YEAST CELL

Abstract

The present invention relates to a yeast cell of the Komagataella genus comprising an orthologous promoter of a methylotrophic yeast cell or a variant thereof inducible by derepression, wherein the orthologous promoter is an orthologous formate dehydrogenase (FMD) promoter of a methylotrophic yeast cell.

Claims

1. A yeast cell of the Komagataella genus comprising an orthologous promoter of a methylotrophic yeast cell or a variant thereof inducible by derepression, wherein the orthologous promoter is an orthologous formate dehydrogenase (FMD) promoter of a methylotrophic yeast cell.

2. The yeast cell according to claim 1, wherein the orthologous promoter is inducible with methanol.

3. The yeast cell according to claim 1, wherein the orthologous promoter is operably linked to a nucleic acid molecule coding for a heterologous or homologous polypeptide.

4. The yeast cell according to claim 3, wherein the heterologous or homologous polypeptide comprises a signal peptide, in particular a secretion signal peptide.

5. The yeast cell according to claim 1, wherein the orthologous promoter originates from a methylotrophic yeast cell selected from the group consisting of the genre Hansenula, Candida, Komagataella, and Pichia.

6. The yeast cell according to claim 1, wherein the methylotrophic yeast cell is selected from the group consisting of Hansenula polymorpha, Candida boidinii, Pichia methanolica, Komagataella pastoris, Komagataella phaffii, Komagataella populi, Komagataella pseudopastoris, Komagataella ulmi, and Komagataella sp. 11-1192.

7. The yeast cell according to claim 1, wherein the orthologous promoter and optionally the nucleic acid molecule coding for the heterologous or homologous polypeptide in the genome and also operably linked to the promoter is optionally present in the genome or as an extrachromosomal nucleic acid construct.

8. The yeast cell according to claim 1, wherein the orthologous promoter comprises nucleic acid sequence SEQ ID No. 1 or a variant thereof.

9. The yeast cell according to claim 8, wherein the variant of SEQ ID No. 1 comprises nucleic acid sequence SEQ ID No. 27: AATGTATCTAAACGCAAACTCCGAGCTGGAAAAATGTTACCGGCGATGCGCGGACA ATTTAGAGGCGGCGAX.sub.1TCAAGAAACACCTGCTGGGCGAGCAGTCTGGAGCACAGT CTTCGATGGGCCCGAGATCCCACCGCGTTCCTGGGTACCGGGACGTGAGGCAGCGC GACATCCATCAAATATACCAGGCGCCAACCGAGTCTCTCGGAAAACAGCTTCTGGAT ATCTTCCGCTGGCGGCGCAACGACGAATAATAGTCCCTCGAGGIGACCGAATATATA IGTGIGGACGGTAAATCTGACAGGGTGTAGCAAACCTAATATTTTCCTAAAACATGCA ATCGGCTGCCCCGCX.sub.2ACGGGAAAAAGAATGACTTTGGCACTCTTCACCAGACTGGG GTGTCCCGCTCGTCTGTGCAAATAGGCTCCCACTGGTCACCCCGGATTTTGCAGAAA AAX.sub.3AGCAAGTTCCGGGGTGTCTCACTGGTGTCCGCCAATAAGAGGAGCCGGCAGG CACGGAGTCTACATCAAGCTGTCTCCGATACACTCGACTACCAX.sub.4CCGGGTCTCTCX.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18CAC, wherein X.sub.1 is adenine or no nucleotide, X.sub.2 is adenine or guanine, X.sub.3 is cytosine or thymine, X.sub.4 is thymine or guanine, X.sub.5 is adenine or cytosine, X.sub.6 is guanine or cytosine, X.sub.7 is adenine or cytosine, X.sub.8 is guanine or cytosine, X.sub.9 is adenine, guanine or cytosine, X.sub.10 is guanine or cytosine, X.sub.11 is guanine or cytosine, X.sub.12 is guanine or cytosine, X.sub.13 is guanine or cytosine, X.sub.14 is adenine or cytosine, X.sub.15 is adenine or cytosine, X.sub.16 is thymine or cytosine, X.sub.17 is guanine or cytosine, X.sub.18 is guanine or cytosine, wherein the CAC end of SEQ ID No. 27 is attached to X.sub.19, and wherein X.sub.19 is a nucleic acid sequence selected from the group consisting of TATAAATACCGCCTCCTTGCGCTCTCTGCCTICATCAATCAAATC (SEQ ID No. 28), TATATAAACTGGTGATAATTCCTTCGTTCTGAGTTCCATCTCATACTCAAACTATATTAAAACTACAACA (SEQ ID No. 29), TATAAATACAAGACGAGTGCGTCCTTTTCTAGACTCACCCATAAACAAATAATCAATAAAT (SEQ ID No. 30) and TATAAATACTGCCTACTTGTCCTCTATTCCTTCATCAATCACATC (SEQ ID No. 31).

10. The yeast cell according to claim 8, wherein the variant of SEQ ID No. 1 comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 48, SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55 and SEQ ID No. 56, preferably selected from the group consisting of SEQ ID No. 35, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 44, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 54, SEQ ID No. 55 and SEQ ID No. 56, more preferably selected from the group consisting of SEQ ID No. 35, SEQ ID No. 39, SEQ ID No. 44, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 54, SEQ ID No. 55 and SEQ ID No. 56.

11. A method for producing a heterologous polypeptide comprising the step of culturing a yeast cell according to claim 1.

12. The method according to claim 11, wherein during the culturing the expression of the heterologous polypeptide is induced or its expression rate is increased by derepressing conditions.

13. The method according to claim 11, wherein during the culturing under derepressing conditions, methanol or an alternative inductor is added.

Description

[0049] The present invention will be defined in greater detail on the basis of the following figures and examples but without being limited to them.

[0050] FIG. 1 shows the fluorescence intensities of a green fluorescent reporter protein (an improved variant of the green fluorescent protein (GFP)) in culturing yeast cells of the Komagataella genus in which a nucleic acid coding for the green fluorescent protein is operably linked to orthologous and endogenous promoters. The orthologous promoters (and endogenous promoters from P. pastoris as reference) were operably linked to the GFP reporter gene and transformed as vectors in P. pastoris. The strains were cultured for 60 hours on minimal medium (BMDl) in microtiter plates with 96 deep wells (deep well plate (DWP)) and then induced with methanol. The fluorescence of the reporter protein and OD 600 (as a measure of biomass) was measured under glucose-repressed conditions (16 h), derepressed conditions (60 h) and measured at various points in time after methanol induction. The fluorescence measurements were normalized with respect to the OD 600 values. Averages and standard deviations of four transformants each are shown in the figure.

