阅读说明：本技术 控制发酵进料速率的方法 (Method for controlling fermentation feed rate ) 是由 J·冷 于 2019-07-11 设计创作，主要内容包括：本发明涉及用于提高发酵的生产率或产量的方法和组合物。所述方法和组合物提供对培养细胞生成的一种或多种化合物进行监测,作为发酵进料速率的反馈控制。(The present invention relates to methods and compositions for increasing the productivity or yield of a fermentation. The methods and compositions provide for monitoring the production of one or more compounds by cultured cells as feedback control of fermentation feed rate.)

1. A method of providing sugars to a microbial culture comprising the steps of:

a. feeding sugar at an initial rate to a microbial culture grown in a fermentation medium;

b. determining the concentration of ethanol in the fermentation medium exhaust;

c. as the ethanol concentration in the off-gas increases, the sugar feed rate decreases; and

d. as the ethanol concentration in the off-gas decreases, the sugar feed rate increases.

2. The process of claim 1, wherein sugar is continuously fed to the fermentation medium.

3. A method according to claim 1 or 2, wherein the concentration of ethanol in the exhaust gas is determined continuously.

4. The method of any one of the preceding claims, wherein the feed rate of the sugar is maintained within ± 25% of the initial feed rate.

5. The method of any one of the preceding claims, wherein the feed rate of the sugar is maintained within ± 15% of the initial feed rate.

6. The method of any one of the preceding claims, wherein the sugar feed rate is maintained within ± 10% of the initial feed rate.

7. The process according to any one of the preceding claims, wherein the feed rate is from 1 to 25 g/L/hr.

8. The method of any one of the preceding claims, wherein the oxygen uptake rate of the microbial culture is maintained.

9. A method according to any one of the preceding claims, wherein the oxygen uptake rate of the microbial culture is maintained by agitation.

10. A process according to any one of the preceding claims, wherein the oxygen uptake rate of the microbial culture is from 1 to 150mmol O₂/L/hr。

11. A method according to any one of the preceding claims, wherein the ethanol concentration in the off-gas is 50-750 ppm.

12. The method according to any of the preceding claims, wherein the ethanol concentration in the off-gas is 100-200 ppm.

13. The method according to any of the preceding claims, wherein the ethanol concentration in the off-gas is 200-300 ppm.

14. The method according to any one of the preceding claims, wherein the ethanol concentration in the off-gas is 250-350 ppm.

15. The method according to any of the preceding claims, wherein the ethanol concentration in the off-gas is 550-650 ppm.

16. The method of any one of the preceding claims, which is carried out for 1-10, 2-9, 3-7, or 4-6 days.

17. The method according to any of the preceding claims, wherein the microbial culture is prokaryotic.

18. The method of any one of the preceding claims, wherein the microbial culture is eukaryotic.

19. The method of any one of the preceding claims, wherein the microbial culture is yeast.

20. The method according to any one of the preceding claims, wherein the microbial culture is saccharomyces cerevisiae (s.

21. The method of any one of the preceding claims, wherein the microbial culture is recombinant.

22. A process according to any one of the preceding claims, wherein the microbial culture produces a water-immiscible compound.

23. The method of any one of the preceding claims, wherein the microbial culture produces an isoprenoid, a polyketide, or a fatty acid.

24. The method of any of the preceding claims, wherein the microbial culture produces sesquiterpenes.

1. Field of the invention

The present invention relates to the use of off-gas monitoring to control or regulate the feed rate of a fermentation medium and an apparatus for the fermentation medium.

2. Background of the invention

The advent of synthetic biology has created a desire to ferment microorganisms to produce biofuels, chemicals and biomaterials from renewable resources on an industrial scale and quality. For example, functional non-natural biological pathways have been successfully constructed in microbial hosts for the production of precursor classes of the antimalarial drug artemisinin (see, e.g., Martin et al, Nat Biotechnol 21: 796-; fatty acid derived fuels and chemicals (e.g., fatty esters, fatty alcohols, and waxes; see, e.g., Steen et al, Nature 463: 559-562 (2010)); polyketide enzymes for the preparation of cholesterol lowering drugs (see, e.g., Ma et al, Science 326: 589-592 (2009)); and polyketides (see, e.g., Kodumal, Proc Natl Acad Sci USA 101: 15573-15578 (2004)). However, the commercial success of synthetic biology will depend in large part on whether the production costs of renewable end products can compete with or outperform the production costs of their corresponding non-renewable products.

Some of the greatest costs of synthetic biology occur in fermentation processes. Most of these costs are applied to the ingredients of the fermentation medium, including carbon sources, nitrogen sources, water, salts, and nutrients. In conventional fermentation, pulsed sugar is fed to the culture until a peak in dissolved oxygen is detected, indicating that excess carbon is consumed. Then, a new pulse of sugar is fed to the culture to start a new cycle. These cycles of pulse feeding and detection of dissolved oxygen peaks are repeated throughout the fermentation process, resulting in inefficient consumption of feed and labor inefficiencies for the practitioner. Methods and compositions that increase fermentation yields will reduce overall costs, thereby making the production of renewable compounds more efficient and competitive.

