PRODUCTION

Technical field

The invention relates to a fermentation medium and a method for fermentation production of erythromycin. More specifically, the present invention relates to a method for fermentation production of erythromycin using a nonionic surfactant.

Background of the Invention

Erythromycin is a macrolide antibiotic that can be used against infections caused by Gram positive bacteria and rickettsial bodies. In addition, erythromycin is an important raw material and intermediate of macrolide antibiotics, such as azithromycin, clarithromycin and roxithromycin, applied clinically in the treatment of bacterial infections.

As a secondary metabolite produced by actinomycetes (especially Saccharopolyspora genus), erythromycin is superfluous to the metabolic activities that are needed for the growth of the organism, and can be excreted into the culture medium.

In industry, erythromycin is mostly produced by fermentation. Research has been done on improvement of erythromycin fermentation process, which focused on screening high- producing strains, optimizing the culture medium and fermentation conditions. With the commercial importance of erythromycin, there is still a need for novel means and process to improve erythromycin production.

Nonionic surfactants are effective emulsifiers in biological systems and have less toxicity to biological systems. It has been reported that some nonionic surfactants can be used to improve microorganism fermentation. Hamedi et al. (JUST 32(1), 2006, P.41-46) studied the effects of various nonionic surfactants on erythromycin production by shake-flask fermentation. Results showed that different surfactants exhibited different effects on morphology of production strain and erythromycin production. For example, Tween 20 and T riton X-100 lysed hyphae; PEG300 and Tween 40 had no negative or positive effects on erythromycin production, and T riton X significantly decreased the production. On the other hand, PEG200, PEG400, PEG600, Tween60, Tween80, and Tween85 increased the production of erythromycin, with PEG400 and PEG600 being superior.

As one type of nonionic surfactants, polyoxyethylene-polyoxypropylene block copolymers were known useful in various fields, for example in industrial cleaning and sanitation applications, food and beverage processing applications, food service and kitchen hygiene applications, domestic detergent applications, pesticide formulation applications, and the like. However use of such polyoxyethylene- polyoxypropylene block copolymers in fermentation of erythromycin production has never been proposed.

Brief summary of the invention

The present invention has surprisingly found that addition of a polyoxyethylene- polyoxypropylene block copolymer as a nonionic surfactant into fermentation medium improves erythromycin production, and the polyoxyethylene-polyoxypropylene block copolymer is much superior to other nonionic surfactant, e.g. polyethylene glycols (PEGs) surfactants, in increasing the erythromycin titer in final broth.

In one aspect, therefore, the present disclosure provides a method for producing erythromycin by fermentation of a production strain, comprising adding a polyoxyethylene- polyoxypropylene block copolymer as a nonionic surfactant to a fermentation medium.

In another aspect, the present disclosure provides a use of a polyoxyethylene- polyoxypropylene block copolymer for fermentation production of erythromycin.

In another aspect, the present disclosure provides a fermentation medium for production of erythromycins comprising a polyoxyethylene-polyoxypropylene block copolymer.

The polyoxyethylene-polyoxypropylene block copolymer as a nonionic surfactant improves the erythromycin titer in fermentation broth at the end of fermentation. Relative to control fermentation without any surfactant supplement, the increase is at least 15%, preferable at least 20%, at least 25%, at least 30%, or at least 35%.

Detailed description of the Invention

The present invention may be understood more readily by reference to the following detailed description and the examples included herein.

Definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

The term "about", when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value.

The term "and / or" when used to connect two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.

As used herein, the term "comprising" or "including" means including the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps. Herein, when the terms "comprising" or "including" are used, unless otherwise indicated, the case of consisting of the mentioned elements, integers, or steps is also covered.

As used herein, the term "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized.

As used herein, the term "fermentation medium" refers to medium used in production bioreactor (fermenter), which contain rich nutrients to support the growth and propagation of the cells of production strain and the production of the product of interest. As used herein, the term "seed culture medium" refers to medium for seed culture to build up the quantity of cells for the inoculation of production bioreactor.

The term "fermentation in industrial scale" (also called large-scale fermentation) refers to fermentation processes with fermenter volumes of greater than or equal to 20 liters.

As used herein, "increasing" erythromycin titer is meant an increased titer of erythromycin produced in the broth at the end of fermentation, for example, after 6-7 days of fermentation, compared to the control fermentation without any surfactant supplement. The increase may be expressed in micrograms per milliliter (pg/ml), or percentage of increase vs. control (%).

Herein, the polyoxyethylene- polyoxypropylene block copolymer is also referred to as "EO-PO block copolymer" for short.

Herein, the average molecular weight, when mentioned for any EO-PO block copolymers, refers to an average molecular weight calculated from the OH numbers measured according to DIN 53240 (1971), wherein the hydroxyl number is determined by reaction with acetic anhydride in pyridine and subsequent titration of the free acetic acid.

Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Detailed description

The present invention is directed to a fermentation medium comprising a polyoxyethylene- polyoxypropylene block copolymer (i.e., an EO-PO block copolymer), and to a method for producing erythromycin wherein an EO-PO block copolymer is added as a nonionic surfactant in a fermentation medium.

Various aspects of the subject matters of the invention are described further below.

EO-PO block copolymers

For the present invention, any EO-PO block copolymers known useful as a nonionic surfactant may be used in the fermentation medium according to the present invention and the method for producing erythromycin according to the present invention.

In a particular embodiment, the EO-PO block copolymers useful as a nonionic surfactant in the fermentation medium and the method for producing erythromycin according to the present invention may have a block arrangement as represented by the formula (I) or (II)

— (CH2 ^CH 2 ⁰ )m(CH(CH ₃ )CH2 ⁰ )n(CH2 ^CH 2 ⁰ ^ wherein m, n and o in formula (I), and p, q and r in formula (II) each represent average numbers of the respective units, and

“ — ” represents a linking to respective remaining residues of the EO-PO block copolymer.

Preferably, m and o in formula (I) are equal to each other, and p and r in formula (II) are equal to each other.

In a preferred embodiment, the EO-PO block copolymers useful as a nonionic surfactant in the fermentation medium and the method for producing erythromycin according to the present invention may be polyoxyethylene- polyoxypropylene-polyoxyethylene block copolymer (hereinafter referred to as EO-PO-EO copolymer) represented by the formula (III): H0(CH ₂ CH20) _U (CH(CH3)CH20) _V (CH2CH ₂ 0) _W H (III)

In a preferred embodiment, the EO-PO block copolymers useful as a nonionic surfactant in the fermentation medium and the method for producing erythromycin according to the present invention may be polyoxypropylene-polyoxyethylene-polyoxypropylene block copolymer (hereinafter referred to as PO-EO-PO copolymer) represented by the formula (IV):

H0(CH(CH ₃ )CH ₂ 0) _x (CH ₂ CH ₂ 0) _y (CH(CH ₃ )CH ₂ 0) _z H (IV) wherein u, v and w in formula (III), and x, y and z in formula (IV) each represent average numbers of the respective units. Preferably, u and w in formula (III) are equal to each other, x and z in formula (IV) are equal to each other.

