Heterotrophic physcomitrium patens cultures and uses thereof

Abstract

The present invention relates to Physcomitrium patens cultures capable of growing under heterotrophic conditions essentially without light or without light, methods of growing, generating and genetically modifying such cultures as well as to methods of using such cultures for producing biotechnological products of interest. The invention also relates to cells, protoplasts, protonemas, gametophytes, sporophytes or spores of such P. patens cultures.

Description

Heterotrophic Physcomitrium patens cultures and uses thereof

[0001] The present invention relates to Physcomitrium patens cultures capable of growing under heterotrophic conditions essentially without light or without light, methods of growing, generating and genetically modifying such cultures as well as to methods of using such cultures for producing biotechnological products of interest. The invention also relates to cells, protoplasts, protonemas, gametophytes, sporophytes or spores of such P. patens cultures.

[0002] The bryophyte Physcomitrium patens (synonym Physcomitrella patens) is a well- established model organism used in research laboratories across the world. Moreover, P. patens is one of the few organisms which are known for highly efficient homologous recombination. This allows the targeted deletion and alteration of genes, which makes the moss a useful tool in biotechnology, for example for the production of biopharmaceuticals. A further advantage of using P. patens for production of biopharmaceuticals is that it is capable of synthesizing proteins with posttranslational modifications closely resembling mammalian modifications while for example eliminating any risk of product contamination with mammalian pathogens.

[0003] However, currently there are limitations to using P. patens for biotechnological applications. One of the drawbacks of using P. patens for biotechnological purposes is that it is growing in principle autotroph, i.e. it requires light for cultivation. This inherently restricts the volume of any bioreactor for P. patens, as light will only be fully available at the culture surface, while penetration of deeper regions of the culture is problematic. Moreover, while conventional and wide spread biotechnological expression systems like bacterial or mammalian expression systems grow heterotrophic, with a broad range of materials and media readily available on the market, the autotrophic mode of growth of P. patens requires custom-made media and materials. P. patens in turn cannot be grown heterotrophic (i.e. without light) over prolonged periods of time (see Fig. 1). This fact additionally significantly increases the costs of using P. patens for biotechnological purposes as compared to conventional heterotrophic expression systems.

[0004] Therefore, there was clearly a need in the art for improved P. patens expression systems which overcome the above-mentioned disadvantages.

[0005] This problem is solved by the subject-matter as set forth in the appended claims and in the description below.

[0006] As will be shown in the following, the inventors of the present invention have surprisingly found that P. patens cultures can be generated, which can grow heterotrophic over long periods of time without light. Moreover, these cultures retain all the established positive features of P. patens, i.e. are highly efficient in integrating foreign DNA in its genome and capable of expressing recombinant proteins. This dramatically increases the utility of P. patens as biotechnological tool and overcomes the problems mentioned above. The inventors have achieved this by subjecting P. patens cultures to mutagenesis in the hope that mutated P. patens cultures would arise which are capable of growing under heterotrophic conditions. Surprisingly, selection for P. patens cultures capable of growing under heterotrophic conditions essentially without light or without light for prolonged periods of time indeed yielded such advantageous cultures.

[0007] Therefore, the present invention relates in a first aspect to a method of growing a culture of the moss Physcomitrium patens (P. patens), wherein the culture is a culture capable of growing under heterotrophic conditions, and wherein the method comprises the step of culturing said P. patens culture under conditions allowing growth of said P. patens culture.

[0008] In a second aspect, the present invention relates to a method of producing a culture of the moss P. patens capable of growing under heterotrophic conditions essentially without light or without light, the method comprising the following steps: subjecting a culture of the moss P. patens (typically not yet capable of growing under heterotrophic conditions essentially without light or without light) to mutagenesis, and selecting the culture of the moss P. patens subjected to mutagenesis in step a) under heterotrophic conditions essentially without light or without light to establish a culture which is capable of growing under heterotrophic conditions essentially without light or without light, and optionally isolating individual clones of the moss P. patens from the culture selected in step b) for growth under heterotrophic conditions essentially without light or without light.

[0009] In a third aspect, the present invention relates to a method of producing a genetically modified culture of the moss P. patens, the method comprising the step of introducing a heterologous nucleic acid into a culture of the moss P. patens, wherein the cultureof the moss P. patens is capable of heterotrophic growth essentially without light or without light.

[0010] In a fourth aspect, the present invention relates to a method of producing a biotechnological product of interest in a culture of the moss P. patens, wherein the P. patens culture is a P. patens culture capable of growing under heterotrophic conditions essentially without light or without light, and wherein the method comprises the following steps: a) expressing a heterologous nucleic acid in said P. patens culture providing for production of said biotechnological product in said P. patens culture, and b) optionally isolating the biotechnological product of interest from said P. patens culture.

[0011] In a fifth aspect, the present invention relates to a P. patens culture capable of growing under heterotrophic conditions essentially without light or without light.

[0012] In a sixth aspect, the present invention relates to a plant cell, protonema, gametophyte, sporophyte or spore of a P. patens culture capable of growing under heterotrophic conditions essentially without light or without light.

[0013] In a further aspect, the present invention relates to the use of P. patens culture capable of growing under heterotrophic conditions essentially without light or without light for producing a genetically modified P. patens culture comprising a heterologous nucleic acid and / or for producing a biotechnological product.