3. Summary of the invention

The present invention provides a process for providing a feed to a microbial culture in a fermentation medium. In some embodiments, the microbial culture fed into the fermentation medium is provided at an initial rate. Determining the concentration of volatile cell products in the off-gas of the fermentation medium. In some embodiments, the feed rate decreases as the concentration of the volatile cell product increases; and when the concentration of the volatile cell product decreases, the feed rate increases. In some embodiments, said feed rate is decreased when said concentration of said volatile cell product is decreased; and said feed rate is increased when said concentration of said volatile cell product is increased. Advantageously, in certain embodiments, the volatile cell product can be rapidly or continuously measured, and the feed rate can be rapidly or continuously adjusted. The process steps described may be carried out using techniques and components apparent to those skilled in the art. Specific techniques and components are described in detail herein.

As described in detail below, the methods and compositions provided herein can increase the productivity of microbial strains by as much as 15% or more. A 15% increase in productivity directly improves the cost and efficiency of such fermentations.

4. Brief description of the drawings

FIG. 1 provides feed rate (g/L/hr), ethanol (ppm), temperature (. degree. C.), pH, volume (L), oxygen uptake rate (mM/L-min), agitation rate (rpm), pO₂(saturation%), air flow (slpm) and average pO₂(degree of saturation%) history.

FIG. 2 provides productivity (g/L/hr) as a function of average off-gas ethanol concentration (ppm) for two different strains.

FIG. 3 provides the concentration of ethanol off-gas for fermentation of yeast strains according to the methods provided herein.

5. Detailed description of the preferred embodiments

5.1 definition

The term "genetically modified" as used herein refers to a host cell comprising a heterologous nucleotide sequence.

The term "heterologous/heterologous" as used herein refers to a substance that is not normally found in nature. The term "heterologous nucleotide sequence" refers to a nucleotide sequence that is not normally found in a given cell in nature. Thus, the heterologous nucleotide sequence may be: (a) is foreign (i.e., is "exogenous" to the cell) to its host cell; (b) naturally occurring in the host cell (i.e., "endogenous/endogenous"), but present in the cell in a non-native amount (i.e., more or less than the amount naturally occurring in the host cell); or (c) occurs naturally in the host cell, but is located outside its native locus. The term "heterologous enzyme" refers to an enzyme that is not normally found in a given cell in nature. The term includes the following enzymes: (a) is exogenous to a given cell (i.e., is encoded by a nucleotide sequence that does not naturally occur in the host cell or does not naturally occur in the given environment of the host cell); and (b) occurs naturally in the host cell (e.g., the enzyme is encoded by a nucleotide sequence that is endogenous to the cell), but is produced in a non-natural amount in the host cell (e.g., greater than or less than the naturally occurring amount).

In another aspect, the term "native" or "endogenous" refers to molecules, in particular enzymes and nucleic acids, which are expressed in the organism from which they originate or are found in nature, irrespective of the expression level, which may be lower, equal or higher than the expression level of the molecule in the native microorganism. It is understood that the expression of a native enzyme or native polynucleotide may be modified in a recombinant microorganism.

The term "production amount" as used herein generally refers to the amount of a compound produced by a host cell provided herein. In some embodiments, production is expressed as the yield of the compound produced by the host cell. In other embodiments, the amount produced is expressed as the productivity of the host cell when the compound is produced.

The term "productivity/productivity" as used herein refers to the amount of compound produced by a host cell, expressed as the amount of compound produced per unit amount of fermentation broth or fermentation medium in which the host cell is cultured (by volume) according to time (per hour).

The term "yield" as used herein refers to the amount of a compound produced by a host cell, expressed as the amount of the compound produced per unit amount of carbon source consumed by the host cell, by weight.

5.2 detailed description of the invention

In one aspect, the invention provides a method for providing a feed compound to a microbial culture. In certain embodiments, the method comprises the steps of: feeding a feed compound at an initial rate to a culture of microorganisms growing in a fermentation medium; determining the concentration of volatile cell products in the fermentation medium off-gas; and adjusting the feed rate of the feed compound to the fermentation medium. In certain embodiments, the feed rate decreases as the concentration of the volatile cell product increases; and when the concentration of the volatile cell product decreases, the feed rate increases. In some embodiments, said feed rate is decreased when said concentration of said volatile cell product is decreased; and the feed rate increases as the concentration of the volatile cell product increases. Advantageously, in certain embodiments, the volatile cell product can be rapidly or continuously measured, and the feed rate can be rapidly or continuously adjusted. In certain embodiments, the volatile cell product is continuously measured and the feed rate is continuously adjusted.