The EO-PO block copolymers useful as a nonionic surfactant in the fermentation medium and the method for producing erythromycin according to the present invention may comprise at least 5% by weight, for example 6% by weight, 7% by weight, 8% by weight, 9% by weight or 10 % by weight or more of oxyethylene units in total. Moreover, the EO-PO block copolymers may comprise 95% by weight or less, for example 90% by weight, 85% by weight, 84% by weight, 83% by weight, 82% by weight, 81% by weight, or 80% by weight or less of oxyethylene units in total. Particularly, the EO-PO block copolymers comprise 5 to 95 % by weight, preferably 5 to 90 % by weight, more preferably 5 to 85% by weight of oxyethylene units in total.

The EO-PO block copolymers useful as a nonionic surfactant in the fermentation medium and the method for producing erythromycin according to the present invention preferably has an average molecular weight of at least 500 g/mol, for example 1 ,000 g/mol, 1 ,500 g/mol, 1 ,600 g/mol, 1,700 g/mol, 1 ,800 g/mol, 1,900 g/mol or 2,000 g/mol or more. Moreover, the EO-PO block copolymers preferably have an average molecular weight of no greater than 15,000 g/mol, for example 14,000 g/mol, 13,000 g/mol, 12,000 g/mol, 11 ,000 g/mol, 10,000 g/mol, 9,000 g/mol or less. Particularly, the EO-PO block copolymers have an average molecular weight in the range of 500 to 15,000 g/mol, preferably 1 ,000 to 15,000 g/mol, more preferably 1,500 to 15,000 g/mol, most preferably 1,500 to 10,000 g/mol. In the context of the present invention, the EO-PO block copolymer may be added into fermentation medium to improve erythromycin production, either at the time of inoculation or preferably at intervals during the fermentation. Therefore, in an embodiment, the EO-PO block copolymer is comprised in basal fermentation medium, or in another embodiment, the EO-PO block copolymer is added into the fermentation broth at growth phase or production phase of fermentation. For example, the EO-PO block copolymer may be added into the broth in early stage of fermentation, or in intermediate or late stage of fermentation. In some embodiment, the EO-PO block copolymer is added after the second or third day of the fermentation. In some embodiments, the EO-PO block copolymer is added between 3-80 hours of fermentation, or preferably 5-75 hours of fermentation. In a preferred embodiment, the EO-PO block copolymer is added between 8-10 hours of fermentation. In a preferred embodiment, the EO-PO block copolymer is added after 10 hours of fermentation, or preferably after 15 hours, or more preferably after 20 or 24 hours. In another preferred embodiment, the EO-PO block copolymer is added between 24-72 hours of fermentation, for example, the EO-PO block copolymer can be added at about 28, 30, 35, 38, 40, 45, 50, 55, 60, 65, 68, 70 hours. In another preferred embodiment, the EO-PO block copolymer is added between 50-70 hours of fermentation. In another more preferred embodiment, the EO-PO block copolymer is added between 35-50 hours of fermentation.

The EO-PO block copolymers useful as a nonionic surfactant in the fermentation medium and the method for producing erythromycin according to the present invention may be prepared with any methods well-known in the art or may be commercially available nonionic surfactants of EO-PO block copolymer type. Suitable commercial nonionic surfactants of EO-PO block copolymer type include, but are not limited to, Pluronic ^® PE series for example Pluronic ^® PE 3100, Pluronic ^® PE 3500, Pluronic ^® PE 4300, Pluronic ^® PE 6100, Pluronic ^® PE 6120, Pluronic ^® PE 6200, Pluronic ^® PE 6400, Pluronic ^® PE 6800, Pluronic ^® PE 7400, Pluronic ^® PE 8100, Pluronic ^® PE 9200, Pluronic ^® PE 9400, Pluronic ^® PE 10100 , Pluronic ^® PE 10300, Pluronic ^® PE 10400, Pluronic ^® PE 10500; and Pluronic ^® RPE series, for example Pluronic ^® RPE 1720, Pluronic ^® RPE 1740, Pluronic ^® RPE 2035, Pluronic ^® RPE 2520 , Pluronic ^® RPE 2525, Pluronic ^® RPE 3110, available from BASF. Preferably, the EO-PO block copolymer is used in the fermentation medium in an amount suitable for improving the erythromycin production, especially increasing the erythromycin titer in broth at the end of fermentation. Preferably, the EO-PO block copolymer is added into the fermentation medium in an amount of about 0.1 g/L to 100g/L, more preferably about 1 g/L to 50g/L, for example, about 1g/L to 25g/L. In some preferred embodiments, the EO-PO block copolymer is added into the fermentation medium in an amount of about 1 g/L to 10g/L, particularly in an amount of about 2g/L-8g/L. For example, the EO-PO block copolymer can be added into the fermentation medium in an amount of about 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, or 10g/L.

In a context of the present invention, the EO-PO block copolymer may be used as the sole surfactant fed in the fermentation, or may be used in combination with another surfactant, for example a nonionic surfactant. The another surfactant may be selected from the group consisting of, polyoxyethylene sorbitan fatty acid esters such as Tween ^® series (for example Tween ^® 40, 60, 80, and 85), PEGs such as PEG 200, PEG 400, PEG 600, PEG 1000 and PEG 2000, ethoxylated soybean oil , isotridecanol alkoxylates and tallow fatty alcohol alkoxylates from BASF. Preferably, PEGs such as PEG 400, PEG 600, PEG 1000 and PEG 2000 may be used in combination with the EO-PO block copolymer.

