NL2022905B1 - Yeast with engineered Molybdenum co-factor biosynthesis - Google Patents

Yeast with engineered Molybdenum co-factor biosynthesis Download PDF

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NL2022905B1
NL2022905B1 NL2022905A NL2022905A NL2022905B1 NL 2022905 B1 NL2022905 B1 NL 2022905B1 NL 2022905 A NL2022905 A NL 2022905A NL 2022905 A NL2022905 A NL 2022905A NL 2022905 B1 NL2022905 B1 NL 2022905B1
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Georges Daran Jean-Marc
Perli Thomas
Thomas Pronk Jacobus
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Univ Delft Tech
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Abstract

The present invention relates to a yeast comprising a Moco pathway gene set that allows said yeast to produce Molybdenum co-factor and its variants bis-Mo-molybdopterin guanine 5 dinucleotide, sulfurated molybdenum co-factor and Mo-molybdopterin cytosine dinucleotide. Preferably the Moco pathway gene set comprises a gene encoding a GTP 3',8—cyclase; a gene encoding a Cyclic pyranopterin monophosphate synthase; a gene encoding a Molybdopterin synthase catalytic subunit; a gene encoding a Molybdopterin synthase sulfur carrier subunit; a gene encoding a Molybdopterin adenylyltransferase; a gene encoding a 10 Molybdopterin-synthase adenylyltransferase and/or a Molybdopterin molybdenumtransferase; and/or a gene encoding a Cysteine desulfurase. Preferably, the Moco-modifying gene set comprises a gene encoding a Molybdenum cofactor guanylyltransferase and a Molybdopterin-guanine dinucleotide biosynthesis adapter protein to form bis-molybdopterin guanine dinucleotide; a Molybdenum co-factor sulfurase to form sulfurated Molybdenum co- 15 factor and/or a Molybdenum co-factor cytidylyltransferase to form Molybdopterin cytosine dinucleotide.

Description

P33965NLO0/MJO Yeast with engineered Molybdenum co-factor biosynthesis Technical field The present disclosure relates to a yeast comprising a recombinant Molybdenum co-factor (Moco) biosynthesis pathway gene set and Moco modifying pathway gene set that allows said yeast to produce Molybdenum co-factor and modified Moco such as bisMGD and sulfurylated Moco respectively.
Background of the disclosure In biochemistry, a metabolic pathway is a linked series of chemical reactions occurring within a cell that connects small molecules {metabolites) with the help of catalytic proteins, enzymes. The set of all enzymes harbored by an organism define the boundaries of the metabolic network and its metabolic identity. Many of the enzymes that catalyze this biochemical reaction catalogue in a living cell require small accessory molecules that can be involved in regulation, promoting correct folding or directly involved in catalysis. The latter group is also referred to as co-factors, a non-protein chemical compound and/or metal ion that is necessary for an enzyme's activity. Consequently, the availability of these essential components, being synthesized or assimilated from the environment, shapes the metabolic landscape of an organism. For instance, certain vitamins are essential micronutrients that an organism needs in small amounts that cannot be synthesized by the organism itself and therefore have to be obtained through diet. In the yeast S. cerevisiae, the standard synthetic medium includes seven of such essential micronutrients. The popular laboratory S. cerevisiae S288C lineage cannot synthesize biotin since it misses the first two genes (B/O7 and B/O6) of the synthetic pathway, but this co-enzyme that is essential for carboxylation reactions (e.g. pyruvate carboxylase Pyc1 and Pyc2, urea carboxylase Dur1,2 or acetyl-CoA carboxylase Acc) can be added in the culture medium and transported by the native biotin transporter Vth1. However, it may happen that expression of enzymes requiring co-factors not produced by the selected host cannot be achieved by supplying the molecule in the culture medium because i) the co-factor might not be commercially available, ii) might be too expensive or iii) might not be imported. In these cases, the microbial strain design should then include the engineering of de novo co-factor biosynthesis or of its transport. The replacement of the native ATP-dependent urease Dur1,2 in S. cerevisiae by the Schizosaccharomyces pombe nickel-dependent, ATP-independent urease required the expression of a specific high affinity Nickel transporter (Nic1) from the same donor, as S. cerevisiae is characterized by a total absence of nickel dependent enzymes and therefore of dedicated transporter. Similarly, the recent introduction of de novo biosynthesis of opioids in S cerevisiae was made possible only after engineering the biosynthesis of tetrahydrobiopterin, also known as sapropterin, co- enzyme of the tyrosine hydroxylase which represents the first committed step into the (S)- reticuline pathway that further leads to morphine and noscapine synthesis. This illustrates that expansion of the metabolic landscape of S. cerevisiae can be associated with the parallel broadening of its co-factor set.
The second-row transition metal molybdenum (Mo) is an essential trace element in all three domains of life, and it is bioavailable as molybdate (MoO.%). Once entered the cell, molybdate is incorporated in a tricyclic pterin-based scaffold, molybdopterin (MPT), to form the molybdenum cofactor (Moco). Moco can be further modified with the replacement of an oxo ligand by a sulfido ligand, forming the mono-oxo Moco (Moco-S) present in the xanthine oxidase family of molybdoenzymes. In addition, prokaryotes can also attach either guanine or cytosine to the molybdopterin molecule to form MPTcytosine dinucleotide (MCD) and MPT guanine dinucleotide (bis-MGD) cofactor respectively. All molybdenum enzymes contain one of the different Moco variants in their catalytic active site with the exception of the bacterial nitrogenase that contains the iron-sulfur cluster-based iron-Mo-cofactor instead. The molybdenum cofactor biosynthetic pathway is very well conserved and have been extensively studied in both prokaryotic and eukaryotic organisms. However, the model eukaryote microorganisms Saccharomyces cerevisiae and Schizosaccharomyces pombe are outstanding exceptions since they cannot synthesise Moco and are devoid of Mo-dependent enzymes.
It is an objective of the present disclosure to solve one or more problems in the prior art.
Summary of the disclosure The present inventors describe the engineering of a yeast, i.e. a Saccharomycotina yeast, particularly a Saccharomycotina yeast naturally devoid of Moco biosynthetic genes and/or Moco modifying enzymes (Table 1), to comprise a pathway leading to synthesis of molybdenum co-factor (Moco), mono-oxo Moco co-factor (Moco-S), MPTcytosine dinucleotide co-factor (MCD) and/or MPT guanine dinucleotide co-factor (bis-MGD). This paves the way to the expression of Moco dependent enzymes, including for example nitrate reductase, formate dehydrogenase, xanthine oxidase, and biotin sulfoxide reductase.
Table 1: list of budding yeast genera that miss the Moco biosynthetic pathway and/or the Moco sulfurase enzyme, and that are preferred in the present disclosure.
Saccharomycetaceae Moco Moco Debaryomycetaceae Moco | Moco eT eee Eremothecium Scheffersomyces Kazachstania Spathaspora Kluyveromyces Suhomyces Lachancea Yamadazyma Naumovozyma Clavispora Torulaspora Pichia Vanderwaltozy Saturnispora ma Zyosaccharomy Ogataea + ces
Saccharomycodaceae Incertae sedis {Ala}
TE ee GN Bornettozyma + - Saprochgete Wickerhamomy & Lipomycetaceae eee ee TTT Lodderomyces + || Debaryomyces + Diutina + Hyphopichia Meyerozyma Millerozyma
In the prior art, it was generally considered that such engineering would not be feasible. One reason is the involvement of iron sulfur cluster containing enzymes which are notoriously known to be transferred in between organisms. Assembly of iron sulfur cluster might require accessory proteins (chaperones) that are species but also protein specific complicating the resolution of the cluster insertion mechanism. To overcome this potential problem, the present inventors identified the Moco pathway of the yeast Ogataea parapolymorpha and transferred this Moco pathway to a Saccharomycotina yeast that naturally does not harbor such native metabolic pathway. The Moco pathway might be further modified to lead to sulfurylated Moco. The pathway might be prolonged to synthesis of a sulfurylated Moco, a Moco guanosine dinucleotide (bis-MGD) or a Moco cytosine dinucleotide (MCD) by expressing a Moco sulfurylase gene, a molybdenum co-factor guanylyltransferase gene and a Molybdopterin-guanine dinucleotide biosynthesis adapter or a Molybdenum cofactor cytidylyltransferase gene respectively.
A Moco producing Saccharomycotina yeast that does not naturally harbor a native pathway or Moco modifying genes according to the present disclosure, for example S. cerevisiae, can combine all beneficial industrial characteristics of such yeast with the ability to express a wide range of new industrially relevant enzymes such as nitrate reductase or formate dehydrogenase. This drastically expands the N and C sources spectra of this organism and offers the opportunity to address new biotechnological challenges.
The work of the present inventors successfully explored the possibility to equip Saccharomycotina yeast naturally devoid of Moco synthesis with such Moco biosynthetic pathway. For this, elucidation of the Moco pathway in Ogataea parapolymorpha was performed and subsequently transferred to S. cerevisiae.
In addition, the expression of the Moco biosynthetic pathway can be coupled to the expression of a Moco-dependent nitrate reductase. Moreover, a high affinity Mo importer can be included since S. cerevisiae is known to lack such a high affinity import system. The present inventors tested the Moco-expressing S. cerevisiae strains for the ability to grow on synthetic media with nitrate as the sole nitrogen source, for the ability to assimilate nitrate at low Molybdate concentration, and also in presence of an extra nitrogen source as ammonium sulfate.
General definitions In the present disclosure, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
5 The term “nucleic acid” {or nucleic acid sequence) refers to a DNA or RNA molecule in single or double stranded form. The nucleic acid may be an isolated nucleic acid, which refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the isolated nucleic acid no longer comprises the nucleic acid sequence naturally flanking the nucleic acid in the natural environment, such as less than 100, 50, 25 or 10 nucleic acids (nucleotides) of the nucleic acid sequence naturally flanking the nucleic acid is present in the isolated nucleic acid. Or for example, the isolated nucleic acid is now in a bacterial host cell or in the plant nuclear or plastid genome, or the isolated nucleic acid is chemically synthesized. The term “gene” means a DNA sequence comprising a region (transcribed region), which is transcribed into a RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5’ leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3’non-translated sequence comprising e.g. transcription termination sites. Next to exons, a gene may also include introns, which are, for example spliced out before translation into protein. It is further understood that, when referring to “sequences” herein, generally the actual physical molecules with a certain sequence of subunits (e.g. nucleotides or amino acids) are referred to.
A "nucleic acid construct” or “vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and which is used to deliver exogenous DNA into a host cell. The vector backbone may for example be a binary or superbinary vector (see e.g. US 5591616, US 2002138879 and WO95/06722), a co-integrate vector or a T-DNA vector, as known in the art and as described elsewhere herein, into which a chimeric gene is integrated or, if a suitable transcription regulatory sequence is already present, only a desired nucleic acid sequence (e.g. a coding sequence, an antisense or an inverted repeat sequence) is integrated downstream of the transcription regulatory sequence. Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like.
The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3- dimensional structure or origin. The protein or polypeptide may be an isolated protein, i.e. a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell. “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using alignment algorithms {when optimally aligned by for example the programs GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps.
Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8
(proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road,
San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the program “needle”). Alternatively, percent similarity or identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc.
As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations (which may be (conservative) substitutions, deletions and/or insertions) per each 100 nucleotides of the reference polypeptide sequence.
In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted and/or substituted with another nucleotide, and/or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
Similarly, by a polypeptide having an amino acid sequence having at least, for example, 985% "identity" to a reference amino acid sequence of SEQ ID NO: 1 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 1. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. Sequence identity can be determined over the entire length of the sequence(s) to be considered. In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
Detailed description of the disclosure The present disclosure relates to a yeast comprising a recombinant Moco pathway gene set that allows said yeast to produce Molybdenum co-factor and/or its variants bis-Mo- molybdopterin guanine dinucleotide, sulfurated molybdenum co-factor and Mo-molybdopterin cytosine dinucleotide. Preferably the Moco pathway gene set comprises a gene encoding a GTP 3',8-cyclase; a gene encoding a Cyclic pyranopterin monophosphate synthase; a gene encoding a Molybdopterin synthase catalytic subunit; a gene encoding a Molybdopterin synthase sulfur carrier subunit; a gene encoding a Molybdopterin adenylyltransferase; a gene encoding a Molybdopterin-synthase adenylyltransferase and/or a Molybdopterin molybdenumtransferase; and/or a gene encoding a Cysteine desulfurase. Preferably, the Moco-pathway gene set further comprises a gene encoding a Molybdenum cofactor guanylyltransferase and a Molybdopterin-guanine dinucleotide biosynthesis adapter protein to form bis-molybdopterin guanine dinucleotide; a Molybdenum co-factor sulfurase to form sulfurated Molybdenum co-factor and/or a Molybdenum co-factor cytidylyltransferase to form Molybdopterin cytosine dinucleotide. In particular, the present disclosure relates to a yeast, e.g. a yeast cell, comprising a (recombinant) Moco pathway gene set which allows said yeast to produce or biosynthesize Molybdenum co-factor (Moco) or a yeast cell, comprising a (recombinant) Moco pathway gene set and/or Moco modifying enzymes which allows said yeast to produce or biosynthesize sulfurylated Moco, bis-MGD or Mo-MCD co-factor.
The yeast according to the present disclosure is preferably a Saccharomycotina yeast, that does not naturally harbor a native Moco biosynthesis pathway or Moco modifying enzymes, or an ascomycete yeast, preferably chosen from the group consisting of Saccharomyces cerevisiae and Yarrowia lipolytica. Preferably, the yeast is from a genus described in Table 1 as not having a Moco biosynthetic pathway and/or the Moco sulfurase enzyme. The yeast is preferably not Ogataea parapolymorpha. The yeast may be chosen from Saccharomycetaceae, in particular chosen from the group consisting of Eremothecium, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces, Naumovozyma, Saccharomyces, Tetrapisispora, Torulaspora, Vanderwaltozyma, Zyosaccharomyces. Additionally or alternatively, the yeast may be chosen from Saccharomycodaceae, in particular Hanseniaspora.
Additionally or alternatively, the yeast may or may not be chosen from Phaffomcetaceae, in particular chosen from the group consisting of Barnettozyma, Cyberlindnera, Wickerhamomyces.
Additionally or alternatively, the yeast may be chosen from Ascoideaceae, in particular Ascoidea. Additionally or alternatively, the yeast may be chosen from Debaryomycetaceae, in particular chosen from the group consisting of Lodderomyces, Debaryomyces, Diutina, Hyphopichia, Meyerozyma, Millerozyma, Scheffersomyces, Spathaspora, Suhomyces, Yamadazyma. Additionally or alternatively, the yeast may be chosen from Metschnikowiaceae, in particular chosen from the group consisting of Clavispora, Metschnikowia.