The feed compound may be any compound that the practitioner deems useful for feeding the fermentation medium. In certain embodiments, the feed compound is selected from the group consisting of carbon sources, nitrogen sources, salts, nutrients, and combinations thereof. In particular embodiments, the feed compound is a carbon source. In certain embodiments, the feed compound is provided from a source selected from the group consisting of molasses, corn steep liquor, cane juice, and beet juice. In certain embodiments, the feed compound is a sugar. In some embodiments, the feed compound is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or one or more combinations thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, xylose, ribose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof. Non-limiting examples of suitable non-fermentable carbon sources include acetate and glycerol.

The volatile cell product can be any off-gassing compound produced by a microbial culture deemed suitable by the practitioner. In a particular embodiment, the volatile cell product is a compound indicative of the metabolic state of the cell in which it is produced. In certain embodiments, ethanol is measured in the exhaust gas. In certain embodiments, the feed rate decreases as the ethanol concentration in the off-gas increases, and the feed rate increases as the ethanol concentration in the off-gas decreases. In certain embodiments, ethanol is measured in the off-gas, and the feed compound is a sugar. In certain embodiments, the feed rate of the sugars decreases as the ethanol concentration in the off-gas increases, and the feed rate of the sugars increases as the ethanol concentration in the off-gas decreases.

The method provided by the invention takes advantage of the following observations: ethanol in the fermentation may be a convenient marker for the consumption of cell feed in the microbial culture. Advantageously, the ethanol in the off-gas can be routinely determined by standard techniques and compositions. In accordance with the findings provided by the present invention, by adjusting the feed rate to maintain a constant or near constant concentration of ethanol in the off-gas, a microbial culture can be grown at a better rate than standard techniques. This can improve efficiency, yield and productivity. As shown in the examples below, the yield is quantitatively improved by the method provided by the present invention.

In certain embodiments, the feed rate is maintained within the range of the initial feed rate. The range may be any range deemed suitable by the person skilled in the art. In certain embodiments, the feed rate is maintained within ± 75%, ± 50%, ± 25%, ± 15%, ± 10%, or ± 5% of the initial feed rate. The feed rate can be adjusted according to standard techniques. In certain embodiments, the feed rate is maintained within the range of 0.01 to 25 g/L/hr. In certain embodiments, the feed rate is maintained within the range of 0.1 to 25 g/L/hr. In certain embodiments, the feed rate is maintained within the range of 1 to 25 g/L/hr. The feed rate is usually expressed as the mass of feed compound per liter of fermentation medium per unit time. In typical embodiments, the flow of feed solution into the fermentation medium is increased or decreased to increase or decrease the feed rate.

In certain embodiments, the oxygen uptake rate of the microbial culture is maintained within the range of the initial oxygen uptake rate. The range may be any range deemed suitable by the person skilled in the art. In certain embodiments, the oxygen uptake rate remains within ± 75%, ± 50%, ± 25%, ± 15%, ± 10%, or ± 5% of the initial oxygen uptake rate. In certain embodiments, the oxygen uptake rate of the microbial culture is maintained between 1 and 150mmolO₂within/L/hr. In certain embodiments, the oxygen uptake rate of the microbial culture is maintained between 10 and 150mmol O₂within/L/hr. In certain embodiments, the oxygen uptake rate of the microbial culture is maintained between 25 and 150mmolO₂within/L/hr. The oxygen uptake rate can be maintained by standard techniques, such as bubbling and/or stirring.

In certain embodiments, the volatile cell products in the waste gas are maintained within a range. The range may be any range deemed suitable by the person skilled in the art. In certain embodiments, the volatile cell product is maintained within ± 75%, ± 50%, ± 25%, ± 15%, ± 10%, or ± 5% of the target amount. In certain embodiments, the volatile cell product is ethanol, and the ethanol in the exhaust gas is maintained at about 600 ppm. In certain embodiments, the ethanol in the off-gas is maintained at 50 to 750 ppm. In certain embodiments, the ethanol in the exhaust gas is maintained at 100-200 ppm. In certain embodiments, the ethanol in the exhaust gas is maintained at 200-300 ppm. In certain embodiments, the ethanol in the exhaust gas is maintained at 250-350 ppm. In certain embodiments, the ethanol in the off-gas is maintained at 550-650 ppm. In certain embodiments, the target ethanol concentration in the off-gas is selected from 100ppm, 150ppm, 200ppm, 250ppm, 300ppm, 350ppm, 400ppm, 450ppm, 500ppm, 550ppm, 600ppm, 650ppm, 700ppm, and 750 ppm.

The amount of the volatile cell product should be maintained according to the techniques described herein. In other words, the feed rate of the fermentation should be adjusted when the amount of volatile cell products deviates from the target value. In certain embodiments, the feed rate should be decreased when the amount of volatile cell product is increased, and the feed rate should be increased when the amount of volatile cell product is decreased. In other embodiments, the feed rate should be increased when the amount of volatile cell product is increased, and the feed rate should be decreased when the amount of volatile cell product is decreased. In a particular embodiment, the volatile cell product is ethanol. In these embodiments, the feed rate should be decreased when the amount of ethanol in the off-gas is increased, and the feed rate should be increased when the amount of ethanol in the off-gas is decreased.