[0014] As used herein, being “capable of growing under heterotrophic conditions” means that the P. patens culture can be grown essentially without light or even entirely without light. The culture is thus capable of metabolizing an external carbon source like glucose and can use it as sole energy source and does not require light as energy source any longer. This does not preclude that the culture is at times cultured under different conditions, because the cultures of the present invention are still capable of autotrophic (i.e. with light) or mixotrophic growth (using both, light and a carbon source for energy purposes). The heterotrophic P. patens cultures of the present invention have been successfully cultured for various months (see Fig. 2) essentially without light, i.e. they were exposed to minimal light only, namely when subculturing was required. In the setup exemplified in the examples subculturing was required for instance after seven days of culturing. Even then light was restricted to a maximum of 1 h of maximum 50 pmol m'2s'1photons (50 pE). Therefore, as used herein, being capable of growing under heterotrophic conditions “essentially without light” implies that the culture iscapable of growing under heterotrophic conditions with no more than 1 h of maximum 50 pmol m'2s'1photons per seven days of cultivation. Due to the long culturing periods demonstrated in the examples section, the inventors of the present invention deem the minimal light exposure during the subculturing process to be of no particular significance for the growth of the P. patens cultures of the present invention, i.e. it is believed that the cultures could be cultured for weeks and months without any light. It is only the process of subculturing which is somewhat difficult to carry out in complete darkness. Furthermore, as used herein, “capable of growing under heterotrophic conditions” is not to be confused with being capable of some residual growth after switching the culture from autotrophic conditions to heterotrophic conditions. Autotrophic growing cultures shifted to heterotrophic conditions may initially be still capable of some growth due to stored energy resources. Such growth will cease over time. Therefore, “capable of growing under heterotrophic conditions” preferably implies that the P. patens cultures is (still) capable of growth (or growing) after 18 days of culture under heterotrophic conditions essentially without light or without light in liquid media. In other words, if a culture of the present invention is inoculated in fresh medium after a preculture for 18 days under heterotrophic conditions (essentially without light or even entirely without light) in liquid media, such culture will show growth as defined herein below.

[0015] A P. patens culture being capable of growing under heterotrophic conditions is preferably capable of growing under heterotrophic conditions for at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more days. The P. patens culture may be capable of growing under heterotrophic conditions for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more weeks. The P. patens culture may even be capable of growing under heterotrophic conditions for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more months. In a particularly preferred embodiment, the P. patens culture is capable of growing under heterotrophic conditions essentially without light in liquid media for at least four, preferably at least six weeks.

[0016] As used herein, “growth” or “growing” of a P. patens culture means an increase in dry biomass [g / L] by at least 100% after 168 hours, preferably after 144 hours, even more preferably after 120 hours, even more preferably after 96 hours, even more preferably after 72 hours, most preferably after 48 hours of culturing. A person skilled in the art will understand that in this context the starting point for “culturing” is the time of inoculation in fresh media. A P. patens culture of the present invention may for example provide for an increase in dry biomass [g / L] by at least 100%, more preferably by at least 200%, more preferably by at least300% or even more preferably by last 400% after 72 hours of culturing. In particular, such “growth” or “growing” may be obtained after culturing the P. patens culture in the dark, i.e. without light or essentially without light. Moreover, such growth or growing rates, in particular a growth or growing rate of at least 100% after 72 hours, may still be obtained when inoculating the P. patens culture in new media after having cultured the culture already for at least 18 days in the dark, i.e. without light or essentially without light. Preferably, said preceding culture for at least 18 days in the dark has been done in suspension culture. Moreover, a P. patens culture capable of growing under heterotrophic conditions essentially without light or without light will preferably exhibit a maximum doubling time of 6 days, more preferably 5 days, even more preferably 4 days, and most preferably of 3 days. A person skilled in the art will be readily familiar with how to determine dry biomass. Preferably, the dry biomass is determined after drying the biomass for 2 hours at 110°C in an oven. “Growth” or “growing” of a P. patens culture and related methods is also understood to refer solely to the increase in dry biomass [g / L] and does not include methods of breeding and crossing P. patens cultures.

[0017] Heterotrophic conditions require an organic carbon source replacing light as energy source. Suitable organic carbon sources for growing a P. patens culture of the present invention are sugars, in particular and without being limited thereto glucose, sucrose, fructose, galactose, arabinose, xylose, maltose, trehalose, amylose, amylopectin, glycogen, ribose and desoxyribose and mixtures thereof. Heterotrophic conditions particularly contemplated by the inventors of the present invention comprise the cultivation of the P. patens culture in presence of glucose, sucrose, fructose and / or mixtures thereof. Cultivation of the P. patens culture in presence of glucose is particularly preferred. A person skilled in the art and familiar with culturing P. patens will be readily aware of suitable culture media for culturing P. patens, which can easily be combined with a carbon-based energy source such as glucose. P. patens may be cultured on solid media (i.e. containing more than 0.5% (w / v) long chain polysaccharides such as agar or gelatine), semi-solid media (containing long chain polysaccharides in the range of 0.2-0.5% (w / v) ) or liquid media (less than 0.2 (w / v) and preferably no long chain polysaccharides). Most preferably, the P. patens culture capable of growing under heterotrophic conditions is cultivated in liquid media. A suitable medium may for example comprise at least one sugar (e.g. glucose), FeSC , KC1, Ca(NO3)2, KH2PO4, MgSC , ideally additionally supplemented with trace elements. A liquid medium which is particularly suitable for culturing a P. patens cultures of the present invention (i.e. which is capable of growing under heterotrophic conditions) comprises preferably aside of glucose KH2PO4, KC1, MgSC ,Ca(NO3)2, FeSC , H3BO3, MnSC , Z11SO4, KJ, NaiMoC , CuSC , and C0CI2. Furthermore, the person skilled in the art will also be readily familiar with devices for culturing P. patens, including suitable types of flasks and bioreactors. The method of growing a culture of P. patens according to the first aspect of the invention (or any other method or use disclosed herein) may thus involve growing said P. patens culture essentially without light or preferably without light in liquid media in a flask or bioreactor.

[0018] As used herein, a P. patens culture may be a monoclonal culture. However, the P. patens culture may also be a polyclonal culture (i.e. a mixture of genetically differing strains). In many scenarios, it is preferable that the P. patens culture is a monoclonal culture. The P. patens culture may for instance be a P. patens culture deriving from a parent P. patens culture which has been subjected to mutagenesis and subsequent selection for growth under heterotrophic conditions according to the second aspect of the invention.