Additionally or alternatively, the yeast may be chosen from Pichiaceae, in particular chosen from the group consisting of Pichia, Saturnispora, (Ogataea). Additionally or alternatively, the yeast may or may not be chosen from Incertae sedis (Ala), in particular Pachysolen.
Additionally or alternatively, the yeast may be chosen from Dipodascaceae, in particular Saprochaete, Yarrowia.
Additionally or alternatively, the yeast may or may not be chosen from Lipomycetaceae, in particular Lipomyces. A Moco pathway gene set refers to set of genes that encode proteins (i.e. enzymes) that are involved in the production or biosynthesis of Molybdenum co-factor. In a preferred embodiment, the Moco pathway gene set according to the present disclosure comprises: - a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:1 and/or encoding a GTP 3',8-cyclase; - a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:2 and/or encoding a Cyclic pyranopterin monophosphate synthase; - a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:3 and/or encoding a Molybdopterin synthase catalytic subunit; - a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:4 and/or encoding a Molybdopterin synthase sulfur carrier subunit; -a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:5 and/or encoding a Molybdopterin adenylyltransferase; and/or - a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:6 and/or encoding a Molybdopterin-synthase adenylyltransferase and/or a Molybdopterin molybdenumtransferase.
The Moco pathway gene set may further comprise - a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:12 and/or encoding a Molybdenum co-factor guanylyl transferase; and/or - a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:13 and/or encoding a Molybdenum-guanine dinucleotide biosynthesis adapter protein. This may allow the yeast to produce sulfurylated Molybdenum co-factor and/or Molybdopterin cytosine dinucleotide. Additionally, any of the following genes may be comprised: -agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:22 and/or encoding Moco sulfurylase - a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:27 and/or encoding Molybdenum cofactor cytidylyltransferase The skilled person knows that genes having a different nucleotide sequence may encode the same polypeptide. For example, it is common general knowledge that codon usage may vary among genes encoding the same polypeptide. Specifically, polypeptide-encoding genes use a triplet code, i.e. a codon code, wherein three bases make up a codon. Because there are four bases (A, C, T, G) possible for each of the three positions in the codon, 64 different codons are possible. However, there are only 20 different amino acids. The overabundance in the number of codons underlies the fact that most amino acids are encoded by more than one codon code. In view thereof, multiple variations of the sequences disclosed above are well within grasp of the skilled person. Preferably, the codon usage in the sequences is optimized for the host organism, preferably a Saccharomycotina yeast, or an ascomycete yeast, preferably chosen from the group consisting of Saccharomyces cerevisiae and Yarrowia lipolytica.
Additionally or alternatively, the Moco pathway gene set according to the present disclosure comprises: - a gene encoding a protein having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:28, wherein the protein preferably is a GTP 3',8-cyclase; - a gene encoding a protein having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:29, wherein the protein preferably is a Cyclic pyranopterin monophosphate synthase; - a gene encoding a protein having at least 50, 70, 75, 80, 85, 90, 95, 98, 97, 98, 99% sequence identity with SEQ ID NO:30, wherein the protein preferably is a Molybdopterin synthase catalytic subunit; - a gene encoding a protein having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:31, wherein preferably the protein is a Molybdopterin synthase sulfur carrier subunit; - a gene encoding a protein having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:32, wherein preferably the protein is a Molybdopterin adenylyltransferase; and/or - a gene encoding a protein having at least 50, 70, 75, 80, 85, 90, 95, 98, 97, 98, 99% sequence identity with SEQ ID NO:33, wherein preferably the protein is a Gephyrin that combines the Molybdopterin-synthase adenylyltransferase and/or the Molybdopterin molybdenumtransferase activity/activities. The skilled person knows that proteins having a different amino acid sequence can have the same activity. It is common general knowledge that it is often possible to substitute a certain amino acid by another one, without loss of activity of the polypeptide. For example, the following amino acids can often be exchanged for one another: Ala, Ser, Thr, Gly (small aliphatic, nonpolar or slightly polar residues)
Asp, Asn, Glu, GIn (polar, negatively charged residues and their amides) His, Arg, Lys (polar, positively charged residues) Met, Leu, lle, Val (Cys) (large aliphatic, nonpolar residues) Phe, Ty, Trp {large aromatic residues)
(refer for example to Schulz, G.
E. et al, Principles of Protein Structure, Springer- Verlag, New York, 1979, and Creighton, T.E., Proteins: Structure and Molecular Principles, W.H.
Freeman & Co., San Francisco, 1984)
Preferred "substitutions" are those that are conservative, i.e., wherein the residue is replaced by another of the same general type.
In making changes, the hydropathic index of amino acids may be considered (See, e.g., Kyte et al., J.
Mol.
Biol. 157, 105-132 (1982). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a polypeptide having similar biological activity.
In making such changes, the substitution of amino acids whose hydropathic indices are within £2 is preferred, those that are within £1 are more preferred, and those within £0.5 are even more preferred.
Similarly, select amino acids may be substituted by other amino acids having a similar hydrophilicity, as set forth in U.S.
Pat.
No. 4,554,101 (herein incorporated by reference in its entirety). In making such changes, as with the hydropathic indices, the substitution of amino acids whose hydrophilicity indices are within £2 is preferred, those that are within £1 are more preferred, and those within £0.5 are even more preferred.
So, multiple variations of the protein sequences disclosed above are also well within grasp of the skilled person.
It is not difficult to determine and evaluate whether a particular protein falling within the terms of the claims confers the technical effect of the invention.
For example, Moco biosynthesis ability may be confirmed by measuring intracellular concentration of Moco, of by verifying if a Moco dependent enzyme has functionality.
The Moco pathway can be seen as four steps wherein the first step typically takes place in the mitochondria in eukaryote.
In this step, GTP is converted to the precursor cPMP which is then exported to the cytosol where it is first sulfurated to form molybdopterin (MPT) and adenylated to form MPT-AMP.
The MPT synthase, catalysing the sulfuration of cPMP is regenerated by a sulfur mobilization route shared with the tRNA thiolation pathway.
Finally, the adenylate group is hydrolysed and molydbdate is inserted into the MPT dithiolene group to form Moco or Mo-MPT.
Moco can be further modified by replacing one oxo ligand by a sulfido ligand, to form the mono-oxo Moco variant (Moco-S), present in both eukaryotic and prokaryotic Moco-enzymes of the Xanthine oxidase family.
Moreover, in prokaryotes, the
Moco molecule can be further modified by the addition of either cytosine or guanosine to form MPT cytosine dinucleotide (MCD) or MPT guanine dinucleotide (MGD) cofactor respectively {see Figure.1). Each form of Moco is inserted into molybdoenzymes which are divided into three families based on the ligands at the Mo atom: the xanthine oxidase (XO) family, the sulphite oxidase (SO) family and the dimethyl sulfoxide (DMSO) reductase family. The XO molybdoenzyme family typically requires MCD at the catalytic site in prokaryotes while Moco- S in eukaryotes. The SO reductase family requires Mo-MPT at the catalytic site. The proteins of the DMSO reductase family typically require instead the bis-MGD cofactor which is formed in a two-step reaction where first the bis-Mo-MPT intermediate is formed and then two GMP moieties are added to the two C4phosphate of bis-Mo-MPT. To date, more than 50 molybdenum enzymes have been purified and characterized while many more gene products have been annotated as putative molybdenum-containing proteins. In general, Mo-enzymes catalyse the transfer of oxygen in the metabolism of carbon, nitrogen and sulfate thanks to the versatile redox chemistry of molybdate. A list of the main molybdoenzymes with relative cofactor is shown in Table 2.
Table 2. Ist of main molvbddoenzymes with relative form of Moco.
Enzymes FormofMocoin the catalytic subunit TMAOreductase =~ bissMGD Formate dehydrogenase =~ bisMGD Formylmethanofuran dehydrogenase ~~ bisMGD
Quinaldine dehydrogenase ~~ mc Quingline-2-oxidoreductasse ~~ MCD Quinoline-d-carboxylate-2-oxidoreductase ~~ MCD Nicotinate hydroxylase =~ mco Sulfite oxidase amity == 0000 In a particularly preferred embodiment, the yeast according to the present disclosure comprises a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:7 and/or encoding a Cysteine desulfurase.
Similarly, the yeast according to the present disclosure may comprise a gene encoding a protein having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:34, wherein the protein preferably is a Cysteine desulfurase.
Additionally or alternatively, the yeast according to the present disclosure comprises a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NQ:8 and/or encoding a molybdate transporter allowing the yeast to import Molybdate with higher affinity than without a molybdate transporter.
Similarly, the yeast according to the present disclosure may comprise a gene encoding a protein having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:35, wherein the protein preferably is a molybdate transporter.
It is further envisaged that the yeast according to the present disclosure in addition or as an alternative comprises a Moco-dependent nitrate assimilation pathway gene set.
A Moco- dependent nitrate assimilation pathway gene set refers to set of genes that encode proteins
(i.e. enzymes) that are involved in the incorporation of inorganic nitrogen into organic compounds, e.g. in the yeast (cell) according to the present disclosure.
In a preferred embodiment, the yeast according to the present disclosure comprises - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:9; - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:10 - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 11 - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 14; and/or - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:15. In a preferred embodiment, the Moco dependent nitrate assimilation pathway gene set comprises one or more of a gene encoding nitrate transporter, a gene encoding nitrate reductase, and a gene encoding nitrite reductase.
Such Moco dependent nitrate assimilation pathway gene set may allow the yeast to grow on nitrate as sole nitrogen source.
In accordance with the present disclosure, it is also foreseen that the present yeast comprises a (recombinant) gene encoding a (Moco dependent) enzyme, for example chosen from the group consisting of: - agene encoding a formate dehydrogenase that allows said yeast to use formate as co-substrate; - agene encoding a xanthine oxidase (enables use of purine as nitrogen source); - a gene encoding a nitrate reductase combined with a gene encoding a chlorite dismutase that allows said yeast to produce intracellular molecular oxygen; - a gene encoding a biotin sulfoxide reductase that allows said yeast to scavenge biotin from peptide bound biotin.
In fact, the yeast according to the present disclosure may additionally or alternatively comprise any Moco dependent enzyme chosen from the following group: TMAO reductase; Biotin sulfoxide reductase; Prokaryotic nitrate reductase; Selenate reductase; Perchlorate reductase; Chlorate reductase; Arsenite oxidase; Formate dehydrogenase; Polysulfide reductase; DMSO reductase; Sufur reductase; Tetrathionate reductase; DMS dehydrogenase; Formylmethanofuran dehydrogenase; Ethylbenzene dehydrogenase; Pyrogallol phloroglucinol transhydroxylase; C25dehydrogenase; Xanthine oxidase; Aldehyde oxidoreductase; Quinaldine dehydrogenase; Quinoline-2- oxidoreductase; Isoquinoline 1-oxidoreductase; Quinoline-4-carboxylate-2-oxidoreductase; Nicotinate hydroxylase; CO dehydrogenase; 4-hydroxybenzoyl-CoA reductase; YedY reductase; Sulfite dehydrogenase; and/or Eukaryotic nitrate reductase. In a preferred embodiment, the yeast according to the present disclosure comprises - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 16; - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 17; - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:18; - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:19; - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:20; - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:21; - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:23; - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:24; - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:25; and/or - agene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:26.
Methods of carrying out the conventional techniques used in methods of the present invention will be evident to the skilled worker, and are disclosed for example in Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).
For example, the gene or nucleic acid/nucleotide sequence comprising said may be comprised in a genetic construct. The genetic construct (or constructs) allows the expression of the protein encoded by the gene. A genetic construct may be comprised in a DNA vector or in a viral vector. Introduction of the nucleic acid or nucleic acids may be via transfection or transduction methods. A genetic construct may be comprised in a DNA vector, e.g. plasmid DNA. A suitable DNA vector may be a transposon. Suitable transposon systems (e.g. class | or class ll based) are well known in the art. When expression of more than one gene is desired, two or more separate genetic constructs can be provided e.g. on a single or two separate DNA vectors. Alternatively, a single genetic construct may also express more than one MRNA encoding more than one protein.
CLAUSES
1. Saccharomycotina yeast that is naturally devoid of a Moco pathway gene set comprising a recombinant Moco pathway gene set that allows said Saccharomycotina yeast to produce Molybdenum co-factor.
2. Saccharomycotina yeast according to clause 1, wherein the Moco pathway gene set comprises - a gene having at least 70% sequence identity with SEQ ID NO:1 and/or encoding a GTP 3',8-cyclase; - a gene having at least 70% sequence identity with SEQ ID NO:2 and/or encoding a Cyclic pyranopterin monophosphate synthase; - a gene having at least 70% sequence identity with SEQ ID NO:3 and/or encoding a Molybdopterin synthase catalytic subunit; - a gene having at least 70% sequence identity with SEQ ID NO:4 and/or encoding a Molybdopterin synthase sulfur carrier subunit; -agene having at least 70% sequence identity with SEQ ID NO:5 and/or encoding a Molybdopterin adenylyltransferase; and/or - a gene having at least 70% sequence identity with SEQ ID NO:6 and/or encoding a Molybdopterin-synthase adenylyltransferase and/or a Molybdopterin molybdenumtransferase.
3. Saccharomycotina yeast according to any one of the previous clauses, wherein the Moco pathway gene set further comprises - a gene having at least 70% sequence identity with SEQ ID NO:12 and/or encoding a Molybdenum co-factor guanylyl transferase; and/or - a gene having at least 70% sequence identity with SEQ ID NO:13 and/or encoding a Molybdenum-guanine dinucleotide biosynthesis adapter protein, which allows said Saccharomycotina yeast to produce sulfurylated Molybdenum co-factor and/or Molybdopterin cytosine dinucleotide.
4. Saccharomycotina yeast according to any one of the previous clauses, wherein the Saccharomycotina yeast further comprises a gene having at least 70% sequence identity with SEQ ID NO:7 and/or encoding a Cysteine desulfurase.
5. Saccharomycotina yeast according to any one of the previous clauses, wherein the Saccharomycotina yeast further comprises a gene having at least 70% sequence identity with SEQ ID NO:8 and/or encoding a molybdate transporter allowing the Saccharomycotina yeast to import Molybdate with higher affinity than without a molybdate transporter.
6. Saccharomycotina yeast according to any one of the previous clauses, further comprising a Moco dependent nitrate assimilation pathway gene set.
7. Saccharomycotina yeast according to clause 6, wherein the Moco dependent nitrate assimilation pathway gene set comprises one or more of a gene encoding nitrate transporter, a gene encoding nitrate reductase, and a gene encoding nitrite reductase.