[0051] FIG. 2 shows the curve of measurements of protein expression over time. Selected strains from FIG. 1 were cultured in shaking flasks. The protein fluorescence (FIG. 2A; ratio RFU/=D600; RFU=relative fluorescence unit), while the OD600 (FIG. 2B) and the amount of glucose (FIG. 2C) were measured over time. The glucose concentration at the start of the measurements was 55.5 mM (10 g/L). The averages (MV) and standard deviations of three transformants each are shown.

[0052] FIGS. 3A to 3C show that the orthologous HpFMD promoter is also capable of upregulating the expression of other reporter proteins such as horseradish per oxidase (HRP) (FIG. 3A), lipase B from Candida antarctica (CalB) (FIG. 3B) and a hydroxynitrile lyase from Manihot esculenta (MeHNL) (FIG. 3C). The strains were cultured in DWPs in minimal medium to the point of glucose depletion after 60 hand then additionally induced with methanol. HRP and CalB enzyme activities were measured in the culture supernatant. The activity of MeHNL was measured using digested cells. Averages and standard deviations of four transformants each are shown.

[0053] FIG. 4 shows reporter protein fluorescence of the HpFMD promoter (P_FMD) and the AOXl promoter (P_AOXl) wild type sequence promoters tested. The strain background is the P. pastoris Bgll KU70. Cultivation was done in deep well plate (DWP). Reporter protein fluorescence and OD600 were measured under glucose derepressed (24 and 48 h) and two different time points of methanol induction (72 and 96 h). The strain harboring the FMD promoter was used as reference strains for testing various promoter variants.

EXAMPLES

Example 1

[0054] Materials and Methods

[0055] Cloning the Promoters

[0056] The orthologous promoters were amplified by means of PCR and cloned before a GFP reporter gene. To do so, the reporter plasmid pPpT4mutZeoMlyI-intARG4-eGFP-Bmristuffer (T. Vogl et al. ACS Synth Biol. 2015, DOI: 10.1021/acssynbio.5b00199; published on 22 Nov. 2015).

[0057] This plasmid is based on the pPpT4 vector, which was described by L. Naatsaari et al. (PLoS One 7 (2012): e39720). The promoters were cloned seamlessly (i.e., without any restriction enzyme cleavage sites or linker sequences between the promoter and the start codon) to obtain the natural context. Primers were designed on the basis of literature references (HpFMD promoters (H. Song et al. Biotechnol Lett 25 (2003):1999-2006; A. M. Ledeboer et al. Nucleic Acids Res 13 (1985):3063-3082), CbAODl promoter (H. Yurimoto et al. Biochim Biophys Acta 1493 (2000):56-63), CbFLDl promoter (B. Lee et al. Microbiology 148 (2000): 2697-704), Pm MODl and MOD2 promoters (C. K. Raymond et al. Yeast 14 (1998):11-23; T. Nakagawa et al. J Biosci Bioeng 91 (2001):225-7; T. Nakagawa et al. Yeast 23 (2006):15-22). The primer sequences used are given in Table A:

TABLE-US-00004 TABLE A Primers for amplification of the orthologous promoters SEQ ID Name Sequence No. HpFMDfwd AATGTATCTAAACG 3 CAAACTCCGAGCTG HpFMDrev GATTTGATTGATGA 4 AGGCAGAGAGCGCA AG HpMOXfwd TCGACGCGGAGAAC 5 GATCTCCTCGAGCT HpMOXrev TTTGTTTTTGTACT 6 TTAGATTGATGTCA CCACCGTGCACTGG CAG PmMODlfwd CGAGATGGTACATA 7 CTTAAAAGCTGCCA TATTGAG PmMODlrev TTTGAGAAATTAAT 8 AGTAAGATTTTTTT TTCGTAAAAGTTTT GATTGAGTTAATTC PmMOD2fwd GGATCCACTACAGT 9 TTACCAATTGATTA CGCCAATAG PmMOD2rev TTTGAATTTTAGTT 10 TTAGATAGATAAAT ATAATTTTCAATCC TGTTATAAAATAGT ATAT CbAODlfwd GGAGTATACGTAAA 11 TATATAATTATATA TAATCATATATATG AATACAATGAAAG CbAODlrev TATTGAAAAATAAT 12 TTTGTTTTTTTTTT TTTGTTTTTTTAAA AGTTCGTTAAAATT CG CbFLDlfwd GGATCCCTTCAACA 13 GCGGAGTCTCAAAC CbFLDlrev TTTTGTGGAATAAA 14 AAATAGATAAATAT GATTTAGTGTAGTT GATTCAATCAATTG AC

[0058] Genomic DNA of the strains Hp (Hansenula polymorpha) DSM 70277, Cb (Candida boidinii) DSM 70026 and Pm (Pichia methanolica) DSM 2147 were isolated and used as templates for the PCR reactions. The PCR products were cloned by TA cloning in the vector pPpT4mutzeoMlyI-intARG4-eGFP-Bmristuffer (see also US 2015/0011407 and T. Vogl et al. (ACS Synth Biol. 2015, DOI: 10.1021/acssynbio.5b00199; published on 22 Nov. 2015)) The control vectors for the P. pastoris endogenous promoters AOXl, CATl and GAP are taken from US 2015/0011407.

[0059] The alternative reporter vectors, containing HRP (isoenzyme A2A; L. Naatsaari et al. BMC Genomics 15 (2014):227), CalB and MeHNL downstream from the corresponding promoters, were taken from US 2015/0011407 or created by installing the eGFP reporter gene that had been cut from the above-mentioned eGFP vectors (restriction enzymes NheI and NotI) and the PCR products of HRP, CalB and MeHNL were installed seamlessly by recombinant cloning. The primers indicated in Table B were used for the PCR amplifications.

