graphs in Fig. 1 and are summarized in Table 6. Volumetric productivity of ethanol ranged from 1.4 g/liter per h for glucose to 0.64 g/liter per h for xylose. Specific productivity of ethanol was highest during the initial growth period for each of the three sugars. The highest productivity was obtained with glucose, 2.1 g of ethanol per g of cell dry weight per h. The highest yield of eth-anot per g of sugar was obtained with xylose, exceeding the maximal theoretical yield for sugar alone. TABLE 4. Expression of Z. mnobilis pyruvate decarboxylase in E. coli strains harboring plo1297 and plo during growth with glucose ATCC strain plo1297 Pyruvate decarboxylase activity" plo h International units per milligram of cell protein. " No data available.

4 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 plo Carbohydrate a % Ethanol (vol/vol) produced by E. coli ATCC strain: (%) Plasmid C Glucose (15)" ploi plo Glucose (15)" ploi ploi Lactose (15)" ploi plo Xylose (8)"' plo plo 'Incubation with plasmids was at 37'C. "Incubation with plasmids was at 30'C. -, No data available. Experiments were conducted with ATCC 11303(pLOI297) to examine ethanol production from arabinose, galactose, and mannose. Ethanol concentrations of 3 to 4% were obtained after 48 h at 30 C but were not investigated further. These sugars are metabolized by pathways similar to those for glucose and xylose and would be expected to produce analogous yields (12). DISCUSSION Brau and Sahm (2) first demonstrated that ethanol production could be increased in recombinant E. coli by the overexpression of Z. mobilis pyruvate decarboxylase, although very low ethanol concentrations were produced. Subsequent studies by Tolan and Finn extended this work by using two other enteric bacteria (E. chrysanthemi [301 and K. planticola [31]) and achieved much higher levels of ethanol from hexoses, pentoses, and sugar mixtures. Alcohol dehydrogenase is also essential for this conversion, and ethanol production in these previous recombinant systems was dependent on native activities in the host organisms. Mutant E. coli which overproduced native alcohol dehydrogenase produced much higher levels of ethanol with Z. mobilis pyruvate decarboxylase than E. coli recombinants with the native activity did (9). The dependence upon host alcohol dehydrogenase activity was eliminated by combining Z. mobilis genes encoding alcohol dehydrogenase II and pyruvate decarboxylase to form a portable, plasmid-borne operon for ethanol production, the PET operon (8, 9). Considering the vast amount of information available concerning the physiology, genetics, and metabolism of E. coli, this organism is an obvious choice for further exploration of ethanol production. E. coli has an extremely wide substrate range which includes hexoses, pentoses, dissacharides, hexuronic acids, sugar alcohols, purines, pyrimidines, and amino acids, among others. Many potentially useful genes have been cloned from other organisms and expressed in E. coli, the most widely used host for molecular genetics. A variety of factors need to be considered in selecting E. coli strains suitable for ethanol production, including substrate range and environmental hardiness (sugar tolerance, salt tolerance, ethanol tolerance, tolerance to low ph, and thermal tolerance). Strain ATCC 9637 (Waksman strain W) appeared superior in terms of environmental hardiness, C) LJD Jo 9-- LLJ C) >1 -- Time (hours) 6 11, :::a-~-~ Al 9 3 1~~~~~~~~~~~~~~~ 3I3 2a~~~~~~~~~~~~~ Time (hours) CD 12 3 a) cn 9 O 6* a-n a_n 3,3 'UL Time (hours) FIG. 1. Growth and ethanol production from 12% glucose (A), 12% lactose (B), and 8% xylose (C). Symbols:, ethanol; - - -, cell mass; 0, strain ATCC 11303(pLO1297); A, strain ATCC 15224(pLO1297) C-,) tn cn s cn Ln =3 C-, CD 'n W _^ Ln nt C> =:

5 VOL. 55, 1989 TABLE 6. Average kinetic parameters for ethanol production by ATCC 11303(pLO1297) and ATCC 15224(pLOI297) Productivity E Sugar (%) Yield" Efficicncy Ethinol' Volumetric' Specific" (Ce) Glucose (12) Lactose (12) Xylose (8) Grams of ethanol per gram of sugar. "Calculated as: (ethanol produced/theoretical maximum from sugair substrate) x 100. Final ethanol concentration in grams per liter. ' Grams of ethanol per liter pei- hour. Grams of ethanol per g of cell dry weight per hour. although ethanol production from glucose was lower than with other strains. ATCC (Luria strain B) and ATCC (Kepes strain ML308) containing ploi297 produced the highest levels of ethanol and exhibited acceptable levels of environmental hardiness. Plasmids were quite stable in these two constructs, and they were selected as the best candidates for further development of ethanol production. Both constructs expressed the high levels of Z. iniobilis pyruvate decarboxylase which are required for efficient ethanol production (9). All major sugar components of plant biomass were converted to ethanol by recombinant E. coli