Method for producing exosomal nucleic acids

Abstract

The present invention relates to a method for increasing nucleic acid production comprising a step of limiting the total amount of sulfur in a culture medium. The invention further relates to the use of a culture medium comprising a limited amount of sulfur for increasing nucleic acid production.

Description

[0001] The present invention belongs to the field of recombinant biotechnology, in particular to the field of nucleic acid production. The present invention relates to a method for increasing nucleic acid production, the method comprising the step of limiting the total sulfur content in the culture medium. The present invention further relates to the use of a culture medium containing a limited amount of sulfur for increasing nucleic acid production.

[0002] The production of protein of interest (POI) or nucleic acid molecule of interest has been completed with many prokaryotic hosts. The most prominent example is bacterium, such as Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, Streptomyces griseus or Corynebacterium glutamicum. In the past few years, a large amount of biopharmaceuticals (such as antibodies or their functional fragments, or nucleic acid molecules) have been produced and increasing biopharmaceuticals are about to be approved for the mankind, but their effective production is still a challenging task. The therapeutic activity dosage of more and more people in need needs a large amount of such proteins or nucleic acid molecules. In addition, due to the need for nucleic acid molecules to produce such as protein in production cells, plasmid rAAV vectors for vaccination, etc., a large amount of such nucleic acid molecules are needed. Therefore, a considerable amount of molecules are needed as active ingredients or as the template for protein production, so that efficient and cost-effective production is worthwhile.

[0003] Bacterial cell expression systems have long been and remain one of the main tools for producing these types of molecules. A key goal of process optimization is to achieve high yields of products with the desired quality at the lowest possible cost, which is often determined by the characteristics of a specific expression construct or system. Many times, batch processes are used, for example, for nucleic acid production or for producing plasmids for vaccination, such as AAV vectors. However, batch processes are labor-intensive because bioreactors require continuous monitoring and must be thoroughly cleaned after each production. When cleaning the bioreactor, production cannot proceed, making the batch process inefficient.

[0004] These disadvantages associated with batch processes can be overcome by applying continuous expression systems and / or fed-batch expression systems instead of batch processes. However, also in continuous processes, high levels of recombinant nucleic acid molecule synthesis may overwhelm the metabolic capacity of the host cells and thus lead to plasmid loss, reduced oxygen transfer, generation of toxic byproducts, formation of inclusion bodies and / or triggering of stress responses, which often impair efficient nucleic acid molecule synthesis.

[0005] To this end, scientists have taken different approaches to deal with these issues when using batch or continuous processes. However, there is still a need for efficient production of proteins or nucleic acid molecules of interest in microbial host cells.

[0006] There is a need to overcome the described and other disadvantages. The present invention therefore addresses these needs and technical objectives and provides a solution as described herein and defined in the claims.

[0007] The present invention relates to a method for increasing the yield of nucleic acids (preferably extrachromosomal nucleic acids) contained in host cells in cell culture, the method comprising the step of limiting the total sulfur content in the culture medium of the cell culture during cell culture so that the total sulfur content, per gram of predetermined cell dry weight of the host cells desired to be obtained in the cell culture, is approximately equal to or less than the amount of sulfur contained per gram dry weight of a reference host cell when preferably grown without total sulfur restriction, thereby increasing the yield of the nucleic acid.

[0008] As used herein, the term limiting the "total sulfur amount in the culture medium (or, in the alternative, the total sulfur demand; thus "amount" and "demand" are used interchangeably herein as they mean the same thing)" during cell culture means that the total sulfur amount, i.e., 100% predetermined as described herein, decreases in the culture medium of the host cells during the cell culture process. For clarity, 100% total sulfur is the amount of sulfur content required to achieve a specific, predetermined (or predefined) dry cell weight or cell concentration of the host cells described herein. How to predetermine (or predefine) the total sulfur amount is described herein.

[0009] It is assumed that the decrease in total sulfur in the cell culture medium during the cell culture process is mainly due to biomass production by the host cells, for example due to growth, cell division, etc.

[0010] The reduction of total sulfur in the cell culture medium can be reduced from 100% to 0% during the cell culture process.

[0011] For example, for 1 g of dry cell mass of E. coli as an exemplary host cell, 8.5 mg of sulfur is required. Therefore, if 50 g of dry cell mass of E. coli as an exemplary host cell is desired, 425 mg of sulfur is required. Therefore, according to the teachings of the present invention, 425 mg of sulfur is considered to be 100% of the total sulfur. Of course, if 100 g of dry cell mass of E. coli as an exemplary host cell is desired, 850 mg of sulfur is required. Therefore, according to the teachings of the present invention, 850 mg of sulfur is considered to be 100% of the total sulfur.

[0012] As shown in the examples and figures, but not limited thereto, the methods of the present invention provide for decoupling the metabolism of host cells from the production of desired nucleic acids (e.g., plasmids). Once sulfur becomes limited, the host cell, preferably a bacterial cell, will no longer be able to synthesize biomass, but will instead produce the desired nucleic acid, e.g., plasmid; see also Figure 1 .

[0013] Surprisingly, when performing the method of the invention, it was observed that when sulfur limitation occurs, the quality, i.e. the proportion, of covalently closed circular (ccc) nucleic acids, such as plasmid DNA (preferably pDNA) of the desired nucleic acid produced by the culture under S limitation increases; see Figure 2 .

[0014] Similarly, when performing the methods of the invention, it is observed that when sulfur limitation occurs, the desired nucleic acid yield, preferably ccc (covalent, closed, circular) DNA productivity of the culture under S limitation is increased; see Figure 3 .

[0015] Furthermore, when performing the method of the invention, it was observed that when sulfur limitation occurs, the yield of the desired nucleic acid, preferably ccc DNA, of the culture under S limitation increases; see Figure 4 .

[0016] Therefore, method of the present invention provides advantageous effects, particularly increases the productive rate of desired nucleic acid, the high quality of desired nucleic acid and / or the beneficial ratio of desired nucleic acid and cell dry mass.Each of these advantageous effects has reduced nucleic acid manufacturing cost and / or improved downstream process, such as the efficiency of the purification of desired nucleic acid.

[0017] "Increased nucleic acid production" as used herein means that the production of nucleic acids by a host cell in a cell culture that is limited by the total amount of sulfur in the culture medium during the cell culture process as described herein produces a higher yield of the nucleic acid compared to a host cell, preferably the same host cell, which is not limited by the total amount of sulfur in the culture medium during the cell culture process, however, preferably during the cell culture process. In other words, the same host cell is not limited in terms of total sulfur during the cell culture process as described herein, compared to a host cell that is limited in terms of total sulfur during the cell culture process as described herein. "Higher yield" includes the yield of the desired nucleic acid, the volumetric yield or mass of the nucleic acid, for example, the amount of ccc DNA in the case of a plasmid. When comparing the yield of a host cell that is limited by sulfur as applied in the methods described herein and compared to a reference host cell as described herein, such a yield can be at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30% higher, relatively speaking.

[0018] The total sulfur amount referred to herein may also be referred to herein as "(required) predetermined total sulfur amount" or "predetermined (required) total sulfur amount", etc. The terms "predetermined" or "predefined" may be used interchangeably herein in the context of total sulfur amount.

[0019] Means and methods for determining the amount and elemental composition of stem cell material of cells contained therein (e.g., host cells referred to herein) are well known in the art; see, for example, Heldal et al., Applied and Environmental Microbiology (1985), 50(5): 1251-1257 or Neidhardt, FC (2006), Escherichiacoli and Salmonella typhimurium, Cellular and Molecular Biology, Part I, Chapter 2 "Chemical Composition of Escherichia coli" (ISBN 0-914826-89-1).

[0020] Alternatively, the host cells can be cultured with different amounts of sulfur, for example three different amounts of sulfur, until a maximum OD600 is reached, the biomass determined gravimetrically, the cell dry mass plotted against the amount of sulfur added, followed by a linear regression that allows calculation of the amount of sulfur per OD unit or dry weight of the host cells; see Examples 5 and Figure 5 Maximum OD600 is reached when OD600 varies over time by a maximum of +0.5 OD600 to -0.5 OD600 units.

[0021] For this purpose, the skilled person is aware of the amount of C source (e.g. glucose) in the culture medium. In the case where sulfur is not limiting, the skilled person is aware of or can readily determine the yield coefficient, e.g. in a chemostat bioreactor in steady state or in a fed-batch process. Thus, the skilled person can determine the (expected) biomass. Thus, the maximum OD600 can also be determined.

[0022] At the same time, the technician sets up two other conditions where the sulfur amount is lower than the sulfur amount used in the non-limiting sulfur condition, whereby the amount of C source, such as glucose, is the same as the amount of glucose used in the non-limiting sulfur condition. As described above, when the maximum OD600 is reached, a linear regression is performed, which allows calculation of the sulfur amount per OD unit or host cell dry weight.

[0023] Using these means and methods, a skilled artisan can predetermine (or predefine) the total sulfur amount (or requirement) as described and referred to herein.