TABLE-US-00005 TABLE B Primers for cloning promoters upstream from various reporter genes SEQ ID Primer Sequence No. pHpFMD-MFalpha- cttgcgctctctgc 15 Gib cttcatcaatcaaa tcatgagattccca tctattttcaccgc tgtc AOXlTT-NotI- caaatggcattctg 16 CalB acatcctcttgagc ggccgcttatgggg gcacgataccggaa caag AOX1TT-NotI- caaatggcattctg 17 HRPA2A acatcctcttgagc ggccgcttaggatc cgttaactttcttg caatcaagtc seq-pHpHMD- actggtgtccgcca 18 149..126fwd ataagaggag pHpFMD-MeHNL cttgcgctctctgc 19 cttcatcaatcaaa tcatg gttactgctcacttc gtcttgattcac AOXlTT-NotI- caaatggcattctga 20 MeHNL catcctcttgagcgg ccgcttaagcgtaag cgtcggcaacttcct g pCATl-MeHNL- cacttgctctagtca 21 Gib agacttacaattaaa atggttactgctcac ttcgtcttgattcac

[0060] The HRP and CalB vectors mentioned in the literature where therefore used as PCR templates (US 2015/0011407 and T. Vogl et al. (ACS Synth Biol. 2015, DOI:10.1021/acssynbio.5b00199; published on 22 Nov. 2015). The MeHNL sequence was optimized for the P. pastoris codon and designed as a synthetic double-stranded DNA fragment with overhangs to the AOXl promoter and terminator (see Table B). This fragment was used as a template for PCRs. The following sequence was used:

TABLE-US-00006 (SEQ ID No. 22) cgacaacttgagaagatcaaaaaacaactaattattgaaagaattcc gaaacgATGGTTACTGCTCACTTCGTCTTGATTCACACTATCTGTCA TGGTGCTTGGATCTGGCACAAGTTGAAGCCAGCATTGGAGAGAGCTG GACATAAGGTTACCGCTCTTGATATGGCTGCATCTGGTATTGATCCT CGTCAAATCGAACAAATCAATTCATTCGACGAGTACTCAGAGCCACT GCTGACCTTCTTGGAAAAGTTGCCTCAAGGTGAAAAGGTGATCATCG TTGGTGAATCCTGTGCTGGATTGAACATTGCCATTGCAGCTGATAGA TATGTCGATAAGATCGCTGCTGGTGTCTTCCACAACTCTCTGTTACC AGATACTGTTCACTCTCCATCTTACACTGTCGAGAAGTTGTTAGAAT CATTCCCAGATTGGAGAGATACTGAATACTTTACTTTCACTAACATC ACTGGAGAGACTATCACCACCATGAAACTTGGATTCGTTTTGTTGAG AGAAAACCTTTTCACCAAGTGTACTGATGGTGAATACGAATTGGCCA AGATGGTTATGAGAAAGGGTTCTTTGTTTCAGAATGTTCTTGCACAA AGACCAAAGTTCACCGAAAAGGGTTACGGTTCTATCAAGAAGGTCTA CATCTGGACTGATCAGGACAAGATCTTCCTGCCAGACTTCCAAAGAT GGCAAATCGCAAACTACAAACCAGATAAGGTCTACCAAGTCCAAGGT GGTGATCACAAGTTACAATTGACCAAGACCGAAGAGGTCGCTCACAT CTTGCAGGAAGTTGCCGACGCTTACGCTTAAgcggccgctcaagagg atgtcagaatgccatttgcctg

[0061] The protein coding sequence here is large and the start and stop codon is shown in bold font, while overhangs to the vector for recombinant cloning are written in lower case letters, EcoRI and NotI, which are cleavage sites typically used for cloning in the pPpT4 vector family, are underlined.

[0062] The same forward primer (pHpFMD-MFalpha-Gib) was also used for PCR amplification of the HRP and CalB genes because the two genes are fused to an MFalpha signal sequence. Genes cloned in the vectors were sequenced by using primers that bind to the AOXl terminator and the respective promoters (seq-pHpHMD149 . . . 126fwd for the HpFMD promoter).

[0063] Strains, Materials, Fluorescence Measurements and Enzyme Assays

[0064] Enzymatic HRP and CalB activity were determined with the substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) and p-nitrophenyl butyrate (p-NPB) according to protocols in Krainer FW (Microb Cell Fact 11 (2012):22).

[0065] For the transformations of all promoter comparisons with GFP, the CBS7435 wild type strain was used. HRP and CalB plasmids were transformed into the mutS strain (L. Naatsaari et al. (PLoS One (2012); 7:e39720) because it has a higher protein expression (F. W. Krainer et al. Microb Cell Fact 11 (2012):22). For MeHNL activity measurements, the cells were lysed by Y-PER digestion according to the manufacturer's instructions (Thermo Fisher Scientific, Y-PERTM Yeast Protein Extraction Reagent) and the activity was measured using a “mandelonitrile cyanogenase assay,” as described by R. Wiedner et al. Comput Struct Biotechnol Jl0 (2014):58-62) (final mandelonitrile concentration 1SmM).

[0066] Results

[0067] Six heterologous promoters of HpFMD, HpMOX, CbFLDl, CbAODl, PmMODl and PmMOD2 genes were tested in P. pastoris. The promoters were compared with the methanol-inducible AOXl promoter, the constitutional GAP promoter and the derepressed/methanol inducible CATl promoter in P. pastoris, namely the orthologous promoters were amplified by genomic DNA PCR and cloned in vectors with GFP as reporter gene. The following promoter sequences were used:

