FERMENTATION PROCESS FOR THE PRODUCTION OF

COENZYME QlO

FIELD

[0001] The present disclosure relates to fermentation processes and media involved in the production of coenzyme QlO (hereafter "CoQio"), and products produced by such processes. As used herein, CoQ ₁ O refers to 2,3 dimethoxy-5 methyl-6 decaprenyl benzoquinone, also known as ubidecarenone and ubiquinone.

BRIEF DESCRIPTION OF THE DRAWINGS

[0002] FIG. 1 is a graph plotting the concentration and growth rate of CoQ ₁ O produced by Rhodobacter sphaeroides cells in a continuous fermentation system. [0003] FIG. 2 is a graph illustrating the effect of agitation on the oxidation reduction potential (ORP) of the medium and the amount of CoQ ₁ O produced in the cells.

[0004] FIG. 3 is a graph showing the effect of glucose limitation on Rhodobacter sphaeroides δcrtE cell growth.

[0005] FIG. 4 is a graph showing the effect of glucose limitation on the concentration of CoQ ₁ O produced in Rhodobacter sphaeroides δcrtE. [0006] FIG. 5 A is a graph showing the effect of oxygen limitation on the concentration of CoQ _1O produced in Rhodobacter sphaeroides with a deletion crtE, ppsR and ccoN gene.

[0007] FIG. 5B is a graph showing the effect of oxygen limitation on Rhodobacter sphaeroides cell growth.

DETAILED DESCRIPTION

[0008] One aspect of the present disclosure relates to a process for producing CoQ ₁ O, which comprises culturing Rhodobacter sphaeroides in a defined medium. Non-limiting examples of Rhodobacter sphaeroides are described in U.S. Patent Application Publication No. 2003/0219798 and PCT International Publication No. WO 2004/047763, the entire contents of which are incorporated herein by reference.

In some embodiments of the present disclosure, the Rhodobacter sphaeroides is a strain that contains a non-functional crtE, ppsR or ccoN, or a non-functional crtE, ppsR and ccoN, or a wild type, or any combination. In other embodiments, the strain contains an exogenous nucleic acid that encodes at least one polypeptide encoding for dxs, dds, ods, sds, dxr, ubiC,4-diphophycytidyl-2C-methyl-D-erythritol synthase, 4- diphosphycytidyl-2C-methyl-D-erythritol kinase, or lytB.

Defined Medium

[0009] As used herein, the term "medium" refers to the aqueous environment in which cells are grown in culture. The medium comprises the physico chemical and nutritional requirements necessary for the survival and growth of the cells in culture. A medium can affect the yield and output of a product of interest such as CoQ ₁ O. A "defined medium" refers to a medium whose chemical composition is quantitatively known. A "defined medium" provides better reproducibility and standardization of fermentation than a complex medium that may contain unknown quantities of such ingredients as amino acids, ions, vitamins or other nutrients. A defined medium also minimizes the number of chemical entities that may reduce the efficiency of downstream purification of the product of interest. In some embodiments, the defined medium comprises Sistrom's medium and about 58 to about 175 mg/L magnesium ion and/or about 1.4 to about 11.2 mg/L iron ion. The components of Sistrom's medium are set forth below in Example 1. In other embodiments, the defined medium further comprises one or more of the following: about 0.6 to about 3.3 mg/L manganese ion, about 0.02 to about 0.1 mg/L copper ion, about 1.1 to about 3.4 mg/L zinc ion, about 0.9 to about 1.5 g/L phosphorous ion, about 0.5 to about 1.1 g/L sodium ion, and about 0.01 to about 0.02 g/L calcium ion. In other embodiments, the defined medium comprises sufficient concentrations of magnesium, iron, manganese, copper, zinc, phosphorus, sodium and/or calcium for growing Rhodobacter sphaeroides.

Temperature andpH

[0010] The process of the present disclosure may be carried out at a temperature of about 27°C to about 36°C, and/or at a pH of about 6.7 to about 7.5. In some embodiments, the temperature is about 30 ⁰ C, and/or the pH is about 7.0 to about 7.3.