containing the ethanol pathway from Z. inobilis. The conversion efficiency of glucose and xylose into ethanol exceeded that for S. erev'isiae (13) and pentose-fermenting yeasts systems ( , 28). Xylose was converted to ethanol by recombinant E. coli with a higher efficiency than glucose by S. (-ei-(eil,sia(e (13). The unusually high ethanol yields with xylose (over 100%7 of theoretical values) may include ethanol derived from the catabolism of complex nutrients. Many amino acids and complex-medium components are catabolized to glycolytic intermediates which are converted to pyruvate. This pyruvate could then be converted to ethanol. The ethanol tolerance of E. c oli (6, 7) is lower than that of S. (erevisiae (6, 19) or Z. mobilis (20, 21). However, it is unlikely that this will be a limitation for ethanol production from biomass. The hydrolysis of biomass by a combination of chemical and enzymatic methods typically yields sugar concentrations well below 12% (1). Recombinant E. (olibased ethanol production would result in a small increase in the costs of product recovery. This would be offset by increased ethanol yield and decreased costs of biomass feedstocks. Although further investigations are needed to optimize ethanol production by recombinant E. coli, the conversion rates observed in batch culture for glucose and lactose fermentation compared favorably with those observed for yeasts (13, 29). The conversion rate of xylose to ethanol equaled or exceeded previous rates by yeasts even in expensive systems using added xylose isomerase ( ). The ability of recombinant E. (coli to convert the diversity of sugars present in biomass to ethanol offers potential for the commercial expansion of fuel ethanol production by using alternative feedstocks which do not have competing value as food. Such expansion could involve a blend of current feedstocks with cellulosic biomass from undervalued agricultural products. ETHANOL PRODUCTION BY E. COLI 1947 ACKNOWLEDGMENTS This work was supported in part by the Florida Agriculturtal Experiment Station and by grants from the Department of Agriculture. Alcohol Fuels Program ( ) and from the Department of Energy. Office of Basic Energy Research (FG ER3574). We ar-e graiteful to the Conselho Nacional de Desenvolvimento Cientifico e Teconologico. Brazil (Processo /87) for providing support for Flavio Alterthum. LITERATURE CITED 1. Beck, M. J Faictor-s affecting efficiency of biomass fermentation to ethanol. Biotechnol. Bioeng. Symp. 17: Brau, B., and H. Sahm Cloning and expression of the structural gene for pyruvate decarboxylase of Zyoinoonas iniobilis in Escherichia (oli. Arch. Microbiol. 144: Conway, T., Y. A. Osman, J. I. Konnan, E. M. Hoffmann, and L. 0. Ingram Promoter and nucleotide sequences of the Zv10nomonas mzob/ilius pyruvate decarboxylase. J. Bacteriol. 169: Conway, T., G. W. Sewell, Y. A. Osman, and L. 0. Ingram Cloning and sequencing of the alcohol dehydrogenase 11 gene from Zvinomona)ns miobdilis. J. Bacteriol. 169: Dombek, K. M., and L. 0. Ingram Determination of intracellular concentrations of ethanol in Saccharon'vccs (cereviisima during fermentation. Appl. Environ. Microbiol. 51:197-2)0(. 6. Ingram, L Microbial tolerance to alcohols: role of the cell membrane. Trends Biotechnol. 4: Ingram, L Effects of ethanol on E.sclherichia coli. p In N. van Uden (ed.). Alcohol toxicity in yeasts and bacteria. CRC Press. Inc.. Boca Raton. Fla. 8. Ingram, L. O., and T. 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6 1948 ALTERTHUM AND INGRAM Selective isolation of cosmid clones by homologous recombination in Escherichia coli. Proc. Natl. Acad. Sci. USA 81: Reynen, M., and H. Sahm Comparison of the structural genes for pyruvate decarboxylase in different Zymomonas mobilis strains. J. Bacteriol. 170: Rosillo-Calle, F., and D. 0. Hall Brazilian alcohol: food versus fuel? Biomass 12: Silhavy, T. J., and J. R. Beckwith Uses of lac fusions for the study of biological problems. Microbiol. Rev. 49: Simon, R., U. Priefer, and A. Puhler A broad host range mobilization system for in i'iio genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology 1: Skoog, K., and B. Hahn-Hagerdal Xylose fermentation. AppI. ENVIRON. MICROBIOL. Enzyme Microbiol. Technol. 10: Slininger, P. J., R. J. Bothast, M. R. Okos, and M. R. Ladisch Comparative evaluation of ethanol production by xylosefermenting yeasts presented high xylose concentrations. Biotechnol. Lett. 7: Terrell, S. L., A. Bernard, and R. B. Bailey Ethanol from whey: continuous fermentation with a catabolite repressionresistant Saccharotnvces cerev'isiae mutant. Appl. Environ. Microbiol. 48: Tolan, J. S., and R. K. Finn Fermentation of D-xylose and L-arabinose to ethanol by Erwinia Chrysanthemni. Appl. Environ. Microbiol. 53: Tolan, J. S., and R. K. Finn Fermentation of D-xylose to ethanol by genetically modified Klebsiella planticola. Appl. Environ. Microbiol. 53: