Process for protein production in methylotrophic yeast host cell.

Abstract

The present invention relates to a method for producing a polypeptide or protein of interest, the method comprising a) providing a methylotrophic yeast host cell comprising a nucleic acid molecule encoding said polypeptide or protein of interest operably linked to a derepressible promoter, b) culturing the host cell of step a) in a culture medium comprising a carbon source to obtain a cell culture, and c) adding at least one nitrogen-containing compound to cell culture of step b) or to the culture medium used in step b) to induce the derepressible promoter to overexpress said polypeptide or protein of interest.

Description

[0001] PROCESS FOR PROTEIN PRODUCTION IN METHYLOTROPHIC YEAST HOST CELL

[0002] TECHNICAL FIELD

[0003] The present invention relates to a novel process for protein production in methylotrophic yeasts.

[0004] BACKGROUND ART

[0005] The most widely used promoter for recombinant protein production in P. pastoris is the strong and inducible A0X1 promoter (PA0X1 ) [1 ,2], The A0X1 promoter is tightly controlled. It is repressed by glucose and highly induced by methanol and only very low transcription occurs upon carbon starvation (derepression). Other carbon sources that repress PA0X1 are glycerol and ethanol.

[0006] In a mixture of either glycerol or glucose with methanol, Pichia pastoris follows a diauxic growth, with C1 utilizing enzymes being repressed [3],

[0007] In contrast, alanine, sorbitol, mannitol and trehalose, which can be used as a sole carbon and energy source, were shown not to repress when methanol is used as inducer, hence can be applied as non-repressing carbon sources in co-feeding strategies either to increase cell growth or to maintain energy during induction without repressing the expression of the recombinant gene. Out of the non-repressing carbon sources mainly sorbitol has been studied, especially regarding co-feeding strategies with methanol and PAOX1 [4,5],

[0008] As carbon source during growth glycerol is most commonly used. Induction of methanol inducible promoters without addition of methanol and interruption of glucose / glycerol repression has been topic of many studies[6],

[0009] Hartner et al. (2008) created an A0X1 promoter library by deletion and duplication of putative transcription factor-binding sites reaching between 6% and >160% of the wild-type promoter activity and was the first to report PAOX1 mutant variants active under derepressing conditions[7].

[0010] Other studies showed carbon source responsive promoters applicable for high level protein production in Pichia pastoris (WO 2013 / 050551 , WO 2017 / 021541 ).

[0011] Similarly, an orthologous promoter (FMD) was shown to be stronger under derepressing conditions than the naturally in Komagataella cells occurring CAT1 and GAP promoters (WO 2017 / 109082A1 ).

[0012] In contrast, EP 4 116 408 combines the use of salts of formic acid (formates) or formic acid together with a non-repressing feeding substrate.

[0013] PCAT1 (“CAT1 promoter”) is a natural monodirectional derepressible promoter, which was first described in 2013 (EP 2 862 933). It can be induced by methanol reaching expression levels comparable to PAOX1 or even beyond. PCAT1 is repressed in the presence of glucose or glycerol and derepressed upon the depletion of these carbon sources. Compared to the A0X1 promoter, which is tightly repressed when glucose or glycerol are present, the CAT1 promoter is a ‘leaky’ carbon source responsive promoter, active under carbon source starvation or limited feed of repressing carbon sources such as glucose or glycerol. PCAT1 expresses at a level of only ~30-40% when used under derepressing conditions compared to methanol induction.

[0014] It has been shown that yeast can use almost 30 distinct nitrogen sources, including amino acids, urea, ammonium, nitrogen bases, and purine derivatives [8,9], While most yeast species prefer glutamine, glutamic acid or ammonium as nitrogen source, they can switch their metabolic pathways if such preferential nitrogen source is not presented and begin to utilize poor nitrogen sources such as urea and proline. The primary amine methylamine has been shown applicable as nitrogen source and to regulate expression of the formaldehyde dehydrogenase promoter which has been shown to have a similar tight regulation as PAOX1

[0010] , In contrast, gene expression of both, A0X1 and CAT1 , has been shown to increase when cells are cultured on media containing glycerol, glutamine and ammonia, when using methanol as inducer.

[0015] Induction of methanol inducible promoters with toxic and flammable methanol is undesirable especially on a large industrial scale for reasons of operational safety. Derepressed promoters for which transcription occurs upon carbon starvation have shown to be favorable alternatives. Yet, many of them do not reach the methanol- induced level when used derepressed.

[0016] As such, there is still an unmet need for novel methods allowing methanol-free induction from derepressed promoters.

[0017] Thus, it is an object of the present invention to provide a novel process for enhanced protein production using low or no methanol. Another object of the present invention is to provide a regulatable promoter which in respect to the presented method can be used for enhanced protein of interest (POI) production.

[0018] SUMMARY OF THE INVENTION

[0019] The present invention relates to a method for producing a polypeptide or protein of interest, the method comprising a) providing a methylotrophic yeast host cell comprising a nucleic acid molecule encoding said polypeptide or protein of interest operably linked to a derepressible promoter, b) culturing the host cell of step a) in a culture medium comprising a carbon source to obtain a cell culture, and c) adding at least one nitrogen-containing compound to cell culture of step b) or to the culture medium used in step b) to induce the derepressible promoter to overexpress said polypeptide or protein of interest.

[0020] It turned surprisingly out that nitrogen-containing compounds can be used to induce derepressible promoters in a methylotrophic yeast host cell to a level which is usually achieved exclusively by adding methanol.

[0021] The addition of nitrogen-containing compounds to a cell culture or cell culture medium used to cultivate the methylotrophic yeast host cell comprising a nucleic acid molecule encoding a polypeptide or protein of interest operably linked to a derepressible promoter results in a significantly increased expression / production of the polypeptide or protein of interest.

[0022] In one aspect the present invention relates to the use of one or more nitrogencontaining compounds as an inducer of a derepressible promoter in a methylotrophic yeast host cell.

[0023] In another aspect the invention allows to reduce the amount of methanol, for instance, in the expression of the polypeptide or protein of interest, by the use of one or more nitrogen-containing compounds.

[0024] In a further aspect the invention allows to completely avoid methanol by the use of one or more nitrogen-containing compounds in a DAO overexpressing strain background.

[0025] Many efforts have been made to establish methanol-free protein production processes in a methylotrophic yeast like Pichia. The inventors surprisingly found that promoters like PCAT1 can be induced by a single nitrogen-containing compound yielding the same or an even higher amount of active protein as compared with methanol. It is the first time nitrogen-containing compounds like alanine are described as inducers of derepressible promoters.

[0026] BRIEF DESCRIPTION OF THE FIGURES

[0027] Fig. 1 shows a comparison of Agl production using PCAT1 with different inducers. Cultivation in 250 ml shake flask (culture volume 50 ml). Media: buffered minimal medium: 1 % (w / v) glucose (D), pH 6.0. Induction: 48 h post inoculation. Inducers: 0.25% (w / v) glycerol (derepression), 1 % (w / v) D-alanine (alanine), 1 % (w / v) D-alanine + 0.1 % (v / v) methanol (alanine / low methanol), 1 % (v / v) methanol (methanol). pNPG activity prior (0 h) and 24 h post induction, 48 h and 72 h post inoculation. Activities as % activity of the methanol induced PAOX1 reference (AOX).

