Ethanol production using engineered mutant E. coli

The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

BACKGROUND OF THE INVENTION 
During glycolysis, cells convert simple sugars, such as glucose, into 
pyruvic acid, with a net production of ATP and NADH. In the absence of a 
functioning electron transport system for oxidative phosphorylation, at 
least 95% of the pyruvic acid is consumed in short pathways which 
regenerate NAD.sup.+, an obligate requirement for continued glycolysis and 
ATP production. The waste products of these NAD.sup.+ regeneration systems 
are commonly referred to as fermentation products (Ingram, L. O., T. 
Conway, D. P. Clark, G. W. Sewell, and J. F. Preston [1987] "Genetic 
Engineering of Ethanol Production in Escherichia coli," App. and Env. 
Microbiol. 53:2420-2425). 
Microorganisms are particularly diverse in the array of fermentations 
products which are produced by different genera (Krieg, N. R., and J. G. 
Holt, eds. [1984] Bergey's manual of systematic bacteriology. The Williams 
& Wilkins Co., Baltimore). These products include organic acids, such as 
lactate, acetate, succinate, and butyrate, as well as neutral products, 
such as ethanol, butanol, acetone, and butanediol. Indeed, the diversity 
of fermentation products from bacteria has led to their use as a primary 
determinant in taxonomy (Krieg and Holt [1984], supra). 
End products of fermentation share several fundamental features. They are 
relatively nontoxic under the conditions in which they are initially 
produced but become more toxic upon accumulation. They are more reduced 
than pyruvate because their immediate precursors have served as terminal 
electron acceptors during glycolysis. The microbial production of these 
fermentation products forms the basis for our oldest and most economically 
successful applications of biotechnology and includes dairy products, 
meats, beverages, and fuels. In recent years, many advances have been made 
in the field of biotechnology as a result of new technologies which enable 
researchers to selectively alter the genetic makeup of some 
microorganisms. 
Escherichia coli is an important vehicle for the cloning and modification 
of genes for biotechnology and is one of the most important hosts for the 
production of recombinant products. In recent years, the range of hosts 
used for recombinant DNA research has been extended to include a variety 
of bacteria, yeasts, fungi, and some eukaryotic cells. The invention 
described here relates to the use of recombinant DNA technology to elicit 
the production of specific useful products by a modified host. 
The DNA used to modify the host of the subject invention is isolated from 
Zymononas mobilis. Z. mobilis is a microorganism with unusual metabolic 
characteristics which is commonly found in plant saps and in honey. Z. 
mobilis has long served as a natural inocula for the fermentation of the 
Agave sap to produce pulque (an alcohol-containing Mexican beverage) and 
as inocula for palm wines. This organism is also used for fuel ethanol 
production and has been reported capable of ethanol production rates which 
are substantially higher than that of yeasts. 
Although Z. mobilis is nutritionally simple and capable of synthesizing 
amino acids, nucleotides and vitamins, the range of sugars metabolized by 
this organism is very limited and normally consists of glucose, fructose 
and sucrose. Substrate level phosphorylation from the fermentation of 
these sugars is the sole source of energy for biosynthesis and 
homeostasis. Z. mobilis is incapable of growth even in rich medium such as 
nutrient broth without a fermentable sugar. 
Z. mobilis is an obligately fermentative bacterium which lacks a functional 
system for oxidative phosphorylation. This organism, like Saccharomyces 
cerevisiae, produces ethanol and carbon dioxide as principal fermentation 
products. Z. mobilis produces ethanol by a short pathway which requires 
only two enzymatic activities: pyruvate decarboxylase and alcohol 
dehydrogenase. Pyruvate decarboxylase is the key enzyme in this pathway 
which diverts the flow of pyruvate to ethanol. Pyruvate decarboxylase 
catalyzes the nonoxidative decarboxylation of pyruvate to produce 
acetaldehyde and carbon dioxide. Two alcohol dehydrogenase isozymes are 
present in this organism and catalyze the reduction of acetaldehyde to 
ethanol during fermentation, accompanied by the oxidation of NADH to 
NAD.sup.+. Although bacterial alcohol dehydrogenases are common in many 
organisms, few bacteria have pyruvate decarboxylase. Attempts to modify Z. 
mobilis to enhance its commercial utility as an ethanol producer have met 
with very limited success. 
