WO1994028154A1

WO1994028154A1 - Methods for increasing carbon conversion efficiency in microorganisms

Info

Publication number: WO1994028154A1
Application number: PCT/US1994/006084
Authority: WO
Inventors: Sandra J. Koehn; Thomas M. Evans; Richard A. Nelson; Paul P. Taylor
Original assignee: The Nutrasweet Company
Priority date: 1993-05-27
Filing date: 1994-05-27
Publication date: 1994-12-08
Also published as: KR950702637A; EP0660875A4; JPH07509375A; AU6960894A; CA2140527A1; EP0660875A1

Abstract

Methods are presented for increasing the carbon conversion efficiency of a microorganism by introduction of DNA encoding enzymes of the glyoxylate cycle into the microorganism.

Description

METHODS FOR INCREASING CARBON CONVERSION EFFICIENCY IN MICROORGANISMS

The present invention generally relates to methods for increasing the carbon conversion efficiency of microorganisms.

Background Of The Invention

In the presence of glucose or another carbon compound (e.g. , a carbohydrate) capable of entry into the tricarboxylic acid cycle (hereinafter referred to as the "TCA cycle") as the sole source of carbon, bacterial cells and other microorganisms utilize the TCA cycle as a primary metabolic pathway. The TCA cycle generates energy sources via the oxidation of acetyl groups which enter the cycle as acetyl CoA molecules. In so doing, the TCA cycle primarily produces energy and various four-carbon intermediates, such as oxaloacetate, which serve as precursors in the synthesis of essential cell components such as amino acids. These four-carbon intermediates result from two decarboxylation reactions in the TCA cycle, both generating carbon dioxide (CO->). The first decarboxylation reaction occurs when the six-carbon metabolit, isocitrate, is converted to the five-carbon compound, α-ketoglutarate, by the enzyme isocitrate dehydrogenase. The second carbon dioxide-producing reaction occurs in the next step of the TCA cycle, wherein α-ketoglutarate is converted to the four-carbon intermediate, succinyl CoA.

As used herein, carbon conversion efficiency generally refers to a ratio of the amount of substrate metabolized to the amount of biosynthetic products formed by a microorganism. The greater the loss of carbon during the process of conversion from substrate to biosynthetic products the greater the inefficiency of carbon conversion. Under conditions when the TCA cycle is a predominant metabolic pathway in a microorganism, carbon conversion is limited by the amount of carbon lost through the aforementioned carbon-dioxide-evolving steps. There exists a need in the art for methods for increasing carbon conversion efficiency under conditions when the TCA cycle would normally be predominant, i.e., when glucose or another substrate is available. The present invention provides methods for increasing carbon conversion efficiency under such conditions.

Brief Summary Of The Invention

The present invention provides methods for increasing carbon conversion efficiency in a microorganism.

In a preferred embodiment of the invention, methods for increasing carbon conversion efficiency are provided, wherein DNA encoding isocitrate lyase, alate synthase, and isocitrate dehydrogenase kinase/phosphatase is incorporated into the microorganism which is then cultivated under appropriate conditions in the presence of a substrate metabolized by the TCA cycle and wherein biosynthetic product may be isolated.

Also in a preferred embodiment of the invention, the DNA encoding isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase comprises a portion of the glyoxylate operon. A microorganism according to the present invention may be any microorganism which normally utilizes the TCA cycle, but may preferably be a bacterial cell, and most preferably an Escherichia coli cell.

In a preferred embodiment of the invention, the DNA encoding isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase may be incorporated in the microorganism in an appropriate vector, such as a plasmid. Also in a preferred embodiment of the invention, the method further comprises calculating the carbon conversion efficiency of a microorganism.

Numerous additional aspects and advantages of the invention are apparent to the skilled artisan upon consideration of the following detailed description which provides presently preferred embodiments thereof.