[0028] Fig.2 shows a comparison of Agl production using PCAT1 with D / L-alanine as inducer in bioreactor. Media: minimal salt medium + PTM1 trace salts, 30% pO2, pH 5.9-6.1. Activity: pNPG activity normalized to biomass (mA / min*(g / kg)’1). Strain: PCAT1-Agl-2. Feeding strategies (Table 1 ): derepressed (glycerol), D / L-alanine (alanine / glycerol).

[0029] Fig. 3 shows a comparison of HRP-SpG production using PCAT1 with different inducers. Cultivation in 250 ml shake flask (culture volume 50 ml). Media: buffered minimal medium: 0.5% (w / v) glycerol, pH 6.0. Induction: 24 h and 48 h post inoculation. Inducers: D-alanine, glycine, L-serine, L-lysine, urea. Final concentration of inducer: 1 % (w / v). ABTS activity 48 h post induction, 72 h post inoculation. Activities as activity units per OD600.

[0030] Fig. 4 shows a comparison of HRP-SpG production using PCAT1 with D- alanine as inducer in bioreactor. Media: basal salt medium (BSM) + PTM1 trace salts, 30% pO2, pH 5.5. Biomass: CWW (cell wet weight, g / L). Activity: Volumetric ABTS activity (U / rnl). Strain: PCAT1-HRP-SpG-1 . Feeding strategies (Table 2): methanol, derepressed (glycerol), D-alanine (alanine / glycerol).

[0031] Fig. 5 shows a comparison of Agl activity in media containing 1 % (w / w) glucose or 1 % (w / v) D / L-alanine in wild type strain background (PCAT1-Agl-3) and in a D-amino acid oxidase (DAO) overexpressing strain (PCAT1-Agl-3-DAO). Cultivation in 96 well micro titer plate (MTP). Media: buffered minimal medium (BMD / BMA). pNPG activity 48 h post induction. pNPG Activity: mA*min’1, mA*0D’1*min’1.

[0032] Fig. 6A shows the average volumetric activity of CBS 2612_PCAT_ass_HRP- SpG_ZeoR_C1 induced by several nitrogen-containing compounds in units per liter (ABTS U / L). Amino acids were used in L-configuration. Samples were measured in quadruplicates. 30 pL of undiluted supernatant were mixed with 150 pL ABTS- solution (Roche) and kinetics were measured at 405 nm for 10 minutes. Wild type: CBS2612 without reporter protein (no activity recorded).

[0033] Fig. 6B shows the average volumetric activity of CBS 2612_PCAT_ass_HRP- SpG_ZeoR_C1 induced by several nitrogen-containing compounds in units per OD600 (ABTS U / OD600). Amino acids were used in L-configuration. Samples were measured in quadruplicates. 30 pL of undiluted supernatant were mixed with 150 pL ABTS-solution (Roche) and kinetics were measured at 405 nm for 10 minutes. Wild type: CBS2612 without reporter protein (no activity recorded).

[0034] Fig. 7A shows that the addition of several nitrogen-containing compounds to a yeast cell suspension increased the overall activity of enzymes produced in the yeast cells under the control of a CAT1 promoter.

[0035] Fig. 7B shows the OD-normalized enzymatic activity of the results depicted in Fig. 7A. For most compounds added, the enzyme activity generated per cell was increased compared to the culture without inducer. Fig. 8A shows that the induction of PCAT1 by the inducers D / L-alanine, urea, L-glycine, L-proline and L-serine, measured by enzymatic activity of Agl1 , is positively correlated to the concentration of the inducer up to 2 % (w / v).

[0036] Fig. 8B shows the OD-normalized enzymatic activity of the results depicted in Fig. 8A. For all compounds tested, the enzyme activity generated per cell is positively correlated to the concentration of the inducer up to 1 % (w / v) and for some up to 5 % (w / v).

[0037] DESCRIPTION OF EMBODIMENTS

[0038] It was found that nitrogen-containing compounds can induce the transcription of nucleic acid molecules operably linked to derepressible promoters in a methylotrophic yeast host cell unexpectedly strong and in the absence of the actual inducer, methanol.

[0039] The nitrogen-containing compounds to induce derepressible promoters in a methylotrophic yeast host cell can be added to a cell culture comprising cells cultivated in a culture medium or to the culture medium that is used for culturing the methylotrophic yeast host cell.

[0040] “Promoter”, as used herein, refers to a regulatory sequence element able to regulate and influence the transcription of an operably linked nucleic acid molecule encoding a polypeptide or protein of interest.

[0041] A “derepressible promoter”, as used herein, refers to a promoter that is less active in presence of a repressing compound. A reduction in concentration of the repressing compound results in the alleviation of the repression from the derepressible promoter and transcription rate increases. When a repressor is bound to a derepressible promoter or when a repressor indirectly represses transcription of nucleic acid molecules operably linked to a derepressible promoter, transcription is substantially decreased as compared to transcription from the derepressible promoter in the absence of repressor. For instance, certain carbon sources can inhibit expression from certain promoters depending on their concentration and removal of repression, for example by carbon source depletion, is generally understood as derepression, with the result of increasing RNA transcript and protein production. Thus, a carbon source can be repressing and / or non-repressing depending on its concentration whereby the concentration at which repressing or non-repressing conditions are met depends on the regulatory element used.

[0042] A derepressible promoter does not necessarily require an inducer for transcription. The wild-type A0X1 promoter, which is induced in methylotrophic yeast cells by methanol, can be considered as a benchmark for a non-derepressible promoter. It is known that the wild-type A0X1 promoter is not significantly induced by the depletion of substances in the culture medium.

[0043] A derepressible promoter in the sense of the present invention is a promoter which shows at least a 10%, preferably at least a 25%, more preferably at least a 50%, increased transcription rate when a repressing substance is depleted in a cell culture.

[0044] “Repressing and / or non repressing carbon source”, as used herein, refers to sugars, including mono- and oligosaccharides, alcohols, or sugar alcohols, which can be used for cell growth and maintenance, and which can suppress or allow transcription depending on their concentration.

[0045] The nucleic acid molecule encoding the polypeptide or protein of interest operably linked to the derepressible promoter can be part of an expression cassette (plasmid or linear), which can be integrated into the chromosome, or be an episomal plasmid within the cell. An expression cassette may comprise a terminator and marker sequence, whereby the marker can be an auxotrophic or antibiotic marker.

[0046] “Operably linked”, as used herein, means that the derepressible promoter of the present invention is fused to a nucleic acid molecule encoding a polypeptide or protein of interest to be able to regulate and influence the transcription of said nucleic acid molecule into RNA, which thereafter is translated into the polypeptide or protein of interest.

[0047] The polypeptide or protein of interest may be a heterologous or homologous polypeptide and may comprise a secretion signal peptide fused thereto. The presence of a signal peptide facilitates or allows the secretion of the polypeptide or protein of interest from inside of the yeast host cell into the culture medium.