[0019] Furthermore, a P. patens culture according to the present invention may exhibit under heterotrophic conditions a phenotype differing from conventional P. patens cultures. Conventional P. patens cultures divide under heterotrophic conditions exclusively asymmetrical and grow in liquid suspension filamentous while P. patens cultures capable of growing under heterotrophic conditions in liquid suspensions tend to divide symmetrical forming round-shaped cells loosely attached as clusters (see Fig. 5). Only upon illumination such culture may show a tendency for asymmetrical division forming firmly attached filamentous cells. Therefore, it is preferred if the P. patens culture capable of growing under heterotrophic conditions essentially without light or without light grows in clusters under such conditions. Vice versa, it is preferred that the P. patens cultures capable of growing under heterotrophic conditions does not form filaments in liquid media when grown under heterotrophic conditions essentially without light or without light.

[0020] A monoclonal P. patens culture growing under heterotrophic conditions essentially without light and useful in the methods of the present invention is the P. patens culture Pp_A_SE-l#008, which has been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7 B, 38124 Braunschweig, Germany) under accession number DSM 35057 on 24 April 2024. Said clone is capable of growing in the dark in the presence of glucose, and divides symmetrical forming round-shaped cells loosely attached as clusters. Upon illumination the culture shows a tendency for asymmetrical division forming firmly attached filamentous cells. Due to a knock-out, theculture lacks expression of the endogenous moss fucosyl- and xylosyltransferase gene. A further monoclonal P. patens culture growing under heterotrophic conditions essentially without light und useful in the methods of the present invention is the P. patens culture Pp_A_SE-l#009, which has likewise been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7 B, 38124 Braunschweig, Germany) under accession number DSM 35058 on 24 April 2024. Said clone is capable of growing in the dark in the presence of glucose, and divides symmetrical forming round-shaped cells loosely attached as clusters. Upon illumination the culture shows a tendency for asymmetrical division forming firmly attached filamentous cells. Due to a knock out, the culture lacks expression of the endogenous moss fucosyl- and xylosyltransferase gene. A further monoclonal P. patens culture growing under heterotrophic conditions essentially without light und useful in the methods of the present invention is the P. patens culture Pp_A_SE-l#010, which has likewise been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7 B, 38124 Braunschweig, Germany) under accession number DSM 35059 on 24 April 2024. Said clone is capable of growing in the dark in the presence of glucose, and divides symmetrical forming round-shaped cells loosely attached as clusters. Upon illumination the culture shows a tendency for asymmetrical division forming firmly attached filamentous cells. Due to a knock out, the culture lacks expression of the endogenous moss fucosyl- and xylosyltransferase gene. A further monoclonal P. patens culture growing under heterotrophic conditions essentially without light und useful in the methods of the present invention is the P. patens culture Pp_A_SE-l#011, which has likewise been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7 B, 38124 Braunschweig, Germany) under accession number DSM 35060 on 24 April 2024. Said clone is capable of growing in the dark in the presence of glucose, and divides symmetrical forming round-shaped cells loosely attached as clusters. Upon illumination the culture shows a tendency for asymmetrical division forming firmly attached filamentous cells. Due to a knock out, the culture lacks expression of the endogenous moss fucosyl- and xylosyltransferase gene. A further monoclonal P. patens culture growing under heterotrophic conditions essentially without light und useful in the methods of the present invention is the P. patens culture Pp_A_SE-l#012, which has likewise been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7 B, 38124 Braunschweig, Germany) under accession number DSM 35061 on 24 April 2024. Said clone iscapable of growing in the dark in the presence of glucose, and divides symmetrical forming round-shaped cells loosely attached as clusters. Upon illumination the culture shows a tendency for asymmetrical division forming firmly attached filamentous cells. Due to a knock out, the culture lacks expression of the endogenous moss fucosyl- and xylosyltransferase gene. A further monoclonal P. patens culture growing under heterotrophic conditions essentially without light und useful in the methods of the present invention is the P. patens culture Pp_A_SE-l#013, which has likewise been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7 B, 38124 Braunschweig, Germany) under accession number DSM 35062 on 24 April 2024. Said clone is capable of growing in the dark in the presence of glucose, and divides symmetrical forming round-shaped cells loosely attached as clusters. Upon illumination the culture shows a tendency for asymmetrical division forming firmly attached filamentous cells. Due to a knock out, the culture lacks expression of the endogenous moss fucosyl- and xylosyltransferase gene. A further monoclonal P. patens culture growing under heterotrophic conditions essentially without light und useful in the methods of the present invention is the P. patens culture Pp_A_SE-l#014, which has likewise been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7 B, 38124 Braunschweig, Germany) under accession number DSM 35063 on 24 April 2024. Said clone is capable of growing in the dark in the presence of glucose, and divides symmetrical forming round-shaped cells loosely attached as clusters. Upon illumination the culture shows a tendency for asymmetrical division forming firmly attached filamentous cells. Due to a knock out, the culture lacks expression of the endogenous moss fucosyl- and xylosyltransferase gene. A further monoclonal P. patens culture growing under heterotrophic conditions essentially without light and useful in the methods of the present invention is the P. patens culture Pp_A_SE-l#015, which has likewise been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7 B, 38124 Braunschweig, Germany) under accession number DSM 35064 on 24 April 2024. Said clone is capable of growing in the dark in the presence of glucose, and divides symmetrical forming round-shaped cells loosely attached as clusters. Upon illumination the culture shows a tendency for asymmetrical division forming firmly attached filamentous cells. The P. patens culture may also be a variant of any of these deposited cultures (DSM 35057, DSM 35058, DSM 35059, DSM 35060, DSM 35061, DSM 35062, DSM 35063, and DSM 35064) comprising additionally one more heterologous nucleic acid sequences (e.g. providing for expression of one moreheterologous recombinant polypeptides), or be a variant of any of these deposited cultures which has a genome exhibiting at least 90%, more preferably, at least 95%, more preferably, at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%%, even more preferably at least 99.5%, even more preferably at least 99.75% sequence identity with the genome of at least one of these deposited cultures. A further monoclonal P. patens culture growing under heterotrophic conditions essentially without light and useful in the methods of the present invention is the polyclonal P. patens culture Pp_Pool_Trophy-l#001, which expresses human lysosomal alpha-glucosidase (GAA) (SEQ ID NO: 1). Said polyclonal culture has been deposited at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7 B, 38124 Braunschweig, Germany) under accession number DSM 35065 on 8 May 2024. Said polyclonal culture is capable of growing in the dark in the presence of glucose, and divides symmetrical forming round-shaped cells loosely attached as clusters. Upon illumination the culture shows a tendency for asymmetrical division forming firmly attached filamentous cells. Due to a knockout, the culture lacks expression of the endogenous moss fucosyl- and xylosyltransferase gene. The P. patens culture may also be a variant of Pp_Pool_Trophy- 1#001 comprising additionally one more further heterologous nucleic acid sequences (e.g. providing for additional expression of one more heterologous recombinant polypeptides), or be a variant of any of this deposited culture which has a genome exhibiting at least 90%, more preferably, at least 95%, more preferably, at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%%, even more preferably at least 99.5%, even more preferably at least 99.75% sequence identity with the genome of this culture. In contrast to the above- mentioned cultures, the parental Pp_A_Doko#069 culture (which was not yet capable of growing under heterotrophic conditions essentially without light or without light for a prolonged period of time, but also lacks expression of the endogenous moss fucosyl- and xylosyltransferase gene due to the knock out) divides under heterotrophic conditions exclusively asymmetrical and grows filamentous.