Detailed Description Of The Invention

If a two-carbon compound, such as acetate, is the sole source of carbon for the microorganism, the two-carbon compound may be utilized in the TCA cycle to produce energy. However, in so doing both carbon atoms are lost as CO₂, thus depleting carbon available for formation of biosynthetic products. Microorganisms adjust to this situation by channeling some isocitrate into the glyoxylate cycle. The interrelation of the TCA cycle and the glyoxylate cycle is depicted in Figure 1. Carbon flow to the glyoxylate cycle is controlled by the bifunctional protein, isocitrate dehydrogenase kinase/phosphatase (hereinafter referred to as "IDH k/p"). The glyoxylate cycle is repressed when glucose is available as a source of carbon. However, when acetate is the sole carbon source, IDH k/p reduces activity of isocitrate dehydrogenase, thus allowing the enzyme isocitrate lyase of the glyoxylate cycle to act upon isocitrate with reduced competition from isocitrate dehydrogenase.

The enzymes of the glyoxylate cycle in Escherichia coli are encoded by genes comprising the glyoxylate operon. The known structural organization of the operon comprise the aceB gene which encodes malate synthase, the aceA gene which encodes isocitrate lyase, and the aceK gene which encodes IDH k/p. Transcription of the operon in its native sequence is under the negative regulation of the iclR gene and, to a lesser extent, X &fadR gene which maps outside the glyoxylate operon in E. coli. Cortay et al , Embo J. , 10:675- 679 (1991).

Two enzymes function in the glyoxylate cycle to convert isocitrate and acetyl CoA to succinate and malate. These are isocitrate lyase, which converts isocitrate to succinate and glyoxylate, and malate synthase, which catalyzes the conversion of acetyl CoA and glyoxylate to malate. The net effect of the glyoxylate cycle is to avoid the carbon dioxide-evolving steps of the TCA cycle, thus generating four-carbon compounds from two-carbon precursors for use in the formation of cellular components. The present invention provides methods for increasing the carbon conversion efficiency of microorganisms, thus resulting in numerous benefits to industrial processes utilizing microorganisms. For example, increasing carbon conversion efficiency allows the use of less substrate to manufacture a given amount of biosynthetic product as compared to cells not treated according to methods of the present invention. Conversely, a given amount of metabolized substrate, such as glucose, will be converted to greater amounts of biosynthetic products under conditions in which carbon conversion efficiency is increased. Increased carbon conversion efficiency may also be utilized to effect increases in the production of a particular amino acid for use in industrial applications. Microorganisms which display increased carbon conversion efficiency may also display increase efficiency in biomass production generally.

Carbon conversion efficiency is a measure of the extent to which carbon from substrates, such as glucose, are incorporated into the intermediates and products of cellular biosynthesis. Carbon conversion efficiency may be measured based on the accumulation of any product or products of cellular biosynthesis and its (their) precursors or on the basis of an increase in biomass with a fixed amount of substrate or on the basis of production of a given amount of biomass utilizing less substrate. Examples of products which may be used to measure carbon conversion efficiency include biomass generally, amino acids, nucleic acids, various structural proteins, enzymes, secondary metabolites and their precursors.

For purposes of the present application, accumulation of L- phenylalanine and its two immediate precursors, phenylpyruvic acid and prephenic acid, are used to demonstrate increased carbon conversion efficiency using methods according to the present invention. Carbon conversion efficiency may be calculated by dividing the total grams of L-phenylalanine, prephenic acid, and phenylpyruvic acid by the total grams of glucose consumed during fermentation of the microorganism. Such a calculation provides an estimate of the extent to which carbon from metabolic precursor molecules, such as glucose, is incorporated into biosynthetic products. Enhanced carbon conversion efficiency means that less carbon is being channeled into undesirable metabolic products. This, in turn, means that the microorganism will more efficiently produce biosynthetic products over time. The use of methods according to the present invention is demonstrated by the following Examples. Example 1 relates to the use of exemplary plasmids comprising glyoxylate operon genes and the incorporation of such plasmids into exemplary host cells. Example 2 provides results of enzymatic assays to confirm proper incorporation and function of a portion of the glyoxylate genes in host cells according to the invention. Example 3 provides culture and fermentation conditions under which carbon conversion efficiency may be measured. Finally, Example 4 relates to an exemplary means of calculating carbon conversion efficiency according to the present invention.

EXAMPLE 1

Escherichia coli, strain ATCC 13281, a tyrosine auxotroph, was used as an exemplary host cell to demonstrate methods according to the present invention. The skilled artisan realizes, however, that numerous host microorganisms may be used in the practice of the invention. Representative host microorganisms are reported in Kornberg et al., Advan. Enzymol., 23:401-470 (1961), incorporated by reference herein.