[0048] The heterologous polypeptide or protein of interest can be any polypeptide or protein and is preferably selected from the group of recombinant proteins including food proteins, feed proteins and cosmetic proteins, and more specifically structural proteins, enzymes, peptides, antibiotics, antibodies, antibody fragments, regulatory proteins, growth factors, hormones, and the like.

[0049] “Overexpress” or “overexpression” of a polypeptide or protein of interest under inducing conditions means that said polypeptide or protein of interest is expressed to an extent of at least 10%, preferably at least 25%, more preferably at least 50%, more preferably at least 75%, more than under non-inducing conditions.

[0050] According to a preferred embodiment of the present invention, the derepressible promoter is repressible by a carbon source, preferably by glucose, glycerol and / or ethanol when applied at a concentration >10 g / L, for instance.

[0051] According to another preferred embodiment of the present invention, the derepressible promoter is a methanol-inducible promoter. Methanol is regularly used as inducer of promoters in methylotrophic yeasts. The method of the present invention allows the use of methanol-inducible promoters under conditions requiring less or even no methanol as inducer of protein production.

[0052] As used herein, an “inducer” is a molecule that binds directly to or indirectly regulates a promoter and activates the transcription of nucleic acid molecule operatively linked to the regulatory sequence element. The use of an inducer during cultivation of the methylotrophic yeast host cell of the invention leads to an increase of the transcription rate by at least 20%, preferably at least 50%, compared to the cultivation without inducer.

[0053] According to a further preferred embodiment of the present invention, the derepressible promoter is a methanol pathway promoter.

[0054] “Methanol pathway promoters” are promoters or promoter regions, which are responsible for the regulation of genes involved in the methanol pathway of methylotrophic yeast cells.

[0055] Particularly preferred is the use of a catalase promoter as derepressible promoter. The CAT1 promoter according to the invention comprises preferably between 100 and 1000 nucleotides, even more preferably comprises 100-800 nucleotides, even more preferably 100-500 nucleotides upstream of the start codon of the CAT1 gene. The CAT1 promoter may comprise or consist of nucleic acid sequence SEQ ID No. 1 , SEQ ID No. 2, of which, regardless of their length, the same functionality can be assumed, or a variant thereof.

[0056] Variants of the promoter, which can be used according to the invention, may comprise deletions, substitutions and insertions in comparison to the naturally occurring promoter. Such promoter variants are at least 80%, preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% identical to the corresponding naturally occurring promoter, whereby % identity refers to the number of identical positions.

[0057] SEQ ID No. 1 :

[0058] CTATGTTGACGGAGAGTGTTGGGTCTAACATCATGGCACATGGTAGAG GAGGTGTTAAGACATCTAGAGAGCGTGATAGAGAGGAAGAGATCAGA GAATACGAGGAGACCAATTTCACTAGATTACCAACTTCGGTTACTGAG AAGTCAAAGAAACAAAAGAAAGATCATAGATTGAACACCTTTGCAGGA GAGGATTGGTCATTCTTTGGCAAGGACAGAGATGAAGACATGAAGAAA AGTGCGAGGAAGAATAAAAATACTGCTTCCTCCGCCTGGGAAAGAGCA AAAAGACGCAGAGGAAACTAAAGTGTGTAATCATATATATAATAAATGA GGAATAATAATTGAATAGAGATTTAACGAGTCGAAGTTTCTGAAATATA CGCACAGTTTATATTTATGATTTTGATATCTAACTACAGTCTTCTCCATA TATTTAACTATAAATAATAAAGTATATAACTCTTATGAAACTGTTTCACC ACATTTTTTTCTACGTAATCGAACTCCGAATGCGGTTCTCCTGTAACCT TAATTGTAGCATAGATCACTTAAATAAACTCATGGCCTGACATCTGTAC ACGTTCTTATTGGTCTTTTAGCAATCTTGAAGTCTTTCTATTGTTCCGGT CGGCATTACCTAATAAATTCGAATCGAGATTGCTAGTACCTGATATCAT ATGAAGTAATCATCACATGCAAGTTCCATGATACCCTCTACTAATGGAA TTGAACAAAGTTTAAGCTTCTCGCACGAGACCGAATCCATACTATGCAC CCCTCAAAGTTGGGATTAGTCAGGAAAGCTGAGCAATTAACTTCCCTC GATTGGCCTGGACTTTTCGCTTAGCCTGCCGCAATCGGTAAGTTTCAT TATCCCAGCGGGGTGATAGCCTCTGTTGCTCATCAGGCCAAAATCATA TATAAGCTGTAGACCCAGCACTTCAATTACTTGAAATTCACCATAACAC TTGCTCTAGTCAAGACTTACAATTAAA

[0059] SEQ ID No. 2:

[0060] TAATCGAACTCCGAATGCGGTTCTCCTGTAACCTTAATTGTAGCATAGA TCACTTAAATAAACTCATGGCCTGACATCTGTACACGTTCTTATTGGTC TTTTAGCAATCTTGAAGTCTTTCTATTGTTCCGGTCGGCATTACCTAATA AATTCGAATCGAGATTGCTAGTACCTGATATCATATGAAGTAATCATCA CATGCAAGTTCCATGATACCCTCTACTAATGGAATTGAACAAAGTTTAA GCTTCTCGCACGAGACCGAATCCATACTATGCACCCCTCAAAGTTGGG ATTAGTCAGGAAAGCTGAGCAATTAACTTCCCTCGATTGGCCTGGACT TTTCGCTTAGCCTGCCGCAATCGGTAAGTTTCATTATCCCAGCGGGGT GATAGCCTCTGTTGCTCATCAGGCCAAAATCATATATAAGCTGTAGACC CAGCACTTCAATTACTTGAAATTCACCATAACACTTGCTCTAGTCAAGA CTTACAATTAAA

[0061] According to a preferred embodiment of the present invention, the inducer is at least one “nitrogen-containing compound”. Surprisingly, nitrogen-containing compounds are suited to strongly induce derepressible promoters.

[0062] “Nitrogen-containing compound”, as used herein, refers to compounds, preferably inorganic and organic compounds, comprising one or more nitrogen atoms. These compounds may also be considered as “nitrogen-containing inducers”, since they are able to induce the derepressible promoter of the present invention.

[0063] According to another preferred embodiment of the present invention, the at least one nitrogen-containing compound comprises at least one amine and / or at least one amide group. According to another preferred embodiment of the present invention, the at least one nitrogen-containing compound is a hydrophobic, neutral or hydrophilic amino acid preferably selected from the group consisting of alanine, glycine, serine lysine, valine, proline, histidine, glutamic acid and arginine, including L-alanine, D- alanine, D / L-alanine, glycine, L-serine, D-serine, D / L-serine, L-lysine, D-lysine, D / L- lysine, L-valine, D-valine, D / L-valine, L-proline, D-proline, D / L-proline, L-histidine, D- histidine, D / L-histidine, L-glutamic-acid, D-glutamic acid, D / L-glutamic acid, L- arginine, D-arginine and D / L-arginine.

[0064] “Amino acid”, as used in the method of the present invention, preferably refers to an a-amino acid possessing an amino moiety located at the a-position relative to a carboxylic group, including glycine which has no asymmetric carbon and proline, though its -NH2 group is part of a heterocycle, and including L-enantiomers, D- enantiomers and racemates.