[0021] As mentioned above, the present invention relates in a first aspect to method of growing a culture of the moss Physcomitrium patens (P. patens), wherein the culture is a culture capable of growing under heterotrophic conditions, and wherein the method comprises the step of culturing said P. patens culture under conditions allowing growth of said P. patens culture. The culture is preferably capable of growing under heterotrophic conditions essentially without light or without light. The method may comprise growing the culture under autotrophic (lightas energy source), mixotrophic (light and organic energy source, in particular glucose) or heterotrophic conditions (in particular in presence of glucose). Preferably, the method comprises cultivating the P. patens culture under heterotrophic conditions essentially without light or without light. The method according to the first aspect of the invention may comprise growing said culture of P. patens in a medium comprising KH2PO4, KC1, MgSC , Ca(NO3)2, FeSCU, H3BO3, MnSC , ZnSC , KJ, Na2MoO4, CuSC , and C0CI2. Preferably, the culture is cultured under heterotrophic conditions essentially without light or without light and in the presence of glucose. Even more preferably, the culture is cultured under heterotrophic conditions essentially without light or without light in the presence of glucose and in liquid media. Examples for suitable media are provided in the examples section. Most preferably, the method according to the first aspect of the invention comprises growing said culture of P. patens under heterotrophic conditions essentially without light or without light in a liquid medium comprising KH2PO4, KC1, MgSO4, Ca(NO3)2, FeSO4, H3BO3, MnSO4, ZnSO4, KJ, Na2MoO4, CuSO4, C0CI2 and glucose. Preferably, the P. patens culture is cultured under heterotrophic conditions for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 days. The P. patens culture may be cultured under heterotrophic conditions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 months. The P. patens culture may even be cultured under heterotrophic conditions for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more months. The P. patens culture grown with the method according to the first aspect of the invention may be monoclonal or polyclonal. Preferably, the culture is monoclonal. The P. patens culture grown with the method according to the first aspect of the invention may be genetically modified. For instance, the culture may lack expression of the endogenous fucosyl- and / or xylosyltransferase genes due to a knock out. Preferably the culture lacks expression of the endogenous fucosyl- and xylosyltransferase gene. Examples of cultures which can be used in the method according to the first aspect of the invention are P. patens cultures as deposited under any of accession numbers DSM 35057, DSM 35058, DSM 35059, DSM 35060, DSM 35061, DSM 35062, DSM 35063, and DSM 35064. The P. patens culture grown with the method according to the first aspect of the invention may also comprise heterologous nucleic acid elements. Such elements can for example be introduced into the genome of the culture by way of homologous recombination. For this reason, the inventors also contemplate to use variants of any of these deposited cultures, where one of these cultures has been transformed with a heterologous nucleic acid, potentially providing for the expression of one more heterologous recombinant polypeptides. An example for such recombinant polypeptide wouldbe human lysosomal alpha-glucosidase (GAA). Other suitable variants are those which exhibit a genome exhibiting at least 90%, more preferably, at least 95%, more preferably, at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%%, even more preferably at least 99.5%, even more preferably at least 99.75% sequence identity with the genome of at least one of these deposited cultures and still retaining the ability of growing under heterotrophic conditions essentially without light or without light.

[0022] As mentioned above, the present invention relates in a second aspect to a method of producing a culture of the moss P. patens capable of growing under heterotrophic conditions essentially without light or without light, the method comprising the following steps: a) subjecting a culture of the moss P. patens (typically not yet capable of growing under heterotrophic conditions) to mutagenesis, and b) selecting the culture of the moss P. patens subjected to mutagenesis in step a) under heterotrophic conditions essentially without light or without light to establish a culture which is capable of growing under heterotrophic conditions essentially without light or without light, and c) optionally isolating individual clones of the moss P. patens from the culture selected in step b) for growth under heterotrophic conditions essentially without light or without light. Mutagenesis can be induced chemically or physically. A preferred means of inducing mutagenesis is by way of using UV radiation. The P. patens culture can for example be irradiated with 254 nm UV light for at least 1 min 30 seconds, at least 1 min 40 seconds, at least 1 min 50 seconds, or at least 2 minutes. Preferably, the culture is irradiated for 2 min 30 seconds. The P. patens culture subjected to mutagenesis in step a) may be a naturally occurring P. patens culture or may be a P. patens culture already genetically modified. For example, a genetically modified culture may exhibit knock-outs of certain genes. Such knock-outs may for example pertain to the endogenous fucosyl- and / or xylosyltransferase genes. A genetically modified culture may also comprise exogenous nucleic acid sequences (DNA) which have been introduced by way of genetic engineering, in particular by way of homologous recombination or non-homologous recombination. The exogenous nucleic acid sequence may encode a polypeptide of interest. Such polypeptide of interest may be of human origin. For instance, the genetically modified culture may comprise a gene encoding for human lysosomal alpha-glucosidase (GAA), such as SEQ ID NO: 1. However, the nucleic acid need not encode a polypeptide of interest. Instead, it may for example also provide for inactivation of endogenous P. patens genes, e.g. by way of a knock-out. The present invention also relates to a P. patens culture capable of growing under heterotrophic conditions essentially without light or without light obtained with the method according to the second aspect of the invention.