The exhaust gas may be measured at any frequency deemed suitable by the practitioner. In an advantageous embodiment, the exhaust gas is measured at high frequency. In a particular embodiment, the off-gas is measured continuously. In certain embodiments, the off-gas is measured at least once per hour, at least once every half hour, at least once every quarter hour, at least once every 10 minutes, at least once every 5 minutes, at least once per minute, at least twice per minute, at least three times per minute, at least four times per minute, or at least ten times per minute. In a preferred embodiment, the off-gas is measured continuously.

The feed rate may be adjusted at any frequency deemed appropriate by the practitioner. In an advantageous embodiment, the feed rate is adjusted at a high frequency. In certain embodiments, the feed rate is continuously adjusted. Typically, the feed rate is adjusted after the volatile cell products in the exhaust gas are measured. In certain embodiments, the feed rate is adjusted at least once per hour, at least once every half hour, at least once every quarter hour, at least once every 10 minutes, at least once every 5 minutes, at least once per minute, at least twice per minute, at least three times per minute, at least four times per minute, or at least ten times per minute. In a preferred embodiment, the feed rate is continuously adjusted.

The present invention provides methods for fermentation of microbial cultures, such as microbial cultures that produce a compound of interest, that provide improved productivity and yield. In certain embodiments, the productivity is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In certain embodiments, the yield is increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In certain embodiments, productivity and yield are improved. In these embodiments, the productivity or yield is increased relative to the same cell line grown under conventional conditions, i.e., without the method of the invention.

The method of the invention is circulated for several times in the whole fermentation process. In some embodiments, the methods of the invention are performed for a period of 3 to 20 days. In some embodiments, the methods of the invention are performed for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days, or more than 20 days.

5.3 cell cultures, media and conditions

Materials and methods for the maintenance and growth of microbial cultures are well known to those skilled in the art of microbiology or fermentation science (see, e.g., Bailey et al, Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986). Depending on the specific requirements of the cell culture, fermentation and process/method, the requirements of the appropriate medium, pH, temperature, and aerobic, microaerobic or anaerobic conditions must be considered.

The processes provided herein can be carried out in a suitable medium (e.g., with or without pantothenate supplementation) in a suitable vessel, including but not limited to a cell culture plate, flask, or fermentor. Furthermore, the process may be carried out on any fermentation scale known in the art to support industrial production of microbial products. Any suitable fermentor may be used, including stirred tank fermentors, airlift fermentors, bubble fermentors, or any combination thereof. In a particular embodiment using Saccharomyces cerevisiae as host cell, the strain may be grown in a fermentor, as described in detail in Kosaric, et al, Ullmann's Encyclopedia of Industrial Chemistry, six Edition, Volume 12, pages 398-.

In some embodiments, the medium is any medium in which the cell culture is viable, i.e., maintains growth and viability. In some embodiments, the medium is an aqueous medium comprising an assimilable carbon source, nitrogen source, and phosphorus source (phosphate sources). Such media may also include appropriate salts, minerals, metals, and other nutrients.

Suitable conditions and suitable media for culturing the microorganisms are well known in the art. In some embodiments, the suitable medium is supplemented with one or more additional agents, such as an inducer (e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressor (e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter), or a selection agent (e.g., an antibiotic that selects for a microorganism comprising the genetic modification).

The concentration of the carbon source (e.g.glucose) in the medium should be such that it promotes cell growth but not so high as to inhibit the growth of the microorganism used. In a preferred embodiment, the carbon source is at an undetectable level (e.g., less than about <0.1g/L) in the fermentation medium. In such embodiments, the culture is carbon-limited and the cultured cells should consume the carbon source immediately when it is delivered. It should be noted that reference to culture component concentrations may refer to initial and/or ongoing component concentrations. In some cases, it may be desirable to deplete the medium of a carbon source during culture.

Sources of assimilable nitrogen that can be used in a suitable medium include, but are not limited to, simple nitrogen sources, organic nitrogen sources, and complex nitrogen sources. Such nitrogen sources include anhydrous ammonia, ammonium salts, and materials of animal, plant, and/or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptones, yeast extract, ammonium sulfate, urea, and amino acids. Typically, the concentration of the nitrogen source in the medium is greater than about 0.1g/L, preferably greater than about 0.25g/L, and more preferably greater than about 1.0 g/L. However, the addition of a nitrogen source to the medium in excess of a certain concentration is disadvantageous for the growth of the microorganism. Thus, the concentration of the nitrogen source in the medium is less than about 20g/L, preferably less than about 10g/L, more preferably less than about 5 g/L. Furthermore, in some embodiments, it may be desirable to deplete the medium of the nitrogen source during culture.