[0024] In the context of the present invention it has surprisingly been found that culturing host cells while limiting the amount of available sulfur in the culture medium leads to a significant increase in nucleic acid production; see e.g. Figure 3 In particular, as has been discovered in the context of the present invention, limiting the amount of sulfur used to grow a desired amount of host cells comprising a nucleic acid molecule to be produced to the amount of sulfur contained in the same amount of a reference host cell (preferably not comprising the nucleic acid molecule) grown in the same medium except without sulfur limitation, results in a significant increase in nucleic acid yield. For example, according to the present invention, if X grams (dry weight) of reference host cells (grown without sulfur limitation) are produced, and said X grams of reference host cells contain 10 g of sulfur, and it is also desired to produce X grams of host cells comprising a nucleic acid molecule to be produced according to the methods provided and described herein, the amount of sulfur in the medium used to culture said host cells comprising said nucleic acid molecule is limited to about 10 g or less.

[0025] The present invention thus provides an efficient and simple method for increasing the yield of a desired nucleic acid, thereby even reducing the amount of resources required to produce the nucleic acid. Without being bound by theory, according to the present invention, it is assumed that by limiting the amount of sulfur in the culture medium, the metabolism of the host cell is reduced in a manner that allows the host cell to increase the yield of nucleic acid molecules, thereby decoupling metabolism from nucleic acid production.

[0026] Preferably, in the methods described and provided in the context of the present invention, the limiting step may comprise

[0027] (a1) predetermining the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and

[0028] (b1) adding to the cell culture at the start of the cell culture a total amount of sulfur per gram of a predetermined dry weight of the host cells (i.e., a desired dry weight), wherein the total amount of sulfur is approximately equal to or less than the amount of sulfur contained in the reference host cells per gram of dry weight.

[0029] As used herein, "at the start" relates to "before the start", so that for example sulfur is added before the cell culture starts.

[0030] The above method, but not limited thereto, can be performed as follows: at the beginning of the cell culture, a total amount of sulfur is added per gram of a predetermined dry weight of the host cells, so that when the host cells reach a predetermined dry weight, the sulfur becomes limited. In practice, once metabolism is decoupled from the production of the desired nucleic acid, the technician predetermines the dry weight of the host cells that are desired to be used for the production of the desired nucleic acid, because the sulfur in the host cells is limited according to the teachings of the present application. To this end, the host cells are grown without sulfur limitation until a predetermined cell dry weight is reached. Because the technician can determine the dry weight of the host cells so grown, and the technician can determine the amount of sulfur contained in the host cells so grown, it is known that the total amount of sulfur that must be added to the host cells at the beginning of the cell culture is so that once the host cells reach a predetermined cell dry weight, the sulfur becomes ideally limited. If so, metabolism will be decoupled from the production of the desired nucleic acid as described and taught herein.

[0031] Alternatively, but also preferably, in the methods described and provided in the context of the present invention, the limiting step may comprise

[0032] (a2) predetermining the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and

[0033] (b2) during the cell culturing process, reducing the amount of total available sulfur in the cell culture per gram dry weight of the host cells to at least or less than the amount of sulfur contained per gram dry weight of the reference host cells.

[0034] To implement alternatives (a2, b2), the skilled person, as in alternatives (a1, b1) above, predetermines the cell dry weight, the amount of sulfur as described above. However, rather than adding a total sulfur amount per gram of the predetermined dry weight of the host cells at the start of the cell culture, sufficient sulfur is added, i.e., sulfur is not a limiting factor, so that sulfur is not limiting. If desired, sulfur addition is reduced during the cell culture. For example, if 10 g of total sulfur is required for a predetermined cell dry weight of the host cells, this amount is not added all at the start of the cell culture, but rather is added over a period of time so that the ratio of available sulfur to host cell dry weight decreases over time, thereby making sulfur a limiting factor.

[0035] Still alternatively, but also preferably, in the methods described and provided in the context of the present invention, the limiting step may comprise

[0036] (a3) predetermining the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and

[0037] (b3) during the cell culturing—from the beginning until the desired amount of host cells containing the nucleic acid is obtained—adding to the cell culture a total amount of sulfur per gram of a predetermined dry weight of the host cells (i.e., the desired dry weight to be obtained), wherein the total amount of sulfur is approximately equal to or less than the amount of sulfur contained in the reference host cells per gram of dry weight.

[0038] To implement alternatives (a3, b3), the skilled person predetermines the dry cell weight, sulfur amount, as described above, as in alternatives (a1, b1) or (a2, b2) above. However, sulfur is added from the beginning until the desired host cell amount is reached. For example, if 10 g of sulfur is required, this 10 g is added during the cell culture until the desired host cell amount is reached, and then no more sulfur is added.

[0039] The first alternative, ie (a1), (b1), is more preferred; see also the Examples.

[0040] Preferably, in the methods described and provided in the context of the present invention, the amount of sulfur contained per gram dry weight of the reference host cell may be about equal to or less than the amount of sulfur contained in the elemental composition per gram dry weight of the reference host cell. In this context, the term "elemental composition" as used herein includes the elemental composition of the host cell, including sulfur and optionally other elements, such as sodium, magnesium, phosphorus, chlorine, potassium, calcium and / or other elements.

[0041] Generally, in the context of the present invention, sulfur can be added to the culture medium or cell culture in all suitable forms which allow the cells to be grown, preferably in a form which allows sulfur uptake into the cells. For example, sulfur can be added in the form of (hydrated) sulfates, such as MgSO4, MnSO4, FeSO4, CuSO4, ZnSO4, (NH4)2SO4 or the respective hydrates, or in the form of dextran sulfate.

[0042] Preferably, in the methods described and provided in the context of the present invention, the supply of nutrients (other than sulfur) to the cell culture can be performed in a linear manner while limiting the total amount of sulfur. However, the supply of nutrients (other than sulfur) to the cell culture can also be performed in an exponential manner while limiting the total amount of sulfur. As already described herein (see, e.g., (b1), (b2) and (b3) above), the limitation of the amount of sulfur can be achieved by controlling the amount of sulfur added to the cell culture at the beginning of the cell culture and / or during the cell culture, so that the total amount of sulfur is approximately equal to or less than the amount of sulfur contained per gram dry weight of the reference host cell as described herein.

[0043] Sometimes, due to the solubility of such sulfur salts, it may be necessary to gradually add sulfur, e.g. in the form of a sulfur salt, such as MgSO4, to the culture medium over a certain time frame. Thus, as used herein, "at the start of the cell culture" encompasses a time frame spanning 0 to 24 hours, e.g. 23 hours, preferably 0 to 20 hours, more preferably 0 to 16 hours, at the start of the cell culture or after the start of the cell culture, respectively, during which the total amount of sulfur per gram of a predetermined dry weight of the host cells (i.e. the dry weight desired to be obtained) is added to the cell culture as described herein.

[0044] like Figure 6A to Fig.6D As shown, when the predetermined total sulfur amount was reduced to 0%, adding more sulfur, such as adding 50% or 25% of the predetermined total sulfur amount, still showed an increase in productivity ( FIG. 6A ), volumetric yield ( FIG. 6B ), and the quality of the desired nucleic acid ( FIG. 6C ) and cell dry mass ( FIG. 6D ) compared to the control. Fig.6D ) remains unchanged. This means that the surprising findings of the present invention can still be observed when the total sulfur content is not kept constant at 0%, but preferably is kept at 50% or less, more preferably 25% or less of the predetermined total sulfur content. Therefore, once the predetermined total sulfur content is reduced to 0%, the surprising effect occurs within the range of preferably 50% to 0% of the predetermined total sulfur content.

[0045] Therefore, in the method of the present invention, when the predetermined total sulfur amount decreases to 0% of the predetermined total sulfur amount in the culture medium, more sulfur is preferably added to the culture medium. This amount is preferably added in the form of feed.

[0046] Starting from the time point when the total sulfur is 0%, one can add more sulfur to the culture medium so as to add an amount of, for example, 50% or less, e.g., 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the total sulfur predetermined as described herein. Such an amount is preferably added in the form of a feed.

[0047] More sulfur may be added once; or may be repeated, e.g., once, twice, three times, four times or more; or may be performed for a (desired) time such that if no more sulfur is added, an amount of e.g., 50% or less, e.g., 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the total sulfur amount predetermined as described herein may remain unchanged or may then be reduced during the cell culture process as described herein. Such an amount is preferably added in the form of a feed.

[0048] Thus, when the total sulfur is 0%, the skilled person may preferably add to the culture medium 50% or less, 45% or less, 40% or less, 35% or less or 30% or less of the total amount of total sulfur predetermined (as described herein). Such amounts are preferably added in the form of feed.

[0049] Thus, when the total sulfur amount is 0%, the skilled person may more preferably add to the culture medium 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less or 1% or less of the total amount of total sulfur previously determined (as described herein). Such amounts are preferably added in the form of feed.

[0050] The skilled person can predetermine the amount of total sulfur required per gram of (desired) cell dry weight as described herein. The skilled person also understands the point in time during the cell culture process at which the total sulfur decreases to 0%.