Fermentation medium

Culturing a microorganism frequently requires that cells are cultured in a medium containing various nutrition sources, like carbon source, nitrogen source, and other nutrients e.g., amino acids, trace elements, minerals, etc., required for growth of those cells and for production of product of interest. Nutrition components suitable for fermentation are generally well known in the art (see, e.g., Peter F Stanbury, et al., Principles of Fermentation Technology, Third Edition, 2017, ELSEVIER SCIENCE & TECHNOLOGY, ISBN: 978-08-099953-1). Culture conditions for a given cell type may also be found in the scientific literature and/or from the source of the cell such as the American Type Culture Collection (ATCC). In one aspect, the present invention provides a fermentation medium for production of erythromycins. In some embodiments, the medium is for cultivation of Sacharopolyspora erythromycins producing microorganism, such as cells of Sacharopolyspora, or more specifically Sacharopolyspora erythraea. In some embodiments, the fermentation medium comprises a polyoxyethylene-polyoxypropylene block copolymer according to the present invention added into it. In some embodiments, the fermentation medium further comprises erythromycins producing microorganism.

The fermentation medium useful in the present invention may be a chemically defined medium or a complex medium. In some embodiments, the fermentation medium according to the present invention comprises carbon source, nitrogen source, and salt ion, and optionally one or more further components selected from the group consisting of oils, trace elements, pH adjuster, and antifoam. In some preferred embodiments, the fermentation medium comprises a vegetable oil, more preferably a soybean oil or a refined cottonseed oil. In some preferred embodiments, the fermentation medium comprises trace elements, preferably at least one selected from the group consisting of Co, Cu, Mo, Mn, Zn, Fe, borate, more preferably, one or more of CuCh, (NH4)dMq7q24, CoCh, Na2B4C>7, FeC .

Carbon source

Carbon sources that can be used for fermentation include various sugars and sugar-containing substances, lipids, organic acids, alcohols, hydrocarbons, as well as hydrolysates of proteins or amino acids. In the present invention, any carbon sources suitable to support the growth and propagation of production microorganism cells and/or the production of metabolites of interest may be used.

Examples of suitable carbon sources that can be mentioned are complex carbon sources such as molasse, corn steep liquor, cane sugar, dextrin, starch, starch hydrolysate, and cellulose hydrolysate, and combinations thereof; and chemically defined carbon sources such as carbohydrates, organic acids, and alcohols, for example, glucose, fructose, galactose, xylose, arabinose, sucrose, maltose, lactose, acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, fumaric acid, glycerol, inositol, mannitol and sorbitol, and combinations thereof.

Utilization rates and efficiencies by microorganisms of different carbon sources are different. In the context of the present invention, a fast-acting, or slow-acting carbon source or a combination thereof may be used. Fast-acting carbon sources refer to the sources of carbon that can be quickly utilized by production strains, such as glucose and other monosaccharides, and glycerol. A slow-acting carbon source refers to a carbon source that requires microorganisms to produce enzymes to decompose. Such slow-acting carbon sources include for example sucrose, lactose, maltose, molasses and other disaccharides and oligosaccharides and polysaccharides such as dextrin and starch. In industrial-scale fermentation, fast-acting carbon sources (such as glucose), slow-acting carbon sources (such as starch), or combination thereof can be used for the production of erythromycin. See, for example, Fan Daidi et al, The improvement of Fermentation Technical Parameters for the Erythromycin Production, Chinese Journal of Biotechnology, Vol. 15, No.1 , January, 1999. Fast-acting carbon sources are conducive to the growth of microorganisms. The utilization rates by microorganisms of slow-acting carbon sources are lower than the fast-acting carbon sources, but conducive to the synthesis of products. Erythromycin biosynthesis can be improved by adjusting the ratio of fast-acting and slow-acting carbon sources in the culture medium. For example, in some embodiments, more fast-acting carbon sources may be used in early stage of the fermentation. In another embodiments, more slow-acting carbon sources may be used in later-stage of fermentation.

In some embodiments, the fermentation medium according to the present invention comprises sugars and sugar-containing substances as sources of carbon. In some embodiments, the fermentation medium according to the present invention comprises a carbon source selected from a monosaccharide (e.g. glucose), a disaccharide (e.g. molasses, sucrose, maltose), an oligosaccharide (e.g. dextrin), a polysaccharide (e.g. starch), or a combination thereof. In a preferred embodiment, the fermentation medium comprises a carbon source selected from the group consisting of glucose, sucrose, dextrin, starch, and a combination thereof. In another preferred embodiment, the fermentation medium comprises a combination of a fast-acting carbon source such as glucose and a slow-acting carbon source such as starch or dextrin.

In some preferred embodiments, one or more of the following carbon sources are present in the fermentation medium at the concentrations (g/l): starch 0.2-55 g/l ((C ₆ HioC> ₅ )n,C:44.4%), glucose 1-25 g/l (C ₆ Hi ₂ C> ₆ ,C:40%), sucrose 10-50 g/l (Ci ₂ H ₂₂ 0n,C:42.1%), corn dextrin 0.1- 40 g/l (Ci ₈ H ₃₂ 0i ₆ ,C:42.9%).

In some embodiments, carbon sources such as glucose syrup or dextrin syrup may be fed into the fermentation broth during the fermentation in fed-batch process, to improve the erythromycin production.

Nitrogen sources

Nitrogen sources that can be used for fermentation include inorganic nitrogen sources and organic nitrogen sources. According to the utilization rates of nitrogen sources by microorganisms, nitrogen sources can also be divided into fast-acting nitrogen sources and slow-acting nitrogen sources. A fast-acting nitrogen source refers to a nitrogen source component that can be directly utilized by microorganism, such as amino nitrogen (such as amino acids) or ammonium nitrogen (such as ammonium salts). Such nitrogen sources may be conducive to the growth of microorganisms. A slow-acting nitrogen source refers to a nitrogen source component that cannot be directly used by bacteria, and requires microorganisms to produce enzymes to decompose before utilization, such as soybean meal and peanut meal. Such slow-acting nitrogen source may be conducive to biosynthesis of the product of interest.

In the context of the present invention, inorganic, organic nitrogen sources, or the combination thereof may be used. In the context of the present invention, a fast-acting nitrogen source, a slow-acting nitrogen source, or a combination thereof may be used.

Illustrative examples of suitable nitrogen sources include protein-containing substances, such as plant proteins, soy meal, corn meal, pea meal, corn gluten, cotton meal, peanut meal, potato meal, casein, gelatins, whey, yeast protein, yeast extract, tryptone, peptone, bacto- tryptone, bacto-peptone, amino acids, and combinations thereof; inorganic nitrogen sources such as ammonia, ammonium, ammonium salts, (e.g., ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, ammonium acetate), urea, nitrate, nitrate salts, or combination thereof; and various amino acids.

In a preferred embodiment, the fermentation medium comprises a fast-acting nitrogen source selected from amino nitrogen and/or ammonium nitrogen, or a slow-acting nitrogen source selected from peptone, yeast extract, corn steep liquor, soybean meal, peanut meal, and/or cottonseed meal, or combination thereof.