TABLE-US-00007 HpFMD: (SEQ ID No. 1) AATGTATCTAAACGCAAACTCCGAGCTGGAAA AATGTTACCGGCGATGCGCGGACAATTTAGAG GCGGCGATCAAGAAACACCTGCTGGGCGAGCA GTCTGGAGCACAGTCTTCGATGGGCCCGAGAT CCCACCGCGTTCCTGGGTACCGGGACGTGAGG CAGCGCGACATCCATCAAATATACCAGGCGCC AACCGAGTCTCTCGGAAAACAGCTTCTGGATA TCTTCCGCTGGCGGCGCAACGACGAATAATAG TCCCTGGAGGTGACGGAATATATATGTGTGGA GGGTAAATCTGACAGGGTGTAGCAAAGGTAAT ATTTTCCTAAAACATGCAATCGGCTGCCCCGC AACGGGAAAAAGAATGACTTTGGCACTCTTCA CCAGAGTGGGGTGTCCCGCTCGTGTGTGCAAA TAGGCTCCCACTGGTCACCCCGGATTTTGCAG AAAAACAGCAAGTTCCGGGGTGTCTCACTGGT GTCCGCCAATAAGAGGAGCCGGCAGGCACGGA GTCTACATCAAGCTGTCTCCGATACACTCGAC TACCATCCGGGTCTCTCAGAGAGGGGAATGGC ACTATAAATACCGCCTCCTTGCGCTCTCTGCC TTCATCAATCAAATC HpMOX: (SEQ ID No. 2) CGACGCGGAGAACGATCTCCTCGAGCTGCTCG CGGATCAGCTTGTGGCCCGGTAATGGAACCAG GCCGACGGCACGCTCCTTGCGGACCACGGTGG CTGGCGAGCCCAGTTTGTGAACGAGGTCGTTT AGAACGTCCTGCGCAAAGTCCAGTGTCAGATG AATGTCCTCCTCGGACCAATTCAGCATGTTCT CGAGCAGCCATCTGTCTTTGGAGTAGAAGCGT AATCTCTGCTCCTCGTTACTGTACCGGAAGAG GTAGTTTGCCTCGCCGCCCATAATGAACAGGT TCTCTTTCTGGTGGCCTGTGAGCAGCGGGGAC GTCTGGACGGCGTCGATGAGGCCCTTGAGGCG CTCGTAGTACTTGTTCGCGTCGCTGTAGCCGG CCGCGGTGACGATACCCACATAGAGGTCCTTG GCCATTAGTTTGATGAGGTGGGGCAGGATGGG CGACTCGGCATCGAAATTTTTGCCGTCGTCGT ACAGTGTGATGTCACCATCGAATGTAATGAGC TGCAGCTTGCGATCTCGGATGGTTTTGGAATG GAAGAACCGCGACATCTCCAACAGCTGGGCCG TGTTGAGAATGAGCCGGACGTCGTTGAACGAG GGGGCCACAAGCCGGCGTTTGCTGATGGCGCG GCGCTCGTCCTCGATGTAGAAGGCCTTTTCCA GAGGCAGTCTCGTGAAGAAGCTGCCAACGCTC GGAACCAGCTGCACGAGCCGAGACAATTCGGG GGTGCCGGCTTTGGTCATTTCAATGTTGTCGT CGATGAGGAGTTCGAGGTCGTGGAAGATTTCC GCGTAGCGGCGTTTTGCCTCAGAGTTTACCAT GAGGTCGTCCACTGCAGAGATGCCGTTGCTCT TCACCGCGTACAGGACGAACGGCGTGGCCAGC AGGCCCTTGATCCATTCTATGAGGCCATCTCG ACGGTGTTCCTTGAGTGCGTACTCCACTCTGT AGCGACTGGACATCTCGAGACTGGGCTTGCTG TGCTGGATGCACCAATTAATTGTTGCCGCATG CATCCTTGCACCGCAAGTTTTTAAAACCCACT CGCTTTAGCCGTCGCGTAAAACTTGTGAATCT GGCAACTGAGGGGGTTCTGCAGCCGCAACCGA ACTTTTCGCTTCGAGGACGCAGCTGGATGGTG TCATGTGAGGCTCTGTTTGCTGGCGTAGCCTA CAACGTGACCTTGCCTAACCGGACGGCGCTAC CCACTGCTGTCTGTGCCTGCTACCAGAAAATC ACCAGAGCAGCAGAGGGCCGATGTGGCAACTG GTGGGGTGTCGGACAGGCTGTTTCTCCACAGT GCAAATGCGGGTGAACCGGCCAGAAAGTAAAT TCTTATGCTACCGTGCAGTGACTCCGACATCC CCAGTTTTTGCCCTACTTGATCACAGATGGGG TCAGCGCTGCCGCTAAGTGTACCCAACCGTCC CCACACGGTCCATCTATAAATACTGCTGCCAG TGCACGGTGGTGACATCAATCTAAAGTACAAA AACAAA CbFLDl: (SEQ ID No. 23) GGATCCCTTCAACAGCGGAGTCTCAAGCAGTG GCTATTATCAGTGTATTTAATTACTGATGCAT TGTATTATAGTGCATACATAGTTAATAATTAC TCTCTGTTATCATTGAAAATTTTGAAATTCTC ACTCTCACGCAGTGCAAAACTTTGCCTAATTG AGTAAGTGGAACGCAATATTTAGGCTACATAT TTTGGATTCCCTTAAGTATGTAATCAAAGATC ATTCATACTGCCATCTTATAATATTGGAGTAT TATTATGTTGCTATACTGTTCTACCTGTTTAT TCTATTGTATGCGTCTAAATCTTTCCATCAGT TTCTATACTATCTTTCGTTTGCAATGAAATAT TACTCCAATTCGCTTGTTTCAACTCGCTTGCC TTCTCTCTTGCCTTCTTTTTTTCTTTTCATTT TATCGTTGTTTAAACGGTATATAAATATGTAA CGTTGTCGCTTAGTTTTGAGAAATCACTTTTG TTGCTCTCAATTCTGTTTTGACATCTTAAGGT TAGTCAATTGATTGAATCAACTACACTAAATC ATATTTATCTATTTTTTATTCCACAAAA CbAODl: (SEQ ID No. 24) GGAGTATACGTAAATATATAATTATATATAAT CATATATATGAATACAATGCAATGAAAGTGAA TATGATAAGATTGAAATAATAACAAACAGCGA TAAATATATCTCAAAATGGAGTTACACAACAA ATAATAATAAAATATAAATTATAAATTATAAA TTATAAAAGAATAAAAAATAAACCCCACTAAT TTATTTTATTAAAAGATAGATTGGTATCTTTA CTTAATAACAATTCTGAAACTTTATTCACTTA ATTTTATTTAACTTATTTAATTTATTTTTACC CCAGTTTTTCAGTACAATGCAGCTCCGAAACT TTATTTGGCTGTGATTTGGCTGTGATTTGGCT GTGATTTGGCTTGGCTTGGCTGGCTGGAATTG TCTCCTGCAGGAATTGCTCGGGGTCCGGTTCT CCCGCTGGCTGGCTATTTGGCGGGCTGGCTAT TTGGCGGGCTGGCTGGCTGGCTGCTCTGCCAT CTGCTGTGGCCACCCCGCATCTCTGGATGCAC GCCGTGCAGCTGGACGTGCGTCTACCCTGCAG CCGTGTGCCTTATTTCCCAATCTCCCAATCTC TCAATCTGCCAGTCAGCCAAAACACCGGCCAG GCAGGCAGGCAGGCAGGCAGGCAGGCAGTGAA GCCTTCCCACGCCCCACTCCGCATAAACATCC CCAGCAGTTTCCCCAGCAGTTTCCCCAGCTTT TCAATTTAATAAAATAGCCTGTTTCTGTTTCT GTTTTATATTATACAATTTTTTATCCTAATAA TTACTCTTTCGGGAATTAAATAATAATTATAT CATATACCCATATCACATTTTACTATATTTAC TATCTATAAATAAATTCATATTATAATATTAA