[0065] According to a further preferred embodiment of the present invention, the at least one nitrogen-containing compound is a compound of the purine or pyrimidine metabolism, preferably selected from the group consisting of thymidine, undine and urea. The urea carboxylase reaction hydrolyses urea-1 -carboxylate to CO2 and ammonia, the latter of which is a preferred nitrogen source of yeasts.

[0066] According to another preferred embodiment of the present invention, the at least one nitrogen-containing compound is an ammonium salt, preferably an inorganic ammonium salt or an organic ammonium salt.

[0067] According to a preferred embodiment of the present invention, the inorganic ammonium salt is selected from the group consisting of di-ammonium hydrogen phosphate and ammonium hydrogen carbonate.

[0068] According to a further preferred embodiment of the present invention, the organic ammonium salt is an ammonium salt of a carboxylic acid, preferably of a C2 to C10, C2 to Cs, C2 to Ce, or C2 to C4, carboxylic acid, more preferably ammonium acetate.

[0069] According to a preferred embodiment of the present invention, the at least one nitrogen-containing compound is added in step c) to the cell culture of step b) or to the culture medium used in step b) to a concentration of 0.005 to 40 g / L, preferably 0.01 to 20 g / L, more preferably 0.02 to 10 g / L, more preferably 0.03 to 7.5 g / L, more preferably 0.04 to 5 g / L, more preferably 0.05 to 2 g / L, more preferably 0.05 to 1 g / L.

[0070] Whereby it makes no difference whether the nitrogen-containing compound is admitted to the cell culture or cell culture medium to a certain concentration, or the yeast has been metabolically engineered to overproduce the respective compound during cultivation. According to another preferred embodiment of the present invention, at least one non-repressing or repressing carbon source is added in step c) to the cell culture of step b).

[0071] The addition of non-repressing carbon sources is particularly advantageous to allow maintenance, growth and proliferation of yeast cells in cell culture. Nonrepressing carbon sources do not significantly influence the promoter activity of the derepressible promoters used in the cells of the present invention. This allows to use non-repressing carbon sources as well as the nitrogen-containing compound as inducers at the same time during cultivation.

[0072] Similarly, repressing carbon sources can be used at a level at which derepressing conditions with respect to the repressing carbon source are met.

[0073] According to a preferred embodiment of the present invention, the at least one non-repressing or repressing carbon source is an alcohol, a sugar, including mono- and oligosaccharides, or a sugar alcohol preferably selected from the group consisting of glucose, glycerol, ethanol, sorbitol, mannitol or trehalose.

[0074] According to a further preferred embodiment of the present invention, the at least one non-repressing or repressing carbon source is added in step c) to the cell culture of step b) to a concentration of 0.01 to 10 g / L, preferably 0.05 to 5 g / L, or at a level at which derepressing conditions with respect to the repressing carbon source are met.

[0075] According to another preferred embodiment of the present invention, methanol is added in step c) to the cell culture of step b).

[0076] It surprisingly turned out that the combined use of nitrogen-containing compounds and methanol as inducers increases the promoter activity of derepressible promoters, preferably of methanol-inducible and derepressible promoters, in a methylotrophic yeast host cell even more than the addition of the nitrogen-containing compound as the sole inducer. It turned out that there is a synergistic effect when both inducers are added to the cell culture or cell culture medium in respect to the expression / production of the polypeptide or protein of interest.

[0077] According to a further preferred embodiment of the present invention, methanol is added in step c) to the cell culture of step b) to a concentration of 0.01 to 12 g / L, preferably 0.01 to 11 g / L, more preferably 0.01 to 10 g / L, more preferably 0.01 to 8 g / L, more preferably 0.01 to 6 g / L, more preferably 0.01 to 5 g / L, more preferably 0.01 to 4 g / L, more preferably 0.01 to 3 g / L, more preferably 0.01 to 2 g / L, more preferably 0.01 to 1 g / L.

[0078] According to another preferred embodiment of the present invention, the methylotrophic yeast host cell is genetically engineered to overexpress D-amino acid oxidase (DAO, EC 1.4.3.3), resulting in enhanced induction of the derepressible promoter, preferably the CAT1 promoter, in the presence of nitrogen-containing compounds, preferably D-amino acids, more preferably D-alanine, compared to wild type strain background.

[0079] It turned surprisingly out that not only methanol co-feeding, but also DAO overexpression synergistically improves expression / production of the polypeptide or protein of interest under the control of a derepressible promoter, preferably the CAT1 promoter, in the presence of nitrogen-containing compounds, preferably D-amino acids, more preferably D-alanine.

[0080] DAO catalyses the oxidative deamination of D-amino acids to the corresponding a-keto acid with strict stereospecificity thereby creating H2O2

[0011] . Similarly, H2O2 is generated by conversion of methanol to formaldehyde, which is known to induce the CAT1 promoter. The principle of enhanced CAT1 promoter induction could be the same for methanol conversion and DAO overexpression, and explain the further improvement seen.

[0081] Overexpression of DAO might have the additional beneficial effect that D- amino acids become more readily available as a nitrogen and energy source for the cells.

[0082] The DAO is an enzyme with D-amino acid oxidase activity and may comprise or consist of amino acid sequence SEQ ID No. 3 or a variant thereof.

[0083] Variants of the DAO, which can be used according to the invention, may comprise deletions, substitutions and insertions in comparison to the naturally occurring sequence. Such variants are at least 80%, preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% identical to the naturally occurring sequence, whereby % identity refers to the number of identical positions.

[0084] SEQ ID No. 3:

[0085] MTDSKYVIIGAGISGLYTAWSLIDKGTGPSDIKVVAEFLPGDQSTLYTSPW AGGNFSLITSTDERSMKFDKFTYTNLHRIQELLGGPECGLDMLPSTEMFE QELDHAKLDSISQYLKEYRPMTKEEMPEGWSGVKFLTWNFNCPLFLANF QKHLAAIGVTFERSKIDHISSVFSPSVDAVFNCTGIGAASLGGVKDENVFP TRGQWWRAPHIRENRFRWRPDSDTYVIPRPFSDGSIVMGGFFQEGNW SGNTYGYETEDILKRGLELYPEIGKRNELKIIREAAGLRPSRKGGVRIEVEH FDQVNGKDRYIVHNYGASGYGYQSGLGMANEATDMYFEAAK According to another preferred embodiment of the present invention, in step c) the at least one nitrogen-containing compound and optionally methanol and / or a nonrepressing carbon source and / or a repressing carbon source is added to the cell culture of step b) preferably when a cell wet weight of at least 20 g / L, preferably of at least 30 g / L, more preferably of at least 40 g / L, more preferably of at least 50 g / L, medium is reached. The cells present in the cell culture of step b) can be methylotrophic yeast host cells overexpressing DAO.

[0086] According to a further preferred embodiment of the present invention, the cells are cultured in a system selected from a group comprising batch cultivation, fed batch cultivation and continuous cultivation.