8. Saccharomycotina yeast according to any one of clauses 6-7, wherein the Moco dependent nitrate assimilation pathway gene set allows said Saccharomycotina yeast to grow on nitrate as sole nitrogen source.
9. Saccharomycotina yeast according to any one of the previous clauses, wherein the Saccharomycotina yeast further comprises a gene encoding a bis-MGD dependent formate dehydrogenase that allows said Saccharomycotina yeast to use formate as co-substrate.
10. Saccharomycotina yeast according to any one of the previous clauses, wherein the Saccharomycotina yeast further comprises a gene encoding a xanthine oxidase.
11. Saccharomycotina yeast according to any one of the previous clauses, wherein the Saccharomycotina yeast further comprises a gene encoding a biotin sulfoxide reductase that allows said Saccharomycotina yeast to scavenge biotin from peptide bound biotin.
12. Saccharomycotina yeast according to any one of the previous clauses, wherein the yeast is an ascomycete yeast, preferably chosen from the group consisting of Saccharomyces cerevisiae and Yarrowia lipolytica.
Brief description of the figures Figure 1: Schematic representation of the molybdenum cofactor biosynthetic pathway. First GTP is converted to cPMP by the heterodimer MoaA/MoaC, this step takes place in the mitochondria in eukaryotic cells. Then MoaD transfers its sulfur mojety to cPMP yielding MPT. MoaD is then recycled by the sulfur transfer by MoeB that was previously sulfurated by IscS. Finally, MPT is first adenylated and then Mo ion is inserted by the heterodimer MogA/MoeA, which in eukaryotes is catalysed by a single enzyme (Gephyrin). Moco can be sulfurated by MOCOS to produce the mono-oxo version (Moco-S) of the cofactor that is needed by enzymes of the xanthine oxidase family. Moreover, Moco can be further modified in prokaryotic cells by the addition of either cytosine (MocA} or guanosine (MobA) to form MCD and bis-MGD respectively. In blue, the orthologous protein in O. parapolymorpha are shown. Figure 2: Sanger sequencing results of purified PCR fragments from each O. parapolymorpha DL-1 mutant strains. gRNA protospacer and PAM sequence are shown in bold and underlined text respectively.
Figure 3: spot assay of S. cerevisiae CEN.PK113-7D, O. parapolymorpha DL-1 (CBS11895) and IMD strains carrying a gene disruption on SM medium with either ammonium sulfate (SMA) or sodium nitrate SMNo) as sole nitrogen source. Strains precultures were grown overnight in 20 ml SMA in 100 ml shake flasks at 30 °C, 200 rpm shaking incubator (Innova 44 Incubator shaker (Eppendorf, Nijmegen, the Netherlands). Cells were harvested by centrifugation at 3000 g for 5 min and supernatant was discarded. Cells were resuspended in sterile demineralized water. Cells were then spun down again and washed twice. At the third resuspension in water, cell densities were normalized to an ODeeo of 1. A volume of 10 pl of normalized cell suspension was spotted on either SMA or SMNo plates. Strains were spotted onthe same plate for each condition and strain disposition in the figure was rearranged by cutting out the relative spots. Plates were incubated in a static incubator at 30 °C and scored after 2 days. As expected, CEN.PK113-7D was able to grow on SMA but not on SMNo. O. parapolymorpha DL-1 (CBS11895) was able to grow on both media. IMD025, strain carrying a disrupted nitrate reductase, was only able to grow on SMA. Mutant strains IMDO19, IMDO020, IMD021, IMD022, IMD023 and IMDO27, carrying a frame-shift mutation in a putative Moco biosynthetic gene, were able to grow on SMA but not on SMNo, indicating that the mutated gene resulted in a defective Moco biosynthetic pathway and therefore, in an inactive Moco-dependent nitrate reductase.
Figure 4. schematic representation of IMA 1777 strain construction, Each expression module was amplified by PCR and 80 bp long unigue homology arms were added to each fragment extremity. Fragments wers co-transformed together with pDUDR113, a plasmid carrying a gRNA targeting the SCA1 locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.
Figure 5: Schematic representation of IMX1778 strain construction. Each expression module was amplified by PCR and 80 bp long unigue homology arms were added to sach fragment B exdiremily. Fragments were cotransformed together with pUDR 119, a plasmid carrying 2 gERNA targeting the SGA1 locus (A). Correct assembly of each junction was verified by diagnostic POR {B}. Primers used for each PUR are shown.
Figure 5: schemalic representation of IMX1778 strain construction. Each expression module was amplified by PCR and 80 bp long unique homoiogy arms were added (0 each fragment extremity. Fragments were co-transformed fogsther with pUDR 119, a plasmid carrying a gRNA targeting the SGA locus (A) Correct assembly of zach junction was verified by diagnostic POR (B). Primers used for each POR ara shown.
Figure 7: schematic representation of IMX1780 strain construction. Each expression module was amplified by POR and 80 bp long unique homology ams were added {o sach fragment sxiremity. Fragments were co-transformed together with pUDR119, a plasmid carrying a gRNA targeting the SCAT locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCE are shown, Figure 8: schemalic representation of IMA1731 strain construction. Each expression module was amplified by PCR and 80 bp long unique homoiogy arms were added (0 each fragment extremity. Fragments were co-transformed together with pUDR 119, a plasmid carrying a gRNA targeting the SGA locus (A) Correct assembly of zach junction was verified by diagnostic POR (B). Primers used for each POR ara shown.
Figure 8: schematic representation of IMX1782 strain construction. Each expression module was amplified by POR and 80 bp long unique homology ams were added {o sach fragment sxirerity. Fragments wers co-transformed together with pUDR119, a plasmid carrying a gRNA targeting the SCAT locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCE are shown, Figure 10: copy number estimation of Chromosome IX for the 3. cerevisiae strains adapted for growth on nitrate as sole nitrogen source, The SGAT locus, where the different pathways where integrated, is located on ChriX at position 178004-179953. All adapted strains show an increase copy number at that locus, up to three copies for IMSO818 and IMS0818.
Figure 11: growth curve of IMS0816 (left) and IMS0817 (right) on synthetic medium with nitrate as sole nitrogen source. OD is shown in squares. Nitrate, nitrite, ammonium, glucose and ethanol concentrations are shown in triangles, reverse triangles, diamonds, circles and empty squares respectively. Thawed aliquots of frozen stock cultures were inoculated in a shake flask containing SMA and incubated overnight at 30 °C 200rpm. This starter culture was used to inoculate two shake flasks containing the same media at an at an initial optical density at 660 nm (ODess) of 0.2, generating duplicates. The flasks were then incubated at 30 °C 200rpm and growth was monitored using a 7200 Jenway Spectrometer (Jenway, Stone, United Kingdom). Specific growth rates were calculated from at least four time points in the exponential growth phase of each culture. At each time point, 2 ml of the liquid culture were centrifuged with a benchtop centrifuge, supernatant was collected used for metabolites analysis. Nitrate, nitrite and ammonium ion concentrations in the supernatant were measured using the HACH (Tiel, Nederlands) cuvette test kits LCK339, LCK341 and LCK304 respectively, following manufacturer instructions. Glucose and ethanol concentrations were measured using supernatant obtained by centrifugation of culture samples via high- performance liquid chromatograph (HPLC) as previously described.’ Error bars represent the standard deviation. (n=2).
Figure 12: growth curve of IMS08186 and IMS017 in 1:100 Mo-SMN. First, strains were grown as a preculture in SMA medium. Then, cells were spun down and washed three times in sterile demineralized water. Washed cells were used to inoculated in a new shakeflask containing SMA lacking Mo (AMo-SMA). The cultures were incubated at 30 °C 200 rpm overnight and once they reached high cell densities, a small amount of broth was transferred into a new shake flask, containing fresh AMo-SMA and incubated at 30 °C 200rpm overnight.
The process was repeated twice in order to make sure that intracellular traces of Mo were depleted. (A) Next, a small amount of cells were transferred into a new shake flask containing SMNs with 1:100 times less Mo concentration (1:100 Mo-SMNs) and incubated at 30 °C 200rpm. After about 12 days, both flasks containing IMS0816 started growing and reached high cell density while both flasks containing IMS0817 did not grow even after =700 h. To whether IMS0816 retained the ability to grow in 1:100 Mo-SMN, cells were transferred in a new shake flask and incubated at 30 °C 200rpm. After about three days, both cultures reached high cell densities again. (B) Cells populations were then transferred in fresh 1:100 Mo-SMNs and let grow to high cell density. The process was repeated 12 times and then the growth rate of the two populations was determined by measuring triplicate samples for each population. (C) Bar plot showing the estimated growth rates of the two adapted strains in 1:100 Mo-SMNs.
Figure 13: Growth curve of IMSOS17 on synthetic medium with 10 mM ammonium nitrate as nirogen source. OD is shown in squares. Nitrate, ammonium, glucose and sthanol concentrations are shown in triangles. reverse triangles, diamonds and circles respectively.
Thawed aliquots of frozen stock cultures were inoculated in a shake flask containing SMNA and incubated overnight at 30 °C 200rpm, This starter culture was used to inoculate two shake flasks containing the same media at an at an initial optical density at 880 nm (Olan) of
(0.2, generating duplicates. The flasks wers then incubated at 30 °C 200mm and growth was monitored using a 7200 Jenway Spectrometer (Jenway, Stone, UK}. Specific growth rates were calculated from at beast four time points in the exponential growth phases of each culture. At seach time point, 2 mil of the liquid culture were centrifuged with a benchiop centrifuge, supsmatant was colleciad used for metabolites analysis. Nitrate, nitrile and ammonium ion concentrations in the supemalant were measured using the HACH (Tiel, Nederlands) cuvette test kits LOKG3, LOKS41 and LOK304 respectively, following manufacturer instructions, Glucose and sthanal concentrations were measured using supernatant oblained by centrifugation of culture samples via high-performance liquid chromatograph (HPLC) as previously described. Error bars represent the standard deviation. (n=3) Figure 14: schematic representation of IMX2134 strain construction. Each expression module was amplified by PCR and 60 bp long unique homology arms were added to each fragment extremity. Fragments were co-transformed together with pUDR514, a plasmid carrying a gRNA targeting the YPRcTAU3 locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.
Figure 15: schematic representation of IMX2133 strain construction. Each expression module was amplified by PCR and 60 bp long unique homology arms were added to each fragment extremity. Fragments were co-transformed together with pUDR514, a plasmid carrying a gRNA targeting the YPRcTAUS locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.
Figure 16: schematic representation of IMX2124 strain construction. Each expression module was amplified by PCR and 60 bp long unique homology arms were added to each fragment extremity. Fragments were co-transformed together with pUDR514, a plasmid carrying a gRNA targeting the YPRCTAU3 locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.
Figure 17: schematic representation of IMX2161 strain construction. Each expression module was amplified by PCR and 60 bp long unique homology arms were added to each fragment extremity. Fragments were co-transformed together with pUDR514, a plasmid carrying a gRNA targeting the YPRCTAU3 locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.