Effective media may contain other compounds, such as inorganic salts, vitamins, trace metals, or growth promoters. Such other compounds may also be present in the carbon, nitrogen or mineral sources in an effective medium, or may be added specifically to the medium.

The medium may also contain a suitable source of phosphorus. Such phosphorus sources include inorganic phosphorus sources and organic phosphorus sources. Preferred phosphorus sources include, but are not limited to, phosphates such as sodium and potassium mono-or dibasic phosphate, ammonium phosphate, and mixtures thereof. Typically, the concentration of phosphate in the medium is greater than about 1.0g/L, preferably greater than about 2.0g/L, and more preferably greater than about 5.0 g/L. However, the addition of phosphate to the medium above a certain concentration is detrimental to the growth of the microorganisms. Thus, the concentration of the phosphate in the medium is generally less than about 20g/L, preferably less than about 15g/L, more preferably less than about 10 g/L.

Suitable media may also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other sources of magnesium may be used at concentrations that contribute similar amounts of magnesium. Typically, the concentration of magnesium in the medium is greater than about 0.5g/L, preferably greater than about 1.0g/L, and more preferably greater than about 2.0 g/L. However, the addition of magnesium to the medium above a certain concentration is detrimental to the growth of the microorganisms. Thus, the concentration of magnesium in the medium is generally less than about 10g/L, preferably less than about 5g/L, more preferably less than about 3 g/L. Furthermore, in some embodiments, it may be desirable to deplete the medium of a magnesium source during culture.

In some embodiments, the medium may also comprise a biologically acceptable chelating agent, such as trisodium citrate dihydrate. In such embodiments, the concentration of the chelating agent in the medium is greater than about 0.2g/L, preferably greater than about 0.5g/L, and more preferably greater than about 1 g/L. However, the addition of a chelating agent to the medium above a certain concentration is detrimental to the growth of the microorganisms. Thus, the concentration of the chelating agent in the medium is generally less than about 10g/L, preferably less than about 5g/L, more preferably less than about 2 g/L.

The medium may also initially include a biologically acceptable acid or base to maintain the desired pH of the medium. Biologically acceptable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and mixtures thereof. Biologically acceptable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the base used is ammonium hydroxide.

The medium may also include a biologically acceptable source of calcium, including but not limited to calcium chloride. Typically, the concentration of the calcium source (e.g., calcium chloride dihydrate) in the medium is in the range of about 5mg/L to about 2000mg/L, preferably in the range of about 20mg/L to about 1000mg/L, and more preferably in the range of about 50mg/L to about 500 mg/L.

In some embodiments, the medium may further comprise trace metals. Such trace metals may be added to the medium as a stock solution, which may be prepared separately from the rest of the medium for convenience. Typically, the amount of this trace metal solution added to the medium is greater than about 1mL/L, preferably greater than about 5mL/L, more preferably greater than about 10 mL/L. However, the addition of trace metals to the medium above a certain concentration is detrimental to the growth of the microorganisms. Therefore, the amount of this trace metal solution added to the medium is generally less than about 100mL/L, preferably less than about 50mL/L, more preferably less than about 30 mL/L. It should be noted that, in addition to the addition of the trace metals to the stock solution, the individual components may also be added separately, each within the ranges corresponding to the amounts of the components specified for the above trace metal solution ranges.

The medium may include other vitamins such as pantothenic acid, biotin, calcium, pantothenate, inositol, pyridoxine-HCl, and thiamine-HCl. Such vitamins may be added to the medium as stock solutions, which may be prepared separately from the rest of the medium for convenience. However, the addition of vitamins to the medium above a certain concentration is detrimental to the growth of the microorganisms.

The fermentation process of the present invention may be carried out in conventional culture modes including, but not limited to, batch, fed-batch, cell recycle, continuous and semi-continuous. In some embodiments, the fermentation is conducted in fed-batch mode. In this case, some components of the medium, including pantothenic acid during the production phase of the fermentation, are depleted during the cultivation. In some embodiments, the culture can be supplemented with relatively high concentrations of such components at the beginning (e.g., during the production phase) such that growth and/or isoprenoid production is supported for a period of time before addition is required. The preferred range of these components is maintained throughout the culture by addition at a level where the culture is depleted. The levels of components in the medium can be monitored, for example, by periodically sampling the medium and determining the concentration. Alternatively, once standard culture procedures are developed, the addition may be performed at specific times throughout the culture period at time intervals corresponding to known levels. As will be appreciated by those skilled in the art, as the cell density of the medium increases, the rate of consumption of nutrients during culture will also increase. In addition, in order to avoid the introduction of foreign microorganisms into the culture medium, can use the field known sterile addition method for adding. In addition, a small amount of a defoaming agent may be added during the culture.

The temperature of the medium can be any temperature suitable for growth and/or isoprenoid production by the genetically modified cells. For example, the culture medium may be placed and maintained at a temperature in the range of about 20 ℃ to about 45 ℃, preferably in the range of about 25 ℃ to about 40 ℃, and more preferably in the range of about 28 ℃ to about 32 ℃ prior to inoculating the culture medium with the inoculum.