TTTATATTCGCTTAATTAAAATGCTCTTTTCC ATCATCATCATCATCATCATCATCACGAGTTT TCGGTTATCAATACTCTTTTCATTAATTTCTA GAATTTCATTATTTATTTTTTATTGACTGGAA ATTTTCAATCAATTTTATTTATTTTTATTTAT TTATTTTCATATTCTTAGATTTAAACTTTTTA GATGACCGCTATTTTACTTACTTACTTACTGT TGTTTTATATTATGATAAGAATTAATTTTCAT ATTTATGATGATGATGATGTAAATTTAACCTA GTATACTATTTTAAAGTTATCACTATCTTTTA GTGCTGGCATTTTTTATTCTATTTTCATATAT GTATATACGTAAATTAAGTATCATCACGCTGC TTACTGTACGTTTAAAATGTGGAGATGGAAAT AGAGATGGGGATGAAGATGAAGATGATGAGAA TTATAAACCATTCATTCATTAATCAATCAATA TAACTTATAAAAAAATTTATATTTAAATGAAT TAATTTCCTTTATTTTAATAATATCGTTAATT CTTTTAAATTCTATTTTATTTTAATTCTTTCT TTATCATAGTTATCATATAACAATTATATAAC ATAGATACACAATTATTATTTCATTATCATAT TATTTTTTAAAATATTGATTATTTTTAAAATA ATATCTTAATTAATTAATTTTTACGAATATAC AAATTTTAACGACTTACTTTTTTTAACGAATT TTAACGAACTTTTAAAAAAACAAAAAAAAAAA AACAAAATTATTTTTCAATA PmMODl: (SEQ ID No. 25) CGAGATGGTACATACTTAAAAGCTGCCATATT GAGGAACTTCAAAGTTTTATCTGTTTTTAGAA TTAAAAGACGATTGTTGTAACAAAACGTTGTG CCTACATAAACTCAAATTAATGGAAATAGCCT GTTTTGAAAAATACACCTTCTTAAGTACTGAC AAAGTTTTGTTAAATGACTATCGAACAAGCCA TGAAATAGCACATTTCTGCCAGTCACTTTTAA CACTTTCCTGCTTGCTGGTTGACTCTCCTCAT ACAAACACCCAAAAGGGAAACTTTCAGTGTGG GGACACTTGACATCTCACATGCACCCCAGATT AATTTCCCCAGACGATGCGGAGACAAGACAAA ACAACCCTTTGTCCTGCTCTTTTCTTTCTCAC ACCGCGTGGGTGTGTGCGCAGGCAGGCAGGCA GGCAGCGGGCTGCCTGCCATCTCTAATCGCTG CTCCTCCCCCCTGGCTTCAAATAACAGCCTGC TGCTATCTGTGACCAGATTGGGACACCCCCCT CCCCTCCGAATGATCCATCACCTTTTGTCGTA CTCCGACAATGATCCTTCCCTGTCATCTTCTG GCAATCAGCTCCTTCAATAATTAAATCAAATA AGCATAAATAGTAAAATCGCATACAAACGTCA TGAAAAGTTTTATCTCTATGGCCAACGGATAG TCTATCTGCTTAATTCCATCCACTTTGGGAAC CGTTCTCTCTTTACCCCAGATTCTCAAAGCTA ATATCTGCCCCTTGTCTATTGTCCTTTCTCCG TGTACAAGCGGAGCTTTTGCCTCCCATCCTCT TGCTTTGTTTCGGTTATTTTTTTTTCTTTTGA AACTCTTGGTCAAATCAAATCAAACAAAACCA AACCTTCTATTCCATCAGATCAACCTTGTTCA ACATTCTATAAATCGATATAAATATAACCTTA TCCCTCCCTTGTTTTTTACCAATTAATCAATC TTCAAATTTCAAATATTTTCTACTTGCTTTAT TACTCAGTATTAACATTTGTTTAAACCAACTA TAACTTTTAACTGGCTTTAGAAGTTTTATTTA ACATCAGTTTCAATTTACATCTTTATTTATTA ACGAAATCTTTACGAATTAACTCAATCAAAAC TTTTACGAAAAAAAAATCTTACTATTAATTTC TCAAA PmMOD2: (SEQ ID No. 26) GGATCCACTACAGTTTACCAATTGATTACGCC AATGTGTTTATTTCACCAAGTAATTACAAAAC TGAGATTTGGTTATGTCATTATGTATTTTCGG CAATGGCTGTAATTTAAACTGGATTAGGGTTA ATTAACGTTTAGCCTACGAAAGCGGCTAGCTT TTATTTCTGCTTTTGTTTTGAGCCCGTTTCTA ATTCCAATCTTTGCAATTTCGTTCCATCTTTT AAAATTAAGTGCTCTTTTCTAATCTGATAAAG ATAAGCCATCGTAGAGTAAGTAAAACAAAATA ATGTACTGTATATTAAGCGGAAAAACTTGGAA AAGTCGTATGATGTTGAAGGAGCAAAGAATGA CTAATATTAGGAGATTTAAGCAAACAATGTTG AGGGGAACAGGACGATTAACCCCTTATAGAGG AAGCGTCTTTGATGTTCGAAGGGGGAGGGGTC AAAAGCACTGAGCAGTGCTAATTAGTAACCAA TTTCTGTAAGCAATGAAACTTGTTGCTATTGG AAATACTATTAAGTAATACAAGGTACAGACTA ATGGGGGTGAGCCGGTAGTTCAGGCTATCTTA TAGACAGACTATTCCGGATTGTCTAATCATTG GTGCACCTGGTTAATAATTATCAGTCAACTCT TTTACGGTGCTGATAGGTCTTTGCGAACTTGC CCTTGTGGAATTTGGTTGTTAATCAAACTGTT CTGTATTTCATGTCATACTACTATTGATATTA TTAATGTTACTTACTCATCTGGCCATTTAACA GGTTTGAAGCTTTAATGCTCTTAACTAACAGC AATCCATCACCGTCAACCTTAACCCCCCTGGT GCTTGCTGTCTTTATCCTTCGTATCTTTTTCA TGTTGCACCGCCCTGTTCCTTATACGGTTGTT CCCCCATAGGCTAACTTCTCTGTTTCCGACCA TCTCTGCAATAACAAAGAATTCTATACGCTTA CACTATAATCATACAATGACTCTACATGCCAT TTTCACTTTACTTACTTGCCATCGGAAGATAC TGAATCAGAAAGCCATAGTAACTACATAACTT CAAAACACACCCTTTTTACAGATTAGTTACAA TTTTGTCAATGTTTGTTTGATAACCCAAGGTG GAACGTTTCCAGTTAGACCTGTTTAATCCAAC TCACTTTACCACCCCAAAACTTTCCTACCGTT AGACAAATACTGGCTAAATCTGACGAAAACAA CCAATCAACAATTGAATCCACTGGGAGGTATC TCTAATCCACTGACAAACTTTGCTAAAACAAG AAAAAGTGGGGGCCTCCGTTGCGGAGAAGACG TGCGCAGGCTTAAAAACACAAGAGAACACTTG GAAGTACCCCAGATTTTTAGCTTCCTACTATT CTGACACCCCCTATTCAAGCACGACGGTGATT GATTCATTCAATTTTGCTGCTCCAATGATAGG ATAAACCCTTTTGGACTTCAATCAGACCTCTG TCCTCCATAGCAATATAAATACCTTCTAGTTG CCCCACTTCCTCTCTCCTGTACTGCCCCAATG AGTGACTTATTCAAGTTACTTTCTCTCTTTTC CTAACAATTAAACAAGAAGCTTTATTATAACA TTAATATACTATTTTATAACAGGATTGAAATT ATATTTATCTATCTAAAACTAAAATTCAAA