Sequences referred to: SEQ ID NO:1 Nucleotide sequence of HPODL_02673 (GTP 3',8-cyclase)
ATGATATCCTCATTACTGCTGAGGCGGCTCCATTCAACGGGTACTTCACAGCACCTGCC ACGGCTTGAAAGACTGAGGCGTATGCCTGTAAGACACTTGAAAGAGTTTCTGACAGACA CGTATGGGCGCAAGCACGACTATCTTCGCATCTCCATCACCGAACGATGTAATCTCCGC TGCGTTTACTGTATGCCTGAGCAAGGCGTTGACTTGTCGCCACCAGAGCACATGCTTAC AACAGAGGAGATAGTCAAACTGGCCACTCTTTTTGCACAGCACGGAGTACGGAAGGTCA GACTGACTGGCGGAGAGCCCACGGTCAGAAAAGATATCGTTGAGCTTGTGGCCAAGCT CAATCAAATTACAGGCATTGAAGAGATCTGTATGACATCTAATGGTCTAGCACTCCATCG AAAATTACCAGATCTTTTCAAGAATGGGCTGACATCGCTCAATTTGAGTCTTGATACTCTT ATCAATGGTAAGTTTCTCTTAATAACGCGACGCAACGGACTCAGCGCAGTAATGAGAAGT TTGAGAACAGCACTTGAATTGGACATTCCCAAGGTGAAAATCAATGTGGTAGTAATGAAG AATCTGAACGAAGATGAGATACTGGATTTCGTTGAACTCAGCAAAAATGATAAAGTCGAG GTTCGATTTATAGAGTACATGCCTTTCGATGGAAACAAATGGTCTACGAATAAGCTGGTC TCATACGAGGACATACTTTCCAATATCAAAGTGAGACATCCGAATATCCAAAGACTCCCC CATAAACATGGTGACACGGCCAAAGTCTACCAAATTCCCGGATTCAAAGGAAAAGTAGG TTTTATAACGTCCATGACTTCGGACTTTTGCAGTACTTGCACACGTTTGAGGATTACTTCT GACGGTAACCTGAAAGTTTGCTTGTTCGATAACACAGAAGTATCTCTGCGAGATATGCTT CGTGCTGGATACAGCGATGATAAATTAATGCAACGCATCGGTGAAGCAGTGAAAAATAA GAAGGAGAAACATGCAGGGATTGACGTATTAGGAGATCAACCCAATAGGCCGATGATTT
TAATTGGAGGA SEQ ID NO:2 Nucleotide sequence of HPODL_02674 (Cyclic pyranopterin monophosphate synthase)
ATGGTTGCAATTCATGAAAAAGAAGACACCCATAGATGTGCTATTGCAGAGGGATCAATC AAGTTCAGCAACCCAGAATCGATGAAATTGCTGCTATCAGAAAGCAACAAGAAGGGAGA TGTGATTTCAATCGCAAGAATCGCAGGGATAATTGCTGTGAAGAAGACCGCAGAGTTGA TTCCTCTCTGTCATCCAATCAGCATCACTGGAATCAAGGTTGATCTGATTCACGACGAGA AAGAGAACTGCATCAAAGTAAACTGTGAGGTGCACTGTAATGGGAAAACCGGCGTCGAA ATGGAAGCATTAACGGGTGCGACGATTTCGTTGCTTACCGTTTATGATATGTGCAAAGCT GTCGACAAAATGATGACGATTAGCGATTGTCGAGTTGTTAAGAAGTCAGGAGGTAAAAG
CGGTGACATAGATCTATCAACCATCTTCAAATAG SEQIDNO:3 Nucleotide sequence of HPODL_00195 (Molybdopterin synthase catalytic subunit)
ATGTCCATCTTTGTAGATATTACTGATAAGCCGCTGGATTCGGCTGAGGTGCTTAATTAT GTTCGGCATCCTCAAGCGGGAGCAATTGTGTATTTTGGAGGCACAACAAGAAATACGTT TGAAGGCAAGGAGGTTGTATCTCTCGCATACGAGGCGCATCCAAGGTTGGCGATCAAAA CTCTCGAGTCCATCGCCCACGAGGCGAAAGCCAAATTTCAGAGTGTTCATAAGATAGCA ATTGTGCACCGCACAGGCGTGGTTCCAGTAGCTACAGAGTCCGTTATGATTGCCGTCAG TTCAACACATCGAAAAGAAGGGTGGCTTTGTGGAGAGTGGGTATTGGAGAAAGTCAAGG AAAGGGCAGAAATCTGGAAGATCGAAAAGTACGCGGACGGAGATAGTGTCTACAAAGAG
AATGACGTTTCTAACGTGCTTAGTCGCACCTAA SEQ ID NO:4 Nucleotide sequence of HPODL_01640 (Molybdopterin synthase sulfur carrier subunit)
ATGGTCGCAGTTGCTATCGAATATTTCGGGCCCGCAAAAACATATACAAACGGCGTGGC GCACGAGAGAGTAGAGCTGACAGAGCCGGCAACGCTTAACACGCTAATTCAGCACGTC GGTCGCTCGTACTCGTCCGAATTCGCTCAATATATCGTCTCCAGCTGTGGAGTCGTGGT CAATGAGGACTACGTGGAAACCGAGCGCATAGGCATCGAATTCTTCGGTAAAAACATTG
CTCTCCAGTCAGGCGACGTGGTAGGCATCATTCCGCCAGTTTCAAGTGGATAA SEQ ID NO:5 Nucleotide sequence of HPODL_03424 (Molybdopterin adenylyltransferase)
ATGACTGTTGGTATCTTGGTTGTATCAGAATCGGTTTCCAGGGGTTTATCGACCGACAAG GTTGTTGACGCATTGAAACAACACCTTGATGGCTTTGAATTGAAGGCTCACAAGGTTGTG CCAGACAAGAAGGAAGATATCCAGGCTGCAGTGGTGGACTGGGTGAAACAGGATTTCAA GCTTATCTTGACCGCTGGCGGAACCGGCTTCACCAAGACTGACATCACACCAGAGGCCA TTGAGCCATTGTTGGACAAAAAGGCGCCTGGACTGGTTCATGCTATGCTGTCCTTTTCTC TTCAAATCACCCCTTTTGCCATGCTTGCTCGGCCGGTAGCTGGTGTTCGCGGAGAATCT TTGATTATCACGCTGCCCGGCTCGCCAAAGGGAGCCACGGAAAATTTCCAGGCAATCAA AGGGGTCATTGGGCACGCACTTTCGCAACTGGGGATTGAAAGCTCAAGATTGCTGCACA AGGAGTCCGGTTCAGGCCATCATCATCATCATCATCATCACCATCATCATGGACACCTTG CCAAACACGAATTGGTCGATTCGGTCGTTGCGAGACATCGAGTCTCGCCCTATCCCACG ATCTCTGTTGATGAAGCATACTCAAGGATCAGAGAGAACACGCCAGCTCCAGAAGTAAT CGAGCTGAGCATTTTGGATCCTCGTCTAGTGGGCAGCGTGGTTGCTGAAAATGTTACTG CCCAGATGGACGTTCCAAATTTTCGCGCAAGTATTGTGGACGGATATGCGATGATCAGC TCTGATGGACCGGGTGTTTACCCTGTTGTTAGTGTTTCGCATGCTTCCAAAAATGACCAG AAAGAGCTGGTTGCAGGACAAATCGCGAGAATCACCACGGGGGCGCCGCTTCCTGAGG GAGCCGACGCCGTAGTGATGGTCGAGGAAACACGACTAGTTGAAACCACTGAGGACGG GAGCGAGGAGAAGCTTGTTGAGATACTGGCGAAAAACGTCAAGACAGGGGAGAACATC AGGGCCATTGGTTCTGACACCAGGAAGGACAGTTTGGTGCTCGCGAAAGGATCAAGAAT CTCGCCAGGAGGAGGAGAAATAGGGGCGTTGGCGAGCGTGGGGGTCAACAAAATTAAA GTGTACCGCAAACCGGTGATTGGGCTGCTATCTACTGGAAATGAACTGCAGGACGTGCA AACCGAAAAATTGTTACATTATGGAGAAATATACGACAGCAACAGACCGACGCTAGTGTC CATAGTGCAGAATTGTGGGTACGAGTTAGTCGACCTCGGAATAGCCTCAGATACCAAGG AGTCTCTTATCAAAATTATAAAGGACGCTGTGGACGTCAAAAAAATTGATTGTTTAATTAC CACTGGAGGAGTGTCCATGGGAGAGCTTGACCTGCTAAAACCGACGATCGAAAATGAAC TTCACGGTACAATCCATTTTGGACGTGTCAGAATGAAGCCTGGCAAGCCAACAACTTTTG CAACAATCGGCAAATCCACCGTTGTCTTTGCTCTACCCGGTAATCCTGCCTCTTCTTCTG TTTGCTACCATCTATTTGTCCTACCTTGTTTGTTGAAATGGCAAGGACTTGAGCCATCAGT TGGCCAAAAATTGCCTACAGAGCCAATTGTTAAGGTCAAGCTTGCAGAGGACTTGAAACT GGACCCACAACGTCCTGAATACCAGCGGGTCAGCATTTGTCAGTCAGACATGGCTCTAG TTGCGAACTCTACAGGGTTCCAAAGAAGCAGCAACATCGGATCTTTTAAGAAGGCCAAC GGACTTGTGTGTCTTCCTGCTGCCAGTGACTTCGGAAAATCGGTAATCGAAGCAGGGAC
GGTGGTGGACGCCATTCTCATCGACCAGATCTATGTGTGA SEQ ID NO:6 Nucleotide sequence of HPODL_00948 (Molybdopterin-synthase adenylyltransferase)
ATGTCTTTGTCCTTAAATGAGTACCTTCGCTATGGGCGACAGCTCATAGTGCCCGAGTTT GGTCTGCAGGGCCAGATCTCGCTGAAGAATTCTCGTGTACTGGTCGTTGGAGCCGGTG GTCTTGGGTGTCCAGCTTTGCAGTATCTTGTTGGAGCTGGTTTTGGCACGGTAGGAATC GTGGATCACGATACCGTCGATATCTCGAACCTTCATAGACAAATCTTGCACACATCAGAA ACAGTGGAGATGTTGAAATGCGAGTCTGCAAAGCTCCAGCTTGCCAAACTCAATCCTTTA GTGCAAATCAATACTCATCCGGTGGCTCTCAGTCCGGATAACAGCTTTGGAATTTTTGAA CAGTATGATATCATTTTGGACTGCACGGACACTCCTGCCACCCGGTATTTGATCAATGAT ACTGCCGTGCTACTTGGTTTGACGGTTATTTCCGGATCTGGTCTCAAGACCGAGGGTCA GTTATCCATTCTCAATTTCAACAACACCGGTCCCTGTTATCGTTGTTTCTATCCAACACCG CCACCTCCTAGCTCTGTGACTGCTTGCAGCGACGGAGGAGTTCTTGGCCCAGTTATCGG GATTATGGGTGTGATGATGGCACTGGAAGCCATTAAGGTGGTTAGTGGCTACTACTTGA GGGAGGATGTGGAGTTTCAACCTTTTTTATCTTTATACAGCGGTTATGGCCCGTTGCAAA GCTTACGGACGTTCAAAATGCGCAGACGATCGCCCAAGTGCCGAGTGTGCAATGCCGG AACGCGAGAAATAACAAGGCACGTGATAGAAACAGAGCTCGACTATGCTGTGTGGTGCG GTAAGATGGACTATAATGTTCTTGAGAAGGATGAAAGAGTATCTGTGGAGCAGCTTTCTG CGCAGAGAGCGCCCTATCTGGTCGATGTGCGAGCCAAAGAACAGTATTCCATTGTCCAT CTGCCAAATTCGATTAATATACCACTCAACGCACTAAAACACATGGACACTCTGGATGTG CCAAAGGAAACTATGATATACGTTATTTGCCGATTTGGAAATGACTCGCAGCTTGCAGTG AAACATTTGAAGAGCATCGGATATGAGAACTGTGTGGATGTCATAGGCGGGCTAACGCA ATGGTCTCGGCAGATAGACCCAAACTTCCCTATTTACTAA
SEQ ID NO:7 Nucleotide sequence of HPODL_02128 (Cysteine desulfurase)
ATGTACAGGTTCAGGATCGGAGCTGGCGTGAGACGCTTTTCCGTCTCTGCGCTGAGAAG GACATCGTCGAGTGGCGTCAAACAGGCAATGCCCACGTCTGGGGCCCAGCCGCCTCAG GGATCATCAATATCTCTCAAGTCCGCAACAAGAGATCCTTCGGAGTATGGCACAAGACC GATATACTTGGATATGCAGGCAACAACGCCGACAGACCCACGTGTGTTGGACAAAATGC TGGAATTTTATACGGGGCTTTATGGCAACCCGCACTCGTCTACCCACGCCTATGGATGG GAGACCGATAAGGCTGTTGAGGAGGCGCGCGCAAACGTCGCTGCCGTGATCGGCGCA GACCCAAAGGAGATTATTTTCACTTCGGGCGCCACCGAATGTAACAACATGGCTCTGAA GGGCGTGGCTCGGTTCTACGGAAAGAGCAAGAAGCACATCATCACCACCCAGACCGAG CACAAGTGCGTTCTGGACTCCTGCAGACATCTGCAGGATGAGGGCTTCGAGGTGACATA TTTGCCGGTCAACTCGGACGGTCTGATCGATTTGGAGCTTTTGGAGAAGTCTATCAGAA AAGATACCTGTTTGGTGTCTGTTATGGCTGTGAATAACGAGATAGGAGTGATCCAGCCTT TGGAGGATATTGGCCGCATCTGTCGCTCGCACAAGGTGTTCTTCCACACCGACGCCGC CCAGGCGTACGGCAAGATACCGATCGACGTCAACAAGTGCAACATTGACCTGATGTCGA TCTCGTCACACAAGATATATGGACCTATGGGTGTTGGTGCCACGTACGTGCGAAGACGG CCTAGAGTCAGACTGGACCCAATCATCAACGGCGGAGGCCAAGAGAGAGGTCTGCGGT CCGGAACTTTGGCTCCTCCGCTTGTTTGTGGSCTTTGGAGAGGCCGCAAGACTGATGGTG CAAGAGTACGATGCAGACCAGGCCCACATCAAGGCCCTAAGCACGAAGCTGATGGATG CCCTSTTGTCTATGGAGCACACACAATTGAACGGATCCCGCATCCATAGATACCCTGGC TGCGTTAACGTTTCCTTTGCGTACATCGAGGGAGAGTCTCTTTTGATGGCATTGAAGGAC ATTGCTCTTTCGTCAGGATCCGCCTGCACGTCCGCGTCTCTTGAGCCTTCGTACGTTTTG CATGCTCTGGGCGCCGACGACGCCCTTGCACACTCATCGATCCGGTTCGGCATCGGCA GATTCACCACCGAGGCCGAGGTCGACTACGTGATCCAGGCCCTGACCGAGAGAGTCAA GTTCTTGCGGGAGCTGAGTCCGCTGTGGGAGATGGTCAACGAGGGAATCGACTTGAAT
TCGATCGAATGGGCAGGACATTGA SEQ ID NO:8 Nucleotide sequence of CrMOT1 (molybdate transporter)
ATGGCCTTGCAAAATGCTTGGCAAAACACAAAAGAAAGAGCTAGAGAAACTTGGGCTCA ATTGACTTGGTCTGAAGTTTCTGGTTCTTTGGGTGATTTGGGTACTTTTTTGCCTTTGTTG ATCGGTTTGGTTCAAAAGGTTCATTTGGACTTGGGTACTACTTTGACTATTACTGGCTTGT ACAACATCATTTCCGGTTGGCAATTCAGAATTCCAATGTGTGTTCAACCCATGAAGACTA TTGCTGCTGTTGCTTTGGCTGGTGGTGCTGCTGGTTTGGATTTGCCACAATTATTGCATG CAGGTTTGTTTGTTGCTGGTTGTGTTGGTTTGTTGGGTGCTTCTCAAGCTATTGATTTGTT CAATTGGTTGGTTCCACCACCAGTTATTAGAGGTGTTCAATTAGCTGTTGGTGTTAAGTT GGCTATGAAGGGTGTTGATATGGCTTTGAGATTGCATGGTGGTCCATCTTCAGGTTGGA GGCCATGGTTGGGTACTGAAGGTTTAGTTGTTGGTGCTGTTGCATTAGCTGCTATGATTG CTACTACTTTACCACCAAGAGCTGCTAGAAGAGGTACTTTGGAAGCTGCTGATGAAGGT GGSTTTGGGTCCAAGACCAACTGATACTGCATTTGAACCATTGCTAAGAAGATTGCCAGCT TGSTTGCGGTGGSTGGTGATAGAGCACCACAAGTTGAAGGTGCTGCAGTTTCTGCTGAAAG AGCTGGTTTGTTAGCTCATGCCGAAGGTGGTGAAAGATCTGGTAATTTGGATGATGGAA CTGAAGCTGGTGTTGGTGCAGCTGCTGGTGGCGSTGSTTGTGSTGGSTGGCGGAGGSTG GCGGTAGAATTCCATCTGCTTTAATTGCAGTTGTTGTTGGTCTAGCTATGGCTGTTTTAC ACAGACCAGGTTTGGTTTGGGAATTGAGATTAGGTCCAACTTTGCCAAGACTGTTAAGAC CATCTTGGCCAGATTTTAAGACTGGTGCTTTGAGAGGTGGTTTACCACAATTGCCATTGA CTACCTTGAACTCCGTTATTGCTGTTACTCAATTGGCTAATGCTTTGTTCGGTGATAAGC CTGAAGCTGAAAGACGTAGATGGCGTCCATCTGCAGTTGCTTTATCCGTTGCTTTGTTGA ATGGTGCCGGSTGTTTGGTTAGGSTGCTATGCCATGTTGTCATGGTGCTGGCGGTTTAGCT GCTCAGTACAAATTTGGTGCTAGAACTGGTCATGCTCCAATTTTGTTAGGTTGTATTAAG GCTGCTTTGGGTTTGTTGTTTGGTGGTTCATTGGTTGTTTTGTTGGAAGCTTTTCCACAG CCATTATTGGGTGCATTATTGACCGTTTCTGGTATTGAATTGGCCTCTGTTGTTAGACATA CAAGATCTCCAAGAGGTTACACTTTCGCTTTGTTAACAGCAGTTGCTATTTTGGCTTTGG ATAACACTGGTACTGGTTTTTTGGSTTGGCTTGGTTGGTGTTGCTGCCGTTGCAGCTTATG AAGGTGCCGTTGCTGCTGCTGCCGCTAGATGGCCAAGAGTTTTTGCTAGAGGTGGTAGA
GCTTAA SEQ ID NO:9 Nucleotide sequence HPODL 02384 (nitrate reductase NiaD)
ATGGACTCTGTTGTCACTGAGGTGACCTATGGTCTGGAGATTAAGAAAATCAAGGAGAT CACGGAGCTGCCTTTTCCAGTCAGGCAAGACTCTCCTTTAAGCGAGGTGCTTCCTACAG ATCTGAAGACCAAAGATAATTTCGTCGCTAGAGATCCTGACCTTCTTAGACTTACTGGTT CACACCCATTCAATTCTGAGCCACCACTGGCAAAGCTCTACGATTCGGGGTTTCTCACTC CAGTGAGTCTTCATTTTGTGAGAAATCACGGCCCCGTTCCTTACGTTCCTGATGAAAATA TTTTGGACTGGGAAGTGTCGATTGAAGGCATGGTTGAAACGCCTTATAAGATCAAACTGT CAGACATAATGGACCAGTTCGATATCTATACAACCCCAGTTACTATGGTCTGTGCTGGAA ACAGAAGAAAGGAGCAGAACATGGTGAAAAAGGGGACCGGTTTCAATTGGGGAGCAGC TGGAACATCTACTTCTCTTTGGACCGGATGCATGCTTGGAGATGTGATAGGCAAAGCCA GACCATCAAAGCGGGCAAGATACATATGGATGGAGGGTGCAGATAACCCGGCAAATGG CGCATACGGCACCTGTGTCCGCCTAAGCTGGGCTATGGACCCTGAACGATGCATCATGA TGGCATACAAACAAAACGGCGAGTGGTTGCATCCTGACCATGGAAAGCCCCTTCGAGTA GTCATCCCCGGTGTCATTGGTGGACGATCAGTCAAATGGCTAAGAAAGTTAGTGGTGAG CGACCGGCCGTCTGAAAATTGGTATCATTATTTTGATAATCGGGTTCTTCCGACCATGGT GACGCCAGAAATGGCCAAAAGTGATGACAGGTGGTGGAAAGACGAGCGATATGCCATC TATGATCTGAACTTGCAAACTATCATTTGCAAGCCCGAAAATCAGCAGGTTATCAAGATTT CAGACGACGAGTACGAAATTGCAGGTTTTGGCTACAATGGAGGTGGGATCAGAATAGGC CGAATCGAAATCAGTCTTGACAAGGGGAAGACCTGGAAGCTGACAGAGATCGACTATCC GGAAGACAGATATAGGGAGGCAGGTTATTTCAGATTGTTTGGCGGACTTGTGAACGTTT GCGACAGAATGAGCTGTCTGTGCTGGTGTTTCTGGAAGCTCAAGGTTCCTCTTTCTGAAT TAGCAACGTCAAAAGATATTCTCGTCCGTGGCATGGATGAGCGTATGGTGGTCCAGCCG CGCACAATGTACTGGAATGTAACGTCCATGTTGAACAATTGGTGGTATCGAGTCGCCATT ATCCGCGAGGGTGACGCTCTTCGATTTGAGCATCCAGTGGTGGCCAACAAGCCTGGCG GTTGGATGGATAGGGTCAAGGCAGAAGGTGGAGATATTTTAGATAATAATTGGGGAGAA GTGGACGATACGGTCAAGCAGGCTGAAAGGAAGCCGCGTGTTGATGAGGATATCGAGA TGATGTGCAACCCGGAGAAAATGGACGTCATTATCAAATACTCAGAGTTTGAAGCACACA AGGACAGCGAGACAGAACCATGGTTTGCTGTCAAGGGCCACGTATTTGATGGTAGCTCG TATTTGGAAGACCATCCAGGAGGTGCCCAGTCGATCTTGATGGTGAGCGGCGAAGACG CTACCGACGATTTCATTGCAATTCACTCATCTTATGCCAAAAAGCTGCTCCCTCCTATGC ACTTGGGAAGACTCGAAGAGGTCAGCTCTGTTACAAAAGTAAAGTCTGTCGAGGAAAAT GTCAAGCGAGAAGTTTTGCTCGATCCGCGAAAATGGCACAAGATAACACTTGCGGAAAA AGAGATTATCTCTTCCGACTCGAGAATATTCAAGTTCGACCTTGAGCATCCAGAACAGCT TATCGGTCTTCCAACGGGTAAACACCTGTTTCTGAGGCTAAAAGATTCATCTGGCAAGTA TGTGATGAGGGCATACACCCCTAAATCGAGCAATTCTTTGCGGGGTCGTCTAGAGATAT TGATAAAGGTTTATTTCCCAAATAGGGAATACCCCAACGGCGGAATTATGACAAATCTTA TCGAAAACCTCCAAGTGGGAAACCAGATCGAGGTCAAAGGACCTGTCGGCGAGTTTGA GTATGTCAAGTGCGGGCACTGCAGTTTCAATAACAAGCCTTATCAAATGAAGCATTTTGT TATGATCTCGGGAGGATCGGGCATCACCCCAACTTACCAGGTTCTGCAAGCTATTTTCA GCGATCCTGAAGATACAACAAGCGTGCAGTTGTTTTTTGGTAATAAGAAAGTTGACGATA TCCTGCTTCGAGAAGAGCTTGACTGTCTACAGATAAAACATCCAGAACAATTCAAAGTTG ATTACTCGCTATCAGATCTGCATCATCTACCGGAGAATTGGAGCGGATTGAAAGGCAGG TTAACATTCAATATTCTGGACAGCTACGTTCAGGGAAAAAATATGGGAGAGTATATGCTA CTGGTATGTGGACCGCCAGGAATGAACGGTGTGGTCGAAAACTGGTGCAAAGCGCGCA
ATTTGGATAAACAGTATGTAGTGTACTTCTGA SEQ ID NO:10 Nucleotide sequence HPODL_02386 (nitrite reductase NirD)
ATGACTTGTTCTGTTCCTCCCTTGCCAGAGGACATCACTCCTCCGGCCGCTAAGAAGAA GCTTGTCATTGTGGGCCTGGGCATGGTCGGCCTGTCATTTCTCGAAAAATTACTTTTGAA CGACTCAAAACTAAATGAGTATAGTATACTGGTTTATGGGGAGGAGCCATATCTTGCTTA TAACCGCGTTGGTCTCACCGAGTATTTCCAGCACCGTGAGTTCAAAAATCTTCTTCTGTC GCCAGAGGAGTTCTACCAGCTACGCGGCGAAAAATGGAACTACGCCATTGATGAGAAG GTGATTGACATAGATCGGCAAGCTCGGACTATCACCACGAACAAAGGAAACAGGGCGTC TTACGATGTGCTTGTTCTTTGCACAGGCTCTACGGCCATCCTTCCTACCGATCTGCTACC ACCTCCTACTAGAAAAAGCTACCGTGAAATGGGATGCTTTGTCTACAGAACCATCGACGA TCTCTATTCAATGATAGATTATTGCCAGGGTGCAAAGAAACAGAGAGCCATTGTCGTTGG TGGTGGGCTTCTCGGTCTAGAAGCAGCCAAAGCTCTCTACGACATGGAGTCGTTTGAGG ATATCACCATTGTCCATAGGTCCCATTGGCTGCTTTCTCAACAAATGGACCAGAAGGGTG GTTCTCTACTTACCAGCAAGGTGAAAGAGCTGGGGATTACTTGTCGAACTGGAACAACT GTGTCAGAGCTGCTTTTTGACGAAGACCAAAATCTGACAGGAGTGAAATACGATAACGG TGAAATTGAAGAATGCTCCCTTCTTTGCTACACTATTGGTATCAAACCAAGAGATGAGCT TACAAGCTGTGGCCTGAATGCGGGCTCAAGGGGCGGATTTAAAGTGAATAACATGTTGC AAACTTCGGATGAAAATATCTATGCGATTGGAGAGTGTGCTTCTTGGAATAACATGACTT TTGGACTGATTGCTCCCGGATATGAAATGGCTGATATACTGGCCTTCAACCTTACTCAGG GAAAACTCCACCAGCCAAAGGAGTTTTCTGAACCTAGTATGGGCACCAGGCTCAAGCTG ATGGGTGTGGATGTTGCATCTTTTGGCGATTTTTTTGCAGACAGAAATGGGCCTAAATGG CTTCCTCGCGGATATGAGAAAGAAGTTCGTGGCCTTGTGTTTGAGGATCCAATAGATGG AACTTACACAAAGTTGCTTTTCACAAAGGATGGCAAGTACATGCTTGGAGGAATTCTGGT TGGCGATACAAGCAACTACACCAAGTTCTCCGCGATGATAAGGAAACCATTACCCAAGT CACCATCCGAGCTTCTGATTGGAAAAGCGGGAGAAGATGACATGGAGAAGCTTTCAGAT GAGACCCAAATATGCTCCTGTCACAATATCACCAAGGGAAAACTTGTGGAAGCTGTGAA AAATGGTTGCAGCAGTCTCGCAGATCTGAAAAAGTGCACTAAGGCGGGAACAGCTTGCG GGGGTTGCGAGCCTACAGTGAAGGTCATTTTTGAAACAGAGGTTAAAAAGTTGGGTGGC AAGGTCTCCAACAACCTATGCGTGCACTTCGACTATTCAAGAGCAGACTTGTTCTCACTG ATTATGGTCAAAAATTTCCGGTCTTTTAGAAGGGTCATGGAGGAGCTGGGAAACAACCC CAGCTCGTCGGGATGTGAAATTTGCAAGCCTACCATTGGCTCCATTCTCTCAACACTATA CAACCGACACCTCTTGAAAAAGGAGGTTCATGGTCTGCAAGACACAAATGACCGATACC TTGGAAACATACAGCGAAACGGCACCTTCTCGGTTGTTCCTCGGATGTCAGCCGGCGAG GTCACTCCGGAAAAGCTTGTTTCTATTGGGCAAATTGCTAAGAAGTACGGTGTCTACACA AAGATCACAGGGGCACAAAGACTGGATTTGTTTGGCGTCAAGAAGAGCGATCTGCCGAA AATATGGAAAGATCTCAACGAAGCTGGCTTCGAGAGTGGACAGGCTTACGGCAAGAGTT TGAGAAACGTCAAGTCCTGCGTTGGATCAACATGGTGTAGATACGGAATTGGAGACTCT GTTGGTTTGGCTGTCAGGTTGGAAGAACGGTACAAAGGCATACGCTCGCCTCATAAAAT GAAGGGGGGCGTGTCTGGGTGTGTTAGAGACTGTGCCGAGTTCCACTCAAAGGACTTT GGTCTCTGCGCTGTGAAGGATGGATTCAACATATATGTTGGTGGAAATGGTGGAATGAA GCCAGCGCACGCGCAGCTTCTTGCTACCAATGTGCGTCCTGACGAGGTGATCCCTATTC TTGACAGATACCTAATGTTTTACATTACCACGGCAGATAGACTGCAGCGGACAGCTCGAT GGCTTGAAAACCTTGATGGTGGAATCGAATATTTGAAGGACGTTATCATCCGCGATAAAT TGGGGATTTGCAAGGATCTGGAAGCCCAAATGCGCCAGCTTGTAGCCGGTTACTACGAT GAGTGGGCCAAGGCCGTTTCTGAAGAGAAAGACAACCCAATTTTCAAGCAATTTGTGAA TACATCTGAGAACCAAGATACTGTGGAGATAGTCAAAGAAAGAGGCCAACCGAGACCGG CAATGTGGCCGGAGAAAGCGGCCAACCAGAAATTCAATGAGATCAAGTGGTCTTCTGTG AGCTGGCAGGAAGTCTGTGAGAGCTCAGATTTGCCTCTAGCAGAGGCAGGGTCCTCGG CAACGGTTTTGGTTGGGGACACCCAAATTGCTCTGTTCAGAACTAGCGAAAACGAGCTC TATTGCTCTCAGAACATGTGCGGACATAAACGTGCCTTTGTGCTAAATCAGGGACTTCTT TCGGAAGATGGTGACAAAAACTGCTACATATCGTGTCCGATGCATAAACGAAATTTTTAC CTCAAGTCCGGTGCCTGCAAAAACGATGAAGCGCTATCTATCGCGACGTTTGAGGTTAA GGAGGAGAATGGCAAAGTGTATGCCAAACTTCCGCCCACAACAGAGCTGGATGAGGTC CTGGGAACTTCTAAATGGAAGGTGACAAGTCAGGAAACAGAGGCTAAACAGGTGCGCAA
GATAACAACGGAGAAAAATTTAGTTGACAAAGCAATATCGTTCGACTGGTAA SEQ ID NO:11 Nucleotide sequence HPODL_02387 (high affinity nitrate transporter NrtB)
ATGCGACTTTCTACCTTATGGGAACCGCCAACGGTGAATCCAAGAAACCTGAAAGCGAC CTCGATACCAATTTTTAACCTGTGGAACGTCTATGGAAGAAACTTCTTTTTCGCGTGGTTT GGGTTTTTTGTGTGCTTTCTTTCCTGGTTTGCTTTTCCGCCTCTTCTTCATGGTATGCTAA AGAAGGACCTAAAGCTCACCGCAGTGGATATATCCAATAATAACATATGTGGACTGACC GGAACTTTACTAGGCAGATTTATTTTGGGGCCCCTTAACGACAAGTATGGOCCCTCGGATT ACTTTGGTAGGCGTGCTGGTTGCAGGAGCAATTCCAACTGCATTTGTTCCTTTGGTTACA AATGTTGCAGGTCTACATGCCATCCGTTTCTTTATCAGCTTCCTAGGCTCCTCGTTTATTT GCTGCTCCCAATTCTGCGCTGTATTTTTTGATAACAATATTATAGGAACAGCAAATGCCG TCTCTGCCGGSTTGGGGAAATGCTGGAGGGGGSTGTGGCATTCTTTGTCATGCCTGCCATT TCAAATGCATTAGAAAAAAGAGGTTACTCTTTACACCATTCGTGGAGCTACTCTTTTGTGA TTGGGCCGTTCTTGATTCTGATGATCACAGCAATTGTGATTTTTGTATTTGGCAGCGACT GCCCGAGAGGCAGATGGTCCCTTCGTGGAGATATCCTTGGAATCAACATGGATAATATG CTCGTGAAGTCTGTCTCTGTCACAAGACACTTCTCTAAGGATGGAGAACTCACTTCTGTC TGTGTTGAGCCTGTTAACGCAATCGATGAGGTTGTGGTTGAGCCAAATCAGGACCAGGA AATTTTTGAAGTCGCAGACATCATAAATGAGGACGAAATCATCGAAGACCCAACTCTCAA AGACGTGGTCAAGATCTGTTTTTCCCCACGCACAATGCTGGTCGGACTTTGCTACATGT GCTCGTTTGGTACTGAGCTTGCAGTGGAGTCTATTATATCCAACCTATTCGGGCAAAAGA TGACAAAGTGGAGCACCTCTAAAGCTGGAGCATGGGGCTCAATGCTTGGACTTCTCAAC GTGGTGACAAGACCAGCCGGAGGAATTATCTCCGATTTCCTATACCAAAGATTCAAGAC CACCAAGGCTAAAAAGTTCTGGATGATATTCACTGGCTTGATGCAGGGAATTTTTTTGAT CTGGGTTGGACTGGTTCCGGAATTGTCCATCGCGGGACTCATAGTGTCGGTTTCGTTTT TGTGTCTTTGGSTTTGAGATGGGAAACGGTGCAAATTATGCCTGTGTTCCGGTTGTGAATA GACACCACAGTGGTATTGTGAGCGGAGTTACGGGAGCAATGGGTAACCTAGGAGGCAT TCTGTTTAGTTTAGTGTTCAGGTACACTATAGCAAATGGAGTGAACAACTACTTCAAGGC GTTCTGGATCATAGGAATTGTTTGCACTGTTGTGAATTTGGCCTGTGTGCTTATTCCAATT
AGGGAGGAGAGGCCTAGGAAAGCGGAAATTTGA SEQID NO:12 Nucleotide sequence EcMobA (Molybdenum cofactor guanylyltransferase)
ATGAATTTGATGACTACCATTACCGGTGTTGTTTTGGCTGGTGGTAAAGCTAGAAGAATG GGSTGGTGTTGATAAGGGTTTGTTGGAATTGAATGGTAAGCCATTGTGGCAACACGTTGC TGATGCTTTAATGACTCAATTGTCCCACGTTGTTGTTAACGCTAACAGACATCAAGAAATC TACCAAGCTTCTGGTTTGAAGGTCATCGAAGATTCTTTGGCTGATTATCCAGGTCCATTG GCTGGTATGTTGTCTGTTATGCAACAAGAGGCTGGTGAATGGTTTTTGTTTTGTCCATGT GATACCCCTTACATCCCACCAGATTTGGCTGCTAGATTGAATCATCAAAGAAAGGATGCT CCAGTTGTTTGGGTTCATGATGGTGAAAGAGATCATCCAACTATTGCCTTGGTTAACAGA GCTATTGAACCCTTGTTGTTGGAGTACTTGCAAGCTGGTGAGAGAAGAGTTATGGTTTTT ATGAGATTAGCTGGTGGTCATGCCGTTGATTTTTCTGATCATAAGGATGCCTTTGTCAAC
GTTAACACTCCTGAAGAATTGGCTAGATGGCAAGAAAAGAGATGA SEQ ID NO:13 Nucleotide sequence EcMobB (Molybdopterin-guanine dinucleotide biosynthesis adapter protein)
ATGGCTGGTAAAACTATGATTCCTTTGTTGGCTTTTGCTGCTTGGTCTGGTACAGGTAAA ACAACTTTGTTGAAGAAGTTGATTCCAGCTTTGTGCGCTAGAGGTATTAGACCAGGTTTA ATCAAACATACCCACCACGATATGGATGTTGATAAGCCAGGTAAAGACTCCTACGAATTG AGAAAAGCTGGTGCTGCTCAAACTATCGTTGCTTCACAACAAAGATGGGCTTTGATGACT GAAACTCCAGATGAAGAAGAATTGGACTTGCAATTCTTGGCCTCTAGAATGGATACTTCC AAGTTGGATTTGATCTTGGTCGAAGGTTTTAAGCACGAAGAAATTGCCAAGATCGTCTTG TTTAGAGATGGTGCTGGTCATAGACCAGAAGAGTTGGTTATTGATAGACATGTTATTGCC GTTGCCTCTGATGTTCCATTGAATTTGGATGTTGCTCTGTTGGATATCAACGACGTTGAA
GGTTTGGCTGATTTTGTTGTTGAATGGATGCAAAAGCAGAACGGCTAA SEQ ID NO:14 Nucleotide sequence KoNasA (assimilatory nitrate reductase, subunit A)
ATGACTGAAACTAGAACTACTTGTCCATACTGTGGTGTTGGTTGTGGTGTTATTGCTTCT AGAGCACCACATGGTCAAGTTTCTGTTAGAGGTGATGAACAACATCCAGCTAATTTCGGT AGATTGTGTGTTAAGGGTGCTGCTTTGGGTGAAACTGTTGGTTTGGAAGGTAGAATGTT GTTCCCTGAAGTTGATGGTGAAAGAGCTACTTGGCCACAAGCTTTGGCTGCTGCTGGTT CTAGATTGAGAGAAATTATTGATAGACATGGTCCACAAGCTGTTGCTTTTTATGCTTCTG GTCAACTGTTGACTGAAGATTATTACGCTGCTAACAAGTTGATGAAGGGTTTTATTGGTG CTGCCAACATTGATACCAACTCTAGATTATGTATGTCCTCTGCTGTTACTGGTTACAAAC GTGCTTTAGGTGCTGATGTTGTACCATGTTCTTACGAAGATGTTGAGAACTCCGATTTGG TTGTTTTGGTTGGTTCTAATGCTGCTTGGGCTCATCCAGTCTTGTATCAAAGATTGGCTC AAGCTAAAAGGGACAACCCACAAATGAGAGTTGTTGTTATTGATCCTAGAAGAACCGCTA CCTGTGATATTGCAGATAGACATTTGGCTTTGGCTCCAGGTTCTGATGGTGGTTTGTTTG TTGGTTTATTGAACGCTATTGCTGCCTCTGGTGCTATTTCTGATGATTTTAATGATGCCCA AAGGGCCTTGACTATTGCTCAAGATTGGGACTTAGATAAGGTTGCTCAATTTTGTGGTTT GCCAAGACAACAAATTGCCGATTTCTACAGAGAATTCATTGCTGCTCCAAGAGCTATTAC CTTGTACACTATGGGTATCAATCAATCCGCTTCTGGTTCTGATAAGTGCAACGCCATTAT TAACGTTCATTTGGCCTGTGGTAAATACGGTAGACCAGGTTGTGGTCCATTTTCTTTGAC TGGTCAACCTAATGCTATGGGTGGTAGAGAAGTTGGTGGTTTAGCTACTATGTTGGCTG CTCATATGAATTTCGAACCAGATGACTTGAGAAGATTGGCAAGATTTTGGGGTTCTGAAA GATTAGCACAAACTCCAGGTTTAACTGGTGTTGAATTATTTGCTGCTATCGGTAGAGGTG AAGTTAAGGCTGTTTGGATTATGGGTACTAATCCAGTTGTTTCCTTGCCAGATTCTCATG CTGTTTCTGAAGCTTTAGCTAGATGTCCATTGGTTATCATCTCAGATGTTGTTGCTGATAC TGATACTGGTAGATTCGCCCATATTAGATTTCCAGCTTTGGCATGGGGTGAAAAATCTGG TACTGTTACCAATTCCGAAAGAAGAATCTCTAGACAAAGGGCTTTTATGCCACCACCAGG TGAAGCTAGAGCTGATTGGTGGATAGTTGCTAGAGTTGCTGAAGCCTTAGGTTTTGGTTC TGCTTTTGCTTGGCAACATCCACATGAAGTTTTTTCTGAACATGCTGCATTGTCCGGTTA CGAAAATGACGGTCAAAGGGCATTTGATATAGGTGGTTTGGCTGATTTGTCAAGAGAAG CTTGGGATGCTTTGGAACCAGTTAGATGGCCAGTTTCTAGATCTGAAGCTGCTTGGTCT GTTCACAAAGGTTGGCATAGAGATGGTAAATTGAGAATGGTTCCAGTTGCTCCACAACCT ACTAGAGCTACTACTGATGCTTTTTACCCACTGATTTTGAACTCCGGTAGAATCAGAGAT CAATGGCATACAATGACTAGAACTGGTGCTGTTCCAAGATTGATGCAACATATTAACGAA CCAGTTGTTGAAGTTGCTCCTGCTGATGCTCAAAGATATCATTTGTTAGAAGGTGAATTG GCCAGAGTCAGATCTCCAAAAGGTGTTATGGTTGCTAAGGTTACTATTGGTGATGGTCAA AGACCTGGTTCTTTGTTCGTTCCTATGCACTGGAACAATCAATTTGCTAGACAAGGTAGA GTCAACAACTTATTGGCTGCAGTTACTGATCCACATTCAGGTCAACCAGAATCTAAACAA ACTGCTGTTGCTATTGCTACTTGGTTGCCAGCTTGGAAGGGTGAATTATTCTCAAGACAA CCAGTTCCATTACCAGCTTCATTGCATTGGAGAAGAAGGGCTGCTCAAGGTATTATTCAT TTGTCTTTAGCTGGTGACACCAGATCAAGAGATTGGTTAGTTGAATGGTGTCAAAGACAA GGATGGCAAATGCAAGTTGCAGAAGGTGGTAAAGTTTGGAATTTGTTAGCTTGGAGAGC TGGTGAATTGATGTTAGGTTGGTGGTCTGATGCTTCTGAACCAGCTATTGATGCTGATTG GATTCATGCTGCTTTTAGAGTTCCACCTCAAAATGCTGCTAGAAGGCATGCTTTGTTATC TGGTAGAAAAGGTGGTGTAGAAATGCCAAGAGGTAGAATTATCTGTTCCTGTTTCTCTGT TGGTGAACGTGCAATTGGTGAAGCCATTGCTGGTGGTTGTAGAACACCAGGTGCATTAG GTGGTAAGTTGAAATGTGGTACTAATTGCGGTTCTTGCATCCCAGAATTGAAAGCTTTAT
TGGCAGCTAAATTGGCCCAAGCCTAA SEQ ID NO:15 Nucleotide sequence KoNasC (assimilatory nitrate reductase, subunit C)
ATGACTAGACAATTGGTTATTATCGGTAACGGTATGGCTGCTACTCCAGGTGAAGCTTTG GTTGAAAGAGATGCTAGAAGATTCTCCATTACCATTATCGGTGATGAACCTAGACAAGCC TACAACAGAATTCAATTGTCTCCAGTTTTGGGTGCTGAAAAAACTGCTGGTGCTACTAGA TTATTGCCAGCTGAATGGTACTCTCAACATAACGTTACAGTTAGAGCTGGTGAAACTGTT ACTGCTGTTGATATGGCTGCAAGAACATTGCAAACTACCGCTGGTGAATTAGGTTGGGA TGAATTGGTTTTTGCTACTGGTTCTTTGCCATTTTTGCCACCATTGCCAGGTATTACTTTG CCACATGTTTTTGCTTTCAGAACCTTGGCTGATGTTGAAGGTATTTTGGCTATTGATGGT CCAGCTGTTGTTATTGGTGGTGGTGTTTTAGGSTGTTGAAGCTGCTGCTGCTTTGAGAAG GCATGGTGATTCTGTTACTTTGTTGCATAGAGGTTCTTGGTTGATGGAACAACAAACTGA TGCTTTCGTTGGTGAACAATTGCAAATGTTGTTGGCTGAAAGAGGTATTGGTTGCGTTAT GGAATCTAGAATTGCTGCCATTGATGAACACCAAGTCTTGTTGGAAGATGGTAGAGCTTT TGCTGCTTCTAGAGTTGTTTTGGCTACAGGTGTTCAACCTAACAAAAGATTGGCAGAAAG ATCTGGTTTGGCTTGTGGTAGAGGTATTTTAGTTGATAGAAGATTGGCTACTGCTCAACC AGGTGTTTCTGCTTTGGGTGAATGTTGTGAAATAGATGGTCAAACCTGGGGTTTAGTTGC TCCATGTTTGAGACAAGCTGAAGTTTTGGCTGATAGATTGTGTGCTATTCCTGGTGAAGA TTTCAGATGGCAAGATTCTGGTACTAGATTGAAGGTTACTGACATCGAATTATTCTCTGC CGGTGAATTGAGAGCTGATGAACAAGATGATGTTTACACTTCTTGGGATCCATTGGATAG ACATTACAGAAGGTTGTTGTTGAGAGATGGTAAATTGAGAGGTGTCTTGTTGTTGGGTGA CTGTTCTTCTGCTGCTCCATTGACTGCTTTGTTGGGTAGACATGGTCCAAGAAGGCAATC
TGGTTTTTCTACACACTTGCAAAGATGTTCTAGAGCCTTGTGGGATAAGTTGAGATGA SEQ ID NO:16 Nucleotide sequence RsBisC (biotin sulfoxide reductase)