Plasmids pCL8 and pCLlOOO were used as the source of genes encoding glyoxylate enzymes. These plasmids contain the aceA, aceB, and aceK genes of the glyoxylate operon in a pBR322 vector and have been described in

Chung et al. , J. Bacteriol. , 170:386-392 (1988), incorporated by reference herein.

Plasmids pCL8 and pCLlOOO were incorporated into E. coli host cells by electroporation. Prior to electroporation, a single colony of ATCC 13281

E. coli cells was inoculated into a 500 ml baffled flask containing 100 mis Luria Broth (Difco, Detroit, MI). The culture was grown on a rotary incubator/ shaker

(New Brunswick, Edison, NJ) for approximately 4 hours at 32° C until it reached mid-log phase, as determined by measuring the culture density as absorbance at 600 nm using a UV/vis spectrophotometer (Hewlett-Packard 8452A diode array spectrophotometer, Hewlett-Packard, Germany). The culture was grown to an optimum optical density of 0.3-0.7 abs. Upon reaching the optimum density range, cells were centrifuged for 10 minutes at 10,000 g and 4° C. The supernatant was then decanted and the cells were washed with 50 mis cold (0° C) distilled water and centrifuged again for 3 minutes at 10,000 g and 4° C. The supernatant was then aspirated so as not to disturb the pellet, which was suspended in distilled water to a final volume of 500 μl.

A 40 μl aliquot of the above-described cell suspension was transferred to a sterile polyvinylchloride (PVC) tube and mixed with 2 μl of either plasmid pCL8 or pCLlOOO. The mixture was then transferred to a cold Bio-Rad Pulser cuvette (Bio-Rad) with a 0.2 cm gap and electroporated with a single pulse using a Bio-Rad Gene Pulser (Bio-Rad) set to 2.5 Kv with 25 μF capacitance. The Bio-Rad pulse controller was set at 200 ohms resistance. Immediately after electroporation, 800 μl of S.O.C., described in Hanahan, J. Mol. Biol, 166:551- 580 (1983), was added to the cuvette and the cultures were allowed to incubate for 30 minutes at 37° C. The cells were then plated on agar containing ampicillin

(200 μg/ml), (Difco) to select for transformants. The plasmids contain an ampicillin resistance marker. Additional transformation procedures are known and available to those skilled in the art and may be used in the practice of the present invention. Positive transformants were re-designated NS3119 (containing pCL8) and NS3120 (containing pCLlOOO). NS3120 (containing plasmid pCLlOOO) was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 on April 27, 1993 and given ATCC Accession No. 69291. In Example 2, positive transformants were tested to confirm that the glyoxylate cycle was operating.

EXAMPLE 2

In order to confirm that host cells described in Example 1 were transformed with functional glyoxylate cycle genes, assays were conducted to determine the activity of glyoxylate cycle enzymes.

In preparation for the enzyme assays, cell extracts were prepared by growing cultures of transformed host cells overnight in M63 medium [Chung, et ah, J. Bacteriol., 770:386-391 (1988), incorporated by reference herein] in a rotary /incubator shaker at 37° C and 300 rpm. A 20 ml aliquot of the culture was transferred into a 50 ml polypropylene centrifuge tube (Corning, Corning NY) and centrifuged for 10 minutes at 10,000 g and 4° C in a Beckman S2-21 centrifuge (Beckman). The pellet was resuspended in 4 ml of 0.1 M potassium phosphate buffer, pH 7.0. The cells were then disrupted using a French Pressure Cell (SLM Instruments, Urbana, IL) at 1000 psi according to the manufacturer's instructions. The resulting cell debris was removed by centrifuging at 1300 g for 8 minutes in an MSE microcentrifuge (MSE, Micro Centaur, U.K.). The supernatants (ly sates) obtained were then stored on ice.