In another preferred embodiment, the fermentation medium comprises one or more nitrogen sources selected from the group consisting of ammonium sulfate, peptone, corn steep liquor, yeast extract, soybean meal, cottonseed meal, peanut meal, corn gluten meal, and amino acids (such as alanine, arginine, serine, cysteine, valine, threonine, methionine, isoleucine, and aspartic acid). Preferably, the fermentation medium comprises one or more of soybean meal, peanut meal, corn steep liquor and peptone.

In a preferred embodiment, one or more of the following nitrogen sources are present in fermentation medium at the concentrations (g/l): peptone 3-45 g/l (for example, total nitrogen: 12.7%; amino nitrogen:3.7%), corn steep liquor 0.05-10 g/l (for example, protein³42%), yeast extract 5-20 g/l (for example, total nitrogen: 10.0%-12.5% _; amino nitrogen: 5.1%), soybean meal 0.5-40 g/l (for example, protein³42%), cottonseed meal 10-20g/l (for example, protein³50%), corn gluten meal 1-25 g/l (for example, protein 20%-70%), peanut meal 3-25 g/l (for example, protein³50%), alanine 0.5-2 g/l (C3H7NO2, C:40.4% _; N:15.7%), arginine 0.5-2 g/ l(C ₆ H ₁₄ N ₄ 0 ₂ ,C: 41.3%; N: 32.2%), serine 0.5-2 g/l (C3H7NO3, C:34.3% _;

N : 13.3%) , cysteine 0.5-2 g/l (C3H7NO2S, C:29.8%; N:11.6%) , valine 1-3 g/l (C ₅ HnN0 ₂ , C:51.3%; N: 12.0%), threonine 1-3 g/l (C4H 9 NO3, C:40.3%, N:11.8%), methionine 1-3 g/l (C ₅ HH0 ₂ NS, C:40.3%; N: 9.4%), isoleucine 1-3 g/l (C ₆ H ₁₃ N0 ₂ , C:55.0%; N:10.7%), aspartic acid 1-3 g/l (C ₄ H ₇ N0 ₄ , C:36.1%; N: 10.5%). In some embodiments, nitrogen source such as ammonium sulfate may be fed into the fermentation broth during the fermentation, to improve the erythromycin production.

Oils

Actinomycetes can use lipids as a carbon source. In the fermentation process, adding lipids such as soybean oil has the functions of antifoaming and replenishing carbon sources, and providing precursors for erythromycin synthesis. See, for example, J. Hamedi, Enhancing of erythromycin production by Saccharopolyspora erythraea with common and uncommon oils, J Ind Microbiol Biotechnol (2004) 31: 447-456.

The oils that can be used according to the invention may be various vegetable oils, for example selected from the group consisted of sunflower, pistachio, cottonseed, melon seed, water melon seed, lard, corn, olive, soybean, hazelnut, rapeseed, sesame, shark, safflower, coconut, walnut, black cherry kernel and grape seed oils. Soybean oil and refined cottonseed oil are preferable oils.

In some preferred embodiments, the fermentation medium may contain soybean oil 0.06-6g/l (e.g. 4 or 5g/l) or refined cottonseed oil 4-1 Og/I (e.g. 5 or 6g/l).

The oils can be added into the fermentation medium at the start of the fermentation, or fed into the broth during the fermentation, for example, in the middle and/or late stages of fermentation.

Trace elements

Some trace elements have been proven to promote the proliferation of production strains and/or the activity of erythromycin biosynthesis-related enzymes (such as enzymes involved in carbohydrate metabolism, TCAand erythromycin biosynthesis, for example, glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, acetone acid carboxylase, pyruvate kinase, methylmalonyl CoA isomerase and methylmalonyl CoA carboxy transferase). Therefore, in some embodiments, trace elements can be added to increase erythromycin fermentation titer. Trace elements that can be used include, but are not limited to, molybdenum ions; zinc ions; manganese ions; magnesium ions, cobalt ions.

In some preferred embodiments, the fermentation medium comprises at least one trace elements selected from the group consisting of Co, Cu, Mo, Mn, Zn, Fe, borate. The trace elements in salts may be present in the medium. The salts may be e.g., alkali metal salts, alkali earth metal salts, chloride salts, ammonium salt, phosphate salts and sulfate salts.

In some preferred embodiments, the fermentation medium comprises one or more of cobalt chloride, copper chloride, ammonium molybdate, sodium tetraborate, manganese chloride, zinc chloride, and iron chloride. In some preferable embodiments, the fermentation medium comprises one or more of the salts: CuCh; (NH4)6Mq7q24; CoCh; Na2B4C>7; FeC , for example, the combination of CuChand (NH4 ₎ 6Mo7C>24; Or the combination of C0CI2, Na ₂ B ₄ C> ₇ , and FeCh.

In some preferred embodiments, one or more of the following salts are present in the fermentation medium at the concentrations (g/l) _: cobalt chloride 0.001-0.1 g/l, copper chloride 0.0001-0.001 g/l, ammonium molybdate 0.00025-0.1 g/l, sodium tetraborate 0.001-0.006 g/l, manganese chloride 0.001-0.1 g/l, zinc chloride 0.01-0.5 g/l, iron chloride 0.001-0.007 g/l.

Salt ions

The fermentation medium according to the present invention contains salt ions. The mineral salts useful for the production may vary according to the medium employed. In addition, organic salts can be used such as betaine, and choline chloride.

Salt ions that can be used in the present invention include, but not limited to, ammonium sulfate, magnesium sulfate, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, Trisodium citrate, Potassium chloride, Betaine, and Choline chloride.

Those salt ions can be present in medium at the following concentrations: ammonium sulfate 0.02-5 g/l, magnesium sulfate 0.2-2 g/l, sodium chloride 0.02-5 g/l, dipotassium hydrogen phosphate 0.8-2g/l, potassium dihydrogen phosphate 0.25-2 g/l, Trisodium citrate 1-4 g/l, Potassium chloride 0.1-1.5 g/l, Betaine 0.1-3g/l, Choline chloride 0.3-1 g/l.

In one embodiment, the medium may contain one or more salt ions selected from the group consisting of NaCI, K2HPO4, KH2PO4, and MgSCU.

Other medium components

Other components that can be comprised in the fermentation medium according to the present invention include, but not limited to, pH adjuster, antifoam, precursor. In some embodiments, the fermentation medium contains pH adjuster, such as CaC0 ₃ , e.g. 0.03-8 g/l. In some embodiments, the fermentation medium contains antifoam. In some embodiments, the precursor for erythromycin biosynthesis, n-propanol, may be fed into the broth during the fermentation, for example, after 1 day of cultivation.