Batch, Fed-Batch and/or Continuous Mode

[0011] The process may be performed in a batch mode, fed-batch mode or continuous mode. In some embodiments, the process is performed in a batch mode. In other embodiments, the process is performed in a fed-batch mode. In yet other embodiments, the process is performed in a continuous mode. Figure 1 illustrates that CoQ ₁ O can be produced in a continuous mode.

[0012] The oxygen concentration and oxygen utilization rate not only regulate the formation of the photosynthetic apparatus in Rhodobacter sphaeroides, it also affects the accumulation of CoQ _1O in the cells. While Rhodobacter sphaeroides grown aerobically typically accumulate about 3000 ppm CoQ ₁ O, much higher concentrations may be obtained when oxygen availability is restricted and the dissolved oxygen (DO) level is kept below the level detectable with DO probes, i.e. DO = 0%. The oxygen uptake rate (OUR) of the cells can be measured. The concentration of CoQlO in the cells can be modulated by changing the cells oxygen uptake rate (OUR). In some embodiments, the OUR is maintained at about 20 to about 120 mmoles/liter/hour. In other embodiments, the OUR is maintained at about 40 to about 100 mmoles/liter/hour. In yet other embodiments, the OUR is maintained at about 50 mmoles/liter/hour or less.

[0013] Dissolved oxygen in the medium is linked to the oxidation-reduction potential (ORP) of the medium. Hence, measures for controlling ORP may also affect DO levels and, ultimately, the growth rate and/or production of CoQ _1O . The present disclosure provides two different ways to control ORP. One involves the direct control of the oxygen supply, and the other involves the addition of antioxidants. [0014] Different oxygen supply rate result in different consumption rates by the organism and the balance between supply and consumption affect the redox potential of the fermentation broth. The oxygen supply can be varied by changes in aeration

and agitation of the broth. When dissolved oxygen levels are very low, it is not possible to measure them with regular DO probes, but by continuously monitoring the

ORP of the medium with appropriate on-line sensors, it is possible to develop automatic control strategies that link the ORP measurement to the aeration or agitation of the medium.

[0015] Oxygen supplies that result in ORP values below -180 mv enhance CoQ _1O production in the cells, while higher values increase biomass formation. By modifying the oxygen supply through agitation and imposing different ORP profiles, cell growth and CoQ concentration can be modulated.

[0016] The addition of one or more antioxidant(s) to the medium is effective in creating or maintaining a particular ORP value under low DO concentration.

Examples of useful antioxidants include, without limitation, non-toxic antioxidants such as L-cysteine, ascorbic acid, dithiothreitol, glutathione and thyoglycolic acid.

Automatic control strategies can also be set up linking the ORP measurement to the feed of the antioxidant, while keeping a constant oxygen supply.

[0017] Accordingly, in some embodiments, the process further comprises regulating CoQ _1O growth rate and/or production by controlling oxygen availability in the medium. The controlling step may comprise:

(i) measuring the DO concentration or the ORP of the medium;

(ii) adding one or more antioxidant(s) and/or varying aeration and/or agitation of the medium based on the DO or ORP measurement of step (i); and

(iii) measuring the oxygen uptake rate and maintaining it within a desired range.

In some embodiments, the oxygen uptake rate is maintained at about 20 to about 120 mmoles/liter/hour, at about 40 to about 100 mmoles/liter/hour, or at about 50 mmoles/liter/hour or less.

[0018] In other embodiments, the process may further comprise maintaining the

DO level of the medium at about 0% throughout the culturing step.

Effect of Carbon

[0019] The inventors have found that the concentration of CoQ _1O in Rhodobacter sphaeroides cells can be increased by as much as two to three-fold when cell growth

is controlled by a carbon source supply. This condition is met in a fermentation process set up as a fed-batch on the carbon source, or as a continuous culture with the carbon source as the limiting nutrient. With such process, CoQ production does not depend on the control of oxygen supply. Indeed, the process may be run under fully aerobic conditions, where the DO level of the medium is maintained above about 20% of saturation throughout the culturing step.