[0068] P. pastoris transformants containing plasmids with CbAODl, PmMODl and PmMOD2 promoters did not have any reporter protein fluorescence (FIG. 1). The CbFLDl promoter exhibited repression on glucose and weak induction by methanol by approximately 10% of the PpAOXl promoter. Both tested H. polymorpha promoters surprisingly retained their natural regulation profile from H. polymorpha and also in Pichia pastoris repression, derepression and methanol induction (FIGS. 1 and 2). The HpFMD promoter surprisingly exceeded the constitutional PpGAP promoter under derepressed conditions and also achieved approximately 75% of the methanol-induced PpAOXl promoter, even without feeding with additional carbon sources. The derepressed expression of the HpFMD promoter exceeded that of the reporter protein fluorescence of the strongest endogenous MUI promoter from P. pastoris (PpCATl) by a factor of approximately 3.5. After methanol induction, the HpFMD promoter exceeded the PpAOXl promoter by a factor of approximately 2. These results on a small scale (FIG. 1) have been confirmed by experiments in shaking flasks (FIG. 2), wherein glucose measurements also show clearly the derepressed regulation profile. A further increase in the technical advantages of the HpFMD promoter can be achieved by an optimized feeding rate in the bioreactor.

[0069] To investigate whether the unexpectedly strong expression of the HpFMD reporter can also be reproduced for other proteins in addition to GFP, the HpFMD promoter was cloned upstream from the coding sequences of other proteins: the secreted proteins horseradish peroxidase (HRP) and Candida antarctica lipase B (CalB) and the intracellular hydroxynitrile lyase from Manihot esculenta (cassava, MeHNL) (FIGS. 3A to 3C).

[0070] With respect to the final yields of active protein in the culture supernatant in the shaking flask experiment, the derepressed expression of all proteins by the HpFMD promoter was equal to the constitutional expression by the GAP promoter and clearly exceeded the derepressed expression by the CATl promoter. Methanol-induced enzyme activities of the HpFMD promoter exceeded the AOXl promoter activity by a factor of 2.5.

[0071] The strong expression the HpFMD promoter could also be observed with four different secreted reporter proteins as well as intracellular reporter proteins (eGFP, HRP, CalB, MeHNL). The orthologous HpFMD promoter even exceeded endogenous promoters in P. pastoris.

[0072] The orthologous promoters interestingly have very low or no sequence identities with promoters in Pichia. A BLAST search of the HpFMD promoter did not yield any significant hits in the Pichia pastoris genome; a direct alignment of the HpFMD promoter with the PpFDHl promoter also did not yield any significant similarities (BLASTN 2.2.32+, Blast 2 sequences, setting for “somewhat similar sequences (blastn)”; molecule type: nucleic acid).

[0073] Such low sequence identity is a desirable property of promoters because these foreign sequences cannot recombine with the identical sequences in the genome of Pichia and therefore cannot be lost, for example, due to homologous recombination events with similar sequences already present in the genome.

[0074] Orthologous promoters may surprisingly be highly useful tools for protein expression, as demonstrated by the higher activities by a factor of as much as 2.5 due to the HpFMD promoter. Unexpectedly, the HpFMD promoter also retained its derepressed regulation profile from H. polymorpha in P. pastoris and thus constitutes the strongest derepressed promoter in P. pastoris. Therefore, efficient production processes free of toxic and highly inflammatory methanol can be made possible.

Example 2: FMD Promoter Variants

[0075] 1. Cloning of Promoters

[0076] The pPpT4mutZeoMlyI-intArg4-EGFP-P_FMD, containing the FMD promoter having SEQ ID No. 1 served as template for PCR amplification of the promoter variants v01 to v22. Primers were designed in a way to introduce point mutations, insertions or different core promoters to the FMD promoter sequence. The promoter variants were amplified in two parts and then assembled with the backbone of the pPpT4mutZeoMlyI-intArg4-eGFP-P_FMD vector, which had been previously cut with the restriction endonuclease Sall. For the generation of the promoter variants v23 to v25 only one part was PCR amplified and the other part was ordered as synthethic DNA. In this case the two DNA fragments were assembled with the backbone of the pPpT4mutZeoMlyI-intArg4-eGFP-P_FMD vector, which had been previously cut with the restriction endonuclease NheI. For the assembly of the DNA fragments with the vector backbone assembly cloning based on sequence homology was used, resulting in a seamless transition from promoter to the reporter gene eGFP.

[0077] 2. P. pastoris Transformations and Screening

[0078] For transformations of the vectors harboring the different promoter variants v01 to v25 into yeast the P. pastoris Bgll KU70 strain was used. Compared to the wild type strain, this strain has two gene knock outs: First, the KU70 gene, which encodes for a protein involved in the non-homologous end joining machinery. By knocking out this gene, homologous recombination events are more likely to happen in P. pastoris. This facilitates targeting of the vectors into a defined locus, in this case the ARG4 locus to avoid unexpected effects by different integration loci in the genome. The second knocked out gene is the AOXl gene (mutS/Bgll strain). By using this knock out strain higher yields of heterologous expressed proteins under the control of a methanol inducible promoter can be achieved (Krainer F W et al. Microb. Cell Fact. 11 (2012) p. 22).