Erythromycin Production Strain

Microorganism Strains useful in the present invention may be any native and genetic engineered microorganism capable of producing erythromycin in their cells, for examples, Saccharopolyspora , Streptomyces, Actinomyces, or E. coli (see, for example US20180016585A1 ).

Some microorganisms useful in the present invention may include strains of Actinobacteria, for example, Pseudonocardiaceae, and more specifically, strains of Saccharopolyspora, e.g., Saccharopolyspora antimicrobia, Saccharopolyspora cavernae, Saccharopolyspora cebuensis, Saccharopolyspora dendranthemae, Saccharopolyspora deserti, Saccharopolyspora erythraea, Saccharopolyspora flava, Saccharopolyspora ghardaiensis, Saccharopolyspora gloriosae, Saccharopolyspora gregorii, Saccharopolyspora halophila, Saccharopolyspora halotolerans, Saccharopolyspora hirsute, Saccharopolyspora hattusasensis, Saccharopolyspora hordei, Saccharopolyspora indica, Saccharopolyspora jiangxiensis, Saccharopolyspora lacisalsi, Saccharopolyspora phatthalungensis, Saccharopolyspora qijiaojingensis, Saccharopolyspora rectivirgula, Saccharopolyspora rosea, Saccharopolyspora shandongensis, Saccharopolyspora spinosa, Saccharopolyspora spinosporotrichia, Saccharopolyspora spongiae, Saccharopolyspora subtropica, Saccharopolyspora taberi, Saccharopolyspora thermophila, Saccharopolyspora tripterygiid.

Saccharopolyspora erythraea (also known as Streptomyces erythraeus , Actinomyces erythreus) is used for industrial-scale production of erythromycin. Multiple Saccharopolyspora strains are available for this purpose, including Saccharopolyspora erythraea strains ATCC 11635, DSM 40517, JCM 4748, NBRC 13426, NCIMB 8594, NRRL 2338. Those strains are within the scope of the present invention. In a preferred embodiment, the production strain used in the present method is a Saccharopolyspora cell, preferably, a cell of Saccharopolyspora erythraea.

Genetic engineered strains and mutant strains for erythromycin production are also known in the art. For example, it has been reported that polar knockout of the methyl malony-CoA mutase (MCM) gene, mutB, improves erythromycin production by the carbohydrate- based and oil-based fermentation of S. erythraea. In addition, engineering of the methyl malonyl-CoA metabolite node through duplication of the mmCoA mutase (MCM) operon leads to a 50% increase in erythromycin production. See, for example, Xiang Zou, et al., Fermentation optimization and industrialization of recombinant Saccharopolyspora erythraea strains for improved erythromycin a production, Biotechnology and Bioprocess Engineering, December 2010, Volume 15, Issue 6, pp 959-968.

A variety of approaches have been used to improve the erythromycin producing microorganism. Present understanding of the genes responsible for the biosynthesis of erythromycin and techniques to inactivate genes in Sac. erythraea have allowed the directed manipulation of the pathway. See for example, WO2018226893A2, describing a method for genomic engineering in Saccharopolyspora spp.

Therefore, various erythromycin-producing microorganisms, variants and mutants produced by means of gene engineering, mutagenesis, strains selection, or other methods are within the scope of the present invention. In some embodiments, preferably, genetically modified or mutant Saccharopolyspora erythraea strains are used in the method according to the present invention.

Fermentation Process

Fermentation methods well known in the art can be applied according to the present invention to ferment the production strains.

Generally, a fermentation process includes seed culture and fermenter fermentation. The seed culture is used to build up an adequate number of cells for the inoculation of a production bioreactor. For this purpose, a seed train can be used, in which cells of production strain are run through multiple-scale cultivation systems (e.g. T-flasks, roller bottles or shake flasks, small scale bioreactor systems and subsequently larger bioreactors). The production bioreactor is inoculated out of the largest seed train scale to perform production of the product of interest.

In some embodiments, therefore, the fermentation process according to the present invention is carried out in multiple stages, for example two or three stages. For example, at the first stage, a relatively small seed culture is grown by inoculation from e.g., spores grown on sporulation medium; and at the second stage, the primary seed culture may be used either to inoculate a second seed culture medium (or in case of two-stage process, into fermentation medium). The second stage seed cultivation may be performed in a 2L or 15L seed tank. At the third stage, the second seed culture is inoculated into fermentation medium in fermenter for production of erythromycin. During the fermentation, preferably, dissolved oxygen is not less than 45%. The nutrient content and the amount of inorganic salts in the primary medium may be controlled to prevent the spores from growing too fast. The secondary medium may contain rich nutrients, and similar to the fermentation medium to facilitate the adaptation of the strain to fermentation. In a preferred embodiment, the fermentation process according to the present invention comprises more than one (e.g., two or three) seed growth stages to scale-up the quantity of the microorganism, so that it can be used as an inoculant for the fermentation phase. However, as is well understood in the art, the number of seed cultures used depends, for example, on the size and volume of the fermentation step. To start the fermentation stage, a portion or all of a seed culture is used to inoculate fermentation medium.

The fermentation may be performed in a batch process. In the batch process, except for aeration and addition of the present surfactant and/or acid or basic pH adjusting agent to the fermentation broth, no other nutrients (substrate) are fed into the broth during the whole process of fermentation.

The fermentation may be performed in fed-batch process. In this process, in addition to the addition of the present surfactant, nutrients may be fed into the broth, such as carbon source (e.g., glucose, dextrin), nitrogen source (e.g., ammonium sulfate), oils (e.g. soybean oil), and/or n-propanol.

The fermentation may be performed in a continuous process. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation allows for the modulation of one or more factors that affect cell growth and/or product concentration.

The fermentation may be carried out in Erlenmeyer flasks or in laboratory as well as in industrial fermenters of various capacities. In a preferred embodiment, the fermentation is in industrial scale. The culture medium in the fermentation may be from 20 L to 300 m ³ , for example, 20L to 1000L, or 1m ³ to 300 m ³ . Preferably, the fermentation has culture medium of at least 20 liters, preferably, at least 50 liters, more preferably at least 300 liters, further preferred at least 1000 liters. In some embodiments, the fermentation has culture medium of at least 5m ³ , 10m ³ , 25m ³ , 50m ³ , 100m ³ , 200m ³ , or up to 300m ³ . The fermentation time, pH, temperature, dissolved oxygen, or other specific fermentation conditions may be applied according to standard conditions known in the art. Preferably, the fermentation conditions are adjusted to obtain maximum yields of the product of interest.