The gene coding for pyruvate decarboxylase in Z. mobilis has been cloned, 
characterized, and expressed in E. coli (Brau, B., and H. Sahm [1986] 
"Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial 
characterization," Arch. Microbiol. 146:105-110; Conway, T., Y. A. Osman, 
J. I. Konnan, E. M. Hoffmann, and L. O. Ingram [1987] "Promoter and 
nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase," J. 
Bacteriol. 169:949-954). The subject invention relates to the creation and 
expression of a novel system coding for the production of ethanol. 
BRIEF SUMMARY OF THE INVENTION 
The invention described here concerns the construction of a unique 
metabolic system for ethanol production which includes the introduction of 
the pyruvate decarboxylase activity into cells with constitutive and 
hyperproducing dehydrogenase gene mutations. The novel pathway utilizes 
the enhanced alcohol dehydrogenase activity of a mutant E. coli combined 
with the pyruvate decarboxylase activity resulting from the introduction 
of the pyruvate decarboxylase gene into the mutant E. coli. This system is 
capable of effectively diverting pyruvate to ethanol during growth under 
both aerobic and anaerobic conditions. 
Also described here are novel strains of E. coli which contain the 
ethanol-producing pathway. 
DETAILED DESCRIPTION OF THE INVENTION 
Certain bacteria and other simple organisms are capable of actively 
metabolizing a wide variety of substrates, including hexoses, pentoses, 
and lactose. The invention described here allows the use of a recombinant 
strain of E. coli for the production of ethanol from under-utilized 
sources of biomass, such as hemicellulose (xylose, arabinose, etc.), which 
represents a major portion of wood and inedible plant parts, and whey 
(lactose), as well as from other biomass sources. 
Described here is a novel system by which cells produce ethanol. The system 
comprises the Z. mobilis gene coding for pyruvate decarboxylase activity, 
together with appropriate regulatory sequences, introduced into cells with 
constitutive and hyperproducing alcohol dehydrogenase gene mutations. The 
regulatory sequences may consist of promoters, inducers, operators, 
ribosomal binding sites, terminators, and/or other regulatory sequences. 
Using the materials and methods described herein, significant amounts of 
ethanol can be produced in recombinants under both aerobic and anaerobic 
conditions. 
The conversion of a host organism to ethanolic fermentation can be used to 
enhance the production of a variety of recombinant products using the 
host's expression system. The maintenance of function in these products is 
related to the pH of the broth during growth in dense culture. The extent 
of this acidification per unit of cell protein is minimized by the 
production of ethanol rather than of organic acids. Oxygen transfer is 
frequently a major limitation during the growth of dense cultures of 
microorganisms, and it is this limitation which results in acid production 
and pH drift of the growth medium. In recombinants expressing the novel 
pathway, part of the pyruvate is diverted from glycolysis to acetaldehyde 
and reoxidizes NADH to produce ethanol, a less damaging product of 
metabolism. Strains containing both functional respiratory chains for 
oxidative phosphorylation and ethanol production enzymes can be grown to 
even higher cell densities because of the operation of both systems during 
the regeneration of NAD.sup.+ and a reduction in acidic waste products. 
Such inherent flexibility results in less stringent process-control 
requirements, as well as increased yields of recombinant products. 
This work demonstrates that recombinants can be developed for commercial 
ethanol production, and it illustrates the feasibility of drastic changes 
in metabolic flow for the future development of novel products from 
microorganisms. In addition, strains containing the novel pathway grow to 
higher cell densities than do the parent organisms under anaerobic 
conditions with glucose and offer the potential for the increased 
production of recombinant products in microorganisms while reducing 
complications associated with acid production. 
MATERIALS AND METHODS 
Organisms and growth conditions. E. coli TC4 (Conway, T., Y. A. Osman, J. 
I. Konnan, E. M. Hoffman, and L. O. Ingram [1987] "Promoter and nucleotide 
sequences of the Zymomonas mobilis pyruvate decarboxylase," J. Bacteriol. 