[0079] P. pastoris Bgll KU70 was transformed with BglII linearized plasmids according to the condensed protocol of Lin-Cereghino et al. (Biotechniques 38 (2005): 44-48). To have reference strains for the screening the same vector as for the promoter variants—but with the non modified FMD promoter of SEQ ID NRl and the AOXl promoter instead—were transformed as well. About 500 ng, which is relatively low amounts of DNA were transformed to avoid multi copy integrations. For example, using 1 μg of a linearized pPpT4_S vector typically only yields single copy transformants (Vogl T et al. ACS Synth. Biol. 3 (2014):188-191).

[0080] For 9 constructs 42 transformants were screened to show the uniformity of the expression landscapes. Since the landscape for all of those tested constructs proved to be uniform, only 16 transformants per construct were picked and cultivated on two different deep well plates (DWP) in the second screening round. DWP cultivations were adapted from the protocol reported by Weis et al. (Weis R et al. FEMS Yeast Res. 5 (2004):179-89). Single colonies were picked and used to inoculate BMD (250 μl) into 96 well DWPs and cultivated for 48 h. Then BMM2 (250 μl) was added to induce the cells for the first the time. The cells were induced another 3 times with BMMl0 (50 μl) after 60, 72 and 84 hours of cultivation in the DWP. Samples were taken and measured after 48, 72 and 96 hours. Samples were taken as followed: 10 μl cell culture was mixed with 190 μl of deionized water in micro titer plates (Nunc MicroWell 96-Well Optical-Bottom Plates with Polymer Base, Black; Thermo Fisher Scientific). eGFP fluorescence measurements were performed using a FLUOstar® Omega plate reader (BMG LABTECH GmbH, Ortenberg, Germany). Fluorescence was measured at 488/507 nm (excitation/emission) and for data evaluation the resulting relative fluorescence units (RFU) me were normalized to the OD600.

TABLE-US-00008 TABLE C Primers and synthetic DNA for generation of FMD promoter variants SEQ ID Name Sequence No. intARG.fwd GCCAATTCTC 60 AATTTGCTAG AGACTCTG P_FMD-v0l.fwd Agaggcggcg 61 Aatcaagaaa cacc P_FMD-v0l.rev Ggtgtttctt 62 gatTcgccgc ctct P_FMD-v02.fwd ctgccccgcG 63 acgggaaaaa gaatg P_FMD-v02.rev Cattcttttt 64 cccgtCgcgg ggcag P_FMD-v03.fwd Ggattttgca 65 gaaaaaTagc aagttccggg P_FMD-V03.rev Cccggaactt 66 gctAtttttc tgcaaaatcc P_FMD-v04.fwd Gtctctcaga 67 gGggggaatg gc P_FMD-v04.rev Gccattcccc 68 Cctctgagag ac P_FMD-v05.fwd Cactcgacta 69 ccaGccgggt ctctc P_FMD-v05.rev Gagagacccg 70 gCtggtagtc gagtg P_FMD-06_fwd CACTCGACTA 71 CCATCCGGGT CTCTCCGAGA GGGGAATGGC ACTATAAATA C P FMD-07 fwd CACTCGACTA 72 CCATCCGGGT CTCTCACAGA GGGGAATGGC ACTATAAATA C P_FMD-08_fwd CACTCGACTA 73 CCATCCGGGT CTCTCAGCGA GGGGAATGGC ACTATAAATA C P_FMD-09_fwd CACTCGACTA 74 CCATCCGGGT CTCTCAGACA GGGGAATGGC ACTATAAATA C P_FMD-lO_fwd CACTCGACTA 75 CCATCCGGGT CTCTCAGAGC GGGGAATGGC ACTATAAATA C P_FMD-ll_fwd CACTCGACTA 76 CCATCCGGGT CTCTCAGAGA CGGGAATGGC ACTATAAATA C P_FMD-12_fwd CACTCGACTA 77 CCATCCGGGT CTCTCAGAGA GCGGAATGGC ACTATAAATA C P_FMD-13_fwd CACTCGACTA 78 CCATCCGGGT CTCTCAGAGA GGCGAATGGC ACTATAAATA C P_FMD-14_fwd CACTCGACTA 79 CCATCCGGGT CTCTCAGAGA GGGCAATGGC ACTATAAATA C P FMD-15 fwd CACTCGACTA 80 CCATCCGGGT CTCTCAGAGA GGGGCATGGC ACTATAAATA C P_FMD-16_fwd CACTCGACTA 81 CCATCCGGGT CTCTCAGAGA GGGGACTGGC ACTATAAATA C P_FMD-17_fwd CACTCGACTA 82 CCATCCGGGT CTCTCAGAGA GGGGAACGGC ACTATAAATA C P_FMD-18_fwd CACTCGACTA 83 CCATCCGGGT CTCTCAGAGA GGGGAATCGC ACTATAAATA C P_FMD-19_fwd CACTCGACTA 84 CCATCCGGGT CTCTCAGAGA GGGGAATGCC ACTATAAATA C P_FMD_rev GAGAGACCCG 85 GATGGTAGTC G P FMD-V20 fwd ctcatactca 86 aactatatta aaactacaaca ATGGCTAGCAA AGGAGAAGAAC TTTTCAC P FMD-V20 rev tgttgtagttt 87 taatatagttt gagtatgagat ggaactcagaa cgaaggaatta tcaccagttta tatagtgccat tcccctctctg ag P_FMD-v2l_fwd gactcacccat 88 aaacaaataat caataaatATG GCTAGCAAAGG AGAAGAACTTT TCAC P FMD-v21 rev atttattgatt 89 atttgtttatg ggtgagtctag aaaaggacgca ctcgtcttgta tttatagtgcc attccccTct ctgag P_FMD-v22_fwd acttgtcctc 90 tattccttca tcaatcacat cATGGCTAGC AAAGGAGAAG AACTTTTCAC P_FMD-v22_rev gatgtgattg 91 atgaaggaat agaggacaag taggcagtat ttatagtgcc attccccTct ctgag Pcore_FMD_v23 atcaagctgt 92 (synthetic ctccgataca DNA) ctcgactacc atccgggtct ctcagagAgg ggaatggcac CGATAGGGCA GAAATATATA AAGTAGGAGG TTGTATACCA AATATACCAA CGCAGTACAA GCAACTCTTG GTTTAAACGG AAGAAACAAT TCTTCGAACA TTTACAACAA AGAAGGTACC GTAACATTAA TAATCGGAAG GGTATGGCTA GCAAAGGAGA AGAACTTTTC ACTGGAGTTG TCCCAATTCT Pcore_FMD_v24 atcaagctgtc 93 (synthetic tccgatacact DNA) cgactaccatc cgggtctctca gagAggggaat ggcacGTAATC TTTCGGTCAAT TGTGATCTCTC TTGTAGATATT TAATAGGACGG CCAAGGTAGAA AAAGATACATA ACTAGTTAGCA AACTTCAATTG CTTAAGTTACA AGTGCAATCCA TATCTTAAAGT TATTACATTAT TTATAATGGCT AGCAAAGGAGA AGAACTTTTCA CTGGAGTTGTC CCAATTCT Pcore_FMD_v25 atcaagctgtc 94 (synthetic tccgatacact DNA) cgactaccatc cgggtctctca gagAggggaat ggcacCCTCCT CTAGGTTTATC TATAAAAGCTG AAGTCGTTAGA ATTTTTCATTT AAAGCATAATC AAACATCTAGA TTCGAATCGAT AAAAAGCAGAT AGAAGTTATTA AGATTATAGGT TACATTCTAGA GTAGTATAGGA AGGTAATGGCT AGCAAAGGAGA AGAACTTTTCA CTGGAGTTGTC CCAATTCT