The cell lysates described above were then tested for isocitrate lyase and malate synthase activity. The isocitrate lyase assay was based on the protocol described in Dixon et al. , Biochem. J., 72:3V (1959) incorporated by reference herein, with 0.1 M potassium phosphate buffer substituted for the tris buffer described in the assay reported in Dixon. The assay is based upon the rate of increase in optical density with increased isocitrate lyase activity as measured by the accumulation of glyoxylic acid phenylhydrazine. To conduct the assay, 0.10 ml of 100 mM phenylhydrazine,

0.05 ml of 20 M cysteine, 0.05 ml of 100 mM MgCl₂, and 0.05 ml of the cell lysate described above were placed in a 1 ml quartz cuvette with 0.10 ml 100 mM potassium phosphate buffer in 1.10 ml distilled water at pH7.2.

The optical density of the above mixture was adjusted to zero in a Hewlett-Packard model 8452A diode array spectrophotometer equipped with a kinetics software package in a Hewlett-Packard operating software package A.02.0. The reaction whereby glyoxylate (and ultimately glyoxylic acid phenylhydrazine) is formed from isocitrate was begun by addition to the cuvette of 0.05 ml 20 mM potassium isocitrate. Absorbance was read at 324 nm and the amount of isocitrate lyase was determined by an increase in optical density.

The results of the isocitrate lyase assay are shown in Table 1, wherein the specific activity of the enzyme is expressed in units per miligram of protein, wherein one unit is defined as the amount of enzyme catalyzing the formation of one micromole of glyoxylic acid phenylhydrazone per minute. As shown in that Table, isocitrate lyase activity increases significantly in cell lysates from transformants containing the glyoxylate cycle genes as compared to the same measurements in cell lysates from untransformed controls.

An assay was also conducted to determine malate synthase activity in transformed and untransformed cell lysates. That assay was conducted according to the procedures in Dixon et al. , Biochem. J. , 72 :3p (1959). The malate synthase assay measures the decrease in optical density with an increase in breakdown of the thio-ester bond in acetyl COA which reacts with malate synthase to form malate in the presence of glyoxylate. The malate synthase assay was conducted by first making a stock solution comprising 4.0 ml 0.1 M tris, 0.50 ml 0.002 M acetyl COA and 0.1 M MgCl₂. A 0.4 ml aliquot of the stock solution was combined with 0.01 ml of the cell lysate in a cuvette. Absorbance was measured in a Hewlett-Packard model 8452A UV/vis diode array spectrophotometer at 232 nm for approximately 2 minutes to ensure that no acetyl CoA deacylase, which may prevent acetyl COA from reacting with malate synthase, was present. Upon achieving a steady baseline absorbance, 0.01 ml of 0.02 M sodium glyoxylate was added to the cuvette. The absorbance was measured every 5 seconds for a total of 200 seconds. The results of the malate synthase assay are also presented in Table

1, wherein specific activity is measured as units per miligram of protein, wherein one unit is defined as the cleavage of one micromole acetyl CoA per minute. As shown in that Table, the activity of malate synthase increases in transformants containing genes of the glyoxylate cycles compared to untransformed controls. The results of both the malate synthase and isocitrate lyase assays confirm that the activity of enzymes of the glyoxylate cycle in transformed host cells is greater than that in untransformed host cells. Example 3 provides fermentation and growth conditions which were used to prepare cultures for carbon conversion efficiency measurements. Table 1

SPECIFIC ACTIVITY

Fermentation

Construction Time (Hrs) Malate Synthase Isocitrate Lyase

E. coli 13281 8 0.066 0.009 Untransformed

24 0.143 0.008

48 0.144 0.054

E. coli 13281 8 0.731 0.164 Transformed With pCL8

24 0.481 0.063

48 0.507 0.16

E. coli 13281 8 1.615 0.428 Transformed With pCLlOOO

24 1.066 0.198

48 1.012 0.393

E. coli 13281 8 0.03 0.003 Untransformed

24 0.098 0.006

48 0.126 0.011

E. coli 13281 8 0.626 0.203 Transformed with pCL8

24 0.475 0.194

48 0.584 0.185 Table 1 (Cont.)