[0023] As mentioned above, the present invention relates in a third aspect to a method of producing a genetically modified culture of the moss P. patens, the method comprising the step of introducing a heterologous nucleic acid into a culture of the moss P. patens, wherein the culture of the moss P. patens is capable of heterotrophic growth essentially without light or without light. Preferably, the nucleic acid is DNA. Preferably, the nucleic is introduced into said P. patens culture by means of homologous recombination or non-homologous recombination (Koprivova et al, 2004; Plant Biotechnology Journal 2, 517-523). The P. patens culture capable of heterotrophic growth essentially without light or without light is the same as defined throughout the application. Examples of cultures which can be used in the method according to the third aspect of the invention are the monoclonal P. patens cultures as deposited under any of accession numbers DSM 35057, DSM 35058, DSM 35059, DSM 35060, DSM 35061, DSM 35062, DSM 35063, and DSM 35064, or variants thereof which exhibit a genome exhibiting at least 90%, more preferably, at least 95%, more preferably, at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%%, even more preferably at least 99.5%, even more preferably at least 99.75% sequence identity with the genome of at least one of these deposited cultures and still retaining the ability of growing under heterotrophic conditions essentially without light or without light. The nucleic acid may encode for example one or more polypeptides. The polypeptide may be a P. patens polypeptide or may be a heterologous polypeptide. The encoded polypeptide may be itself a biotechnological product of interest, such as an enzyme, an antibody or fragment thereof, a receptor or fragment thereof, a coagulation factor or fragment thereof, an interferone etc. Preferably, the polypeptide is in such scenario a human polypeptide such as GAA (preferably according to SEQ ID NO:1). Alternatively, the nucleic acid may also encode a polypeptide indirectly leading to production of biotechnological product of interest in P. patens. In such scenario, the encoded polypeptide may for example be a natural P. patens polypeptide, such as a transcription factor or pathway regulator protein. The expression of such polypeptide may then provide for improved or increased production of all kinds of biotechnological products, in particular of P. patens metabolites, including small molecules such as sesquiterpenes (e.g. taxadien, sclareol, artimisinin, patchoulol or santalene) and other terpenes. In a further scenario, the nucleic acid does not encode a polypeptide at all. Instead, it may for example provide for inactivation of endogenous P. patens genes, e.g. by way of a knock-out. The present invention also relates to a P. patens culture capable of growing under heterotrophic conditions essentiallywithout light or without light obtained with the method according to the third aspect of the invention.

[0024] In further, fourth aspect, the present invention relates to a method of producing a biotechnological product of interest, such as a recombinant polypeptide, in a culture of the moss P. patens, wherein the P. patens culture is a P. patens culture capable of growing under heterotrophic conditions essentially without light or without light, and wherein the method comprises the following steps: a) expressing a heterologous nucleic acid in said P. patens culture providing (directly or indirectly) for production of said biotechnological product of interest (e.g. recombinant polypeptide of interest) in said P. patens culture, and b) optionally isolating the biotechnological product (e.g. the recombinant protein of interest) from said P. patens culture. As discussed above for the third aspect of the invention, the biotechnological product of interest may be any product which can be produced in P. patens, including recombinant polypeptides and organic compounds such as metabolites. In the context of the present invention, the biotechnological product of interest is preferably GAA. The P. patens culture capable of growing under heterotrophic conditions essentially without light or without light is as defined throughout herein, of course with the additional proviso that it needs to comprise a heterologous nucleic acid, i.e. is a genetically modified organism. An example of such culture is the polyclonal P. patens culture Pp_Pool_Trophy-l#001 expressing GAA.

[0025] As mentioned above, the present invention relates in a fifth aspect to a P. patens culture capable of growing under heterotrophic conditions essentially without light or without light. The P. patens culture capable of heterotrophic growth essentially without light or without light is the same as defined throughout the application. Such culture can be obtained in particular with the method according to the second aspect of the invention. Specific examples of P. patens culture according to the invention are the monoclonal P. patens cultures as deposited under any of accession numbers DSM 35057, DSM 35058, DSM 35059, DSM 35060, DSM 35061, DSM 35062, DSM 35063, and DSM 35064. Another example is the polyclonal P. patens culture deposited under accession number DSM 35065.

[0026] As mentioned above, the present invention relates in a sixth aspect to a plant cell, protoplast, protonema, gametophyte, sporophyte or spore, wherein the plant cell, protoplast, protonema, gametophyte, sporophyte or spore is a cell, protoplast, protonema, gametophyte, sporophyte or spore of a P. patens culture according to the fifth aspect of the invention. Preferably, the plant cell, protoplast, protonema, gametophyte, sporophyte or spore is anisolated cell, protoplast, protonema, gametophyte, sporophyte or spore of a P. patens culture according to the fifth aspect of the invention. A person skilled in the art will be readily aware of how to obtain a plant cell, protoplast, protonema, gametophyte, sporophyte or spore from a P. patens cultures according to the present invention, i.e. capable of growing under heterotrophic conditions essentially without light or without light.