[0051] The reason is that the total sulfur amount decreases during the cell culture, which, translated into time points, is presumably mainly due to biomass production by the host cells, e.g. due to growth, cell division, etc.

[0052] As mentioned above, the skilled person can predetermine the total sulfur amount required per gram of (desired) cell dry weight as described herein. The skilled person also understands the point in time during the cell culture process at which the total sulfur amount decreases to 0%. Therefore, the skilled person can also predetermine the time at which the total sulfur amount can be reduced to, for example, 50%.

[0053] In other words, the addition of more sulfur may be performed when the predetermined total sulfur amount is 0%, or may be performed when the predetermined total sulfur amount is preferably reduced to 50% or less of the predetermined total sulfur amount, more preferably reduced to 25% or less.

[0054] Therefore, when the total sulfur level is 0%, in the alternative of adding more sulfur described above, more sulfur can be added before the total sulfur level drops to 0% during the cell culture process.

[0055] Therefore, in the method of the present invention, when the predetermined total sulfur amount decreases to 50% or 25% or less of the predetermined total sulfur amount in the medium, more sulfur is preferably added to the medium.

[0056] Starting from this amount, the skilled person can add sulfur to the culture medium so as to reach an amount of, for example, 50% or less, such as 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less or 1% or less of the total sulfur predetermined as described herein.

[0057] More sulfur can be added once; or can be repeated, for example, once, twice, three times, four times or more; or can be performed for a (desired) time such that if no more sulfur is added, an amount of, for example, 50% or less, for example, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less of the total sulfur amount predetermined as described herein can remain unchanged or can then decrease during the cell culturing as described herein.

[0058] Therefore, during the cell culture process, the reduction of the total sulfur amount in the cell culture medium can preferably be reduced from 100% to 50% or less, 45% or less, 40% or less, 35% or less or 30% or less.

[0059] More preferably, during the cell culture process, the reduction of total sulfur in the cell culture medium can be preferably reduced from 100% to 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, for example 0%.

[0060] In fact, as explained above, it is apparent that the surprising effect of the present invention, as described herein, occurs within a range of preferably between 50% and 0%, more preferably between 25% and 0%, of the predetermined total sulfur amount. Therefore, it seems reasonable that the total sulfur amount does not necessarily have to be reduced to 0% before the surprising effect occurs, but rather the surprising effect has occurred when the predetermined total sulfur amount may preferably be between 50% and 0%, more preferably between 25% and 0%, of the predetermined total sulfur amount.

[0061] As used herein, a "reference host cell", especially in the context of the methods of the present application, is a host cell identical to the host used in the methods described herein for increasing the production of a desired nucleic acid, the term "identical" preferably referring to another cell of the same type, species and / or strain as the host cell comprising the nucleic acid to be produced. However, the reference host cell is not sulfur-limited, especially when used in the methods of the present application.

[0062] Preferably, the reference host cell can be grown in the same culture medium as the host cell used in the method for increasing the production of the desired nucleic acid as described herein. The term "same culture medium" preferably relates to a cell culture medium having the same composition as the cell culture medium used for growing the host cells comprising the nucleic acid to be produced, except for the sulfur content.

[0063] Preferably, the reference host cell can be grown under the same conditions as the host cell used in the method described herein for increasing the yield of a desired nucleic acid, for example with regard to temperature, pressure, gas atmosphere and / or mixing rate.

[0064] Preferably, the reference host cell does not contain the desired nucleic acid, particularly when used in the method of the present application. However, in an alternative, the reference host cell may preferably contain the desired nucleic acid, particularly when used in the method of the present application. The latter alternative is preferred over the first alternative.

[0065] Therefore, preferred reference host cells are not sulfur-restricted and do not contain the desired nucleic acid, particularly when used in the methods of the present application. Alternatively, however, preferred reference host cells are not sulfur-restricted and contain the desired nucleic acid, particularly when used in the methods of the present application.

[0066] The latter mentioned alternatives to the reference host cell are preferred over the first mentioned alternatives to the reference host cell.

[0067] Therefore, more preferred reference host cells

[0068] (i) as described herein, the same host as used in the method for increasing the production of a desired nucleic acid as described herein;

[0069] (ii) not limited by sulfur, particularly when used in the process of the present application; and

[0070] (iii) comprises a desired nucleic acid, particularly when used in the methods of the present application.

[0071] In addition, the more preferred reference host cell

[0072] (iv) can be grown in the same culture medium as the host cell used in the methods described herein for increasing production of a desired nucleic acid; and / or

[0073] (v) can be grown under the same conditions as the host cell used in the methods described herein for increasing the production of a desired nucleic acid, e.g. with respect to temperature, pressure, pH, gas atmosphere and / or mixing rate.

[0074] Such more preferred reference host cells may also be referred to herein as a "control," such as a "control host cell," "control culture," or in the Examples, a "control bacterial culture," and the like.

[0075] Preferably, in the methods described and provided in the context of the present invention, predetermining the dry cell weight of the host cells can be achieved by obtaining a sample of the cell culture medium of a defined volume, harvesting the host cells, drying the host cells, and weighing the dried host cells. Means and methods for determining the amount of dry cell material and elemental content of cells contained therein are well known in the art; see, for example, Heldal et al., Applied and Environmental Microbiology (1985), 50(5): 1251-1257.

[0076] Preferably, in the methods described and provided in the context of the present invention, limiting the total sulfur in the culture medium of the cell culture can occur when the host cells are in logarithmic phase. Means and methods for determining whether a cell culture is in logarithmic phase are known in the art, including measuring the optical density (OD) of the cell culture; for example, see https: / / www.implen.de / od600-diluphotometer / od600 / .

[0077] Preferably, in the methods described and provided in the context of the present invention, limiting the total sulfur in the culture medium of the cell culture may occur when the dry weight of the host cells is at least about 5 g / liter of culture medium, preferably at least about 10 g, or at least about 20 g / liter of culture medium, such as 25 g / liter of culture medium, 30 g / liter of culture medium, 35 g / liter of culture medium, 40 g / liter of culture medium, 45 g / liter of culture medium or 50 g / liter of culture medium.

[0078] Preferably, in the methods described and provided in the context of the present invention, the extrachromosomal nucleic acid can be a bacmid, a cosmid, a plasmid or a minicircle. The plasmid can be circular or linear. If circular, the plasmid is preferably covalent, closed, circular (ccc). As described herein, "nucleic acid" also includes RNA molecules. Therefore, the extrachromosomal nucleic acid can also be an RNA molecule, for example in linear or circular form.

[0079] Preferably, the plasmid may be a low copy number, medium copy number or high copy number plasmid, preferably a high copy number plasmid. Examples of high copy number plasmids are plasmids with a pMB1 derived replication origin (ori), ColE1 ori, pUC or F1 ori. A high copy number plasmid with a pUC ori is preferred.

[0080] Preferably, in the methods described and provided in the context of the present invention, the host cell can be a eukaryotic (e.g., fungus) or a prokaryotic (e.g., bacterial) host cell, preferably a prokaryotic cell. A preferred prokaryotic cell is an E. coli cell.

[0081] In the context of the present invention, examples of eukaryotic host cells include fungal cells, such as yeast and microalgae (eg, Chlorella), and protozoa.

[0082] As used herein, the term "host cell" preferably refers to a nucleic acid molecule that has been (or can be) introduced into the non-natural host cell, i.e., a cell that has been (or can be) genetically engineered to the host cell. A preferred example of a prokaryotic host cell is Escherichia coli. However, it is also possible to envision Pseudomonas species, Salmonella species, Bacillus species, Lactobacillus species, Corynebacterium species, Bacillus species or Actinomyces species in the context of the present invention. It should be understood that such terms not only mean a specific host cell, but also mean the progeny of such a cell. In the successive passages, some modifications may occur due to mutations or environmental influences, so such progeny may not actually be equivalent to the parental cell, but it is still included in the scope of the term "host cell" used herein. Recombinant host cells can be isolated cells, preferably cells grown in culture. In a preferred embodiment of the present invention, the bacterial host cell is Escherichia coli. In a more preferred embodiment, the bacterial host cell is an Escherichia coli K lineage, such as Escherichia coli DH5a or more preferably Escherichia coli JM108. The E. coli may also be from a B lineage, such as E. coli BL21, REL606 or a W lineage or any other derivative of E. coli. The bacterial host cells described herein may be produced by means and methods well known in the art.

[0083] Preferably, the prokaryotic host cell may have an inactive recA and / or endA gene. The inactivation of recA and / or endA may be due to a total or partial deletion or due to a mutation. The technician knows to find the respective recA and / or endA genes in the prokaryotic host, for example using E. coli recA or endA as a query sequence in a database or as a probe in a wet lab experiment, respectively.

[0084] Preferably, in the methods described and provided in the context of the present invention, the host cell may be a recombinant host cell.