169:949-954) and plasmid-containing derivatives were used in the present 
study. Plasmids containing the pyruvate decarboxylase gene (pLOI276) have 
been described previously (Conway and Osman et al. [1987], "Promoter and 
nucleotide sequences. . . ," supra). 
In order to practice the invention, it is also necessary to have 
microorganisms with the capacity for producing high levels of alchol 
dehydrogenase. For example, the E. coli strains DC862adhC and DC863adhC 
adHR have hyperproducing alcohol dehydrogenase gene mutations. These 
microorganisms have been deposited with the American Type Culture 
Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 USA. The 
deposits were made on Dec. 29, 1988. The cultures were assigned the 
following accession numbers by the repository: 
______________________________________ 
Accession 
Culture number Deposit date 
______________________________________ 
E. coli DC862adhC 
ATCC 53846 December 29, 1988 
E. coli DC863adhC adHR 
ATCC 53847 December 29, 1988 
______________________________________ 
The subject cultures have been deposited under conditions that assure that 
access to the cultures will be available during the pendency of this 
patent application to one determined by the Commissioner of Patents and 
Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The 
deposits are available as required by foreign patent laws in countries 
wherein counterparts of the subject application, or its progeny, are 
filed. However, it should be understood that the availability of the 
deposits does not constitute a license to practice the subject invention 
in derogation of patent rights granted by governmental action. 
Further, the subject culture deposits will be stored and made available to 
the public in accord with the provisions of the Budapest Treaty for the 
Deposit of Microorganisms, i.e., they will be stored with all the care 
necessary to keep them viable and uncontaminated for a period of at least 
five years after the most recent request for the furnishing of a sample of 
the deposits, and in any case, for a period of at least 30 (thirty) years 
after the date of deposit or for the enforceable life of any patent which 
may issue disclosing the cultures. The depositor acknowledges the duty to 
replace the deposits should the depository be unable to furnish a sample 
when requested, due to the condition of the deposits. All restrictions on 
the availability to the public of the subject culture deposits will be 
irrevocably removed upon the granting of a patent disclosing them. 
The existence of microorganisms which produce high levels of alcohol 
dehydrogenase has been recognized and described in Clark, D. P. and M. L. 
Rod (1987) J. Mol. Evol. 25:151-158. 
Strains and growth conditions. Plasmids pUC18 and pUC19 (Yanisch-Perron, 
C., J. Vieira, and J. Messing [1985] "Improved M13 phage cloning vectors 
and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors," 
Gene 33:103-119), pLOI204 (Conway, T., M. O. -K. Byung, and L. O. Ingram 
[1987] "Expression vector for Zymomonas mobilis," Appl. Environ. 
Microbiol. 53:235-241), and pLOI295 (Ingram et al. [1987], supra) have 
been previously described. 
Cultures were grown at 37.degree. C. in Luria broth (Luria, S. E. and M. 
Delbruck [1943] "Mutations of bacteria from virus sensitivity to virus 
resistance," Genetics 28:491-511) containing 50 g of glucose per liter. 
Cells for enzyme analyses and inocula for fermentation studies were grown 
in tubes (13 by 100 mm) containing 3 ml of broth at 37.degree. C. in a 
tube rotator. Overnight cultures were diluted 100-fold into fresh medium. 
Aerobic cultures (50 ml of broth in 250 ml flasks) were shaken in a 
reciprocating water bath (120 oscillations per min). Anaerobic cultures 
were grown in stoppered serum bottles (100 ml of broth in 130 ml bottles) 
with gyratary agitation (150 rpm) in a 37.degree. C. incubator. Anaerobic 
cultures were vented with a 25-gauge needle to allow escape of gaseous 
fermentation products. 
Growth was monitored spectrophotometrically with a Spectronic 70 
spectrophotometer (Bausch & Lomb, Inc., Rochester, N.Y.) at 550 nm. 
Disposable culture tubes (10 by 75 mm) were used as cuvettes. One 
absorbance unit under our conditions contained approximately 0.25 mg of 
cellular protein per ml. Growth was measured at A.sub.550 ; 1.0 absorbance 
unit is equivalent to 0.25 mg of total cell protein per ml. 