[0020] Accordingly, in some embodiments the process is performed in a fed batch mode or in a continuous mode and the process further comprises maintaining the DO level of the medium above about 20% of saturation throughout the culturing step, and regulating growth rate and/or CoQ _1O production by controlling a carbon source supply to the medium. Non-limiting examples of a carbon source include glucose, other hexoses, carboxylic acids and alcohols.

[0021] In other embodiments, the process is performed in a fed batch mode and the carbon source is added to the medium at an appropriate feed rate. The optimum rate depends on the medium composition and it can be modulated to increase process productivity. In yet other embodiments, the carbon source is a water solution with a concentration of about 25 % to about 60 % w/w.

[0022] In further embodiments, the process is performed in a continuous mode wherein both carbon source concentration and feed rate can be optimized to improve process productivity. In yet further embodiments, the carbon source is added to the medium at a dilution rate of about 0.08 to about 0.12 h ^"1 .

Effect of Magnesium

[0023] A study of a synthetic basic medium for Rhodobacter sphaeroides (Sistrom's medium described in Example 1) showed that by increasing the concentration of magnesium, it was possible to increase the cell concentration in the fermentation broth by several fold. Thus, in some embodiments the process further comprises adding magnesium ion at a concentration of about 58 to about 175 mg/L to the medium. In other embodiments, the process further comprises adding MgSO ₄ -VH ₂ O to the medium at a concentration of about 1.2 g/1.

Effect of Iron

[0024] Growth of Rhodobacter sphaeroides to high cell density in magnesium- fortified media produced cells with lower CoQ ₁ O concentration than Rhodobacter sphaeroides grown to a lower cell density by limitation of the oxygen supply. This effect may be overcome by supplying the media with high concentrations of iron, which was found to affect CoQ _1O accumulation. When cells are grown to about 30 g/1 DCW, iron ion at a concentration of about 1.4 to about 11.2 mg/L may be added to the medium to avoid a limitation in CoQ production. Thus, optimizing iron concentration and the timing of its supply to the medium may enhance CoQ _1O production. In some embodiments the process further comprises adding iron ion at a concentration of about 1.4 to about 11.2 mg/L to the medium. In other embodiments, the process further comprises adding iron ion at a concentration of about 1.4 to about 4.5 mg/L to the medium.

Effect of Other Minerals

[0025] The concentration of other minerals in the medium may also affect CoQ ₁ O production. Accordingly, in some embodiments the process further comprises regulating CoQ _1O production by varying magnesium, iron, phosphorus, copper, manganese, molybdenum, zinc, sodium, calcium and/or nickel concentration(s) in the medium. In other embodiments, the process further comprises regulating CoQ ₁ O production by varying magnesium, iron and/or manganese concentration(s) in the medium. In yet other embodiments, the process further comprises adding about 58 mg/L to about 175 mg/L Mg ion to the medium. In yet other embodiments, the process further comprises adding to the medium about 1.2 g/L MgSO ₄ ^' 7H ₂ O, about 1.4 to about 11.2 mg/L Fe, and/or about 0.01-0.02 g/L CaCl ₂ ^' 2H ₂ O and/or about 0.6- 3.3 mg/L manganese ion

[0026] Another aspect of the present disclosure relates to a process for producing CoQ ₁ O, which comprises:

(i) culturing Rhodobacter sphaeroides in a medium; and (ii) adding to the medium a magnesium source, an iron source and/or a manganese source at sufficient concentration(s) to increase CoQ ₁ O production relative

to a control process wherein a suboptimal concentration of a magnesium source, iron source and/or manganese source is added. Except for the noted differences, a "control process" is identical to the process of the present disclosure. In some embodiments, the medium is a defined medium as described above.