[0085] The skilled artisan is aware of genetic engineering techniques known in the art to generate bacterial host cells for use in the systems or processes of the present invention. For example, various kits are available for genetic engineering of bacterial host cells to randomly or targetedly integrate nucleic acids comprising nucleotide sequences into the bacterial genome; see, for example, Zhang et al. (1998), Nature Genetics 20, 123-128 or Sharan et al. (2009), Nature Protocols 4(2), 206-223. The skilled artisan is also aware of techniques for transforming bacterial host cells, as well as any other cloning techniques that can be used to generate extrachromosomal elements such as plasmids, cosmids, bacmids, minicircles, etc.

[0086] In one embodiment of the present invention, the method described and provided in the context of the present invention may be a method of culturing the host cell in a fermenter in a fed-batch or continuous manner, preferably in a fed-batch manner.

[0087] In fed-batch culture or continuous culture host cells as described herein, the feeding can be linear or exponential. Thus, the fed-batch culture as described herein can include linear feeding or exponential feeding. Likewise, the continuous culture as described herein can include linear feeding.

[0088] The method described and provided in the context of the present invention is based on decoupling the growth of host cells from the production of nucleic acids of interest in a two-stage process ("growth decoupling"). In the inventive system and process of the present invention, the growth (i.e., reproduction) of host cells can be spatially separated from the production of nucleic acids of interest. In a bioreactor, i.e., a seed bioreactor, host cells can be kept under conditions ideal for their reproduction. The production of nucleic acids of interest will not be induced or only a small amount of nucleic acids of interest will be induced. The host cells from the seed reactor can be continuously transferred to the second bioreactor, i.e., the production bioreactor. The growth of the host cells can then be suppressed, and the production of nucleic acids of interest can be induced in the production bioreactor. This concept is referred to as "growth decoupling" in this article. This allows continuous production. A portion of the host cells or the supernatant / culture medium of the production reaction can be continuously taken out from the production bioreactor, and nucleic acids of interest can be harvested from the host cells. The portion taken out from the production bioreactor can preferably be continuously replaced with fresh host cells from the seed bioreactor. Therefore, this technology allows the continuous production of nucleic acids of interest in relatively small bioreactors, while still having high yield / productivity, and thus improving the STY (space-time yield) of nucleic acids of interest.

[0089] Harvesting and separating nucleic acids such as plasmid DNA are well known to those skilled in the art. Generally, the production of nucleic acids of the present invention includes utilizing the system or process of the present invention to cultivate microorganism / bacteria culture, and then optionally harvesting and lysing microorganism / bacteria host cells, and optionally purifying nucleic acid molecules such as plasmid DNA. The first step of processing is usually to decompose microorganism / bacteria host cells by lysis, preferably alkaline lysis. In a preferred embodiment of the present invention, the lysis reaction is carried out according to methods known in the art using an alkaline lysis solution containing a detergent. Typical lysis solutions are composed of NaOH (0.2M) and SOS (1%), but other alkaline solutions and other detergents (see, for example, WO 97 / 29190) can also be used. After lysis, it is the neutralization of the alkaline solution. In principle, this step can be carried out according to methods known per se, preferably according to a gentle method that can be operated in a continuous and automated mode. In a preferred embodiment, in the neutralization step, the cell solution of lysis is mixed with a neutralization solution. Generally, a buffer solution with an acidic pH and a high salt concentration is used for neutralization. Preferably, the solution is composed of 3 M potassium acetate (KAc) of pH 5.5. However, other neutralizing salts may also be used or added. Nucleic acid molecules, such as plasmid DNA, may then be precipitated by adding an aqueous solution containing about 70% ethanol. The nucleic acids may be pelleted by centrifugation and washed by aspirating the supernatant, resuspending the pellet containing the nucleic acids in an aqueous solution containing about 70% ethanol, and then pelleting again by centrifugation. After washing, the pelleted nucleic acids may be dried.

[0090] As an alternative or supplement to this process, a nucleic acid binding resin in the form of a column can also be used. Before capture / purification by the resin, it may be necessary to adjust the parameters of the solution (such as salt composition, conductivity, pH value) to ensure the binding of the desired biomolecule to the chromatographic support (usually resin) (in the sense of the present invention, this step is referred to as "adjustment step"). The simplest adjustment procedure is to dilute the clarified lysate with water or a low-salt buffer, especially when the chromatographic resin in the subsequent capture step is realized by anion exchange chromatography (WO 97 / 29190). In addition, especially when hydrophobic interaction chromatography is used as the first purification step, a high concentration salt solution can be added and the precipitation that may be produced by filtering or centrifugation (if a certain salt concentration is exceeded in the solution, the precipitation is present) (WO 02 / 04027). In the case of using high concentration ammonium sulfate; This treatment reduces the RNA content (WO 98 / 11208). In order to capture and purify, several steps are applied to obtain highly purified biomolecules that meet pharmaceutical requirements. As for the previous steps, enzymes, detergents and organic solvents should be avoided. Separation and purification are carried out according to methods known in the art, in particular by a combination of different chromatographic techniques (anion exchange chromatography AIEC, hydrophobic interaction chromatography HIC, size exclusion chromatography (SEC), ultra (diafiltration), filtration or precipitation and extraction). A method that can be advantageously used, in particular for obtaining a plasmid DNA (pDNA) for therapeutic applications, comprises a combination of two steps based on different chromatographic principles, wherein any one of the two steps is selected from hydrophobic interaction chromatography (HIC), polar interaction chromatography (PIC) and anion exchange chromatography (AIEC), and wherein at least in one of the two steps, preferably in both steps, the chromatographic support is a porous monolithic bed, preferably a monolithic column in the form of a rigid monolith based on methacrylate. Suitable monolithic columns are commercially available from BIA Separations / Sartorius under the trademark CIMmultus®. The purification process can advantageously be carried out with a chromatographic support in the form of a single monolithic bed, which comprises a tube-in-a-tube system, with the outer tube and the inner tube carrying different functional parts. In such a system, one of the monolithic tubes represents the support for the chromatography principle of one step and the other tube represents the support for the chromatography principle of another step. Preferably, the capture / purification step can be operated in batch mode or quasi-continuous or continuous mode using techniques such as circular chromatography, rotating disk chromatography or simulated mobile chromatography.

[0091] As used herein, "bioreactor" or "reactor", including "seed bioreactor" or "production bioreactor", refers to a reaction vessel used for fermentation production of cells and biosynthetic products, which can range in size from a benchtop fermenter to an industrial tank. Bioreactors preferably allow for automatic regulation of the flow of oxygen, culture medium and other nutrients, and maintenance of temperature and pH; they preferably minimize the potential for contamination and can produce higher densities of cells than those produced in traditional cultures.

[0092] As used herein in the processes and systems of the present invention, "seed (bio)reactor" refers to a bioreactor used for the propagation of host cells. In a seed bioreactor, the microbial or bacterial host cells are in an uninduced / unrestricted state with respect to sulfur limitation that inhibits the growth of the host cells. Otherwise, propagation in the seed bioreactor is impossible.

[0093] A "production (bio)reactor" as used herein in the processes and systems of the invention relates to a bioreactor for the production of a nucleic acid molecule of interest.

[0094] Bioreactors often include various sensors for monitoring important parameters such as pH, dissolved oxygen or temperature and devices for controlling these parameters. Therefore, the seed bioreactor preferably includes a device for adjusting pH. The at least one production bioreactor preferably includes a device for adjusting pH. The device for adjusting pH may include a pH probe, a device for recording pH probe signals and / or a device for adding acid or alkali to the culture medium, preferably a device for adding alkali. The seed bioreactor preferably includes a device for adjusting dissolved oxygen. The at least one production bioreactor preferably includes a device for adjusting dissolved oxygen. The device for adjusting dissolved oxygen may include a pO2 probe, a control system for adjusting pO2 by pressure, ventilation rate, oxygen addition, stirring control and / or the like. The seed bioreactor preferably includes a device for adjusting temperature. The at least one production bioreactor preferably includes a device for adjusting temperature. The device for adjusting temperature may include a thermometer, a heating device, a cooling device, wherein the temperature control performed by the heating and / or cooling device is preferably performed by heating and / or cooling the housing of the bioreactor. The seed bioreactor preferably includes a gas inlet and a gas outlet and a device for adjusting gas flow. The at least one production bioreactor preferably includes a gas inlet and a gas outlet and a device for regulating gas flow. The at least one production bioreactor preferably includes a biomass sensor. The biomass concentration in the seed bioreactor can be regulated by a feed inflow and / or a biomass effluent. The biomass concentration in the at least one production bioreactor can be regulated by a feed inflow, a biomass inflow and / or a biomass effluent. The gas flow can be regulated in the at least one production bioreactor. The gas flow can be regulated in the at least one seed bioreactor. The seed bioreactor and the production bioreactor are connected or coupled to allow microorganisms or bacterial host cells to be transferred from the seed bioreactor to the production bioreactor. The flow of the culture medium containing the host cells from the seed bioreactor to the production bioreactor is preferably controlled. This control can be carried out, for example, by applying a chemostat or a turbidostat. Therefore, the system of the present invention preferably also includes (c) a device for operating the seed bioreactor and the production bioreactor as a chemostat or a turbidostat connected. Preferably, the seed bioreactor effluent serves as the inflow of the production bioreactor.