[0081] 3. Results

[0082] The results of the reporter protein fluorescence of the HpFMD promoter (P_FMD) and the AOXl promoter (P_AOXl) wild type sequence promoters tested are shown in FIG. 4.

[0083] a) FMD Promoter Variants—Point Mutations and Single Nucleotide Insertion

TABLE-US-00009 TABLE D Relative promoter activities of all promoter variants containing point mutations and single nucleotide insertions. Relative fluorescence values (RFU) of the eGFP reporter protein were measured and these values were normalized to the OD600. These RFU/OD600 values were normalized to the RFU/OD600 value of the parental HpFMD promoter variant (wt = SEQ ID No. 1) sequence resulting in relative promoter activities. The strains were cultivated in DWPs cultivation on BMD1 media (24 and48 h) and subsequently induced with methanol (72 and 96 h). 72 h 96 h 24 h 48 h induced with induced with derepressed derepressed methanol methanol wt 1.0 ± 0.53 v13 0.62 ± 0.058 v09 0.56 ± 0.031 v09 0.56 ± 0.031 v12 1.0 ± 0.51 v12 0.63 ± 0.071 v14 0.56 ± 0.073 v14 0.56 ± 0.073 v13 1.1 ± 0.58 v14 0.67 ± 0.088 v12 0.57 ± 0.028 v12 0.57 ± 0.028 v09 1.2 ± 0.47 v11 0.70 ± 0.062 v11 0.58 ± 0.028 v11 0.58 ± 0.028 v14 1.3 ± 0.52 v09 0.69 ± 0.088 v13 0.59 ± 0.029 v13 0.59 ± 0.029 v11 1.3 ± 0.37 v15 0.75 ± 0.062 v15 0.69 ± 0.051 v15 0.69 ± 0.051 v04 1.3 ± 0.49 v04 0.83 ± 0.083 v04 0.74 ± 0.036 v04 0.74 ± 0.036 v19 1.3 ± 0.48 v08 0.87 ± 0.047 v06 0.77 ± 0.049 v06 0.77 ± 0.049 v16 1.3 ± 0.41 v07 0.81 ± 0.071 v07 0.83 ± 0.076 v07 0.83 ± 0.076 v15 1.4 ± 0.14 v16 0.91 ± 0.082 v08 0.83 ± 0.056 v08 0.83 ± 0.056 v08 1.4 ± 0.46 v02 0.94 ± 0.10  v16 0.88 ± 0.024 v16 0.88 ± 0.024 v07 1.5 ± 0.49 v19 0.96 ± 0.053 v02  0.9 ± 0.066 v02  0.9 ± 0.066 v02 1.5 ± 0.60 wt 1.0 ± 0.13 v19 0.97 ± 0.079 v19 0.97 ± 0.079 v18 1.5 ± 0.59 v03 1.1 ± 0.11 v03 0.99 ± 0.080 v03 0.99 ± 0.08  v03 1.7 ± 0.62 v01 1.1 ± 0.12 wt  1.0 ± 0.088 wt  1.0 ± 0.088 v06 1.7 ± 0.73 v06  1.1 ± 0.069 v01 1.04 ± 0.066 v01  1.0 ± 0.066 v17 1.8 ± 0.63 v18 1.1 ± 0.11 v17 1.06 ± 0.056 v17  1.1 ± 0.056 v01 1.8 ± 0.66 v17 1.2 ± 0.15 v18 1.08 ± 0.12  v18 1.1 ± 0.12 v10 1.9 ± 0.65 v10 1.3 ± 0.16 v05  1.1 ± 0.061 v05  1.1 ± 0.061 v05 2.4 ± 0.64 v05 1.4 ± 0.17 v10  1.2 ± 0.066 v10  1.2 ± 0.066

[0084] b) FMD Promoter Variants—Core Promoter Exchanges

TABLE-US-00010 TABLE E Relative promoter activities of all promoter variants containing with an exchanged core promoter. Relative fluorescence values (RFU) of the eGFP reporter protein were measured and these values were normalized to the OD600. These RFU/OD600 values were normalized to the RFU/OD600 value of the parental HpFMD promoter variant (wt = SEQ ID No. 1) sequence resulting in relative promoter activities. The strains were cultivated in DWPs cultivation on BMD1 media (24 and 48 h) and subsequently induced with methanol (72 and 96 h). 72 h 96 h 24 h 48 h induced with induced with derepressed derepressed methanol methanol v23 0.36 ± 0.30  v25 0.29 ± 0.067 v25 0.24 ± 0.032 v25 0.42 ± 0.032 v25 0.53 ± 0.31  v24 0.42 ± 0.056 v24 0.41 ± 0.054 v24 0.58 ± 0.022 v24 0.59 ± 0.44  v23 0.54 ± 0.070 v23 0, SO ± 0.063  v23 0.60 ± 0.074 wt 1.0 ± 0.44 v22 0.96 ± 0.097 v21 0.76 ± 0.074 v21 0.92 ± 0.06  v21 1.9 ± 0.90 v21 1.0 ± 0.14 v22 0.78 ± 0.089 v22 0.99 ± 0.051 v22 2.8 ± 0.36 wt  1.0 ± 0.098 wt  1.0 ± 0.132 wt  1.0 ± 0.134 v20 3.7 ± 0.65 v20 1.6 ± 0.14 v20  1.4 ± 0.173 v20  1.5 ± 0.072