SPECIFIC ACTΓVITY

Fermentation

Construction Time (Hrs) Malate Synthase Isocitrate Lyase

E. coli 13281 8 0.947 0.369 Transformed with pCLlOOO

24 1.11 0.261

48 1.073 0.257

E. coli 13281 8 0.099 0.008 Untransformed

24 N.D. N.D.

51 0.037 0.028

E. coli 13281 8 1.119 0.37 Transformed with pCLlOOO

24 0.664 0.434

51 1.086 0.871

EXAMPLE 3

In order to determine carbon conversion efficiency, transformed cells were fermented as follows.

Fermentations were conducted using 20 L fermentors (LSL Biolafitte, France). The temperature, pH, agitation, and air flow were kept constant at 32° C, 7.2, 500 rpm, and 11 L/minutes, respectively. Glucose feed, dissolved oxygen, and other parameters were varied and may be set at values determined by the skilled artisan.

The fermentor contained 10 L of K12 medium [Konstantinov et al. , J. Fermentation and Bio Engineering, 70(4):253-260 (1990), incorporated by reference herein] supplemented with 1 g/L yeast extract and was sterilized at 121° C for 30 minutes. The fermentor was then inoculated with 1 L of the cell culture containing either the untransformed strains or the glyoxylate cycle transformants. The back pressure of the fermentor was regulated at 10 psi and the dissolved oxygen concentration was initially 100% and was allowed to reach a value of 15% where it was maintained throughout the fermentation. A 70% (weight per volume) glucose solution was initially added until the fermentor contained 3.0 g/L glucose. The glucose was then continuously added into the fermentor to maintain glucose in excess during exponential growth. The final biomass in the fermentor was limited by tyrosine (E. coli strain ATCC 13281 is a tyrosine auxotroph). The concentration of glucose was then allowed to decrease to approximately 0.2 g/L and was maintained at that concentration for the rest of the fermentation. The total fermentation time was 48 hrs. The concentrations of glucose, L-phenylalanine, prephenic acid, phenylpyruvic acid, and acetate were then measured to determine carbon conversion efficiency as taught in Example 4. EXAMPLE 4

Carbon conversion efficiency relates to the ratio of carbon (glucose or another carbon source) metabolized to the amount of carbon ultimately incorporated in biosynthetic precursors and end products. It is apparent to any skilled artisan that numerous biosynthetic precursors and end products may be used as a basis for the calculation of carbon conversion efficiency. Examples of such compounds are amino acids, especially the aromatic amino acids, nucleotides, various enzymes, and structural proteins. By way of example, the methods of calculating carbon conversion efficiency are exemplified herein using L-phenylalanine and its two immediate precursors as a measure of the incorporation of carbon from the precursor, glucose. The amount of L-phenylalanine, prephenic acid, and phenylpyruvic acid and acetate which accumulated in the fermentor and the amount of glucose consumed were measured in order to calculate carbon conversion efficiency as provided below.

A. L-phenvIalanine Production

The accumulation of L-phenylalanine was measured using flow- switching High Performance Liquid Chromatography. A flow-switching column allows faster clearance of phases not containing L-phenylalanine through the column. High performance liquid chromatography is well-known in the art and the skilled artisan is aware of the application of numerous such techniques to determine the concentration of any biosynthetic product, such as L-phenylalanine. The chromatographic apparatus comprised two Waters (Waters, Milford, MA) Model 600 A solvent delivery systems, an Applied Biosystems (Foster City, CA) 983 programmable detector, a Waters Model 710B auto sampler and model 680 automatic gradient controller, all used according to the manufacturer's instructions. A Vici (Valco Instruments, Houston, TX) 10-part column-switching valve was used according to the manufacturer's instruction to achieve flow reversal and a Fisions Multichrome Data Collection System (Danvers, MA) was used to analyze the data.

A 10 μl aliquot of sample obtained from fermentation broth containing E. coli transformed with glyoxylate cycle genes or from broth containing untransformed E. coli cells was injected onto a Supelco (Bellefonte,

PA) LC-18 DB column (97.5 mm x 4.5 mm) at a flow rate of 1.5 ml/minutes Detection was at 214 nm and retention time was 4.1 minutes. External standards of 0.1, 0.5, and 1.0 g/ml were diluted with the HPLC mobile phase, and a standard curve was developed according to procedures known in the art. The mobile phase for the HPLC runs and for the standards comprised 5 % acetonitrile,

95% 0.005 M pentane sulfonic acid (sodium salt) at pH 2.5.