[0095] The bioreactor of the present invention, i.e. the seed bioreactor and / or the production bioreactor, may be any bioreactor suitable for the purposes of the present invention. In one embodiment, the seed bioreactor is a stirred tank bioreactor. In one embodiment, the seed bioreactor is a plug flow bioreactor. In one embodiment, the production bioreactor is a stirred tank bioreactor. In one embodiment, the production bioreactor is a plug flow bioreactor.

[0096] As used herein according to the present invention, "growth" of (host) cells as used herein generally means an increase in cell number due to cell division, unless otherwise indicated herein.

[0097] As used herein, "continuous fermentation" may be related to operating conditions, wherein a liquid culture medium, such as a culture or nutrient medium, or a cell broth is added to a fermentor tank, while the culture fluid is released from the fermentor tank. The inflow of the liquid culture medium is preferably constant or intermittent at a certain rate. The outflow of the culture fluid is preferably at the same rate, so that the amount of liquid in the fermentor tank remains substantially constant. During the continuous fermentation, the biomass preferably remains substantially unchanged. Under a stable state, fermentation conditions such as nutrient concentration, product concentration, constant pH, biomass can be maintained as required. The bioreactor for continuous fermentation can be a stirred tank bioreactor or a tubular bioreactor. "Continuous fermentation tank" as used herein is preferably a fermentor tank suitable for continuous fermentation.

[0098] For example, the (initial) start of the continuous process according to the present invention can be carried out as follows. Both the seed bioreactor and the production bioreactor can be started in the form of batch culture, followed by batch feeding until the necessary biomass concentration is reached. Therefore, during the start of the process of the present invention or in other words during the start, the host cells are preferably (in both the seed bioreactor and the production bioreactor) in an uninduced state with respect to the limitation of sulfur in the culture medium. When the required cell density (e.g., logarithmic phase) is reached, the production in the production bioreactor can be induced by limiting the amount of sulfur in the culture medium, in other words: the production bioreactor is now in an induced state with respect to the amount of sulfur. In addition, the continuous process can be started in the following way: the host cells are transferred from the seed bioreactor to the production bioreactor, for example, by the culture medium starting to flow into the seed bioreactor, thereby producing a flow from the seed bioreactor outlet to the production bioreactor inlet. Therefore, in the continuous process of the present invention, cell growth is preferably inhibited in the production bioreactor.

[0099] Equally, also as envisioned in the context of the present invention, fed-batch method can be applied.Fed-batch process is well known in the art and also illustrated in this article, and generally comprises wherein in the same bioreactor, carries out different steps (for example, feed supplement and cultivation) and wherein product (for example, nucleic acid molecule) is retained in the bioreactor until the process of cultivating ends.Batch fed fermentation may be preferred for the cultivation and cultivation process of the specific amount or concentration of concrete nutrients or culture components that should be controlled wherein.In one embodiment of the invention, batch fed fermentation is applied in the method described and provided herein.

[0100] Preferably, in the methods described and provided in the context of the present invention, the host cell may comprise a recombinant extrachromosomal nucleic acid (eg, a plasmid, a bacmid, a cosmid or a minicircle, preferably a plasmid).

[0101] In the context of the present invention, the nucleic acid molecule to be produced can be a recombinant extrachromosomal nucleic acid (e.g., a plasmid, a bacmid, a cosmid or a minicircle, preferably a plasmid). In one embodiment of the present invention, the nucleic acid (e.g., a recombinant extrachromosomal nucleic acid, such as a plasmid, a bacmid, a cosmid or a minicircle, preferably a plasmid) comprises a promoter sequence.

[0102] As used herein, "promoter sequence" is a non-coding expression control sequence, preferably inserted near the starting point of the expression cassette coding sequence, and regulates its expression. In a simple but substantially correct way, it is the interaction between the promoter and various specialized proteins called transcription factors that determines whether a given coding sequence can be transcribed and ultimately translated into the actual protein encoded by the gene. Those skilled in the art will recognize that any compatible promoter can be used for recombinant expression in a host cell. The promoter itself can be preceded by an upstream activation sequence, an enhancer sequence, or a combination thereof. These sequences are known in the art to be any DNA sequence that shows strong transcriptional activity in cells, and are derived from genes encoding extracellular or intracellular proteins. Those skilled in the art will also recognize that termination sequences and polyadenylation sequences can be suitably derived from the same source as the promoter.

[0103] As used herein, the term "inducible promoter" refers to a promoter that regulates the expression of an operably linked gene or functional RNA in response to the presence or absence of an endogenous stimulus or an exogenous stimulus. Such stimuli can be, but are not limited to, chemical compounds or environmental signals, such as temperature changes. Examples of inducible promoters include, but are not limited to, pBAD, OR2-OR1-PR, pltetO, pllacO, PesR, plac, lacUV, tac promoter, pPrpB, pTetO, FixK2, pltetO-1, or PcpcG2.

[0104] Nucleic acid molecules (e.g., recombinant extrachromosomal nucleic acids, such as plasmids, bacmids, cosmids or minicircles, preferably plasmids) to be produced according to the invention described and provided herein are capable of autonomous replication in a host cell (e.g., vectors with a replication origin that function in a host cell). Nucleic acid molecules can have linear, circular or supercoiled configurations and can be complexed with other nucleic acids or other materials for certain purposes.

[0105] Nucleic acid molecules to be produced according to the present invention described and provided herein (e.g., recombinant extrachromosomal nucleic acids, such as plasmids, bacmids, cosmids or minicircles, preferably plasmids) can also be suitable as vectors. The vector generally contains transcription control elements suitable for driving transcription, such as promoters, enhancers, polyadenylation signals, transcription pauses or termination signals, as elements of expression cassettes. For the correct expression of the polypeptide, suitable translation control elements are preferably included in the vector, such as preferably an optimized 5' non-translated region leading to a ribosome binding site (RBS), such as a Kozak sequence for protein translation initiation, suitable for recruiting ribosomes and stop codons to terminate the translation process. Specifically, the nucleotide sequence serving as a selective marker gene and the nucleotide sequence encoding a protein of interest can be transcribed under the control of a transcription element present in an appropriate promoter. The resulting transcript of the selective marker gene and the resulting transcript of the protein of interest carry a functional translation element that promotes the substantial level of protein expression (i.e., translation) and appropriate translation termination. In one embodiment of the invention, the selective marker used is an auxotrophic marker gene used in a host strain, and the host strain contains a knockout or deletion of the auxotrophic marker gene. The vector may contain a polylinker (multiple cloning site, MCS), a short DNA segment containing many restriction sites, which is a standard feature of many plasmids used for molecular cloning. Multiple cloning sites usually contain more than 5, 10, 15, 20, 25 or more than 25 restriction sites. The restriction sites in an MCS are usually unique (i.e., they appear only once in a particular plasmid). MCS is often used in procedures involving molecular cloning or subcloning.

[0106] In the context of the present invention described and provided herein, one type of vector can be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments can be introduced via connection or by unrestricted cloning. Other nucleic acid molecules (e.g., recombinant extrachromosomal nucleic acids, such as plasmids, bacmids, cosmids or minicircles, preferably plasmids) to be produced according to the present invention described and provided herein that may also be suitable as vectors include cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) or minichromosomes. Another type of vector is a viral vector, in which additional DNA segments can be linked to the viral genome. An expression cassette can be inserted into an expression vector as a DNA construct. The DNA construct can be made by recombining synthetic DNA molecules, genomic DNA molecules, cDNA molecules or a combination thereof. The DNA construct is preferably made by connecting different fragments to each other according to standard techniques known in the art. An expression cassette can be a part of an expression vector. Preferably, the expression vector is a DNA vector. The vector can conveniently contain sequences that promote the correct expression of genes of interest and antibiotic resistance genes. These sequences generally include, but are not limited to, promoter sequences, transcription start sites, transcription termination sites, and polyadenylation functional sequences as described herein. The expression cassette may comprise an enhancer and / or an intron. Typically, an intron is placed at the 5' end of an open reading frame. Therefore, in order to increase the expression rate, an intron may be included in the expression cassette for expressing a polypeptide of interest. The intron may be located between a promoter and or a promoter / enhancer element and the 5' end of the open reading frame of the polypeptide to be expressed. Several suitable introns are known in the prior art and can be used in conjunction with the present invention. The expression cassette or vector according to the present invention present in the host may be integrated into the genome of the host, or may be maintained outside the chromosome in some form. In addition, the expression cassette may comprise an appropriate transcription termination site. This is because continued transcription from an upstream promoter to a second transcription unit may inhibit the function of a downstream promoter, a phenomenon known as promoter blocking or transcription interference. This event has been described in both prokaryotes and eukaryotes. The correct placement of a transcription termination signal between two transcription units can prevent promoter blocking. Transcription termination sites are well characterized, and it has been demonstrated that their incorporation into expression vectors has a variety of beneficial effects on gene expression.