ATGGGTGTTGAACCATTTGCTCATGATCCAGCTCCATCTGAATTGATTCATTCTGTTCCA GCTTGTGGTTCTCCTGAGAGAAGAGTTATGAGGCCAATGGTTAGAGAAGGTTGGTTGGC TGATAGACAACATTCTGATAGAAGAGGTAGAGGTAGGGAAAGATTTTTGCCAGTTTCTTG GGATGCTGCTTTGGATTTGGTTGCTGGTGAAATTAGAAGAGTTTCTGCTGATCATGGTAA CGCTGCTATTTTTGCTGGTTCTTATGGSTTGGACTTCTTGCGGTAGATTTCATCATGCTTCT ACCTTGTTGAAGAGGATGTTGAACTTGGTTGGTGGTTTTACTGGTCATGTTGATACCTAT TCTATTGCTGCTGGTCCAGTTATTTTGAGACATACTTTGGGTGATGATAGAGCATGTGGT GGTCAAGCTAATACCTTGGATTCAATTGCTGAACACTCTCAAACCTTGGTTGTTTTTGGT GCTATGTCTCCAAGAACTGCTCAATCTGAAGCTGGTGGTATTGGTGCTCATCATTTGGAA ACTTACTTGAGAAGAATCGTCGAAAGAGGTGTTAGAGTCATTTTGGTTTCTCCATTGAAG GATGATTTGCCAGATTGGGTTGCTGCAGAATGGTGGCCAATTAGACCAAATACTGATAC CGCTTTGATGTTGGGTTTAGCCGGTGAAATAGTTAGATCTGGTAGACAAGACTCTGATTT CTTGGCTAGATGTACTTCTGGTTCTGAATTATACTTGGCCTACTTGAGAGGTGAAGGTGA TGGTAGACCAAAAGATGCTGAATGGGCTTCTACTATTACTGGTTTGCCAGCTGAAGCTAT TAGAGCTTTGGCTGGTGATTTGCCTAGAACTAGATCTATGTTGACTGTCTCTTGGTCTTT ACAAAGGGCTCATCATGGTGAACAACCATTTTGGGCTGCTTTAGGTTTAGCTGCTGTTAT TGGTCAAATTGGTAGACCTGGTGGTGGTGTTGGTTATGGSTTACGGSTTCTTTAGGTGGTG TAGGTGCTCCTTTTACTATTGGTAAATCTCCAGCTATGTCCCAATTGTCTAAGCCAATCAA TTCCTTCATTCCAGTTGCCAGAATCTCCGATATGTTGTTGAATCCAGGTGGTCCATATTCT TACGAAGGTGAAGATAGAAGATACCCAGATATCAGATTGGTGTATTGGTCTGGTGGTAAT CCATTCCATCATCACCAAGATTTGAACAGATTGTCTGAAGCTTGGACTAGACCAGAAACC ATTATAGTTCAAGATCCAATGTTCACTGCTACCGCTAAAAGAGCTGATATAGTTTTACCAG CCTCCACCTCTATTGAAAGAAATGATTTGGCAGGTAACAAGAGGTCCGATTTCATTTTGG CTATGGGTCAAGCCATTGCTCCATTGGGTGAAGCTAGATCTGATTTCGATATTTTCAACG CCTTGTCTGGTAAATTGGGTGTTGCTGCTGCTTTTAATGAAGGTAGAGATGAGATGGGTT GGATCAGACACTTGTATGAAGAATCTAGAAACCACGCTCAAAGACATCATCACTTTGAAA TGCCAGATTTCGAAACTTTTTGGGCTCAAGGTCATGCTCCATGTCCAGTTCAAAGAGATC ATACCTATTTGGCTGCTTTCAGAGAAGATCCAGGTGCACATCCATTGGATACTGAATCTG GTTTGATAGTTTTGGGTTCTGCTACTTTAGCCAGATTAGGTTATGCTGATTGTGGTCCAC ATCCAGCTTGGATTGAACCAGCTGAATGGTTGGGTAAAGCTCAAGCTGGTGAATTGCAT TTGATTTCTCATCAACCTAAGGGCAGATTGCACTCTCAATTGGAAACTGCTGAAGCTTCT TTAGCTGGTAAACGTGAAGGTCGTGATGAAGTTATGTTGCATCCAGATGATGCTTCCGTT AGAGGTATTGCAGATGGTCAAACTGTTAGATTGTGGAATGCTAGAGGTGCTTGTTTGGCT ACTGCTCAAGTTACTGATTCAGTTGCAGCTGGTGTTGCTATTTTGCCAACTGGTGCTTGG TTTACTCCAGCAGAAGCTGAAGGTCCAGAATTGTCAGGTAATCCAAATGTTTTGACCTTG GACATTGGTTCCTCTGCTTTTGGTCAAGGTTGTTCTGCTCATACTTGCTTGGTTAGAATT GAAGCACATGCTGGTGATGCCGGTGATGCTGTTAGAATCTATGATGCTCATTTGGCAGC
AATCTTGCCAACTTAA SEQ ID NO:17 Nucleotide sequence RcFdsA (Formate dehydrogenase, alpha subunit)
ATGAAGGATTTGATTATCCCACCATTGGATTGGACTCAAGATATGGGTACTCCAAAAAGA GAAGGCGCTCCAGTTCATTTGACTATTGATGGTGTTGAAGTTACTGTTCCAGCTGGTACT TCTGTTTTGAGAGCTGCTGCTGAAGCTGGTATTTCTATTCCAAAATTGTGTGCCACCGAT AACGTTGAACCAGTTGGTTCTTGTAGATTGTGCATGGTTGAAATCGAAGGTATGAGAGGT ACTCCTACTTCTTGTACTACTCCAGTTGCTCCAGGTATGAGGGTTCATACTCAAACTCCA CAATTGCAAAAGTTGAGAAGAGGTGTTATGGAGTTGTACATCTCTGATCATCCATTGGAC TGTTTGACTTGTGCTGCTAATGGTGATTGTGAATTGCAAGACATGGCTGGTGCTGTTGGT TTGAGAGAAGTTAGATATCAAGCTAAGGATACCCATTTCGCTAGAAGAGATGCTACTGGT CCAAATCCAAGATATATCCCAAAGGATAACAGCAACCCATACTTCTCTTATGATCCAGCT AAGTGTATTGTCTGCATGAGATGTGTTAGAGCTTGCGAAGAAGTTCAAGGTACTTTTGCT TTGACTGTTATGGGTAGAGGTTTCGATGCTAGAATTTCACCAGCTGCTCCAGATTTTTTG TCCTCTGATTGTGTTTCTTGTGGTGCTTGTGTTCAAGCTTGTCCAACTGCTACTTTGGTTG AAAAGTCCGTTGAAAGAATTGGTACTCCTGAAAGAAAGGTTGTTACTACCTGTGCTTACT GTGGTGTTGSTTSTTCTTTTGAAGCTCATATGTTGGGTGATCAGTTGGTTAGAATGGTTC CTTGGAAAGGTGGTGCTGCAAATAGAGGTCATTCTTGTGTTAAGGGTAGATTCGCTTATG GTTACGCTACTCATCAAGACAGAATTTTGAAGCCAATGATCAGGGATAAGATTACCGATC CTTGGAGAGAAGTAAATTGGACTGAAGCTTTGGATTTCACTGCTACTAGATTGAGAGCTT TGAGAGATTCTCATGGTGCTGATGCTTTGGGTGTTATTACTTCTTCTAGATGCACTAACG AGGAAACCTATTTGGTTCAAAAATTGGCTAGAGCCGTTTTCGGTACAAACAACACTGATA CTTGTGCTAGAGTTTGTCATTCTCCAACAGGTTACGGTTTGAAGCAAACTTTTGGTACAT CTGCTGGTACTCAAGATTTCGATTCTGTTGAAGAAACCGATTTGGCCTTGGTTATTGGTG CTAATCCAACAGATGGTCATCCAGTTTTTGCCTCCAGATTGAGAAAAAGATTAAGAGCTG GTGCCAAGTTGATCGTTGTTGATCCAAGAAGAATTGACTTGTTGAACACTCCACATAGAG GTGAAGCTTGGCACTTGCAATTGAAACCAGGTACTAATGTTGCAGTTATGACTGCTATGG CTCATGTTATCGTTACCGAACAAATTTTCGACAAGAGATTCATCGGTGATAGATGCGATT GGGATGAATGGGCTGATTATGCTGAATTTGTTGCTAACCCAGAATATGCTCCAGAAGCT GTTGAATCTTTGACTGGTGTTCCAGCAGGTTTGTTGAGACAAGCTGCTAGAGCTTATGCT GCTGCTCCAAATGCTGCTATCTATTATGGTTTAGGTGTCACCGAACATTCTCAAGGTTCT ACTACTGTTATTGCCATTGCTAACTTGGCTATGATGACTGGTAATATTGGTAGACCAGGT GTTGGTGTTAATCCTTTAAGAGGTCAAAACAACGTCCAAGGTTCTTGTGATATGGGTTCT TTTCCACATGAATTCCCAGGTTACAGACACGTTTCTGATGATGCTACAAGAGGTTTGTTT GAAAGAACATGGGGTGTTACCTTATCTTCTGAACCAGGTTTGAGAATCCCAAACATGTTG GATGCTGCTGTTGAAGGTAGATTCAAAGCCTTGTATGTTCAGGGTGAAGATATCCTACAA TCTGATCCAGATACCAGACATGTTTCAGCTGGTTTGGCTGCTATGGATTTGGTTATAGTT CACGACTTGTTCTTGAACGAAACTGCTAATTACGCCCATGTTTTTTTGCCAGGTTCTACCT TTTTGGAAAAGGATGGTACTTTCACCAATGCCGAAAGAAGAATCAACAGAGTTAGAAGAG TTATGGCTCCAAAAGCAGGTTTTGCTGATTGGGAAGTTACTCAAATGTTGGCTAATGCTT TAGGTGCTGGTTGGCATTATACTCATCCATCTGAAATTATGGCTGAAATTGCTGCAACTA CTCCTGGTTTTGCCGCTGTTACTTACGAAATGTTAGATGCTAGAGGTTCTGTTCAATGGC CATGTAACGAAAAAGCACCTGAAGGTTCTCCAATCATGCACGTTGAAGGTTTTGTTAGAG GTAAGGGCAGATTCATTAGAACTGCTTATTTGCCAACTGACGAAAAAACCGGTCCAAGAT TTCCTTTGTTGTTGACCACTGGTAGAATCTTGTCTCAGTATAATGTTGGTGCTCAAACTAG AAGAACCGAAAACACTGTTTGGCATGGTGAGGATAGATTGGAAATTCATCCAACTGATGC TGAAACCAGAGGTATTAGAGATGGTGACTGGGTTAGATTGGCTTCTAGAGCCGGTGAAA CTACTTTAAGAGCTACTGTTACTGATAGAGTTTCTCCAGGTGTAGTTTACACTACTTTCCA TCATCCTGATACTCAAGCCAACGTTGTTACAACAGATACTTCTGATTGGGCTACTAATTGT CCAGAGTACAAAGTTACTGCTGTTCAAGTTGCTGCTTCTAATGGTCCATCTGATTGGCAA
CAAGATTACGCTGCTCAAGCTGCCGCTGCTAGAAGAATAGAAGCTGCAGAATGA SEQ ID NO:18 Nucleotide sequence RcFdsB (Formate dehydrogenase, beta subunit)
ATGAAGATTTGGTTGCCATGTGATGCTGCTGCTAAAGCTTGTGGTGCTGAAGCTGTTTTG GCTGCTTTGAGATTGGAAGCTGAAAAAAGAGGTGGCGCTTTGGATATTGCTAGAAATGG TTCTAGAGGTATGATCTGGTTGGAACCTTTGTTGGAAGTTGAAACTCCAGCTGGTAGAAT TGGTTTTGGTCCAATGACACCAGCTGATGTTCCAGCTTTGTTTGATGCTTTGGAATCTCA TCCAAAGGCCTTGGGTTTAGTTGAAGAAATTCCATTCTTCAAGAGACAGACCAGATTGAC TTTTGCTAGATGTGGTAGAATCGAGCCATTGTCTTTGGCTCAATTTGCTGCTGCAGAAGG TTGGGCTGGTTTGAGAAAAGCTTTGAAAATGACTCCAGCCGAAGTTGTTGAAGAAGTTTT GGCTTCTGGSTTTAAGAGGTAGAGGTGGTGCTGGTTTTCCAACTGGTATTAAGTGGCGTA CTGTTGCTGCAGCTCAAGCTGATCAAAAGTACATTGTCTGTAACGTTGATGAAGGTGACT CTGGTTCTTTTGCTGATAGAATGTTAATCGAAGGTGACCCATTCTGTTTGGTTGAAGGTA TGGCTATTGCTGGTCATGCTGTTGGSTGCTACTAGAGGTTATGTTTACATCAGATCAGAAT ACCCAGATGCCATTGCTGTTATGAGAGCTGCTATTGCTATGGCTAAACCATTTTTGGCTG AAGCCGGTTTTGAAATGGAAGTTAGAGTTGGTGCCGGTGCTTATGTTTGTGGTGAAGAA ACTTCTCTGTTGAACTCTTTGGAAGGTAAGAGAGGTACTGTTAGAGCTAAACCACCATTG CCAGCTTTGAAGGGTTTGTTTGGTAAACCTACTGTCGTGAACAACTTGTTGTCTTTAGCT GCTGTTCCATGGATTATTGCTCATGGTGCAAAAGCTTACGAATCCTTTGGTATGGATAGA TCCAGAGGTACTATTCCATTGCAAATTGGTGGTAATGTAAAGAGAGGTGGTTTGTTCGAA ACTGGTTTCGGTATTACTTTGGGTGAATTGGTCGAAGATATTTGTGGTGGTACTGCTTCT GGTAGACCAGTTAAGGCTGTTCAAGTTGGTGGTCCATTGGGTGCTTATCATCCAGTTTCT GATTACCATTTGCCATTCTGCTACGAACAATTCGCTGGTCAAGGTGGTCTAGTTGGTCAT GCAGGTTTGGTTGTTCATGATGATACAGCTGATATGTTGAAGTTGGCTAGATTCGCTATG GAATTCTGCGCTATTGAATCTTGTGGTACTTGTACCCCATGTAGAATAGGTGCAGTTAGA GGTGTTGAAGTCATTGATAGAATTGCTGCTGGTGATGCTTCAGCTATGCCATTATTGGAT GATTTGTGTCAGACTATGAAGTTGGGTTCTTTGTGTGCTTTAGGTGGTTTTACTCCATATC CAGTTCAATCCGCTATTAGACATTTTCCAGCAGATTTTCCATGTGCTAGAGAAGCTGCTG
AATGA SEQ ID NO:19
Nucleotide sequence RcFdsG (Formate dehydrogenase, gamma subunit)
ATGACTGATACTGCTAGATTGAGAGCTATTTTGGCTGCTCATAGAGGTAGAGAAGGTGCT TTGTTGCCAATATTGCATGATGTTCAAGCTGCCTTTGGTTTCATTCCAGAAGATGCTTAC GCTCCAATTGCTGCTGATTTGGGTTTGACTAGAGCTGAAGTTGCTGGTGTTGTTGGSTTTT TACCATGATTTTAGAAAAGCTCCAGCTGGTAGACACGTTATTAAGTTGTGTAGAGCCGAA GCTTGTCAAGCTATGGGTATGGATGCTGTTCAAGCAAGATTGGAATCTGCTTTGGGTTTA AGATTGGGTGATTCTTCTGAAGCTGTTACCTTGGAAGCTGTTTACTGTTTAGGTTTGTGT GCTTGTGCTCCAGCAGCTATGGTTGATGATAGATTGGTTGGTAGATTGGATGCTGCTGC