[0087] It is particularly preferred to combine a batch cultivation method and / or a fed batch cultivation method, where any carbon source or carbon source mixture suitable for cell growth is used, in combination with either of the following cultivation methods:

[0088] 1 ) a fed batch in which the nitrogen-containing compounds are supplemented as single inducers or a combination thereof.

[0089] 2) a fed batch in which the at least one nitrogen-containing compound is supplemented together with a non-repressing carbon source selected from the group of sorbitol, mannitol, trehalose or the like.

[0090] 3) a fed batch in which the at least one nitrogen-containing compound is supplemented together with a repressing carbon source at a concentration at which derepressing conditions are met selected from the group of glucose, glycerol, ethanol or the like.

[0091] 4) a fed batch in which the at least one nitrogen-containing compound is supplemented together with low levels of methanol.

[0092] “Low levels of methanol”, as used herein, refers to 1 / 10 of the usually applied methanol levels of 1 % (v / v).

[0093] According to another preferred embodiment of the present invention, the methylotrophic yeast host cell is selected from the group consisting of the genera Komagataella, Pichia, Candida and Ogataea, wherein methylotrophic yeast host cells of the genus Komagataella are particularly preferred.

[0094] According to a further preferred embodiment of the present invention, the methylotrophic yeast host cell is selected from the group consisting of Komagataella pastoris, Komagataella kurtzmanii, Komagataella phaffii, Komagataella populi, Komagataella pseudopastoris, Komagataella ulmi and Komagataella sp. 11-1192, wherein Komagataella phaffii is particularly preferred.

[0095] The present invention is further illustrated by the following examples, however, without being restricted thereto. EXAMPLES

[0096] Example 1 : Alanine as inducer of PCAT1 using alpha-glucosidase as reporter enzyme

[0097] As reporter enzyme alpha-glucosidase (ABE96517, Agl) was used. Codon optimized Agl was cloned in frame into plasmid pPpT5a-PCAT1 and plasmid pPpT5a-PAOX1 using the restriction sites Xho\ and Not. Plasmid pPpT5a builds on plasmid pPpT4 as published under NCBI Accession number JQ519689.1. Pichia pastoris strain CBS7435 was transformed with BglW linearized plasmids, i.e. , pPpT5a-PCAT1 -Agl and pPpT5a-PAOX1 -Agl, following a protocol described in

[0012] , Transformants were obtained after 48 hours of incubation on selective agar-plates containing 100 pg / ml zeocin. Transformants were cultivated in shake flask using 50 ml BMD, 120 rpm and 28°C. BMD media contained 200 ml KPi (1 M) buffer pH6, 100 ml 10xYNB, 600 ml H2O, 100 ml 10% (w / v) glucose (D) and 2 ml 500x Biotin.

[0098] Cultures were typically grown for 48 hours and induced one-time with inducer. D-alanine was used in a final concentration of 1 % (w / v), methanol in a final concentration of 1 % (v / v) and in case of alanine / low methanol, 1 % (w / v) D-alanine and 0.1 % (v / v) methanol were used. Agl expression using different inducers was evaluated 24 hours post induction using pNPG assay. pNPG activity assay was performed by adding 40 pl 10 mM 4-Nitrophenyl a-D- glucopyranoside (pNPG; in dF ) stock solution to premixed and preheated 34°C master mix (110 pl dF + 20 pl Citric acid-Na2PO4-Buffer pH7) and 30 pl supernatant. Absorbance was measured every 15 seconds at 405 nm (at 34°C) for a total of ten minutes.

[0099] Addition of alanine resulted in active enzyme comparable to the methanol induced reference (PAOX1 ), while induction using a mixture of alanine / low methanol further boosted activity levels, reaching 150% relative activity (Fig. 1 ). For comparison, derepression alone (spiking glycerol to 0.25% w / v) resulted in only 70% of the activity of the methanol induced reference.

[0100] The applicability of using alanine as inducer for Agl production was demonstrated in a bioreactor. A derepressed and alanine-induced process were evaluated in comparison. PCAT1 driven expression allows comparing different conditions with one strain. Accordingly, strain PCAT1 -Agl-2 was studied using two feeding strategies, i.e., derepressed and alanine-induced (Table 1 ), thereby avoiding copy number effects or bias from different integration loci.

[0101] For cultivation minimal salt medium was used and contained 40 g / kg glycerol 2.0 g / kg citric acid ■ H2O, 12.4 g / kg (NH4)2HPO4, 16.4 g / kg K2SO4, 13ml / kg 17% (v / v) H3PO4. Final pH 5.9-6.1. Medium was supplemented with 1 ml / kg PTM1 trace salt solution and 5ml / kg 2M MgSO4■ 7 H2O stock solution. PTM1 containing (per 1000g in ddH2O) 6.0 g CuSO4■ 5 H2O, 0.8 g KI, 3.0 g MnSO4H2O, 0.2 g Na2MoO4■ 2 H2O, 0.02 g H3BO3, 0.5 g CaSO4■ 2 H2O, 42.0 g ZnSO4■ 7 H2O, 65.0 g FeSO4■ 7 H2O, 0.2 g Biotin, 5ml / kg cone H2SO4.

[0102] Following feed solutions were used:

[0103] Feed 1 - 80% (w / w) glycerol

[0104] Feed 2 - 80% (w / w) glycerol + 3.2% (w / w) D / L-alanine All feeds (1 -3) were supplemented with 4 ml / kg PTM1 .

[0105] Table 1 Feeding strategies bioreactor (2000 ml bioreactor, 1600 ml working volume)

[0106] The clone PCAT1 -Agl-2 was used to demonstrate the induction of PCAT1 with alanine. Fermentation regimes consisted of a batch phase with glycerol and a subsequent fed-batch phase. Clone PCAT1 -Agl-2 was twice cultivated under derepressed conditions and under alanine-induced conditions with 3.2 % (w / w) alanine in the feed. The activity of the glucosidase Agl was measured using the 4- Nitrophenyl a-D-glucopyranoside (pNPG) assay. The increase of absorbance at 405 nm (A / min) represents the glucosidase activity.

[0107] At harvest of the two derepressed bioreactors, dry cell weight normalized pNPG activities were 1006 mA / min*(g / kg)’1and 1024 mA / min*(g / kg)’1. At harvest of the two alanine-induced bioreactors, dry cell weight normalized pNPG activities were 1409 mA / min*(g / kg)’1and 1410 A / min*(g / kg)’1(Fig. 2).

[0108] The example shows that alanine can be used to induce PCAT1 . Compared to the derepressed conditions, the DCW normalized enzyme activity in the supernatant reached 139% with alanine induction.

[0109] Example 2: Amino acids and urea as inducer of PCAT1 using horseradish peroxidase as reporter enzyme

[0110] HRP-SpG

[0013] was used as reporter protein. Streptococcal protein G (SpG) binds immunoglobulin G from various mammalian species with high affinity. It is produced by Streptococcus spp. of the Lancefield group G and conjugated to HRP, horseradish peroxidase, it is commonly used in immunohistochemical applications.