[0027] In a further aspect, the present invention relates to the use of P. patens culture capable of growing under heterotrophic conditions essentially without light or without light for producing a genetically modified P. patens culture comprising a heterologous nucleic acid and / or for producing a biotechnological product, such as a recombinant protein or P. patens metabolite. The respective culture may be used in this context in a method according to the third or fourth aspect of the invention.

[0028] For sake of completeness it is noted that what ever has been set forth herein for a specific aspect of the invention will likewise be applicable to other aspects of the invention. For instance, the method of growing a culture of P. patens according to the first aspect of the invention may comprise growing a P. patens producing a biotechnological product (e.g. expressing a recombinant polypeptide), which essentially reflects the method of the fourth aspect of the invention. Therefore, unless technically indicated otherwise, any embodiment disclosed herein for one specific aspect of the invention will likewise be applicable to any of the other aspects of the invention.

[0029] The term "comprising", as used herein, shall not be construed as being limited to the meaning "consisting of" (i.e. excluding the presence of additional other matter). Rather, "comprising" implies that optionally additional matter may be present. The term "comprising" encompasses as particularly envisioned embodiments falling within its scope "consisting of" (i.e. excluding the presence of additional other matter) and "comprising but not consisting of" (i.e. requiring the presence of additional other matter), with the former being more preferred.Figures

[0030] In the following a brief description of the appended figures will be given. The figures are intended to illustrate aspects of the present invention in more detail. However, they are not intended to limit the overall scope of the invention.

[0031] Fig. 1 illustrates that a conventional P. patens culture (Pp_A_Doko#069) does not exhibit any significant growth (increase in dry biomass (g / L)) when cultured without light. The amount of dry biomass essentially remains the same over a culture period of almost 30 days. Dry biomass was assessed every seven days (dots) of culture. Importantly, no subculturing was performed due to lack of growth.

[0032] Fig. 2 illustrates growth as evidenced by an increase in dry biomass (g / L) for a P. patens culture after irradiation and selection under heterotrophic conditions over a period of time of more than 500 days. Dots represent the amount of dry biomass before (maximum values) and after (minimum values at 0.1 g / L) subculturing.

[0033] Fig. 3 illustrates growth as evidenced by an increase in dry biomass (g / L) after 3 days of culture in a fresh volume of 20 ml for 8 different P. patens cultures obtained after cloning of a P. patens culture previously irradiated and selected for growth under heterotrophic conditions, i.e. in the dark and in presence of glucose. The 8 cultures (from the left to the right) are Pp_A_SE-l#008 (DSM 35057), Pp_A_SE-l#009 (DSM 35058), Pp_A_SE-l#010 (DSM 35059), Pp_A_SE-l#011 (DSM 35060), Pp_A_SE-l#012 (DSM 35061), Pp_A_SE-l#013 (DSM 35062), Pp_A_SE-l#014 (DSM 35063) and Pp_A_SE-l#015 (DSM 35064). Culture Pp_A_Doko#069 on the right is the parental culture, which died after 3 days of culture without light. Prior to inoculation in the fresh medium all cultures had been cultured under heterotrophic conditions essentially without light for at least 18 days (see example 2).

[0034] Fig. 4 illustrates the production of human GAA (SEQ ID NO: 1) in five different polyclonal P. patens cultures. The x-axis gives the parent strain of the polyclonal culture, all of which were P. patens strains capable of growing under heterotrophic conditions according to the present invention. All cultures had been incubated for three days under heterotrophic conditions, except for the polyclonal culture deriving from parent strain Pp_A_SE-l#011, which had been cultured for 4 days. The indicated mean concentration of GAA in mg / L and standard deviation (sd) is the mean and sd of 3 replicates.

[0035] Fig. 5 illustrates the morphology of a P. patens culture (Pp_A_Doko#069) growing under autotrophic conditions with a filamentous phenotype (top) and a P. patens culture of the present invention (Pp_A_SE-l#013) growing in clusters under heterotrophic conditions (bottom).Examples

[0036] In the following, specific examples illustrating embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific examples described herein. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description and the examples below. All such modifications fall within the scope of the appended claims.Example 1: Development of Heterotrophic Strains via Adaptive Laboratory Evolution (ALE)

[0037] P. patens strain Pp_A_Doko#069 is a genetically modified organism derived from the wild type moss Physcomitrium patens. Two genes, coding for the fucosyl- and the xylosyltransferase have been knocked out using homologous recombination. For gene knockout an appropriate homologous PCR product containing point mutations was derived from Physcomitrium patens DNA. Said parent strain was subjected to UV mutagenesis by irradiating with 254 nm UV light (NU-6, Herolab, Wiesloch, Germany) for 2 min 30 s. Afterwards, the mutagenized tissue was cultivated dark in 180 mL liquid media (see General Conditions for cultivation below). Depending on the growth, the culture was split initially every 14 days and at the end every 3 to 4 days. Subculturing was performed as described below. At day 478 of ALE the adapted culture was cloned. For this purpose, the moss tissue was digested to liberate protoplasts, which were purified and re-cultivated as described in Koprivova et al. (2004; Plant Biotechnology Journal 2, 517-523). The eight clones with fastest growth rate under heterotrophic conditions were identified and stored as cellbanks (Schulte & Reski, Plant Biol (Stuttg). 2004 Mar-Apr;6(2): 119-27). Respective frozen cultures were deposited at the Leibniz- Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7B, 38124 Braunschweig, Germany).

[0038] The eight strains are identified in table 1 below.