[0107] The terms "5'" and "3'" used herein refer to the convention for describing the nucleotide sequence features related to the position and / or event direction (5' to 3') of genetic elements, such as the transcription of RNA polymerase or the translation of ribosomes carried out in the 5' to 3' direction. Synonyms include upstream (5') and downstream (3'). By convention, nucleotide sequences, gene maps, vector cards and RNA sequences are drawn from left to right with 5' to 3', or the 5' to 3' direction is indicated by an arrow, wherein the arrow points to the 3' direction. Therefore, when following this convention, 5' (upstream) indicates the genetic element positioned towards the left hand side, and 3' (downstream) indicates the genetic element positioned towards the right hand side.

[0108] As used herein, the term "expression" preferably means the transcription of a nucleotide sequence. The nucleotide sequence preferably encodes a protein. Therefore, the term also includes the production of mRNA (as a transcription product of the nucleotide sequence) and the translation of the mRNA to produce a corresponding gene product, such as a polypeptide or protein.

[0109] In another embodiment of the methods described and provided in the context of the present invention, the method may be a method for producing a recombinant extrachromosomal nucleic acid (eg, a plasmid, a bacmid, a cosmid or a minicircle, preferably a plasmid).

[0110] The present invention also relates to the use of a culture medium comprising a predetermined amount of total sulfur per gram of dry weight of host cells desired to be obtained in said cell culture, which is approximately equal to or less than the amount of sulfur per gram of dry weight of a reference host cell, preferably when grown without limitation of total sulfur, for increasing the yield of nucleic acids, preferably extrachromosomal nucleic acids, comprised by said host cells in a cell culture.

[0111] As used herein, unless explicitly defined otherwise, the terms "nucleic acid" or "nucleic acid molecule" are used synonymously, and may also include "oligonucleotide", "nucleic acid chain", etc., and mean a polymer comprising one, two or more nucleotides arranged in a single-stranded or double-stranded nucleotide chain.

[0112] Generally, as used according to the present invention, nucleic acid molecules can especially include DNA molecules, cDNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribo-oligonucleotides or PNA molecules, preferably DNA molecules or cDNA molecules. In addition, the term "nucleic acid molecules" can refer to DNA or RNA or its hybrid or any modification thereof known in the art (see, for example, modification examples in US5525711, US 471 1955, US 5792608 or EP 302175). The polynucleotide sequence can be single-stranded or double-stranded, linear or circular, natural or synthetic, and without any size restriction. For example, the polynucleotide sequence can be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozyme RNA or the DNA (Gamper, Nucleic Acids Research, 2000, 28, 4332 - 4339) encoding such RNA or chimera. The polynucleotide sequence can be the form of a vector, a plasmid or viral DNA or RNA. Also described herein are nucleic acid molecules complementary to the above nucleic acid molecules and nucleic acid molecules capable of hybridizing with the nucleic acid molecules described herein. The nucleic acid molecules described herein can also be fragments of nucleic acid molecules in the context of the present invention. In particular, such fragments are functional fragments. Examples of such functional fragments are nucleic acid molecules that can serve as primers.

[0113] According to the present invention, nucleic acid molecules can be chemically or biochemically modified, or can contain non-natural or derived nucleotide bases, which will be readily understood by those skilled in the art. Such modifications include, for example, tags, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), overhangs (e.g., polypeptides), intercalators (e.g., acridine, psoralens, etc.), chelators, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). Also included are synthetic molecules that simulate the ability of polynucleotides to bind to a specified sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, molecules in which peptide linkages replace phosphate bonds in the molecular backbone. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpin-shaped, circular, or in a locked conformation.

[0114] Embodiments that characterize the invention are described herein, shown in the drawings, exemplified in the examples, and reflected in the claims.

[0115] It must be noted that, as used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an agent" includes one or more of such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art, which steps and methods may be modified or substituted for the methods described herein.

[0116] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only routine experiments. Such equivalents are intended to be encompassed by the present invention.

[0117] The term "and / or" wherever used herein includes the meanings of "and," "or," and "all or any other combinations of the elements connected by the term."

[0118] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% or 2% of a given value or range, and also includes the corresponding exact numerical values.

[0119] Unless the context requires otherwise, throughout this specification and the claims that follow, the word "comprise" and variations should be understood to imply the inclusion of the stated integer or step or groups of multiple integers or steps, but not the exclusion of any other integer or step or groups of multiple integers or steps. When used herein, the term "comprise" may be replaced with the term "contains" or "includes", or, as is sometimes the case herein, with the term "having".

[0120] When used herein, "consisting of excludes any element, step or ingredient not specified in the claim elements. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0121] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of may be replaced with either of the other two terms.

[0122] It should be understood that the present invention is not limited to the particular methodology, protocols, reagents, etc. described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the claims.

[0123] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include plural terms and plural terms shall include singular terms. The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art. Typically, the terms used in conjunction with biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization techniques described herein are well known and commonly used terms in the art.

[0124] Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, J, Greene Publishing Associates (1992, and 2002 Supplement); Handbook of Biochemistry: Section A Proteins, Vol. 1, 1976, CRC Press; Handbook of Biochemistry: Section A Proteins, Vol. II, 1976, CRC Press. The nomenclature and laboratory procedures and techniques used in conjunction with the molecular and cell biology, protein biochemistry, enzymology, and medicinal chemistry described herein are those well known and commonly used in the art.

[0125] All publications and patents cited throughout this specification (including all patents, patent applications, scientific publications, manufacturer specifications, specifications, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as allowing the present invention to antedate this disclosure without authorization by virtue of prior invention. If material incorporated by reference contradicts or is inconsistent with this specification, this specification supersedes any such material.

[0126] The present invention also relates to the following items:

[0127] 1. A method for increasing the yield of nucleic acids, preferably extrachromosomal nucleic acids, contained in host cells in cell culture, the method comprising the step of limiting the total sulfur content in the medium of the cell culture during cell culture so that the total sulfur content, per gram of predetermined cell dry weight of the host cells desired to be obtained in the cell culture, is approximately equal to or less than the amount of sulfur contained per gram dry weight of a reference host cell when preferably grown without total sulfur restriction, thereby increasing the yield of the nucleic acid.

[0128] 2. The method of item 1, wherein the limiting step comprises

[0129] (a1) predetermining the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and

[0130] (b1) adding to the cell culture a predetermined total amount of sulfur per gram of dry weight of the host cells at the start of the cell culture, wherein the total amount of sulfur is approximately equal to or less than the amount of sulfur contained in the reference host cells per gram of dry weight.

[0131] 3. The method of item 1, wherein the limiting step comprises

[0132] (a2) predetermining the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and

[0133] (b2) during the cell culturing process, reducing the amount of total available sulfur in the cell culture per gram dry weight of the host cells to at least or less than the amount of sulfur contained per gram dry weight of the reference host cells.

[0134] 4. The method of item 1, wherein the limiting step comprises

[0135] (a3) predetermining the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and

[0136] (b3) adding to the cell culture during the cell culturing—from the beginning until a desired amount of host cells comprising the nucleic acid is obtained—a total sulfur amount per gram of dry weight of the host cells, a predetermined amount, which is approximately equal to or less than the amount of sulfur contained in the reference host cells per gram of dry weight.

[0137] 5. A method as described in any one of items 1 to 4, wherein when the predetermined total sulfur amount decreases to 0% of the predetermined total sulfur amount in the culture medium, more sulfur is added to the culture medium.

[0138] 6. A method as described in any one of items 1 to 4, wherein when the predetermined total sulfur amount decreases to 50% or 25% or less of the predetermined total sulfur amount in the culture medium, more sulfur is added to the culture medium.

[0139] 7. The method of any of the preceding items, wherein the amount of sulfur contained per gram dry weight of the reference host cell is about equal to or less than the amount of sulfur contained in the elemental composition per gram dry weight of the reference host cell.

[0140] 8. The method of any of the preceding items, wherein the reference host cell is identical to the host, except that it does not contain the nucleic acid.

[0141] 9. The method of any of the preceding items, wherein the reference host cell is grown in the same culture medium as the host cell, except that the total sulfur amount is limited.

[0142] 10. The method according to any one of the preceding items, wherein the predetermining of the dry cell weight of the host cells is achieved by obtaining a sample of the cell culture medium of a defined volume, harvesting the host cells and drying the host cells.

[0143] 11. The method of any of the preceding items, wherein limiting the total sulfur in the culture medium of the cell culture occurs when the host cells are in logarithmic phase.

[0144] 12. The method of any of the preceding items, wherein the total sulfur in the culture medium of the cell culture is limited when the host cells have a dry weight of at least about 5 g / liter of culture medium, preferably at least about 10 g or at least about 20 g / liter of culture medium.

[0145] 13. The method of any of the preceding items, wherein the extrachromosomal nucleic acid is a bacmid, a cosmid, a plasmid or a minicircle.