The pH of the medium can be controlled by adding an acid or base to the medium. In this case, when ammonia is used to control the pH, it can also be conveniently used as a nitrogen source in the medium. Preferably, the pH is maintained from about 3.0 to about 8.0, more preferably from about 3.5 to about 7.0, and most preferably from about 4.0 to about 6.5.

In some embodiments, the concentration of the carbon source, e.g., glucose, of the culture medium is monitored during the culturing. The glucose concentration of the medium can be monitored using known techniques, for example, using a glucose oxidase assay or high pressure liquid chromatography, which can be used to monitor the glucose concentration in the supernatant (e.g., the cell-free component of the medium). As described elsewhere herein, the feed rate of the carbon source is adjusted according to the methods provided herein. Using aliquots of the original medium may be desirable because the concentration of certain nutrients (e.g., nitrogen source species and phosphorus source species) in the medium may be maintained simultaneously. Likewise, the trace metal concentration can also be maintained in the culture medium by adding aliquots of trace metal solution.

5.4 cells

The cell culture may comprise any cell deemed useful by one skilled in the art. Cells useful in the compositions and methods provided herein include archaeal cells, prokaryotic cells, or eukaryotic cells. In certain embodiments, the cell is recombinant, comprising one or more heterologous nucleic acids. In certain embodiments, the cell is a host cell comprising one or more heterologous nucleic acids encoding one or more enzymes capable of catalyzing the production of a compound of interest.

Suitable prokaryotic cells include, but are not limited to, any of a variety of gram-positive, gram-negative, or gram-variant bacteria. Examples include, but are not limited to, cells belonging to the genera: agrobacterium, Alicyclobacillus, Anabaena, Clostridium, Corynebacterium, Enterobacter, Azotobacter, Bacillus, Brevibacterium, Chromobacterium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Mesorhizobium, Methylobacterium, Microbacterium, Schidium, Schizophyllum, Pseudomonas, Rhodobacterium, Salmonella, Staphylococcus, Pseudomonas, Salmonella, Staphylococcus, Pseudomonas, Bacillus, streptomyces (Streptomyces), Synechococcus (Synnecoccus) and Zymomonas (Zymomonas). Examples of prokaryotic strains include, but are not limited to: bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Brevibacterium ammoniagenes (Brevibacterium ammoniagenes), Brevibacterium ammoniaphilum (Brevibacterium immariophilum), Clostridium beijerinckii (Clostridium beigericum), Enterobacter sakazakii (Enterobacter sakazakii), Escherichia coli (Escherichia coli), Lactococcus lactis (Lactobacilli), Rhizobium loti (Mesorhizobium loti), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas mellonella (Pseudomonas mevalonii), Pseudomonas putida (Pseudomonas megateri), Pseudomonas putida (Pseudomonas putida), Rhodobacter capsulatus (Rhodococcus capsulatus), Rhodococcus globiformis (Rhodococcus Rhodobacter sphaeroides), Salmonella typhimurium (Salmonella typhimurii), Shigella (Shigella intramura), Shigella (Shigella enteric bacteria (Shigella), Salmonella typhimurium (Shigella enteric bacteria (Shigella), Salmonella typhi, Shigella (Shigella typhi), and Shigella typhi). In a particular embodiment, the cell is an Escherichia coli (Escherichia coli) cell.

Suitable archaeal cells include, but are not limited to, cells belonging to the genera: aeropyrum (Aeropyrum), Archaeoglobus (Archaeglobus), Halobacterium (Halobacterium), Methanococcus (Methanococcus), Methanobacterium (Methanobacterium), Pyrococcus (Pyrococcus), Sulfolobus (Sulfolobus), and Thermoplasma (Thermoplasma). Examples of archaeal strains include, but are not limited to: archaeoglobus fulgidus (Archaeoglobus fulgidus), Halobacterium sp, Methanococcus jannaschii (Methanococcus jannaschii), Methanobacterium thermoautotrophicum (Methanobacterium thermoautotrophicum), Thermoplasma acidophilum (Thermoplasma acidophilum), Thermoplasma volcanium (Thermoplasma volcanum), Pyrococcus perniciosus (Pyrococcus horikoshii), Pyrococcus profundae (Pyrococcus abyssi), and Aeropyrum pernix (Aeropyrum pernix).