The results of HPLC analysis of L-phenylalanine concentration in fermentation broths are presented in Table 2.

B. Phenylpyruvic Acid Production Phenylpyruvic acid is the immediate precursor of phenylalanine in the phenylalanine biosynthetic pathway. Phenylpyruvic acid concentration provides, in part, a measure of carbon conversion because, at any point in time, not all the carbon from glucose which has been transferred to the biosynthesis of cellular components will be in the form of L-phenylalanine. Some of the carbon will be in the form of the immediate precursors of phenylalanine. Thus, measurement of those precursors in addition to phenylalanine provides a more accurate measure of carbon conversion than would phenylalanine measurements alone.

Phenylpyruvic acid concentration was measured in samples taken from fermentation broths containing either transformed or untransformed E. coli cells as described above by reverse-phase High Performance Liquid Chromatography at a detection wavelength of 214 nm, 0.2 AUFS (Absorbance Units Full Scale) using a Whatman RAC II Partisil 5 ODS-3 10 cm column (Whatman, Hillsboro, OR) according to the manufacturer's instructions. An isocratic method was used, with a mobile phase comprising 10 mis of 1 % 0.5 M pentane sulfonic acid solution, 950 ml water, and 40 ml acetonitrile, pH.2.5. A volume of 10 μl was injected onto the column at a flow rate of 2 ml/minutes and the run time was 10 minutes Phenylpyruvic acid standards were also run at concentrations of 0.02, 0.08, and 0.4 mg/ml.

The results are shown in Table 2, wherein phenylpyruvic acid concentration is shown as grams/liter for the various samples, including untransformed controls.

C. Prephenic Acid Production

Prephenic acid is the immediate precursor of phenylpyruvic acid in the pathway leading to synthesis of phenylalanine. The concentration of prephenic acid was determined by High Performance Liquid Chromatography at a detection wavelength of 220 nm, 0.2 AUFS, using a Supelco LC10 column (Supelco). An isocratic method was used, with the mobile phase comprising 10% acetonitrile in

0.1 M ammonium phosphate buffer, pH 6.7 with 0.005 M tetrabutylammonium phosphate. A 10 μl volume of each sample was injected onto the column at a flow rate of 1.5 ml/minutes. Equivalent-size standards were also run at concentrations of 0.02, 0.15, 0.2 mg/ml. The results are shown in Table 2, wherein prephenic acid is labelled PA.

D. Glucose Concentration

The amount of glucose concentration of the feed stock was measured using High Performance Liquid Chromatograph equipped with a refractive index detector and a column heater. A 400 mg sample was obtained from the glucose being fed into the fermentor and diluted to 100 ml. A Biorad

HPX-878 column equilibrated to 85° C was used with a mobile phase comprising Milli-Q water (Millipore, Bedford, MA). The flow rate was 0.6 ml/minutes and the run time was 20 minutes. A 20 μl volume of sample was injected on the column at time zero. The column was equilibrated for 30 minutes until a stable base line was reached. External standards were also run using Glucose obtained from Sigma Chemical (G-5500, Sigma St. Louis, MO).

The results are presented in Table 2 and are used in the calculation of carbon conversion efficiency below.

E. Acetate Production

During the fermentation process glucose may be broken down to form acetate. While such glucose will be detected as being consumed, it should not properly be incorporated into calculations of carbon efficiency according to the presently-claimed methods. The reason for this is that any glucose used to produce acetate is not available to directly produce biosynthetic products. Therefore, acetate production should be calculated in order to form a basis for subtracting glucose which is diverted from pathways generating biosynthetic products and intermediates as noted above.

Ion chromatography was used to detect acetate in fermentation broths according to the procedure set forth in the Dionex (Sunnvale, CA) Ion Chromatography Cookbook, Issue I, p. 11-16. A 50 μl aliquot of sample was injected on an HPIcE-ASl column (Dionex) with a flow rate of 0.8 ml/min. The eluant was 1.0 mM octane-sulfonic acid in 2% 2-propanol. The regenerant was 5 mM tetrabutyl ammonium hydroxide. Standards were also run at concentrations of 0.02, 0.1, 0.2 mg/ml.