[0027] Another aspect of the present disclosure relates to a product produced by a process of the present disclosure. In some embodiments, the process comprises culturing Rhodobacter sphaeroides in a defined medium. In other embodiments, the process comprises:

(i) culturing Rhodobacter sphaeroides in a medium; and

(ii) adding to the medium a magnesium source, an iron source and/or a manganese source at sufficient concentration(s) to increase CoQ ₁ O production relative to a control process wherein suboptimal concentration(s) of a magnesium source, iron source and/or manganese source is added. The control process is identical to the process of the present disclosure except for the magnesium source, iron source and/or manganese source concentration(s). In other embodiments, the medium is a defined medium as described above.

[0028] Another aspect of the present disclosure relates to a process for producing coenzyme Q _1O , which comprises:

(i) culturing Rhodobacter sphaeroides in a medium; and

(ii) controlling glucose supply to the medium.

[0029] In some embodiments, the controlling step comprises performing the fermentation in a continuous mode by adding a glucose feed at a dilution rate of about 0.08 to about 0.12 h ^"1 . In other embodiments, the medium is a defined medium as described above.

[0030] Another aspect of the present disclosure relates to a process for producing coenzyme Q _1O , which comprises:

(i) culturing Rhodobacter sphaeroides in a medium; and

(ii) heat treating the Rhodobacter sphaeroides.

[0031] In some embodiments, the heat treating step is carried out at a temperature of about 5O ⁰ C to about 9O ⁰ C. In other embodiments, the heat treating step is carried

out at a temperature of about 5O ⁰ C to about 9O ⁰ C for about one to two hours. In other embodiments, the medium is a defined medium as described above. [0032] Another aspect of the present disclosure relates to a process for producing coenzyme Q ₁ O, which comprises:

(i) culturing Rhodobacter sphaeroides in a medium; and (ii) measuring and maintaining the oxygen uptake rate of the Rhodobacter sphaeroides at about 20 to about 120 mmoles/liter/hour. In some embodiments, the oxygen uptake rate is maintained at about 40 to about 100 mmoles/liter/hour. In other embodiments, the oxygen uptake rate is maintained at about 50 mmoles/liter/hour or less. In yet other embodiments, the medium is a defined medium as described above. [0033] Yet another aspect of the present disclosure relates to a product prepared by any of the processes described above.

[0034] It will be apparent to one of ordinary skill in the art that specific embodiments of the present disclosure may be directed to one, some or all of the above-indicated aspects as well as other aspects, and may encompass one, some or all of the above- and below- indicated embodiments, as well as other embodiments. [0035] Other than in the working examples, or where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified by the term "about". Accordingly, unless indicated to the contrary, such numbers are approximations that may vary depending upon the-desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding techniques.

[0036] While the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the working examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. As used herein, "comprising" means

"including" and the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise.

[0037] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES

Example 1 : Production of CoQ ₁ O by Rhodobacter sphaeroides in a Defined Medium [0038] A frozen stock vial of Rhodobacter sphaeroides 35053 with a deletion in the crtE gene was inoculated into 300 ml Sistrom's medium with 10 g/1 glucose and supplemented with 5 g/1 yeast extract. The flask was incubated at 30°C at 250 rpm for 20-24 hours. The culture was transferred into a fermentor with 3 liters of Sistrom's medium with 40 g/1 glucose and 5 g/1 yeast extract. Sistrom's medium contained in g/L the following components:

KH ₂ PO ₄ 2.72

(NH ₄ ) ₂ SO ₄ 0.5

NaCl 0.5

MgSO ₄ -7H ₂ O 0.3

CaCl ₂ -2H ₂ O 0.0334

EDTA 0.2

ZnSO ₄ -7H ₂ O 0.01095

FeSO ₄ -7H ₂ O 0.007

MnSO ₄ -H ₂ O 0.00154

CuSO ₄ -5H ₂ O 0.000392

Co(NO ₃ ) ₂ -6H ₂ O 0.000284

H ₃ BO ₃ 0.000114

(NH ₄ ) ₆ Mo ₇ O ₂₄ -4H ₂ O 0.0002

Nicotinic acid 0.075

Thiamine.HCl 0.0375

Biotin 0.000075

[0039] The pH was controlled at 7.3 and the DO at 40%. 150 ml was removed from this fermentor and used to inoculate a 3 liter fermentor that contained Sistrom's medium with 4% glucose. At the beginning of the fermentation, aeration was