[0146] 14. The method of any of the preceding items, wherein the plasmid is a high copy number plasmid, such as a high copy number plasmid having a pMB1 derived replication origin (ori), ColE1 ori, pUC or F1 ori.

[0147] 15. The method of any of the preceding items, wherein the host cell is a fungal or prokaryotic host cell.

[0148] 16. A method as described in any of the preceding items, wherein the host cell is a recombinant host cell.

[0149] 17. The method according to any one of the preceding items, wherein the method is a method for continuously culturing the host cell in a fermenter.

[0150] 18. A method as described in any of the preceding items, wherein the host cell comprises a recombinant extrachromosomal nucleic acid (e.g., a plasmid).

[0151] 19. The method according to any one of the preceding items, wherein the method is a method for producing a recombinant extrachromosomal nucleic acid (eg, a plasmid).

[0152] 20. Use of a culture medium comprising a predetermined amount of total sulfur per gram of dry weight of host cells desired to be obtained in said cell culture, said total sulfur amount being approximately equal to or less than the amount of sulfur per gram of dry weight of a reference host cell preferably when grown without limitation of said total sulfur amount, for increasing the yield of nucleic acids, preferably extrachromosomal nucleic acids, comprised by said host cells in a cell culture.

[0153] Attached photos

[0154] The accompanying drawings show:

[0155] Figure 1 Shown is the cell dry mass (CDM) of the bacterial culture according to the inventive method, directly compared with the control bacterial culture without S restriction. It can be seen that the CDM of the control culture (without S restriction) is constantly increasing, while the CDM of the culture under S restriction decreases about 23 hours after the start of cultivation (including the start of feed supplement). At this stage, S becomes limited and the bacterial cells no longer obtain biomass, but produce desired nucleic acids, such as plasmids. Therefore, once S becomes limited, it can be said that metabolism is decoupled from the production of desired nucleic acids (such as plasmids).

[0156] In the figures, "lin / S-lim" means a process in which sulfur supply was limited and a linear feed scheme was used; "lin / control" means the same linear feed scheme but without sulfur limiting conditions; "average" reflects that the data points refer to triplicates, and the data are reflected as the average.

[0157] Figure 2 The plasmid DNA (pDNA) quality of bacterial cultures according to the method of the present invention is shown, directly compared with control bacterial cultures without S restriction. It can be seen that the quality of pDNA produced by the culture under S restriction increases significantly from about 25.5 hours to 27 hours, especially after 27 hours, while the quality of pDNA produced by the control culture continues to decrease.

[0158] exist Figure 2In the figures, “lin / S-lim” means a process in which sulfur supply is limited and a linear feed scheme is used; “lin / control” means the same linear feed scheme but without sulfur limiting conditions; “average” reflects that the data points refer to triplicates, and the data are reflected as the average.

[0159] Figure 3 The productivity of covalently closed circular (ccc) DNA of bacterial cultures according to the methods of the present invention is shown, in direct comparison with control bacterial cultures without S limitation. It can be seen that when S becomes limiting, ccc DNA productivity increases significantly, while productivity decreases in control cultures without S limitation.

[0160] exist Figure 3 In the figures, “lin / S-lim” means a process in which sulfur supply is limited and a linear feed scheme is used; “lin / control” means the same linear feed scheme but without sulfur limiting conditions; “average” reflects that the data points refer to triplicates, and the data are reflected as the average.

[0161] Figure 4 The volumetric yield of ccc DNA achieved by bacterial cultures according to the methods of the present invention is shown, in direct comparison with control bacterial cultures without S limitation. It can be seen that when S becomes limiting, the ccc DNA yield increases, while the yield decreases in the control culture without S limitation.

[0162] exist Figure 4 In the figures, “lin / S-lim” means a process in which sulfur supply is limited and a linear feed scheme is used; “lin / control” means the same linear feed scheme but without sulfur limiting conditions; “average” reflects that the data points refer to triplicates, and the data are reflected as the average.

[0163] Figure 5 An illustrative example of a sulfur / demand calculation is shown.

[0164] The sulfur / demand calculation is described in Example 5.

[0165] Figure 6A to Fig.6D It was shown that when the predetermined total sulfur amount was reduced to 0%, adding more sulfur, such as adding 50% or 25% of the predetermined total sulfur amount, still showed an increase in productivity ( FIG. 6A ), volumetric yield ( FIG. 6B ), and the quality of the desired nucleic acid ( FIG. 6C ) and cell dry mass ( FIG. 6D ) compared to the control. Fig.6D ) remains unchanged.

[0166] The present invention is further illustrated by the following examples. However, the examples and specific embodiments described herein are not to be construed as limiting the present invention to these specific embodiments. Example

[0167] Materials and methods

[0168] Fed-batch fermentation

[0169] For fed-batch fermentation, E. coli cells, preferably E. coli JM108, were grown in a 2.1 L (1.0 L net volume, 0.5 L batch volume) computer-controlled bioreactor (DASGIP parallel bioreactor system, Eppendorf AG, Germany). The bioreactor was equipped with a pH probe (Hamilton Bonaduz AG, Switzerland) and an optical DO probe (Hamilton Bonaduz AG). The pH was maintained at 7.0 ± 0.1 by adding 12.5% ​​ammonia solution (Carl Roth, Germany); the temperature was maintained at 37 ± 0.5 ° C. The dissolved oxygen (O2) level was stabilized above 30% saturation by controlling the agitator speed, aeration rate and aeration composition. Foaming was suppressed by adding 2 mL of 1:10 diluted Struktol J673A antifoam suspension (Schill+Seilacher, Germany) to the batch medium and by automatic addition of 1:10 diluted Struktol J673A controlled by a conductivity operated level sensor. For inoculation of the bioreactor, a seed culture (25 mL batch medium inoculated with 250 µL MCB in a 250 mL baffled glass flask, shaken at 180 rpm at 37°C) was used. The seed culture was incubated until a final OD600 of 2-4 was reached and a defined volume was aseptically transferred to the bioreactor to reach an initial ODini of 0.015.

[0170] The fermentation process was designed to obtain a final amount of 50 g CDM, of which 1.51 g was obtained in a batch volume of 500 mL and 48.5 g was obtained in the feeding phase by adding another 500 mL of feed medium. The amount of glucose for the specific medium was calculated based on a yield factor (Yx / s) of 0.303 g / g and added as C6H12O6·H2O. For the medium preparation, all chemicals were purchased from Carl Roth GmbH (Germany) unless otherwise stated.

[0171] The specific pDNA productivity was compared between two different media compositions: one with sulfur source limitation (S-limited) and one without sulfur source limitation (control).

[0172] S-restricted medium

[0173] Depending on the grams of CDM formed in the batch stage, the following components were added followed by sterile filtration: For batch medium preparation, the following components were added followed by sterile filtration: 0.05 g yeast extract (Bacto yeast extract), 1.00 g NH4Cl, 0.12 g MgSO4*7 H2O, 2.72 g KH2PO4, 3.69 g Na2HPO4*2 H2O, 5 g glucose monohydrate, 0.1 g L-proline, 0.1 g L-isoleucine, 0.05 mL of 1% thiamine-HCl solution, 1.0 g citric acid (anhydrous), 8.34 mL trace element solution. Based on the grams of CDM formed during the feeding phase, sterile filtered feed medium consisted of the following components: 2.72 g KH2PO4, 3.69 g Na2HOP4*2 H2O, 169.94 g glucose monohydrate, 3.0 g L-proline, 3.0 g L-isoleucine, 1.0 g citric acid (anhydrous), 1.50 mL of 1% thiamine HCl stock solution, 25.0 mL trace element solution, 0.42 g MgCl2*7H2O; a total of 9.6 mL of 200 g / L MgSO4*7 H2O stock solution was pulsed into the bioreactor (3.2 mL at the start of feeding, 3.2 mL 7 hours after the start of feeding, and 3.2 mL 15 hours after the start of feeding).

[0174] Control medium:

[0175] Depending on the grams of CDM formed in the batch stage, the following components were added, followed by sterile filtration: For batch medium preparation, the following components were added, followed by sterile filtration: 0.05 g yeast extract (Merck), 1.00 g NH4Cl, 0.12 g MgSO4*7 H2O, 2.72 g KH2PO4, 3.69 g Na2HOP4*2 H2O, 5 g glucose monohydrate, 0.1 g L-proline, 0.1 g L-isoleucine, 0.05 mL of 1% thiamine-HCl solution, 1.0 g citric acid (anhydrous), 8.34 mL trace element solution. Based on the grams of CDM formed during the feed phase, the sterile filtered feed medium consisted of the following components: 3.60 g MgSO4*7 H2O, 2.72 g KH2PO4, 3.69 g Na2HOP4*2 H2O, 164.94 g glucose monohydrate, 3.0 g L-proline, 3.0 g L-isoleucine, 1.0 g citric acid (anhydrous), 1.50 mL of 1% thiamine HCl stock solution, 25.0 mL trace element solution. The trace element solution was prepared in 5M HCl and contained (g / L): 4.41 g CaCl2*2H2O, 3.34 g FeSO4*7H2O, 1.43 g CoCl2*6H2O, 1.03 g MnSO4*H2O, 0.15 g CuSO4*5H2O, 0.17 g ZnSO4*7H2O.