Results are included in Table 2 and used in the exemplified calculations below. Table 2

CONCENTRATION G L

Carbon Conversion

Fermentor Fermentation Total Glucose (g) Efficiency

Construction Volume (L) Time (Hrs) L-PHE PPA PA (L-PHE+PA+PPA) Acetate Consumed (%)

E. coli 13281 14.98 8 0.00 0.00 0.14 0.14 Untransformed

24 3.13 0.41 11.63 15.17

48 7.20 0.60 14.03 21.83 2.26 2451.8 13.62

E. coli 13281 16.365 8 0.00 0.00 0.16 0.16 Transformed With pCL8

24 2.93 0.92 16.04 19.89

48 7.35 1.30 17.03 25.68 19.33 3294.91 14.9

E. coli 13281 16.065 8 0.00 0.00 0.12 0.12 Transformed With pCLlOOO

24 2.34 0.41 12.43 15.18

Table 2 (Cont.)

CONCENTRATION G L

Carbon Conversion

Fermentor Fermentation Total Glucose (g) Effidency

48 6.77 1.10 22.55 30.42 13.3 3095.15 17.61

E. coli 13281 13.01 8 0.01 0.00 0.08 0.09 Untransformed

24 2.56 0.26 7.61 10.43

48 5.88 0.37 9.67 15.92 0.66 2099.25 9.66

E. coli 13281 12.66 8 0.01 0.00 0.09 0.10 Transformed with pCL8

24 3.21 0.25 5.64 9.10

48 6.38 0.28 10.26 16.92 0.37 2303.19 9.59

E. coli 13281 13.11 8 0.01 0.00 0.16 0.17 Transformed with pCLlOOO

Table 2 (Cont.)

CONCENTRATION G L

Carbon Conversion

Fermentor Fermentation Total Glucose (g) Effidency

24 2.46 0.24 10.43 13.13

48 6.11 1.81 15.55 23.47 2.86 2844.18 11.04

E. coli 13281 13.67 8 0.00 0.00 0.00 0.00 Untransformed

24 4.3 0.24 10.43 14.97

51 8.12 0.373 11.3 19.793 0.551 2570 10.71

E. coli 13281 13.90 8 0.00 0.00 0.00 0.00 Transformed with pCLlOOO

24 3.14 0.234 9.2 12.574

51 6.62 0.375 13.08 20.057 0.569 2370 11.95

F. Calculation Of Carbon Conversion Efficiency

Carbon conversion efficiency of transformed and untransformed cells was calculated as the total combined grams of L-phenylalanine, phenylpyruvic acid, and prephenic acid produced at 48 hrs. of fermentation divided by the total grams of glucose consumed during that fermentation. If acetate accumulates during the fermentation, carbon conversion efficiency may be calculated by subtracting 180.16 g (1 mole) of glucose for every 121 g (2 moles) of acetate produced and then calculating as indicated above. The carbon conversion efficiencies for various batches of transformed and untransformed host cells are shown in Table 2. As is evident in that Table, carbon conversion efficiency significantly increased in cells which had been transformed with glyoxylate cycle genes.

The results obtained in one fermentation run are used herein to demonstrate the calculation of carbon conversion efficiency according to the invention. Specifically, the results obtained in fermentation run 4 in Table 2, wherein E. coli cells had been transformed with plasmid pCLlOOO as described above. As noted in Table 2, the total phenylalanine (6.11 g) prephenic acid (15.55 g), and phenylpyruvic acid (1.81) was 23.47 g/L in a fermentation volume of 13.11 L. Additionally, 2.86 g/L acetate were produced and 2,844.18 grams of glucose were consumed during fermentation.