maintained at 1 wm and agitation at 480 rpm. At 24 hours, the aeration was reduced to 0.3 wm and agitation to 240 rpm so that all the dissolved oxygen would be consumed and the probe read zero. At 68 hours, the fermentation was stopped and the dry cell weight was measured at 5.4 g/1 and the CoQ ₁ O concentration in the cells was 6,572 ppm.

Example 2: Production of CoQ ₁ O by Rhodobacter sphaeroides in a Continuous Fermentation System

[0040] Rhodobacter sphaeroides ATCC 35053 with a deletion in the crtE gene was grown in a continuous fermentation system with pH controlled at 7.3 with 2N NH ₄ OH. The temperature was maintained at 3O ⁰ C, dissolved oxygen at 40% and aeration at 0.3 wm. The dilution rates were adjusted from 0.08 to 0.12 hr/1. The medium used was as described in Example 1, but with 0.6 g/1 MgSO ₄ -7H ₂ O and 10 g/1 glucose.. The Rhodobacter sphaeroides strain produced CoQ _1O in a continuous fermentation system, as shown in Figure 1. The CoQlO concentration in the cells was higher at the lower dilution rates applied.

Example 3 : Production of CoQ _1O by Rhodobacter sphaeroides in a Fed Batch Fermentation System

[0041] Rhodobacter sphaeroides ATCC 35053 with a deletion in the crtE gene was grown as in Example 1, except that the medium contained 1.2 g/1 MgSO ₄ -7H ₂ O, 0.5 g/1 CaCl ₂ -2H ₂ O and 5 g/1 YE. Starting glucose was 40 g/1. The fermentation was carried out at various agitation speeds to modify the oxygen supply rate. This affected the growth of the cells and the glucose consumption, so glucose was added to the fermentation as needed to avoid a limitation. The amount of glucose fed at a particular time varied from 5 g/1 to 20 g/1. The biomass and CoQ _1O concentrations are shown in Table 1. This example demonstrates that CoQ ₁ O can be produced under a variety of glucose feeding conditions.

TABLE 1 Fed Batch Fermentation Results

Example 4: Effect of Magnesium (Mg) on Biomass and CoQ ₁ O Production [0042] Previous reports have shown the use of a complex medium supplemented with corn steep liquor (CSL) and 5 g/L Mg ₃ (PO ₄ ) ₂ to be beneficial for growth and CoQ ₁ O production in some strains of Rhodobacter spheroides (Sakato et.al, Biotechnol. Appl. Biochem., 16, 19-28, 1992). The effect of this medium on Rhodobacter sphaeroides 35053 was tested and compared with Sistrom's medium as described in Example 1 , but supplemented with additional Mg salt. The fermentation was run as described in Example 1 , but the limitation in oxygen supply was imposed earlier in the run. The results obtained indicate that CSL was not the preferred source of nutrients, and that Mg was the key ingredient to increase the biomass concentration in the fermentor, and that higher biomass was obtained with the addition of a soluble salt of Mg, than when CSL was used. Supplementation with 40 g/L of CSL produced similar results to those obtained at 20 g/L. A comparison of biomass and CoQ ₁ O concentrations is shown in Table 2.

TABLE 2

Example 5 : Effect of Temperature on Biomass and CoQ _1O Production [0043] Microorganisms typically grow and make products at an optimal temperature. The effect of temperature on growth and the production of CoQlO in different mutants of Rhodobacter spheroides was measured. The results obtained for the strain with a crtE deletion are shown in Table 3. The fermentation was run as in Example 1 but with medium supplemented with 5 g/L yeast extract. Growth and CoQ production were very similar within the temperature range of 27°C to 36°C.