[0039] Table 1 : Heterotrophic cultures of P. patens and morphology when cultured under heterotrophic conditions without lightExample 2: Heterotrophic Growth and dry biomass determination

[0040] Strains of P. patens were maintained on solid media by transferring pin- sized (approx. 10+2 mg fresh weight) biomass material on fresh agar plates monthly. To start liquid suspension cultures, pin-sized biomass material of each strain from a petri dish with solid media was used to inoculate 160 mL fresh liquid media in a 500 mL shake flask. After 7 + 1 days the culture was homogenized using an ultraturrax dispersing tool (S25N18G, IKA, Staufen, Germany) for 40 s and 20,000 rpm. After further 7 + 1 days the culture was homogenized again as described previously and the dry biomass of a 10 mL sample was determined in order to normalize inoculum (0.05 g / L final dry biomass) for subculturing into 160 mL fresh media. After further 4 days the culture was homogenized as before and subcultivated with a final dry biomass of 0.1 g / L and a volume of 20 mL fresh media in tubespins (Tubespin 50 mL bioreactor, TPP, Trasadingen, Switzerland) in triplicates for each strain. After 3 days the whole culture was used for dry biomass determination.

[0041] For dry biomass determination biomass, a sample was collected on glass fibre filters and dried in an oven at 110 °C overnight. For biomass normalization drying time was reduced to 2 h.

[0042] Cultivation conditions: Culture media was prepared as described previously (e.g. Niederau et al., Plant Cell Rep. 2024 Jan 22;43(2):43) with the addition of 25 mM glucose and and where desired 1.5 % / w / v) agar for solidification. Plates were incubated at ambient temperature (22-24 °C), suspension cultures at 25 °C and shaken at 120 rpm for shake flasks or 180 rpm for tubespins. All vessels were kept dark by wrapping with aluminum foil. During subculturing illumination was restricted to maximum one hour with maximum 50 pE.Example 3: Recombinant Protein Production

[0043] The nucleotide sequence coding for human lysosomal alpha-glucosidase (GAA) was synthesized by Invitrogen GeneArt Gene Synthesis Services (Thermo Fisher Scientific) and cloned into an expression vector containing genetic elements providing for strong expression and secretion into the apoplast as described previously (see Niederau et al., Plant Cell Rep. 2024 Jan 22;43(2):43). Briefly, for recombinant protein production 8616 bp of synthetic linear DNA was integrated into the moss genome. The linear DNA codes for the human alpha- glucosidase, the green fluorescent protein (GFP) from Aequorea coerulescens and the neomycin phosphotransferase II from Escherichia coli. Heterotrophic suspension cultures were cultivated autotrophic 3-4 weeks before protoplasts were used for PEG-mediated transformation as described in Koprivova et al. (2004; Plant Biotechnology Journal 2, 517— 523). After antibiotic selection clones were cultivated heterotrophic as described above. Determination of dry biomass as described above (inoculation of 0.1 g / L, three days of cultivation in 20 ml) yielded 0.3 g / L of mean dry biomass.

[0044] Productivity in 20 mL culture volume was determined in triplicates via ELISA / enzyme assay as described in Hintze et al. (2020, Int J Mol Sci 21, 2642).

[0045] One polyclonal culture (Pp_Pool_Trophy-l#001) has been deposited as frozen culture at the Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Inhoffenstr. 7B, 38124 Braunschweig, Germany).

[0046] The pooled heterotrophic culture is identified in table 2 below.

[0047] Table 2: Heterotrophic culture of P. patens expressing human lysosomal alphaglucosidase:Example 4: General Conditions for cultivation

[0048] Heterotrophic strains and cultures according to the present invention may be cultivated in the dark in the following liquid media at pH 5.8: 250 mg / L KH2PO4, 250 mg / L KC1, 250 mg / L MgSO4, 1 g / L Ca(NO3)2, 12.5 mg / L EeSO4, 50 pM H3BO3, 50 pM MnSO4 x H2O, 15 pM ZnSO4x 7 H2O, 2.5 pM KJ, 0.5 pM Na2MoO4x 2 H2O, 0.05 pM CuSO4x 5 H2O, 0.05 pM C0CI2 x 6 H2O, 25 mM glucose. The interval of transfer to fresh medium is preferably 7 days.

[0049] Axenic cultures grow heterotrophic in 500 mL shake flasks at 22-25 °C while shaking with 120 rpm. To ensure heterotrophic conditions shake flasks are wrapped in aluminium foil. Preferably, every 3 to 8 days, most preferably every 7 days a culture should get homogenized using a dispersing tool and is then diluted 30 times with fresh media. During subculturing illumination should be limited to maximum 1 h of maximum 50 pE of visible light.

[0050] Alternatively, heterotrophic strains and cultures according to the present invention may be cultivated in the dark in the following solid media at pH 5.8: 250 mg / L KH2PO4, 250 mg / L KC1, 250 mg / L MgSO4, 1 g / L Ca(NO3)2, 12.5 mg / L EeSO4, 50 pM H3BO3, 50 pM MnSO4x H2O, 15 pM ZnSO4 x 7 H2O, 2.5 pM KJ, 0.5 pM Na2MoO4x 2 H2O, 0.05 pM CuSO4x 5 H2O, 0.05 pM C0CI2 x 6 H2O, 1.5% (w / v) agar, 25 mM glucose. The interval of transfer to fresh medium is preferably 4 weeks.