[0176] When the culture in the batch medium entered the stationary phase, feeding was started. A fed-batch protocol with linear substrate feeding (0.26 g / min, corresponding to 13.91 mL / hour) was used for 35 hours (five generations). During the S-limited fermentation, the provided sulfur was consumed 23 hours after the start of feeding.

[0177] All incubations were performed in triplicate, except for those shown in Figure 6. All graphs and tables use mean values.

[0178] Offline analysis

[0179] Bioreactors for offline analysis (OD600, CDM, product) were sampled during the fed-batch phase. OD600 was measured using an Ultrospec 500 pro spectrophotometer (Amersham Biosciences, UK), and samples were diluted with phosphate-buffered saline to reach the linear range of the measurement. For CDM determination, 1 mL of cell suspension was transferred to a pre-weighed 2.0 mL reaction tube and centrifuged at 16,100 rcf at 4 °C for 10 min using an Eppendorf 5415 R centrifuge. The supernatant can be transferred to another reaction tube and stored at -20 °C for further analysis. As a washing step, the cell pellet was resuspended in 1.8 mL RO-H2O, centrifuged, and the supernatant discarded. Afterwards, the pellet was resuspended in 1.8 mL RO-H2O and finally dried at 105 °C for 24 h. The reaction tubes were cooled to room temperature in a desiccator before they were weighed again.

[0180] For pDNA product analysis, estimate the sampling volume of the cell suspension corresponding to 20 mg CDM by directly measuring OD600. Transfer the calculated amount to a 2.0 mL reaction tube and centrifuge at 16,100 rcf for 10 minutes at 4°C. Discard the supernatant and store the cell pellet at -20°C.

[0181] pDNA analysis

[0182] The content of pDNA in ccc conformation was determined using AIEX-HPLC (CIMac™ pDNA-0.3 analytical column, 1.4 µm; BIA Separations doo, Slovenia). For HPLC analysis, cell decomposition was performed by alkaline lysis method (Birnboim and Doly, 1979). The obtained lysate was directly analyzed by HPLC (Agilent 1100 with quaternary pump, diode array detector (DAD). The coefficient of variation of the values ​​obtained from three biological replicates was less than 10%.

[0183] Example 1 - Cell Dry Mass (CDM)

[0184] exist Figure 1 As can be seen in the graph, the CDM of the control culture (without S limitation) continues to increase, while the CDM of the culture under S limitation (i.e., the culture according to the method of the present invention) decreases around 23 hours after the start of the culture. At this stage, S becomes limited and the bacterial cells no longer obtain biomass, but instead produce the desired nucleic acid, such as a plasmid. Therefore, once S becomes limited, it can be said that metabolism is decoupled from the production of the desired nucleic acid (e.g., a plasmid).

[0185] Example 2 - Plasmid DNA (pDNA) Quality

[0186] exist Figure 2 As can be seen from the graph, the mass of the plasmid DNA (pDNA) produced by the culture under S limitation, i.e., the culture according to the method of the present invention, increased significantly at about 25.5 hours, while the mass of the pDNA produced by the control culture continued to decrease.

[0187] Example 3 - Productivity of covalently closed circular (ccc) DNA

[0188] In direct comparison with control bacterial cultures without S limitation, the productivity of covalently closed circular (ccc) DNA in bacterial cultures according to the methods of the present invention increases significantly when S becomes limiting; see Figure 3 In contrast, in control cultures without S limitation, ccc DNA productivity was reduced.

[0189] Example 4- Volumetric Yield of ccc DNA

[0190] In direct comparison with control bacterial cultures without S restriction, bacterial cultures according to the methods of the present invention achieve an increased volumetric yield of ccc DNA, whereas in control cultures the volumetric yield is reduced; see Figure 4 .

[0191] Example 5 - Method for determining sulfur requirements for growth

[0192] - Plate cells on agar plates and grow them overnight

[0193] - Prepare, for example, 120 ml of sulfur-free medium for a shake flask; for example, "medium for S limitation" as described herein

[0194] - Add different amounts of sulfur stock solution, such as MgSO4 or (NH4)2SO4, to the shake flask, e.g. 0, 25 µl, 50 µl, 100 µl, etc. of sulfur stock solution

[0195] - Inoculate the plated cells (taking care to minimize sulfur from the pre-culture)

[0196] - Allow cells to grow for at least 24 hours or until maximum OD600 is reached; maximum OD600 is reached when the OD600 variation over time is at most +0.5 OD600 to -0.5 OD600 units

[0197] - Determination of the biomass content of each shaker by gravimetric analysis

[0198] The sulfur content / demand can be calculated as follows:

[0199] - Plot cell dry mass (CDM) versus calculated / added sulfur content in shake flasks

[0200] - Perform linear regression

[0201] - Can calculate grams of sulfur per gram of biomass

[0202] - Sulfur requirement is, for example, 0.0085 g S / g CDM, e.g. Figure 5 Illustrative example of sulfur requirement calculation.

Claims

1. A method for increasing the yield of a nucleic acid, preferably an extrachromosomal nucleic acid, contained in a host cell in a cell culture, the method comprising the step of limiting the total sulfur content in the medium of the cell culture during the cell culture process such that the total sulfur content, per gram of predetermined dry cell weight of the host cells desired to be obtained in the cell culture, is approximately equal to or less than the amount of sulfur contained per gram dry weight of a reference host cell when preferably grown without total sulfur restriction, thereby increasing the yield of the nucleic acid.

2. The method of claim 1, wherein the limiting step comprises (a1) predetermining the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and (b1) adding to the cell culture a predetermined total amount of sulfur per gram of dry weight of the host cells at the start of the cell culture, wherein the total amount of sulfur is approximately equal to or less than the amount of sulfur contained in the reference host cells per gram of dry weight.

3. The method of claim 1, wherein the limiting step comprises (a2) predetermining the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and (b2) during the cell culturing process, reducing the amount of total available sulfur in the cell culture per gram dry weight of the host cells to at least or less than the amount of sulfur contained per gram dry weight of the reference host cells.

4. The method of claim 1, wherein the limiting step comprises (a3) predetermining the amount of dry cell weight of the host cells desired to be obtained in the cell culture; and (b3) adding to the cell culture during the cell culturing—from the beginning until a desired amount of host cells comprising the nucleic acid is obtained—a total sulfur amount per gram of dry weight of the host cells, a predetermined amount, which is approximately equal to or less than the amount of sulfur contained in the reference host cells per gram of dry weight.

5. The method of any one of claims 1 to 4, wherein when the predetermined total sulfur amount decreases to 0% of the predetermined total sulfur amount in the culture medium, more sulfur is added to the culture medium.

6. The method of any one of claims 1 to 4, wherein when the predetermined total sulfur amount decreases to 50% or 25% or less of the predetermined total sulfur amount in the culture medium, more sulfur is added to the culture medium.

7. The method of any of the preceding claims, wherein the amount of sulfur contained per gram dry weight of the reference host cell is about equal to or less than the amount of sulfur contained in the elemental composition per gram dry weight of the reference host cell.

8. The method of any one of the preceding claims, wherein the reference host cell is identical to the host cell, except that it does not comprise the nucleic acid.

9. The method of any one of the preceding claims, wherein the reference host cell is grown in the same culture medium as the host cell, except that the total sulfur is limited.

10. The method of any one of the preceding claims, wherein predetermining the dry cell weight of the host cells is achieved by obtaining a sample of the cell culture medium of a defined volume, harvesting the host cells and drying the host cells.

11. The method of any one of the preceding claims, wherein limiting the total sulfur in the culture medium of the cells occurs when the host cells are in log phase.

12. The method of any one of the preceding claims, wherein the total sulfur in the culture medium of the cell culture is limited when the host cells have a dry weight of at least about 5 g / liter of culture medium, preferably at least about 10 g or at least about 20 g / liter of culture medium.

13. The method of any one of the preceding claims, wherein the extrachromosomal nucleic acid is a bacmid, a cosmid, a plasmid or a minicircle.

14. The method of any one of the preceding claims, wherein the host cell is a fungal or prokaryotic host cell.

15. The method according to any one of the preceding claims, wherein the method is a method for continuous cultivation of the host cell in a fermenter.

16. A method as claimed in any one of the preceding claims, wherein the host cell comprises a recombinant extrachromosomal nucleic acid, such as a plasmid.

17. Use of a culture medium comprising a predetermined amount of total sulfur per gram of dry weight of host cells desired to be obtained in said cell culture, said total sulfur amount being approximately equal to or less than the amount of sulfur contained per gram of dry weight of a reference host cell, preferably when grown without limitation of said total sulfur, for increasing the yield of a nucleic acid, preferably an extrachromosomal nucleic acid, comprised by said host cells in a cell culture.

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