TABLE 3

[0044] Using Rhodobacter sphaeroides ATCC 35053 with deletions in the crtE, ppsR, and ccoN gene, biomass growth and CoQ _1O production occurred at both 30 and 36°C, with the higher temperature being more favorable, as seen in Table 4.

TABLE 4

Example 6: Effect of Magnesium (Mg) concentration on Rhodobacter Growth and CoQ _1O Accumulation.

[0045] The effect of Mg concentration on Rhodobacter growth and CoQ ₁ O accumulation was tested using the Sistrom's medium from Example 1. This medium contained 29 mg/L of Mg (ion). Mg supplementation was tested in flasks at a range of 29 to 200 mg/L, and further tested in fermentors at between 58 and 175 mg/L. The effect was tested at different levels of oxygen supply to determine the impact of Mg on biomass growth and CoQ ₁ O accumulation. To achieve low O ₂ supply, aeration was set at 0.3 wm and agitation was fixed at 375 rpm. To achieve high O ₂ supply,

aeration was set at 0.3 wm and agitation was fixed at 425 rpm. The results are shown in Table 5.

TABLE 5

[0046] The results indicate that Mg is a key medium component for Rhodobacter growth; while growth is limited by the oxygen supply, increasing the concentration of Mg has no impact. However, when the oxygen supply is increased, Mg quickly becomes limiting and increasing its concentration results in biomass accumulation to higher cell densities.

Example 7: Effect of Iron (Fe) on Rhodobacter Growth and CoQ ₁ O Accumulation [0047] The results presented in Example 6 show that while increasing Mg concentrations was key to increasing biomass in the fermentor, it did not improve specific CoQ _1O production, which actually went down at higher biomass concentrations. Further tests revealed that the availability of Fe was critical for the accumulation of CoQ ₁ O. The effect of Fe concentrations between 1.4 and 4.5 mg/L are shown in Table 6.

TABLE 6 Effect of Fe Concentrations on R. Sphaeroides ATCC 35053 δcrtEδppsRδccoN

[0048] It was also demonstrated that when higher cell densities were obtained, it was necessary to increase the iron concentration accordingly to maintain the specific CoQ ₁ O production. Iron concentrations up to 20 mg/L have been used in other experiments, with positive results.

Example 8: Effect of Oxygen Availability and Supply Rate on Biomass and CoQ _1O Production

[0049] While it was known that low oxygen levels increased the formation of photosynthetic apparatus in Rhodobacter, the effects on CoQ _1O production were not known. Oxygen generally supports the production of more biomass in organisms grown aerobically than in organisms grown anaerobically or with limited oxygen. As shown in Table 7, in high oxygen conditions, production of Rhodobacter cells was favored but CoQ _1O production was greatly reduced. When oxygen was limited by maintaining agitation at 350 rpm, CoQ _1O production increased while biomass production decreased.

TABLE 7 Effect of Oxygen Supply on Biomass and CoQio Production in R. Sphaeroides

[0050] Further studies were conducted to examine the effect of oxygen supply rate on the accumulation of CoQ _1O in the biomass. At oxygen supply rates below 30 mmoles/L/h, biomass growth was limited below the optimum supported by the

medium, reaching between 20 and 25 g/L, with CoQ _1O accumulation of between 16000 and 17000 ppm. In studies where the oxygen was supplied at rates that allowed consumption of up to 70 mmols/L/h but the culture was still oxygen limited, or in aerobic cultures, higher biomass was achieved although the concentration of CoQ _1O in the cells decreased accordingly. Further mineral supplementation did not overcome this effect. These results are shown in Table 8.