Suitable eukaryotic cells include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. In some embodiments, yeasts useful in the methods of the invention include yeasts that have been deposited by the microorganism collection (e.g., IFO, ATCC, etc.) and belong to the genera: saccharomyces (Aciclulococcus), deinocystia (Ambrosiozyma), Strobilanthus (Arthroascus), Arxiozyma, Ashbya (Ashbya), Babjevia, Bensenula (Bensingenia), Botryaascus, Botryozyma, Brettanomyces (Brettanomyces), Bullera (Bullera), Bullera (Bulleromyces), Candida (Candida), Saccharomyces (Citeromyces), Corynebacterium (Clavispora), Cryptococcus (Cryptococcus), Melanomyces (Cystofilodinium), Debaryomyces (Debaryomyces), Dekkera (Dekkera), Dipodospora (Dipodospora), Saccharomyces (Saccharomyces), Saccharomyces (Gepodium), Saccharomyces (Gepodocarpus (Hypocrea), alkaline-cinerea), hormoascus, Pichia stipitis (Hyphophora), Issatchenkia (Issatchenkia), Kloeckera (Kloeckera), Kluyveromyces (Kluyveromyces), Kondoa, Kuraishi, Kluyveromyces (Kurtzmanomyces), Asparagus (Leucospora), Lipomyces (Lipomyces), Loudeomyces (Loreomyces), Malassezia (Malassezia), Metschnikowia, Moraxella (Mrakia), Saccharomyces genuinalis (Myzxomyces), Rhodotorula (Nadsonia), Nakazaea, Neurospora (Neospora), Saccharomyces (Saccharomyces), Saccharomyces cerevisiae), Saccharomyces tectorum (Saccharomyces), Rhodosporidium (Pichia), Rhodosporium (Rhodosporium), Rhodosporidium (Rhodosporium), Rhodosporium (Rhodosporium), Rhodosporidium (Rhodosporidium), Rhodosporidium (Phaeosporium), Rhodosporium (Rhodosporium), Rhodosporium (Rhodosporidium), Rhodosporium (Phaeosporium), the genus Zingiber (Saitoella), Sakaguchia, Saturnospora, Schizosaccharomyces (Schizosaccharomyces), Schwanniomyces (Schwanniomyces), Trichosporon (Schwanniomyces), Sporidiobolus (Sporobolomyces), Protospora (Sporospora), Courospora (Stephaniaascus), Stemonaspora (Sterigmatomyces), Pediobolus (Steriginospora), Symbiostaphina (Symphora), Symphomycotsis, Torulopsis, Torulaspora (Torulaspora), Trichosporon (Trichosporon), Trichosporon (Trichosporomyces), Yahoo (Zygosaccharomyces), Zygosaccharomyces (Zygosaccharomyces), and the like.

In some embodiments, the host is Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Saccharomyces brueckii (Dekkera bruxellensis), kluyveromyces lactis (Kruyveromyces lactis, previously known as lactic acid yeast (Saccharomyces lactis)), kluyveromyces marxianus (kluyveromyces marxianus), Saccharomyces pombe (Arxula adensis), or Hansenula polymorpha (Hansenula polymorpha) (now known as Pichia angusta). In some embodiments, the cell is a strain of Candida (Candida), such as a strain of Candida lipolytica (Candida lipolytica), Candida guilliermondii (Candida guilliermondii), Candida krusei (Candida krusei), Candida pseudotropicalis (Candida pseudotropicalis) or Candida utilis (Candida utilis).

In a particular embodiment, the cell is Saccharomyces cerevisiae (Saccharomyces cerevisiae). In some embodiments, the cell is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) selected from the group consisting of Saccharomyces Baker's yeast, CBS7959, CBS7960, CBS7961, CBS7962, CBS7963, CBS7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1. In some embodiments, the cell is a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) selected from the group consisting of PE-2, CAT-1, VR-1, BG-1, CR-1, and SA-1. In a particular embodiment, the strain of Saccharomyces cerevisiae is PE-2. In another particular embodiment, the strain of Saccharomyces cerevisiae is CAT-1. In another particular embodiment, the strain of Saccharomyces cerevisiae is BG-1.

In some embodiments, the cell is a microorganism suitable for industrial fermentation. In particular embodiments, the microorganism is adapted to survive high solvent concentrations, high temperatures, extended substrate utilization, nutrient limitation, osmotic stress caused by sugars and salts, acidity, sulfite and bacterial contamination, or combinations thereof, which are recognized stress conditions for industrial fermentation environments.