[0051] Particularly preferred embodiments of the invention are set forth in items 1 to 16 below:1. Method of growing a culture of the moss Physcomitrium patens (P. patens), wherein the culture is a culture capable of growing under heterotrophic conditions essentially without light or without light, and wherein the method comprises the step of culturing said P. patens culture under conditions allowing growth of said P. patens culture.2. The method of item 1, wherein said P. patens culture comprises a heterologous nucleic acid.3. Method of producing a genetically modified culture of the moss P. patens, the method comprising the step of introducing a heterologous nucleic acid into a culture of the moss P. patens, wherein the culture of the moss P. patens is capable of growing under heterotrophic conditions essentially without light or without light.4. The method of item 3, wherein the nucleic acid is introduced into said culture of P. patens by way of homologous recombination or non-homologous recombination.5. The method according to any one of items 2 to 4, wherein the heterologous nucleic acid encodes a polypeptide, preferably a human polypeptide.6. Method of producing a biotechnological product of interest in a culture of the moss P. patens, wherein the culture is a culture capable of growing under heterotrophic conditions essentially without light or without light, and wherein the method comprises the following steps: a) expressing a heterologous nucleic acid in said P. patens culture providing for production of said biotechnological product of interest in said P. patens culture, and b) optionally isolating the biotechnological product of interest from said P. patens culture.7. The method according to item 5 or item 6, wherein the polypeptide or biotechnological product, respectively, is human lysosomal alpha-glucosidase (GAA).The method according to anyone of the preceding items, wherein said P. patens culture is capable of growing under heterotrophic conditions essentially without light for at least four weeks. The method according to anyone of the preceding items, wherein growth or being capable of growing is defined as increase in dry biomass [g / L] by at least 100% after 72 hours of culturing. The method according to anyone of the preceding items, wherein the method comprises culturing said P. patens culture under heterotrophic conditions essentially without light or without light. The method of item 10, wherein the method comprises culturing said P. patens culture in presence of glucose, sucrose, fructose, galactose, arabinose, xylose or mixtures thereof, preferably wherein the method comprises culturing said P. patens culture in presence of glucose. The method of item 10 or item 11, wherein the method comprises culturing said P. patens culture in liquid media. P. patens culture capable of growing under heterotrophic conditions essentially without light or without light. Plant cell, protoplast, protonema, gametophyte, sporophyte or spore, wherein the plant cell, protonema, gametophyte, sporophyte or spore is an isolated cell, protoplast, protonema, gametophyte, sporophyte or spore of a P. patens culture according to item 13. The method according to any one of items 1 to 10, or the P. patens culture of item 13 or the plant cell, protoplast, protonema, gametophyte, sporophyte or spore of item 14, wherein the endogenous fucosyl- and / or xylosyltransferase gene is knocked out in said P. patens culture.The method according to any one of items 1 to 12, or the P. patens culture of item 13 or the plant cell, protoplast, protonema, gametophyte, sporophyte or spore of item 14, wherein said P. patens culture is a P. patens culture as deposited under any of accession numbers DSM 35057, DSM 35058, DSM 35059, DSM 35060, DSM 35061, DSM 35062, DSM 35063, DSM 35064 and DSM 35065 or is a variant of any of these deposited cultures comprising additionally one or more heterologous nucleic acid sequences, or is a variant of any of these deposited cultures which has a genome exhibiting at least 95% sequence identity with the genome of at least one of these deposited cultures.

Claims

Claims1. Method of growing a culture of the moss Physcomitrium patens (P. patens), wherein the culture is a culture capable of growing under heterotrophic conditions essentially without light or without light, and wherein the method comprises the step of culturing said P. patens culture under conditions allowing growth of said P. patens culture, wherein growth or being capable of growing is defined as increase in dry biomass [g / L] by at least 100% after 168 hours of culturing.

2. The method of claim 1, wherein said P. patens culture comprises a heterologous nucleic acid.

3. Method of producing a genetically modified culture of the moss P. patens, the method comprising the step of introducing a heterologous nucleic acid into a culture of the moss P. patens, wherein the culture of the moss P. patens is capable of growing under heterotrophic conditions essentially without light or without light.

4. The method of claim 3, wherein the nucleic acid is introduced into said culture of P. patens by way of homologous recombination or non-homologous recombination.

5. The method according to any one of claims 2 to 4, wherein the heterologous nucleic acid encodes a polypeptide.

6. The method according to claim 5, wherein the heterologous nucleic acid encodes a human polypeptide.

7. Method of producing a biotechnological product of interest in a culture of the moss P. patens, wherein the culture is a culture capable of growing under heterotrophic conditions essentially without light or without light, and wherein the method comprises the following steps: a) expressing a heterologous nucleic acid in said P. patens culture providing for production of said biotechnological product of interest in said P. patens culture, andb) optionally isolating the biotechnological product of interest from said P. patens culture.

8. The method according to claim 5, claim 6 or claim 7, wherein the polypeptide or biotechnological product, respectively, is human lysosomal alpha-glucosidase (GAA).

9. The method according to anyone of the preceding claims, wherein said P. patens culture is capable of growing under heterotrophic conditions essentially without light for at least four weeks.

10. The method according to anyone of the preceding claims, wherein growth or being capable of growing is defined as increase in dry biomass [g / L] by at least 100% after 72 hours of culturing.

11. The method according to anyone of the preceding claims, wherein the method comprises culturing said P. patens culture under heterotrophic conditions essentially without light or without light.

12. The method of claim 11, wherein the method comprises culturing said P. patens culture in presence of glucose, sucrose, fructose, galactose, arabinose, xylose or mixtures thereof.

13. The method of claim 12, wherein the method comprises culturing said P. patens culture in presence of glucose.

14. The method of claim 11, claim 12 or claim 13, wherein the method comprises culturing said P. patens culture in liquid media.

15. P. patens culture capable of growing under heterotrophic conditions essentially without light or without light, wherein the endogenous fucosyl- and / or xylosyltransferase gene is knocked out in said P. patens culture.

16. Plant cell, protoplast, protonema, gametophyte, sporophyte or spore, wherein the plant cell, protonema, gametophyte, sporophyte or spore is an isolated cell, protoplast, protonema, gametophyte, sporophyte or spore of a P. patens culture according to claim 15.

17. The method according to any one of claims 1 to 14, or the P. patens culture of claim 15 or the plant cell, protoplast, protonema, gametophyte, sporophyte or spore of claim 16, wherein said P. patens culture is a P. patens culture as deposited under any of accession numbers DSM 35057, DSM 35058, DSM 35059, DSM 35060, DSM 35061, DSM 35062, DSM 35063, DSM 35064 and DSM 35065 or is a variant of any of these deposited cultures comprising additionally one or more heterologous nucleic acid sequences, or is a variant of any of these deposited cultures which has a genome exhibiting at least 95% sequence identity with the genome of at least one of these deposited cultures.

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