[0111] Codon optimized HRP-SpG was cloned in frame into plasmid pPpT5a-PCAT1 using the restriction sites Xho\ and Not. Plasmid pPpT5a builds on plasmid pPpT4 as published under NCBI Accession number JQ519689.1 . Pichia pastoris strain CBS2612 was transformed with BglW linearized plasmid pPpT5a-PCAT1 -HRP-SPG following a protocol described by

[0012] , Selection and cultivation were performed as described, however using 1 % (w / v) glycerol instead of 1 % (w / v) glucose (D) for cell growth during cultivation.

[0112] ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)-assay was used to quantify enzyme activity. 30 pL of supernatant were mixed with 150 pL of ABTS solution (Roche). Absorbance was measured every 30 seconds at 405 nm for a total of ten minutes.

[0113] Several clones were found active upon cultivation in BMG1 and methanol induction. Clone PCAT1 -HRP-SPG-1 was found best and used for evaluation of inducers.

[0114] The applicability of using nitrogen-containing compounds as inducers for HRP- SPG production using PCAT1 was demonstrated in shake flask. Clone PCAT1 -HRP- SPG-1 was cultivated in 50 ml BMG 0.5 % (w / v) at 120 rpm and 28°C for 24 h. Induction was performed by spiking D-alanine, glycine, L-serine, L-lysine and urea 24 and 48 hours post inoculation to a final concentration of 1 % (w / v). HRP-SPG expression using different inducers was evaluated 48 hours post induction using ABTS assay.

[0115] Use of alanine as inducer resulted in an activity of 0.00170 U OD’1(activity units per biomass). Surprisingly, strong induction was also found when using urea as inducer (0.00125 U OD-1), showing that compounds of the purine or pyrimidine metabolism can also induce PCAT1. Although to a lesser extent, induction was also observed when adding glycine, serine and lysine, reaching a level of 0.0005, 0.0003 and 0.0006 U OD’1, respectively (Fig. 3).

[0116] The applicability of using alanine as inducer for HRP-SPG production was demonstrated in a bioreactor. A methanol induced, and a derepressed process were evaluated in comparison. PCAT1 driven expression allows to compare different conditions from one strain. Accordingly, strain PCAT1 -HRP-SPG-1 was studied using three feeding strategies, i.e., methanol-induced, derepressed and alanine-induced (Table 2), thereby avoiding copy number effects or bias from different integration loci.

[0117] For cultivation basal salt medium was used and contained 0.17 g / L CaSO4 ■ 2 H2O, 2.32 g / L MgSO4■ 7 H2O, 2.86 g / L K2SO4, 2.00 g / L KOH, 0.22 g / L NaCI, 40 g / L glycerol, 12.75 mL / L 85% H3PO4.

[0118] PTM1 trace salt solution 1 contained per 100 ml 0.016 g Nal, 0.04 g Na2MoO4 ■ 2 H2O, 0.004 g H3BO3, 0.146 g C0CI2 ■ 6 H2O, 0.04 g Biotin. PTM1 trace salt solution 2 contained per 100 ml 1.2 g CuSO4 ■ 5 H2O, 0.59 g MnCL ■ 4 H2O, 4.0 g ZnCL, 13.0 g FeSO4 ■ 7 H2O, 1 .0 g H2SO4 cone. Following feed solutions were used:

[0119] Feed 1 - 100% methanol

[0120] Feed 2 - 50% (w / v) glycerol or sorbitol

[0121] Feed 3 - 50% (w / v) glycerol + 15% (w / v) D-alanine

[0122] BSM and all feeds (1-3) were supplemented with 4.3 ml / L PTM1 trace salt solution containing 1 +1 PTM1 trace salt solution 1 and PTM1 trace salt solution 2.

[0123] Table 2 Feeding strategies bioreactor (5000 ml, 4500 ml working volume)

[0124] ‘Adaptation to feed rates was done manually in small increments. Feed rates are given as average values.

[0125] The clone PCAT1-HRP-SPG-1 was used to demonstrate the induction of PCAT1 with alanine. Fermentation regimes consisted of a batch phase with glycerol and a subsequent fed-batch phase. Clone PCAT1-HRP-SPG-1 was cultivated under methanol-induced, derepressed and under alanine-induced conditions. Activity units of the HRP were calculated using ABTS assay, based on the increase of absorbance at 405 nm (A / min).

[0126] 75 U ml’1were reached for the methanol induced process after a total cultivation time of 112.5 h (Fig. 4 A), while a process based on derepression yielded 32 U ml’1only (after 125 h of cultivation; Fig. 4 B), which however is in accordance with literature data. In contrast, by using alanine as inducer a titer like in the methanol-induced process was reached, specifically 70 U ml’1after 112.5 h of cultivation (Fig. 4 C), demonstrating the scalability of the novel methanol-free process.

[0127] The example shows that alanine can be used to induce PCAT1 . Compared to the derepressed conditions, enzyme activity in the supernatant reached 219% with alanine induction, and an activity level comparable to the methanol-induced process.

[0128] Many efforts have been made to establish methanol-free processes in methylotrophic yeast like Pichia. It is surprising that promoters like PCAT1 can be induced by a single nitrogen-containing compound yielding the same or an even higher amount of active protein as compared with methanol. It is the first time alanine is described as inducer of a derepressible promoter.

[0129] Example 3: Increased inducing effect of alanine on PCAT1 by overexpression of D-amino acid oxidase

[0130] It was hypothesized that the H2O2 released during conversion of D-alanine contributes to the activation of PCAT 1 . Hence, it was tested whether the overexpression of DAO (SEQ ID No. 3) could increase the inducing effect of alanine, especially D-alanine or D / L-alanine. The gene of DA01 was cloned into plasmid pPpT5 (which builds on plasmid pPpT4 as published under NCBI Accession number JQ519689.1 .), leading to the construct pPpT5_PCAT1-DAO1 . In this plasmid, the gene of DA01 is under control of PCAT1 . As recipient strain, P. pastoris PCAT1-Agl- 3 was used, which harbored two copies of the expression cassette PCAT1-Agl. The strain was transformed with linearized pPpT5_PCAT1-DAO1 and transformants were selected on YPD agar plates containing zeocin. The resulting strains (PCAT1-Agl-3- DAO) expressed both the Agl gene and DA01 under control of PCAT1 . As a control, the strain PCAT1-Agl-3 (no overexpression of DAO) was streaked out to generate single colonies on agar. Two media were prepared containing either glucose (BMD: 200 mM potassium phosphate buffer, pH 6.0; 1.34 % (w / v) yeast nitrogen base; 0.00004 % (w / v) biotin; 1 % (w / w) glucose) or glucose and alanine (BMA: 200 mM potassium phosphate buffer, pH 6.0; 1.34 % (w / v) yeast nitrogen base; 0.00004 % (w / v) biotin; 0.5% (w / w) glucose; 1 % (w / v) D / L-alanine). In a 96 well micro titer plate (MTP), colonies were first picked in 500 pL YPD (PCAT1-Agl-3) or YPD containing 100 pg / mL zeocin (PCAT1-Agl-3-DAO). Cultures were cultivated overnight and then 25 pL were transferred to two new MTPs containing 250 pL BMD. After 24 h, MTP1 was supplemented with 250 pL BMD and MTP2 was supplemented with 250 pL BMA. Using the same pre-culture made it possible to directly compare the alanine inducing effect between PCAT1-Agl-3 and PCAT1-Agl-3-DAO. Cells were cultivated for another 48 h, after which the OD was measured and the Agl glucosidase activity was determined using the pNPG assay.