In some embodiments, the cell is engineered to produce C₅An isoprenoid. These compounds are derived from one isoprene unit, also known as hemiterpenes. An illustrative example of a hemiterpene is isoprene. In other embodiments, the isoprenoid is C₁₀An isoprenoid. These compounds are derived from two isoprene units, also known as monoterpenes. MonoterpeneIllustrative examples of (d) are limonene, citronellol (citranellol), geraniol, menthol, perillyl alcohol, linalool, thujone (thujone), and myrcene. In other embodiments, the isoprenoid is C₁₅An isoprenoid. These compounds are derived from three isoprene units, also known as sesquiterpenes. Illustrative examples of sesquiterpenes are blattarone B, ginkgolide B, amorphadiene, artemisinin, artemisinic acid, valencene, nootkatone, epi-cedrol, epi-aristolocene, farnesol, gossypol, sanonin, blattarone, forskolin and patchoulol (also known as geraniol). In other embodiments, the isoprenoid is C₂₀An isoprenoid. These compounds are derived from four isoprene units, also known as diterpenes. Illustrative examples of diterpenes are ricinene, eleutherobin, paclitaxel, prostratin, pseudopterosin, and taxadiene (taxadiene). In other embodiments, the isoprenoid is C₂₀₊An isoprenoid. These compounds are derived from four or more isoprene units, including: triterpenes (C derived from 6 isoprene units)₃₀Isoprenoid compounds), such as arbutine e (arbusside e), brucine, testosterone, progesterone, cortisone, digitoxin and squalene; tetraterpenes (C derived from 8 isoprenoids)₄₀Isoprenoid compounds), such as beta-carotene; and polyterpenes (C derived from more than 8 isoprene units)₄₀₊Isoprenoid compounds) such as polyisoprene. In some embodiments, the isoprenoid is selected from the group consisting of: abietadiene (abiitadiene), amorphadiene, carene, alpha-farnesene, beta-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchouli alcohol, beta-pinene, sabinene, gamma-terpinene, terpinolene, and valencene. Isoprenoid compounds also include, but are not limited to, carotenoids (e.g., lycopene, alpha-and beta-carotene, alpha-and beta-cryptoxanthin, bixin, zeaxanthin, astaxanthin, and lutein), steroidal compounds, and isoprenoids modified with other chemical groups such as mixed terpene alkaloids and coenzyme Q-10The compounds of (a).

In some embodiments, the cell is engineered to produce a polyketide. In certain embodiments, the polyketide is selected from the group consisting of polyketide macrolides, antibiotics, antifungal compounds, cytostatic compounds, anticholesterolemic compounds, antiparasitic compounds, anticoccidial compounds, animal growth promoters, and pesticides.

In some embodiments, the cell is engineered to produce a fatty acid.

Useful cells are described in WO 2015/095804, WO 2015/020649 and WO 2014/144135, the contents of each of which are incorporated herein by reference in their entirety.

5.5 recovery of the Compound

Once the compound is produced by cell culture, it may be recovered or isolated for subsequent use using any suitable separation and purification method known in the art. In some embodiments, the organic phase comprising the compound is separated from the fermentation by centrifugation. In other embodiments, the organic phase comprising the compound is isolated from the fermentation spontaneously. In other embodiments, the organic phase comprising isoprenoid is separated from the fermentation by adding a demulsifier and/or a nucleating agent to the fermentation reaction. Illustrative examples of demulsifiers include flocculants and coagulants. Illustrative examples of nucleating agents include droplets of the isoprenoid itself and organic solvents such as dodecane, isopropyl myristate, and methyl oleate.

The compounds produced in these cells may be present in the culture supernatant and/or bound to the cells. In embodiments where the compound binds to cells, the recovery of the isoprenoid can include methods of permeabilizing or lysing the cells. Additionally or concurrently, the compounds in the culture medium may be recovered using recovery methods including, but not limited to, chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization, and crystallization.

In some embodiments, the compound is separated from other products that may be present in the organic phase. In some embodiments, separation is achieved using adsorption, distillation, gas-liquid extraction (stripping), liquid-liquid extraction (solvent extraction), ultrafiltration, and standard chromatographic techniques.

6. Examples of the embodiments

6.1 example 1

This example provides a method showing increased cell density (OD) of a determined yeast cell culture₆₀₀) An exemplary method of (1).

In the continuous feed process, both strains were evaluated using ethanol in the off-gas at target concentrations of 150, 300 and 600 ppm. Biomass (bioglass) is produced using a typical pulse feed process. After about 48 hours, the generation phase of the continuous feed was started. During the generation, the Oxygen Uptake Rate (OUR) was controlled at 110mmol/L/h without dissolved oxygen peak detection. The process flow trend for a continuous feed process using ethanol off-gas is shown below in fig. 1.

Fig. 1 provides an mfcs (biopat) trace for a continuous feed process. The off-gas ethanol concentration (blue) was maintained at 150ppm by adjusting the feed rate (green). OUR (red) was kept constant by adjusting the stirring (dark green). Dissolved oxygen is maintained at a near zero level (black).

A time interval of 3-6 days was chosen to illustrate the process. As shown in fig. 3, both strains showed a tendency to increase in productivity with an increase in the off-gas ethanol concentration set value, and there was no difference in yield. This figure shows that productivity increases with increasing average ethanol concentration in the exhaust. The data also show that the CFSC process typically produces an average exhaust gas concentration of 220-. By controlling the ethanol off-gas concentration at a higher level by adjusting the feed rate, both strains can be made to provide higher productivity.

FIG. 3 provides the average ethanol off-gas concentration of strain A in the standard CFSC process and the continuous feed process. The CFSC process produced an average of 200-225ppm ethanol in the off-gas, which was well below the upper set point for the continuous feed experiment.