Preferably, the temperature of the fermentation broth during fermentation is about 25°C to 40°C, preferably about 28°C to 35°C, more preferably, about 34°C. Preferably, the pH of the fermentation broth during fermentation is controlled at pH 6.0 to 7.5, for example from pH 6.3 to 7.5, preferably from pH 6.5 to 7.3, more preferably from 6.7 to 7.3. Preferably, the fermenter pressure during fermentation is controlled at 0.02 to 0.08MPa, preferably 0.03 to 0.05MPa. Preferably, the biomass concentration in the fermentation broth at the stationary phase during fermentation is controlled at 20 to 50 g/L cell dry weight.

Preferably, fermentation is carried out with stirring and aeration. The rate of aeration may be expressed as air volume/culture volume/min (vvm, m ³ /(m ³ *min)). In a preferred embodiment, during the fermentation, the aeration rate is controlled at 0.5-3vvm (preferably, 2.0-3.0vvm), and agitation rate at 120-350rpm (preferably, 180-300 rpm).

In some embodiments, the fermentation time may be for 50-200 hours, preferably, 100-200 hours, for example, about 130-170 hours.

In a preferred embodiment, the fermentation consists in culturing the production microorganisms in a previously sterilized liquid culture medium under aerobic conditions at a temperature ranging from 28 to 37°C (preferably at about 34°C) over a period of time varying from 3 to 9 days (preferably 6-7 days) and at a pH value controlled in the range of pH 6.3 to 7.5. Further preferably, the fermentation is performed under the fementer pressure of 0.02 to 0.08MPa (preferably 0.03-0.05MPa), with aeration rate of 0.5-3vvm (preferably, 2.0-3.0vvm), and agitation rate of 120-350 rpm (preferably, 180-300 rpm).

Production of erythromycin may be monitored by removing samples from the fermentation, and assaying by any known methods, for example, colorimetry, chromatography. For example, the titers of erythromycin produced in a shake flask or bioreactor cultivations may be determined using chemical assay or bioassay, with commercially available erythromycin as a standard. In one example, the quantitative determination of the total erythromycins present in the fermentation broths is performed by colorimetric method, which depends on reacting erythromycins with phosphoric acid and monitoring the absorbance at 485 nm. Regarding more details of the colorimetric method, see, for example, Zhang-Yu et al., Determination of erythromycin in fermentation broth, China Brewing, 2011, Vol.5.

Fermentation may continue until the yield has been maximized, for example, for 6-7 days. After that, the product erythromycin can be obtained by recovery from the fermentation broths, concentration from crude solution and purification, using any of conventional methods in the art.

The invention is further illustrated in the following examples which are not intended to be in any way limiting to the scope of the invention as claimed.

Examples

The following examples only serve to illustrate the invention. The numerous possible variations that are obvious to a person skilled in the art also fall within the scope of the invention.

Unless otherwise stated the following experiments have been performed by applying standard equipment, methods, chemicals, and biochemicals as used in genetic engineering and fermentative production of chemical compounds by cultivation of microorganisms.

Material and Methods

Chemical products: (all commercially available from BASF)

PEG-1: Polyethylene glycol having an average molecular weight of 400;

PEG-2: polyethylene glycol having an average molecular weight of 600;

PEG-3: polyethylene glycol having an average molecular weight of 1000;

PEG-4: polyethylene glycol having an average molecular weight of 2000; EO-PO copolymer-1: EO-PO-EO block copolymer having an average molecular weight of 6500, and having 50% by weight of EO unit;

EO-PO copolymer-2: EO-PO-EO block copolymer having an average molecular weight of 3500, and having 10% by weight of EO unit;

EO-PO copolymer-3: EO-PO-EO block copolymer having an average molecular weight of 8000, and having 80% by weight of EO unit;

EO-PO copolymer-4: EO-PO-EO block copolymer having an average molecular weight of 2900, and having 40% by weight of EO unit

EO-PO copolymer-5: PO-EO-PO block copolymer having an average molecular weight of 2150, and having 20% by weight of EO unit;

EO-PO copolymer-6: PO-EO-PO block copolymer having an average molecular weight of 2650, and having 40% by weight of EO unit;

EO-PO copolymer-7: PO-EO-PO block copolymer having an average molecular weight of 3100, and having 20% by weight of EO unit;

EO-PO copolymer-8: PO-EO-PO block copolymer having an average molecular weight of 3500, and having 10% by weight of EO unit;

Surfactant-1: Ethoxylated (10EO) soybean oil.

Surfactant-2: C -Oxo alcohol + 8 EO

Surfactant-3: Ethoxylated (11 EO) fatty alcohol (Cie-Cie)

Erythromycin-producing Strain

Saccharopolyspora erythraea was used for erythromycin production in the Examples.

Method of determination of erythromycin titers in fermentation broth Erythromycin titer in fermentation broth is determined by colorimetric method. Briefly, 0.8 ml diluted fermentation broth (with estimated titer of 300-700ug/ml) is pipetted into 10ml colorimetric tube. 4ml of 10mol/L phosphoric acid solution is added and mixed uniformly. The mixture is put into 80 ^° C water bath for 3min, and then cooled in water batch. After adding 10mol/L phosphoric acid solution up to 10ml and mixing uniformly, the absorbance of this mixed solution is measured at 485 nm with a spectrophotometer. 0.8ml_ of the same diluted fermentation broth but untreated in 80 ^° C water bath is used as control. Spectrophotometer is calibrated against the control. The standard curve of erythromycin titer (absorbance at 485nm versus concentration in ug/ml) is plotted using erythromycin standard sample at concentrations of 100ug/ml, 200ug/ml, 300ug/ml, 500ug/ml and 800ug/ml. The erythromycin titer of fermentation broth is determined by reference to the standard curve (y =0.00070x- 0.00539, R ² = 0.00702).

Preliminary Screen Experiments To investigate the effects of different kinds and concentrations of surfactants on erythromycin production, shake-flask fermentation assay and 5L-fermenter fermentation assay were conducted to screen suitable surfactants for the erythromycin production.

Medium for the assays

Plate medium contained (%): Starch, 10; NaCI, 3; corn syrup, 13; (NhU^SCU, 3; CaCCh, 3; agar, 20. The pH was adjusted to 7.0 before sterilization.