TABLE 8 Use of Oxygen Uptake Rate to Control Production of CoQio

Example 9: Effect of glucose limitation

[0051] The effect of glucose was tested using a Rhodobacter spheroides crtE ^~ strain, in fully aerobic fermentations (DO maintained above 20%), with the medium described in Example 1 (Sistrom's medium) under conditions of glucose excess (high initial glucose) and glucose limitation (glucose fed continuously while concentration in the fermentor was maintained below 0.05 g/L). The same medium supplemented with 1.2 g/L MgSO ₄ 7H ₂ O, 0.067 g/1 CaCl ₂ 2H ₂ O, and 0.035 g/1 FeSO ₄ 7H ₂ O was also tested under conditions of glucose limitation. Figures 3 and 4 show the results obtained with respect to cell growth and CoQlO accumulation. Using a basic synthetic medium (Sistrom's medium) under glucose excess, about 10 g/1 dry biomass was produced with 2400 ppm CoQ. When glucose was fed as the limiting substrate in the same medium, 7 g/1 cells were obtained but the CoQ concentration increased up to 7300 ppm. When the medium was optimized for cell growth (Sistrom's medium fortified with Mg, Ca and Fe), 24 g/1 biomass were produced under glucose limitation and the CoQlO concentration in the cells was still above 6900 ppm.

Example 10: CoQlO Fermentation

[0052] Rhodobacter sphaeroides with a deletion in the crtE, ppsR, and ccoN gene was taken from a frozen stock vial and was propagated first in 300 ml and then at 3 liters as described in Example 1. The 3 liter fermentation was then inoculated into another 3 liter fermentor that contained the medium described in example 1 except that it contained 80 g/1 glucose, 1.2 g/1 MgSO ₄ VH ₂ O, 0.067 g/1 CaCl ₂ 2H ₂ O, and 0.035 g/1 FeSO ₄ VH ₂ O. The aeration was set at 0.5 lpm and the agitation was fixed at 800 rpm in order to obtain oxygen limitation. The fermentation temperature was maintained at 36°C and pH was controlled at 7.3 by the addition of 2N NH ₄ OH. The oxygen uptake rate was maintained between 80 and 110 mmoles/liter/hour and an additional 80 g/1 glucose was added at 24 hours after the start of fermentation. Samples were withdrawn periodically and optical density and CoQlO were measured and the results are shown in Figure 5.

Example 11 : Effect of Other Nutrients on Biomass and CoQ _1O Production [0053] The medium composition was examined with respect to several nutrients. Using the protocol described in Example 10, the effect of the concentrations of Mn, Cu, Zn, P, Na and Ca were studied. The minerals were studied in two groups: one tested Mn, Cu and Zn, and the other P, Na and Ca. Eight fermentations were set for each group. The concentration of each of the three minerals was varied according to a full factorial experiment. These concentrations covered a range around those in the basic medium described in Example 1. The ranges determined for each mineral were:

Mn: 0.6 - 3.3 mg/L

Cu: 0.02 - 0.1 mg/L

Zn: 1.1 - 3.4 mg/L

P : 0.9 - 1.5 g/L

Na: 0.2 - 0.5 g/L

Ca: 0.01 - 0.02 g/L

[0054] Different nutrient combinations proved to have different effects on the concentration of biomass and the accumulation of CoQ _1O . The concentration of CoQ _1O in the cells was most significantly affected by the concentration of manganese (Mn), sodium (Na) and calcium (Ca) in the medium. The availability of phosphorus

(P) affected the biomass concentration, while zinc (Zn) and copper (Cu) concentrations had only minor impact and their effect depended on the concentrations of the other minerals.

Example 12: Effect of Heat Treatment on CoQ _1O Concentration [0055] The CoQ _1O is entrained as part of the biomass and downstream separation technology is generally used to purify the material. This has effects on the economics of the process. The inventors have found that one way to improve the recovery of CoQ _1O from cell biomass is to heat treat the cells. A fermentation of R. sphaeroides ATCC 35053 with a deletion in the crtE, ccoN and ppsR genes was conducted and the cells were harvested. CoQ ₁ O concentrations were measured before the cells were heat treated and after heat treatment at 50 ⁰ C, 70 ⁰ C and 90 ⁰ C for one to two hours. As seen in Table 9, the heat treatment improved the recovery of CoQ _1O .

TABLE 9