[0131] As can be seen in Fig. 5, the overexpression of DAO1 has no impact on Agl activity in BMD. Agl activity is higher in media containing alanine than in media containing only glucose. The strain without DAO overexpression produced 35% more Agl in BMA than in BMD, while the DAO overexpressing strain produced 84% more Agl in BMA than in BMD. The overexpression of DAO increased the inducing effect of alanine on PCAT1 . This effect can be observed not only regarding the volumetric activity, but also the OD-normalized activity. Example 4

[0132] Additional nitrogen-containing inducers of PCAT1 using horseradish peroxidase as reporter enzyme

[0133] The reporter strain pPpT5a-PCAT1-HRP-SPG was chosen to evaluate additional inducers. The tested inducers included thymidine, undine, glycine, L- arginine, L-histidine, L-glutamic acid monosodium salt monohydrate, L-serine, L- proline, L-valine, L-lysine, pyruvic acid and citric acid. All cultivations were performed in deep well plates at 28 °C and 320 rpm. Media components were as follows: BMG1 (200 mM potassium phosphate buffer pH 6, 1.34% (w / v) yeast nitrogen base, 1 % (w / v) glycerol, 0.4 pg / mL biotin) and buffered minimal inducer (200 mM potassium phosphate buffer pH 6, 1 .34% (w / v) yeast nitrogen base, 2 or 10% (w / v) of the respective inducer, 0.4 pg / mL biotin). A preculture of the strain was grown in 50 ml BMG1 medium in shake flask for two days at 28°C and 150 rpm. A deep well plate was filled with 250 pL BMG1 medium each well and was inoculated with 20 pL of the preculture to start the cultivation. The batch phase lasted for 72 hours. Cultures were induced after 72 and 96 hours of cultivation with buffered minimal inducer to a final concentration of the inducer of 1 % (w / v). Induction at 72 hours was performed using 250 pL of buffered minimal inducer 2%. Induction at 96 hours was performed using 50 pL of buffered minimal inducer 10%. After 120 hours of cultivation, ODeoo values were measured (1 :20 dilution), and cells were harvested by centrifugation. To test the HRP activity ABTS-assay was performed with the undiluted supernatant. 30 pL of supernatant were mixed with 150 pL ready-to-use ABTS-solution (Roche, # 11684302001 ) and kinetics were measured at 405 nm for 10 minutes using a BMG Labtech SPECTROstar Nano microplate reader.

[0134] Since the growth in the presence of the inducer can play a decisive role, the U / ODeoo values (Fig. 6B) are discussed in the following, while U / L are summarized in Fig. 6A. In this experimental setup, induction with glycine as inducer gave the best results, reaching an HRP activity level of 0.00193 U / ODeoo. Surprisingly, the nucleosides thymidine and uridine also performed well as inducers, reaching 0.00189 and 0.00162 U / ODeoo, respectively. In addition to serine, also valine, proline and lysine were confirmed as inducers of PCAT1 , reaching activities between 0.0009- 0.0014 U / ODeoo. Low induction was observed for histidine (0.00046 U / ODeoo) and even lower for arginine and glutamic acid (0.00015-0.00018 U / ODeoo), while induction with pyruvic acid and citric acid resulted in no enzyme activity.

[0135] Example 5: Additional nitrogen-containing inducers of PCAT1 using alpha-glucosidase as reporter enzyme The following nitrogen-containing substances were tested for their potential to induce PCAT1 : di-ammonium hydrogen phosphate, ammonium hydrogen carbonate, ammonium acetate, urea, D / L-alanine, L-arginine, L-glutamic acid monosodium, L- glycine, L-lysine mono hydrochloride, L-proline and L-serine. The reporter strain PCAT1-Agl-2, described in example 1 , was chosen to test the effect of these inducers on the activity of PCAT1 . The strain was streaked out on selective YPD agar plates containing zeocin. A single colony was used to inoculate a 5 mL YPD zeocin test tube that was cultivated at 28°C and 200 rpm for 7 h. The optical density (OD) was measured and a shake flask with 50 mL BMD (200 mL KPi (1 M) buffer pH6, 100 mL 10xYNB, 600 mL H2O, 100 ml 10% (w / v) glucose (D) and 2 mL 500x Biotin) was inoculated with an OD of 0.4. The shake flask was incubated at 28°C and 180 rpm for 18 hours until reaching an OD of 18. The cells were harvested by centrifugation and the pellet was resuspended in fresh BMD medium to reach a final OD of 50. BMD media containing different inducers at a concentration of 1 % (w / v) were prepared and inoculated with the cell suspension to a starting OD of 1. As a control, BMD without inducer was inoculated as well. The different media containing inducers and cells of the strain PCAT1-Agl-2 were each aliquoted to 12 wells of 96 well micro titer plates. The micro titer plates were incubated at 30°C and 300 rpm. After 48 h, the OD was measured and the alpha-glucosidase activity was determined using the pNPG assay. The activity was normalized to the control BMD without inducer. This analysis was done with the volumetric activity and the OD-normalized activity.

[0136] As can be seen in Fig. 7A, the addition of several nitrogen-containing compounds increased the overall activity. The strongest positive effects were measured for ammonium salts (di-ammonium hydrogen phosphate, ammonium hydrogen carbonate, ammonium acetate), urea and the amino acids D / L-alanine, L- arginine, L-glycine, L-proline and L-serine. This increase in activity was in some cases supported by increased cell growth. Therefore, the OD-normalized activity was compared in Fig. 7B. The strongest increases in activity per cells were achieved by addition of di-ammonium hydrogen phosphate, ammonium hydrogen carbonate, urea, D / L-alanine, L-arginine, L-glycine, L-proline and L-serine. This example demonstrates that PCAT1 can be induced by several nitrogen-containing compounds, especially different amino acids.

[0137] Example 6: Concentration dependent effects of nitrogen-containing inducers of PCAT1 using alpha-glucosidase as reporter enzyme

[0138] The following substances were used to demonstrate a concentration dependent induction of PCAT1 : urea, D / L-alanine, L-glycine, L-proline and L-serine. The reporter strain PCAT1-Agl-2, described in example 1 , was chosen to test different concentrations of these compounds for the induction of PCAT1 . The strain was streaked out on selective YPD agar plates containing zeocin. A single colony was used to inoculate a 5 mL YPD zeocin test tube that was cultivated at 28°C and 200 rpm for 7 h. The OD was measured and a shake flask with 50 mL BMD (200 mL KPi (1 M) buffer pH6, 100 mL 10xYNB, 600 mL H2O, 100 mL 10% (w / v) glucose (D) and 2 mL 500x Biotin) was inoculated with an OD of 0.4. The shake flask was incubated at 28°C and 180 rpm for 18 hours until reaching an OD of 13. The cells were harvested by centrifugation and the pellet was resuspended in fresh BMD to reach a final OD of 50. BMD media containing different inducers at the concentrations 0.1 % (w / v), 0.5% (w / v), 1 % (w / v), 2% (w / v) and 5% (w / v) were prepared and inoculated with the cell suspension to a starting OD of 1 . As a control, BMD without inducer was inoculated as well. The different media containing inducers and cells of the strain PCAT1-Agl-2 were each aliquoted to 12 wells of 96 well microtiter plates. The micro titer plates were incubated at 30°C and 300 rpm. After 48h the OD was measured and the alpha-glucosidase activity was determined using the pNPG assay. The activity was normalized to the control BMD without inducer. This analysis was done with the volumetric activity and the OD-normalized activity.