Genetic techniques. Recombinants were selected on solid media (1.5% agar) 
containing 2 g of glucose per liter and appropriate antibiotics. 
Recombinants containing functional ethanologenic genes from Z. mobilis 
were identified by their growth as oversized colonies on Luria agar plates 
containing glucose and were confirmed by their poor growth on Luria agar 
plates lacking glucose and by the expression of alcohol dehydrogenase on 
aldehyde indicator medium. 
Enzyme assays. Cells were disrupted, heat-inactivated, and assayed for 
pyruvate decarboxylase activity (thermostable) as described previously 
(Conway and Osman et al. [1987] "Promoter and nucleotide sequences. . . ," 
supra). Cells were prepared and assayed for alcohol dehydrogenase II 
activity in the direction of ethanol oxidation as described previously, 
except that cells were washed and disrupted in 30 mM potassium phosphate 
buffer to which solid ferrous ammonium sulfate (final concentration, 0.5 
mM) and sodium ascorbate (10 mM) had been freshly added as described by 
Neale et al. (Neale, A. D., R. K. Scopes, J. M. Kelly, and R. E. H. 
Wettenhall [1986] "The two alcohol dehydrogenases of Zymomonas mobilis: 
purification by differential dye ligand chromatography, molecular 
characterization and physiological role," Eur. J. Biochem. 154:119-124). 
Analysis of fermentation products. Fermentation products were determined in 
clarified broth with a Millipore/Waters high-performance liquid 
chromatograph (Millipore Corp., Bedford, Mass.) equipped with a refractive 
index monitor and an electronic integrator. Separations were performed on 
an Aminex HPX-87H column (300 by 7.8 mm) purchased from Bio-Rad 
Laboratories, Richmond CA, at 65.degree. C. at a flow rate of 0.25 ml/min 
(100 .mu.l injection volume). Peaks were identified by using authentic 
standards. The two peaks eluting before glucose and the later unknown peak 
eluting at 45.4 to 45.8 min are components of uninoculated medium.

Following are examples which illustrate procedures, including the best 
mode, for practicing the invention. These examples should not be construed 
as limiting. All percentages are by weight and all solvent mixture 
proportions are by volume unless otherwise noted. 
EXAMPLE 1 
Strain construction 
The sizes of the structural gene coding for pyruvate decarboxylase is 1.7 
kilobases, and this gene encodes a protein with a molecular weight of 
60,000. This gene is located on derivatives of pUC18 under the control of 
the lac promoter. A plasmid containing the pyruvate decarboxylase gene was 
created as described in Conway et al. (Conway, T., Y. A. Osman, J. I. 
Konnan, E. M, Hoffmann, and L. O. Ingram [1987] "Promoter and nucleotide 
sequences of the Zymomonas mobilis pyruvate decarboxylase," J. Bacteriol. 
169:949-954). Clones were selected for resistance to ampicillin and for 
the presence and expression of alcohol dehydrogenase activity on a newly 
developed pararosaniline-ethanol indicator plate which detects the 
production of aldehydes. Clones containing the indicated construction grew 
poorly on the surface of Luria agar plates (aerobic) in the absence of 
added glucose but grew to much higher densities than the plasmid-free 
strain on agar plates containing 2% glucose. Recombinants containing the 
pyruvate decarboxylase gene were readily detected as larger, more opaque 
colonies on Luria agar plates (aerobic) containing glucose. 
EXAMPLE 2 
Expression of Z. mobilis genes in E. coli 
Pyruvate decarboxylase was expressed at high levels in E. coli under the 
control of the lac promoter singly. During growth of E. coli in the 
presence of glucose, the specific activities of the Z. mobilis enzymes 
declined by approximately 50%, which is consistent with glucose repression 
of the lac promoter. 