The above data are used to calculate carbon conversion efficiency, wherein the grams of glucose which were used to produce acetate in the fermentation are calculated as follows:

Hole* ΛcβtΛtβ prod, during terment-tlon - 13 * *^t ^" P¹^- _{x 60 D} "^J _e* _tΛtβ) * IβiBβπtβi vol. (L)

moles ac ^O . ^iβy prJueo^gej

Grams of glucose used to prod, acetate Λ ' etate prod, x IB

1 mole I Carbon conversion efficiency may then be calculated as follows using the above data:

_CE _ f [g/L (p e) * g/L(PPA) * g/UPA) ] x fermentation volume ( ) ^*j _{χ 10Q} [ g (glucose consumed) - g( lucose used to prod, acetate) j

To calculate carbon conversion efficiency from the foregoing exemplary data, 180.16 g (1 mole) of glucose is subtracted for every 121 g (2 moles) acetate produced to give the total amount of glucose actually consumed. Doing so reveals that 63.11 g of glucose were used to produce acetate, leaving 2,781.07 g of glucose consumed. Thus, carbon efficiency expressed as a percentage is 11.04, the total grams of phenylalanine, phenylpyruvic acid and prephenic acid divided by the total glucose consumed.

11.04 = ⁽6-1 + 15-55 + 1.81) x 13.11 ₁₀₀ 2844.18 - 63.11

Host cells which had been transformed with genes encoding enzymes of the glyoxylate cycle utilized carbon in a significantly more efficient manner as compared to untransformed controls. This effect is likely due to the fact that transformed cells utilized the glyoxylate cycle to bypass the CO₂-evolving steps of the TCA cycle, thus making available greater amounts of carbon for incorporation into biosynthetic products. The greater efficiency of carbon utilization further results in increases in the synthesis of those products.

While the present invention has been characterized in terms of a preferred embodiment thereof, it is readily apparent to the skilled artisan that increased carbon conversion efficiency may also be obtained through use of host cells and plasmids other than those presently disclosed. For example, a host cell or plasmid comprising the pheA gene reported in U.S. Patent No. 5,120,837 is expected to produce greater quantities of L-phenylalanine when such a host cell or plasmid is used in methods according to the present invention than would host cells of plasmids without the pheA gene. In addition, it is apparent to the skilled artisan that the use of specific promoters and other constituents of transcription and translation, such as, e.g. , a tac promoter [Russell, et al., Gene, 20:231-243 (1982)], may improve upon the carbon conversion efficiency, biosynthetic product yield, or biomass resulting from practice of the presently-claimed invention.

Mutations in the glyoxylate genes may also be used to obtain increases in carbon conversion efficiency. For example, a mutated aceK gene, which possesses primarily kinase activity, may be used in methods according to the invention. The foregoing parameters are known in the art and may be varied according to the use to which the presently-claimed methods are put.

While the present invention has been described in terms of its preferred embodiments, the skilled artisan realizes that numerous modifications may be made. For example, there are numerous microorganisms which may serve as host cells of the invention and numerous means of incorporating glyoxylate cycle genes into a host cell. Thus, the present invention should only be limited by the scope of the appended claims.

Claims

We claim:

1. A method for increasing the carbon conversion efficiency of a microorganism, comprising the steps of:

(a) incorporating in a microorganism, said microorganism having a functional glyoxylate operon, DNA encoding isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase;

(b) fermenting said microorganism in a suitable fermentation medium containing a carbon compound capable of entry into a tricarboxylic acid cycle; and (c) selecting one or more biosynthetic product(s) from said fermentation medium.

2. The method according to claim 1, wherein the DNA encoding isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase comprises a portion of the glyoxylate operon.

3. The method according to claim 1, wherein said microorganism is a bacterial cell.

4. The method according to claim 3, wherein said bacterial cell is an Escherichia coli cell.

5. The method of claim 1, wherein said incorporating step comprises transformation of said microorganism with a vector comprising said

DNA encoding isocitrate lyase, malate synthase, and isocitrate dehydrogenase kinase/phosphatase.

6. The method according to claim 1 , wherein said DNA encoding isocitrate dehydrogenase kinase/phosphatase is mutated to encode an isocitrate dehydrogenase kinase/phosphatase which possesses primarily kinase activity.

7. A method for producing an amino acid comprising the steps of: (a) incorporating in a microorganism, said microorganisms having a functional glyoxylate operon, DNA encoding isocitrate lyase, malate synthase, and isocitrate dehydrogenase;

(b) fermenting said microorganism in a suitable fermentation medium containing a carbon compound capable of entry into a tricarboxylic acid cycle; and

(c) isolating the amino acid from said fermentation medium.

8. The method according to claim 7, wherein said amino acid is selected from the group consisting of phenylalanine, tyrosine, and tryptophan.