[0139] As can be seen in Fig. 8A, the addition of increasing amounts of urea, D / L- alanine, L-glycine, L-proline and L-serine further increase the volumetric activity of the alpha-glucosidase Agl1 up to concentrations of 2% (w / v). In the case of L-proline, it is further increased up to a concentration of 5% (w / v). This increase in activity was in part supported by increased cell growth. Therefore, the OD-normalized activity was compared in Fig. 8B. For all five compounds tested, the alpha-glucosidase activity generated per cell was increased with higher amounts of the inducer up to a concentration of at least 0.5% (w / v), in some cases 1 % (w / v) or even 5% (w / v). This experiment demonstrated the concentration dependent induction of PCAT1 by the nitrogen-containing compounds urea, D / L-alanine, L-glycine, L-proline and L-serine.

[0140] In summary it was shown that the methanol-inducible PCAT1 can surprisingly also be strongly induced by several nitrogen-containing compounds, especially different amino acids such as alanine, glycine and serine. It was also shown that the inducing effect is positively correlated with the concentration of the inducer. Overexpression of DAO increased the inducing effect of alanine on PCAT1 . LITERATURE

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[0010] D. Resina, A. Serrano, F. Valero, P. Ferrer, Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter, J. Biotechnol. 109 (2004) 103-113. https: / / doi.Org / 10.1016 / J. JBIOTEC.2003.10.029.

[0011] S.H. Klompmaker, A. Kilic, R.J. Baerends, M. Veenhuis, I. J. van der Klei. Activation of a peroxisomal Pichia pastoris D-amino acid oxidase, which uses d- alanine as a preferred substrate, depends on pyruvate carboxylase. FEMS Yeast Res. 10 (2010) 708-16. https: / / doi.10.1111 / j.1567-1364.2010.00647.x.

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[0152]

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Claims

CLAIMS:1 . A method for producing a polypeptide or protein of interest, the method comprising a) providing a methylotrophic yeast host cell comprising a nucleic acid molecule encoding said polypeptide or protein of interest operably linked to a derepressible promoter, b) culturing the host cell of step a) in a culture medium comprising a carbon source to obtain a cell culture, and c) adding at least one nitrogen-containing compound to cell culture of step b) or to the culture medium used in step b) to induce the derepressible promoter to overexpress said polypeptide or protein of interest.

2. Method according to claim 1 , wherein the derepressible promoter is a methanolinducible promoter, preferably a methanol pathway promoter.

3. Method according to claim 1 , wherein the derepressible promoter is repressible by a carbon source, preferably by glucose, glycerol and / or ethanol.

4. Method according to claim 2 or 3, wherein the derepressible promoter is the CAT1 promoter or a variant thereof, preferably comprising or consisting of SEQ ID No. 1 or SEQ ID No. 2.

5. Method according to any one of claims 1 to 4, wherein the at least one nitrogencontaining compound comprises at least one amine and / or at least one amide group.

6. Method according to any one of claims 1 to 5, wherein the at least one nitrogencontaining compound is a hydrophobic, neutral or hydrophilic amino acid, preferably selected from the group consisting of alanine, glycine, serine, lysine, valine, proline, histidine, glutamic acid and arginine.

7. Method according to any one of claims 1 to 6, wherein the at least one nitrogencontaining compound is a compound of the purine or pyrimidine metabolism, preferably selected from the group consisting of thymidine, undine and urea.

8. Method according to any one of claims 1 to 7, wherein the at least one nitrogencontaining compound is an ammonium salt, preferably an inorganic ammonium salt or an organic ammonium salt.

9. Method according to claim 8, wherein the inorganic ammonium salt is selected from the group consisting of di-ammonium hydrogen phosphate and ammonium hydrogen carbonate.

10. Method according to claim 8, wherein the organic ammonium salt is an ammonium salt of a carboxylic acid, preferably of a C2 to C10 carboxylic acid, more preferably ammonium acetate.11 . Method according to any one of claims 1 to 10, wherein the at least one nitrogencontaining compound is added in step c) to the cell culture of step b) or to the culture medium used in step b) to a concentration of 0.005 to 40 g / L, preferably 0.01 to 20 g / L, more preferably 0.02 to 10 g / L, more preferably 0.03 to 7.5 g / L, more preferably 0.04 to 5 g / L, more preferably 0.05 to 2 g / L, more preferably 0.05 to 1 g / L.

12. Method according to any one of claims 1 to 11 , wherein at least one nonrepressing or repressing carbon source is added in step c) to the cell culture of step b) to a concentration of 0.01 to 10 g / L, preferably 0.05 to 5 g / L, or at a level at which derepressing conditions with respect to the repressing carbon source are met.

13. Method according to claim 10, wherein the at least one non-repressing or repressing carbon source is an alcohol, a sugar, including mono- and oligosaccharides, or a sugar alcohol preferably selected from the group consisting of glucose, glycerol, ethanol, sorbitol, mannitol or trehalose.

14. Method according to any one of claims 1 to 13, wherein methanol is added in step c) to the cell culture of step b) to a concentration of 0.01 to 12 g / L, preferably 0.01 to 11 g / L, more preferably 0.01 to 10 g / L, more preferably 0.01 to 8 g / L, more preferably 0.01 to 6 g / L, more preferably 0.01 to 5 g / L, more preferably 0.01 to 4 g / L, more preferably 0.01 to 3 g / L, more preferably 0.01 to 2 g / L, more preferably 0.01 to 1 g / L.

15. Method according to any one of claims 1 to 14, wherein in step c) the at least one nitrogen-containing compound and optionally methanol and / or a non-repressing carbon source and / or a repressing carbon source is added to the cell culture of step b) preferably when a cell wet weight of at least 20 g / L, preferably of at least 30 g / L, more preferably of at least 40 g / L, more preferably of at least 50 g / L, medium is reached.

16. Method according to any one of claims 1 to 15, wherein D-amino acid oxidase (DAO) or a variant thereof is additionally overexpressed in said methylotrophic yeast host cell, wherein said D-amino acid oxidase preferably comprises or consists of SEQ ID No. 3 or a variant thereof.

17. Method according to any one of claims 1 to 16, wherein the methylotrophic yeast host cell is selected from the genera Komagataella, Pichia, Candida and Ogataea, preferably selected from the group Komagataella pastoris, Komagataella kurtzmanii, Komagataella phaffii, Komagataella populi, Komagataella pseudopastoris, Komagataella ulmi and Komagataella sp. 11-1192.

18. Use of one or more nitrogen-containing compounds as an inducer of a derepressible promoter in a methylotrophic yeast host cell.

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