EXAMPLE 3 
Fermentation of glucose by recombinant strains 
Expression of the pyruvate decarboxylase gene in mutant E. coli resulted in 
the production of ethanol as the primary fermentation product during 
anaerobic growth. The parent strain produced succinate (1.5 mM), lactate 
(18 mM), and acetate (7 mM) as major fermentation products. With pLOI276 
carrying the gene coding for pyruvate decarboxylase, an ethanol peak is 
clearly evident. This higher level of ethanol results from the combined 
activities of the pyruvate decarboxylase from Z. mobilis and the mutant E. 
coli alcohol dehydrogenase. Thus, the fermentation of this organism was 
converted to the equivalent of those of S. cerevisiae and Z. mobilis. 
High cell densities are also achieved during mixed growth conditions with 
moderate agitation or stirring of culture vessels in which gas exchange is 
not restricted. Under these conditions, a final pH of 6.3 or above was 
observed, depending upon the extent of aeration. 
EXAMPLE 4 
Growth of recombinant strains containing the pyruvate decarboxylase enzyme 
from Z. mobilis 
Shifting the catabolism of glucose to the production of ethanol also 
affected growth yield and pH drift of the growth medium. Although 
fermentation products are relatively nontoxic, they may accumulate to 
toxic levels during fermentation. During anaerobic growth in bottles 
containing Luria broth containing 10% glucose, the plasmid-free strain 
achieved a final density of 0.25 mg of cell protein per ml after 48 hr, 
with a final pH of 4.4. The cell density increased by twofold in the 
strain carrying pLOI276 (carrying the gene coding for pyruvate 
decarboxylase), with a final pH of 4.5. 
EXAMPLE 5 
Effects of ethanologenic enzymes on the acidification of broth during 
growth 
The pH fell rapidly during the first 6 hr of growth of strain TC4 lacking a 
plasmid but declined more slowly in derivatives containing the 
ethanologenic enzymes. Although the recombinants reached a higher final 
cell density, the pH of the broth from the recombinants grown under both 
anaerobic and aerobic conditions for 24 hr was less acidic than that of 
the broth from strain TC4 lacking ethanologenic enzymes. 
The reduced rate and extent of acidification in recombinants accompanied by 
increased cell growth suggested that the fall in pH was a major factor 
limiting growth even under highly aerobic conditions. This hypothesis was 
supported by an 85% increase in the final cell density of strain TC4 
(lacking a plasmid) grown in medium supplemented with a 1/10 volume of 1M 
sodium phosphate buffer (pH 7.0). Lower levels of buffer addition resulted 
in intermediate levels of growth. 
EXAMPLE 6 
Effects of ethanologenic enzymes on fermentation products 
Under aerobic conditions, acetate was the primary fermentation product that 
accumulated during the growth of strain TC4 lacking a plasmid in rich 
medium, with no detectable ethanol. The amount of acetate produced was 
drastically reduced in strains containing the pyruvate decarboxylase 
enzyme from Z. mobilis, and ethanol appeared as the major fermentation 
product. 
Under anaerobic conditions, lactate was the principal fermentation product 
that accumulated during 24 hr of growth of strain TC4 lacking a plasmid in 
rich medium containing glucose, with lesser amounts of acetate, succinate, 
and ethanol being present. Lactate production was dramatically reduced in 
strains containing the pyruvate decarboxylase enzyme and was accompanied 
by the production of substantial quantities of ethanol. It is likely that 
this lower level of accumulated ethanol was caused by the reduction in 
total cell mass produced under these anaerobic conditions, thus reducing 
the volumetric rate of ethanol production. 
The extent of ethanol production under anaerobic and aerobic conditions was 
directly related to the level of expression of the Z. mobilis 
ethanologenic gene. 
Derivatives of E. coli TC4 containing plasmids which express the 
ethanologenic enzymes from Z. mobilis grew to higher cell densities than 
did the parent organism lacking a plasmid. The increase in the final cell 
density, the extent to which ethanol accumulated in the medium, and the 
reduction in the rate of acidification of the culture broth during growth 
all correlated with the level of expression of pyruvate decarboxylase 
enzyme. Heterologous promoters were used to express the gene in order to 
minimize potential problems associated with transcriptional regulation. 
It should be understood that the examples and embodiments described herein 
are for illustrative purposes only and that various modifications or 
changes in light thereof will be suggested to persons skilled in the art 
and are to be included within the spirit and purview of this application 
and the scope of the appended claims.