Shake-flask seed medium contained (g/l): Starch, 40; Tryptone, 20; NaCI, 4; dextrin, 20; glucose, 10; KH ₂ PO ₄ , 0.2; MgSC> ₄ .7H ₂ 0, 0.25; CaCCh, 6. The pH was adjusted to 7.0 before sterilization.

Fermentation medium contained (g/l): Glucose, 22; K2HPO4, 1.2782; KH2PO4, 0.6391; MgSCU 7H2O, 1 ; alanine, 0.686; arginine, 0.5472; cysteine, 0.6251 ; serine, 0.587; trisodium citrate, 2.2841; trace elements, 10 mL/L. The pH was adjusted to 7.0 before sterilization.

Culture conditions and culture methods Plate culture

The strain stored in the glycerin tube was cultured on sterilized medium of a plate, at 34 ^° C for 10 days.

Shake flask seed culture

In 500 ml shake flask containing 50 ml shake-flask seed medium, spores grown on 1 cm ² of the plate medium was inoculated, and cultured at 34 ^° C for 48 h at 220 rpm.

Shake-flask fermentation

1 ml of the cultured shake-flask seed culture was transferred into a 500 ml shake flask containing 50 ml of fermentation medium, and incubate at 220 rpm and 34 ^° C for 144 h.

5L-fermenter fermentation

6 bottles of seed cultures were harvested and pooled, and then transferred into a 5 L fermenter containing 2.8 L fermentation medium. The temperature of the fermentation was controlled at about 34 ^° C. The dissolved oxygen in the fermentation broth was kept greater than 40% by controlling the agitation speed and aeration.

Shake-flask assay

The surfactants shown in Table A below were selected for 500 ml shake-flask fermentation. 5g/L of surfactants were fed into the broth on the second day of fermentation. After 5 days of fermentation, the erythromycin titers in fermentation broth were measured. The fermentation without any surfactant supplement was used as control. The results are shown in Table A.

Table A. Erythromycin titers in shake-flask assay for surfactant screen

The EO-PO copolymer (EO-PO Copolymer-7) was tested at different concentrations in 500 ml shake-flask fermentation assay. Three concentrations (1 g/L, 5g/L, and 20g/L) of the EO-PO block copolymer were fed into the broth on the second day of fermentation. After 5 days of fermentation, the erythromycin titers in fermentation broth were measured. The fermentation without any surfactant supplement was used as control. The results are shown in Table B. Table B. Erythromycin titers in shake-flask assay by EO-PO copolymer-7 at different dosages

The above results in shake-flask assay suggest erythromycin titers can be improved by addition of EO-PO copolymers, at a relatively broad dosage range.

5L-fermenter fermentation assay

The surfactants shown in Table C below were selected for 5L-fermenter fermentation. 5g/L of surfactants were fed into the broth on the second day of fermentation, and after about 24 hours of fermentation. After 156 hours of fermentation, the erythromycin titers in fermentation broth were measured. The fermentation without any surfactant supplement was used as control. The results are shown in Table C.

Table C. Erythromycin titers in 5L-fermenter fermentation assay for surfactant screen

Based on the above preliminary screen assays, the present EO-PO block copolymer surfactants were further investigated in 5L or 50L fermentation, to verify their effects on the erythromycin production. Example 1

Spores of S. erythraea was grown on a plate of sporulation medium. Then, spores on 1 cm ² medium was inoculated in 500 ml Erlenmeyer flask containing 50ml seed medium, and incubated at 34 ^° C for 48hr at 220rpm. The composition of seed medium was (g/l): starch, 40; peptone, 20; NaCI, 4; dextrin, 20; glucose, 10; KH2PO4, 0.2; MgSCU 7H2O, 0.25; CaCCh, 3. The pH was adjusted to 7.0 before the sterilization of medium.

10% (v/v) of the seed culture was inoculated into 5 L fermenter containing 2.5L basal fermentation medium. The composition of the basal fermentation medium was (g/l): starch, 50; yeast extract, 5; NaCI, 2; (NhU^SCU, 2; CaCCh, 5; soybean meal, 35; soybean oil, 4; antifoam, 0.3. The pH was adjusted to 7.0 before the sterilization of medium.

Fermentation was done at pH 6.7-7.5, with aeration rate of 3.0vvm, fermenter pressure of 0.05MPa, fermentation temperature of 34 ^° C, agitation rate of 250 rpm. During the fermentation, the biomass concentration at the stationary phase was kept at 40-50g cell dry weight. 5 g/L sterilized surfactant was added into the fermenter at 54 hours of fermentation. The fermentation without addition of any surfactant supplement was used as control. The fermentation broth was discharged from the fermenter at 156 hours. The titers of erythromycins in broths at discharge and the percentage increase versus control are shown in Table 1.

Table 1. Titers of erythromycins in broths and the percentage increase versus control

As shown in Table 1, addition of the present surfactants increased the erythromycin titers. In addition, the increases in titers induced by EO-PO copolymer (29% to 36%) were about 2 to 3 times of that induced by PEG-based surfactants (9.1% to 13%).

Example 2

Spores of S. erythraea was grown on a plate of sporulation medium. Then, spores on 1 cm ² medium was inoculated in 500 ml Erlenmeyer flask containing 50ml seed medium, and incubated at 34 ^° C for 48hr at 220rpm. The composition of seed medium was (g/l): starch, 40; peptone, 20; NaCI, 4; dextrin, 20; glucose, 10; KH2PO4, 0.2; MgSCU 7H2O, 0.25; CaCCh, 3. The pH was adjusted to 7.0 before the sterilization of medium.

10%(v/v) of the seed culture was inoculated into 5 L fermenter containing 2.5 L basal fermentation medium. The composition of the basal fermentation medium was (g/l): starch, 50; corn dextrin, 20; cottonseed meal, 15; soybean meal, 35; KH2PO4, 1.5; K2HPO4, 0.6; MgS0 ₄ 7H ₂ 0, 1.5; CaCOs, 4; CuCI ₂ .2H ₂ 0, 0.00030; (NH ₄ )6Mo ₇ 0 ₂₄ 4H ₂ 0, 0.00030; soybean oil, 5; antifoam, 0.3. The pH was adjusted to 7.0 before the sterilization of medium.

Fermentation was done at pH 6.7-7.5, with aeration rate of 2.0vvm, fermenter pressure of 0.03MPa, fermentation temperature of 34 ^° C, agitation rate of 180 rpm. During the fermentation, the biomass concentration at the stationary phase was kept at 20-50g cell dry weight. 5g/L sterilized surfactant was added into the fermenter at 64 hours of fermentation. The fermentation without addition of any surfactant supplement was used as control. The fermentation broth was discharged from the fermenter at 156 hours. The titers of erythromycins in broths at discharge and the percentage increase versus control are shown in Table 2.

Table 2. Titers of erythromycins in broths and the percentage increase versus control

As shown in Table 2, with different fermentation medium adopted, the surfactant of the present invention still resulted in an increased erythromycin titer, and was superior to conventional surfactants of the PEGs.

Example 3

20 L fermenter was used for seed cultivation, with fermentation temperature of 34 ^° C , aeration rate of 0.8vvm, fermenter pressure of 0.04MPa, agitation rate of 200 rpm. The composition of the seed medium was (g/l): starch, 40; peptone, 20; NaCI, 4; dextrin, 20; glucose, 10; KH ₂ PO ₄ , 0.2; MgSCU 7H2O, 0.25; CaCCh, 3. The pH was adjusted to 7.0 before the sterilization of medium.

10%(v/v) of the seed culture was inoculated into 50 L fermenter containing 25L basal fermentation medium. The composition of the basal fermentation medium was (g/l): starch, 50; peptone, 45; corn gluten meal, 15; peanut meal, 10; NaCI, 2; (NhU^SCU, 1.5; CaCCh, 5;refined cottonseed oil, 5. The pH was adjusted to 7.0 before the sterilization of medium.

Fermentation was done at pH 6.7-7.3, with aeration rate of 3.0vvm, fermenter pressure of 0.04MPa, fermentation temperature of 34 ^° C, agitation rate of 250 rpm. During the fermentation, the biomass concentration at the stationary phase was kept at 40-50g cell dry weight. 5g/L sterilized surfactant was added into the fermenter at 8 hours of fermentation. The fermentation without any surfactant supplement was used as control. The fermentation broth was discharged from the fermenter at 156 hours. The titers of erythromycins in broths at discharge and the percentage increase versus control are shown in Table 3.

Table 3. Titers of erythromycins in broths and the percentage increase versus control

As shown in Table 3, with process scaling-up, the surfactant of the present invention also resulted in an increased erythromycin titer, and was superior to conventional PEG-based surfactants.

Example 4

20L fermenter was used for seed cultivation, with fermentation temperature of 34 ^° C, aeration rate of 0.8vvm, fermenter pressure of 0.04MPa, agitation rate of 200 rpm. The composition of the seed medium was (g/l): starch, 40; peptone, 20; NaCI, 4; dextrin, 20; glucose, 10; KH2PO4, 0.2; MgSC> ₄ .7H ₂ 0, 0.25; CaCCh, 3. The pH was adjusted to 7.0 before the sterilization of medium.

10%(v/v) of the seed culture was inoculated into 50 L fermenter containing 25L basal fermentation medium. The composition of the basal fermentation medium was (g/l): glucose, 20; corn dextrin, 25; peptone, 40; corn steep liquor, 6; Valine, 1.23; Threonine, 1.38; Methionine, 1.37; Isoleucine, 1.53; K2HPO4, 1.73; KH ₂ P0 ₄ , 0.94; MgS0 ₄ 7H ₂ 0, 2 _; CoCI ₂ 6H ₂ 0, 0.008; Na ₂ B ₄ C> ₇ 10H ₂ O, 0.07; FeC 6H ₂ 0, 0.007; betaine, 1.5; refined cottonseed oil, 5; antiform, 0.3. The pH was adjusted to 7.0 before the sterilization of medium.

Fermentation was done at pH 6.7-7.3, with aeration rate of 2.5vvm, fermenter pressure of 0.03MPa, fermentation temperature of 34 ^° C, agitation rate of 250 rpm. During the fermentation, the biomass concentration at the stationary phase was kept at 20-40g cell dry weight. 5g/L sterilized surfactant was added into the fermenter at 45 hours of fermentation. The fermentation without any surfactant supplement was used as control. The fermentation broth was discharged from the fermenter at 156 hours. The titers of erythromycins in broths at discharge and the percentage increase versus control are shown in Table 4.

Table 4. Titers of erythromycins in broths and the percentage increase versus control As shown in Table 4, with the same process scale-up as in Example 3, but with different fermentation medium and different surfactant feeding time, the surfactant of the present invention again resulted in an increased erythromycin titer. Example 5

A three-stages fermentation process was used for erythromycin production.

2L fermenter was used for a first seed cultivation, with fermentation temperature of 34 ^° C , aeration rate of 0.8vvm, fermenter pressure of 0.04MPa, agitation rate of 200 rpm. The composition of the first seed medium was (g/l): starch, 40; peptone, 20; NaCI, 4; dextrin, 20; glucose, 10; KH ₂ PO ₄ , 0.2; MgSCU 7H2O, 0.25; CaCCh, 3. The pH was adjusted to 7.0 before the sterilization of medium.

All of the first seed culture was transferred into 20L fermenter containing 10L second seed medium for the seed culture expansion, with fermentation temperature of 34 ^° C, aeration rate of lOvvm, fermenter pressure of 0.05MPa, agitation rate of 200 rpm. The composition of the second seed medium was (g/l): corn flour, 28; K2HPO4, 0.4 _; NaCI, 0.9; corn gluten meal, 18; groundnut meal, 15; glucose, 6; CaCCh, 2; corn steep liquor, 7; antifoam, 0.3. The pH was adjusted to 7.0 before the sterilization of medium.

10% (v/v) of the second seed culture was inoculated into 50 L fermenter containing 25L basal fermentation medium for the third-stage fermentation culture. The composition of the basal fermentation medium was (g/l): starch, 30; yeast extract, 5; dextrin, 40; soybean meal, 30; KH ₂ PO ₄ , 5; (NH ₄ ) ₂ SC> ₄ , 2; CaCCh, 6; choline chloride, 0.6; soybean oil, 5. The pH was adjusted to 7.0 before the sterilization of medium.

Fermentation was done at pH 6.7-7.3, with aeration rate of 2.8vvm, fermenter pressure of 0.05MPa, fermentation temperature of 34 ^° C, agitation rate of 300 rpm. During the fermentation, the biomass concentration at the stationary phase was kept at 40-50g/L cell dry weight. 5g/L sterilized surfactant was added into the fermenter at 38 hours of fermentation. The fermentation without any surfactant supplement was used as control. The fermentation broth was discharged from the fermenter at 156 hours. The titers of erythromycins in broths at discharge and the percentage increase versus control are shown in Table 5. Table 5. Titers of erythromycins in broths and the percentage increase versus control

As shown in Table 5, with a three-stage process, the surfactant of the present invention resulted in a significantly increased erythromycin titer.