TGTTGCTGGTATAGTTGCTGAATTGGGTGCTTAA SEQ ID NO:20 Nucleotide sequence RcFdsD (Formate dehydrogenase, delta subunit)
ATGTCTGATGATAAGATTATCAGAATGGCCAATCAAATTGCTGCTTTCTTTGCTGTTCAAC CAGGTGATAGAGCTGGTCCAGTTGCTGCTCATATTTCTGAAAATTGGTCTGCTCCAATGA GAGCTGCTTTGTTGGCTCATGTTGCTGCACAATCTCCAGGTTTGGATCCATTGGTTATTG
CTGCTGCTCCACAAATTAGACCAGTTCCAGCTTAA SEQ ID NO:21 Nucleotide sequence RcFdhD (Sulfur carrier protein FdhD)
ATGTCTTTGCCAGCTGGTGCTGTTACTGTTCCATTACCAGGTGGTGCTAGAGCTGTTTTG GCTGAAGAAGTTCCAGTTGCTTTGGTTTTTGATGGTGTTACTCAAGCTGTTATGATGGCT TCTCCTGTTGATTTGGAAGATTTCTTGTTGGGTTTCGCTTTGACCGAAGGTATGATTGCT GATAGAGCTGAATTATTGAGACACGAAGTTGTCAGACAACCACAAGGTATTGAATTGAGA GGTTGGTTGGCTGCTCCAGCAGGTCAAAGATTTGCTGCTAGACGTAGAGCTATGGCTGG TCCAGTTGGTTGCGGTTTGTGTGGTTTGGATTCTTTGGCTGCTGTTTTAAGACCATTGCC AAGAGCACCAAGAGGTGGTGCCCCACCACCATTGGCTGATGGTGCTTTAGCTGCTTTGA GAGCTGGTCAATCTTTACAAGATGCTGTTAGATCTGTTCATGCTGCTGGTTTTTGGGATG GTGCTCAAATGAGAGCTTTAAGAGAAGATGTTGGTAGACATAACGCCTTGGATAAGTTG GCTGGTGCATTGGCTGGTCAAGGTATAGATGCAGCTGCTGGTGCCTTGGTTTTGACTTC TAGATTGTCTGTAGATTTGGTTCAAAAGGCTGCTATGATTGGTGCCAGAGTTTTGATTGC TCCATCTGCTCCAACTGCTTTGGCTGTTGCTGAAGCTCAAGCTGCAGGTTTGGCTTTAAT
TGCTAGAGGTCCAGATGGTCCAACATTATACACTGAAACTGAAGCCGAATGA SEQ ID NO:22 Nucleotide sequence DbMOCOS (Molybdenum cofactor sulfurase)
ATGACTGTTTCTGAGTTCTACCCGCTACCAGTTTCAGAGATAAGAGAGAAGTACTACCCC AATTTGGCCAATCAAACATATCTAGATCATGCTGGAACAACTGTGTACTCATCACTCACAT TAGATAAAATCCATGAGGTATTATCCAAAACGCTTCTTGCAAATCCGCACTCCCTATCTTC AGCATCACGCGATACTGCCAGTCTTGTCGAGGAAACCAGATACAAGATTTTAAGCATATT TCACGCCGATCCGGCGGAATATGACATTGTCTTTTCTCTAAATGCAACACACGCAATAAA GATTGCTGCCTCCCTTATCCAGGATGCCGCAGAAAGTTCATTCAACTATTATTACAACAT TAACTGTCACACTTCGTTGATTGGGTTGCGAACTTTAGCCGCCAAATACGCAACTTTTGA CGATATATCCAGTTTTGAGCCAGTTGAAGACAAAGATGGCAACCATCCAGCACTTAACTT TGTCTCGTGGACGGGGCAGTCGAATTTCAACGGCCAGAAGTTTCCTCTTGGCTGGTGTA AGGAGTTTAGAAGGCGATTGGATCATTGCTACACACTTTATGATGCCTCTGCATTGTCAA CATCCGATCCTCCCGACTTAAGTGATGCAAACAGCTCGCCAGACTTTGTGGTGATGTCA TTCTACAAGATTTTTGGGATGCCAGATATAGGAGCGCTCATTTTAAGACGTTCAACTGCA AAACAGCTCGTTGAAAAAAGAAGATACTTTGGTGGAGGCACAATTGATGCTCTCACGATT GAGGAGCCGTTTTGCCGCCGGAGTAAGCAATTGCATCAAAGCCTAGAAGACGGAACGA TTCCGGTACATGCAATTTTGGAATTGTCCGTGGCGATTGATTCGCATTATCAGATTTTCG GGTCTTTTAACAGCATTCGTTTGCACACGGATGAAATCAGAAAATACGCCATTTGCAAGT TGAAGCAGCTGAAATATGGCAACACTGGGCGCAGAATGCTGCAGATTTATGATTGGCCA GGTGCCAAGCACGGCCCTATTATTGCATTTTCGCTTTTATCGCCAGCAGGAGACCCGAT TGGGTATTATGGCTTTGGGAAGCTTGCCTCAGCTCGAAATATTTCGCTAAGAACGGGAA CATTGTGCAATATAGGCGGAATTCAGAAGTTTCTCGATCGCACAAATGAGGATATAAGGC AAGACTACGAAAAAGGGCACAAGTGCGGTGATATATTGGATATAATCGATGGAAAACCA ACCGAACTTGTAGTTAAAAGCTTGACGGTATATCCAATAAAATCGTGTCCAGGTTATCGA ATACCGGAAGGCCGAAAATGGAAATTGACCAAGCACGGTTTCGAGTTCGATCGCAGTTT TGTGCTTCTCGATTTGCTGACGCAAAAGCCTCTTTTGCTAAAGAATAATCCCAGAATGGC ACTTTTAGATTGTCGTGTGGATCCAGAAAAGCATATGCTCTATGTGAGAGATAAAAGAGG CGGTAATAAGCTTTGGGTCAGTACCAACATCCGGAGGTACAAAACCAAGCAGATGGGAG ATTTCATAGCGATTTCTGAAAGAAAGATGGTCAAATTTTTCAGTGATGTCATGAGCATAG GCTGCACTTTGGCTGGGTTTGTTACTGAGAAGCAGATGCAGAATAAAACTGCTTTCTTAC TAGTAAATGAGAGAAGTATGCGACAGGTTTCGAAAGATGATTCTCTCATTTCAAGGTTCA GAGCCAACATTGTTGTCGATTCGGCACATCCTTACATTGAGGACAAGTTGTCCGTTCTAA CAGACATGGACAGCGGAGTTGTGCTCAAGAAAAGATGCAAATGTGACAGATGCTACATG ATTACAGTGTCTGATAAGGGGTCACGTGATCCGTCGTTGCTTGTTGAGCTTTCAAAGGA GCGAAAACAGAAGGGAAAAGTCTATTTTGGGGTGAATATCGATGTTGAGAATGTTGGTTA
CAGGTACATGAGAGTTGGGGATCGCATTGTTGGAGAGGAGTGA SEQ ID NO:23 Nucleotide sequence DbXDH (Xanthine dehydrogenase/oxidase)
ATGGCACCAATAGCGGTCGAACCCAGCCCATATGATGGCACAGCAAACAATGATCTTAA AGATATTCAATTCACAGATAGCATAAGATTTTACCTCAACGATAAACTCCAGGTGGTGAA AAATCCAGATCCGGAGGAGCGACTCATTGACTACATCAGAAATGAGGCTGATCTCAGAG GCACAAAAGAAGCATGTTCGGAATCGGGATGTAATGCTTGCTCTGTTACCATTGCATCCA TTTCATACACAGACACGGATTATCCAGAGCGCCCACAGGTTCACTATCGGTCCGTAAATT CATGTGTTACTCCTTTGATACTTGCAGATGGCAAGCAAGTGATCACAGTTGAAGGTGTTG GCTCGTCTAAAAACCCACATCCAGTTCAGGAGAGAATTGCTAAATTCCACGGTTCACAGT GTGGATTTTGCACACCTGGTTTTGTGATGTCTTTGTATGCGTTGCTAAGGGAGAAAAATG GCCATGTGTCGGTTGCAGAAATCGATGAAGCATTGGAATCGAATTTGTGCAGATGCACG GGCTACATGCCAATCTATGATGCGGCATATTCGTTTGCGTACGATTCTGATAATTACAAC AGGGAGAAGATCAGACCTTTCCTCAAGAAGAAGGACACGAGTTTTGAGACTGGAAGTGA CTTGTATGGCGGTTCAGTGTGTGCGTTAGGAACCAAATGCTGCAGATACAAGTCTGGAA AAGAGAAAGCTGACGAAGAATGTGATAAGTCTGCATCCAATTCGGACATGGAGATCGAT ATGAACAAAATATTCACACCAAACGGTCTGCCATTGAAGCCATACAATCCGGCAGCAGAT TTGCCGTTCCCTCTTAAACTTTCCAGGATTAGCCCTAAGCCAATTTGCTACGGAAATGAG CGAAAGGTGTGGTTTCGCCCCGTGACTAAAGAACAATTTCTACAGATATACAGAATCTAT CCGGATGCGAAAATTGTCGCCGGAGCATCGGAGGTTCAGATCGAGGTGAAGTTCAAGG CTGCCAATTATAAGGTGAATATTTATGCTGGGGACGTCAAGGAACTCAAAGGATGGTCAT ACAAGAAGGGGAAGGGTTTAACAATTGGAGGTGATATTCCATTGATCGAGTTAGAATCTA TATGCGGTGATTTAGCAAAACGGTTGGGTAGAACTGCAGCTGGACAGACATACAACGCA ATTGAGGAGCAGTTGAAGGTCTTTGCTTCTAAAGCTGTTCGTAATGTGGCCACTCCGGC AGGTAATATCGTGACAGCATCGCCAATTGCGGATCTAAATCCAATATTTGTGGCTTGTGG TGCAATCATAACGGCGGAAAAACTGACTGAGGATGGTAAACTAGAAAAGACGCACATTG ATATGCGTGACAACTTCTTTACTGGGTACAGGCGGCACAAGTTACCTACATCATCGTTGA TTACCGAGATCTTCATTCCAGATACTGCAGATAACGAATACATCCACTGCTATAAGCAGT GTAAGCGGAAAGATGACGATATCTCCATTGTCACTGCCTGCTTGAGAATGGAGCTAGAT GACGAAGGAAATGTGCTCGATTCAACATTAGTCTACGGTGGTATGGCTCCTATAACCAA GAACTCACCAAAGGCCGAAAAAACTATCAAGGGGAAGAATATTTACAACTCATCATTTAA CGAGGAATGCTGTAAGTGCCTTAGTGAAGATGATTATAAGATGCCGTACGGCGTCCCTG GCGGTGCTGCCTCGTATAGAAGATCCCTAACATTGTCCTTCTTCTACAAATTTTGGCAGT ACGTTTTGGCCACAGCGCCAATTCCCAAGGCTAACGTTGCCACAATCCAGTGTAGGGAT GCCATTCTGGATGTTGATTCGTTGAGTGAAGTGACAAGGGTCCAAAAGCATGGCTACAG AGAAATGAACACTCCAGGGCACAAGACTGGAATCATTGGCAAGCCTATCGTTCATGTGA ATGCAATAAAACAGGCAACAGGCGAAGCTCAGTATACCAATGATATTCCACCATTGCACC GCGAACTATTTGGTGTGCAGGTGATGTCTGAAAAGGCGCATGCTAAGATTCTCTCCGTG GACTGGAGTGAGGCCTTGGAAGTGGAATCAGTTGTTGGATACGTGGACATTAACGACTT GCCTAACAAGGAGGCCAATCTTTGGGGCAATTTACCCTTTGGAAAGGAACCTTTCTTTGC AGATGGTGAGGTGTTCTTTGTTGGCCAGGCTATTGGTGTTATCCTTGCCTCAAGTAAAGA GAGGGCCTATGAGGCATCTAGAAAAGTTAGAGTTGTGTACGATGAGCTTCCCCGAATCA TCTCCGTTGAGGACGGTGTGCGACAGAAGTCGTTCTTTCCAGACAGGAGAGAAGTCAAG CTTGGGGATTGGGAATCTGCATTCAAGAACAGTAAATATTATCTTGAGAACACTGCGAGA TTATCGGCCCAGGAGCATTTCTACTTCGAGGTTCAGAACTGCCTCGTTATACCACAGGAA GGTGGTGAGCTCAAGGTGTATTCTTCCACACAAAATCCTACCGAGACCCAGTTGTGTGC CGCACAGGTCACGGGCGTTCCTGCAAACAGAGTCATATGCCGTGTTAAAAGACTCGGAG GTGGATTTGGTGGTAAGGAGACCAGATCTATCCAACTTTCGAGTTTGGCGGCAGTTGCA GCCAGAAAGTTTAATCGTCCAGTCAGGTTGGAACTCAACAGAAGTGAAGACATGAAAAC TTCCGGTGAAAGACATCCATTTTTGGTAAAGTACCGGGCCTCGTTGGACGAAGATTTAAA GTTCACTGGATTGGACATGGTTCTTTATGCGAATGCTGGTTGGTCGATGGATTTGACTCG TGGTGTCATTGACAGATCCGTTCTCCATGCATCAAATGCTTATTACATTCCTAATGCTCG GGTTTGTGGCATACCAGTGAAGACCAACATTGCCTCTAACACGGCATATCGGACGTTTG GTGCCCAGGCTGGATTCTATGCGATTGAGTCTGTTGTGACGGAGTTTGCCGAGAAGCTT GGTGTGGATCCGGAGGAAATAAGAAGAAGGAATTACTTGAAGCCAAATTGTGGTGAGGT GTTCCCATACAAGCAGGTAGTTGGCGAAGACATCACGATTTCCAACGTGGTGGATGAAA ACTTGAAGGAGTGCAACTACAAGAAGATGAAGCAGGAAATCAATGAGTTCAACAAGCATT CAAAATGGATAAAGCGTGGTATAGCACAAATTCCCGCAGTGTTTGGTGTTTCGTTCGGTG TTCTTTTCTTGAATCAGGCTGGTGCTTTAGTCCACATTTATAACGATGGCTOCTGCTTGAT TTCAACCGGTGGTGTTGAGATTGGCCAGGGTATTAGTACCGTGATGAGGATGATTGCAG CTGAAGAATTGGGCGTTCCGTTTGACAAGATATTCTTGAGTGAGACTTCTACCGAATGTG TTCCTAACACATCATCTACTGCAGCTTCTTCCGGATCCGATTTGAACGGAATGGCGTTAA AGGATGCATGCATGAAGCTTAACAAGCGTTTAAAGCCGGTGAAGGATGCAATCACGAAG GAAAAAGGTGACAAATGGACGTGGGAGGAGTTGATCACAAAGGCCTACTTGGACAGAGT CTCATTGAGTGCAACTGGATTCTACAAGACGCCAGAAATCGGTTTTGAGTGGGGAGATG AGAATCCTAAACCCGCCTTTTTCTATCACACTCAGGGATCCGCTGTATCTGTAGTCGAAG TAGATACGTTAACGGGTGATTGGAGTTGCTTGGAGTCACACATCAAGATGGACTGTGGA CGGCCGTTAAATAAAGCAATCATATACGGTCAAATTGAAGGAGCTTTCATCCAGGGAATG GGCTACTTTACCATGGAACAGTCATTATGGTTAAGCAGGACAGGAGGGCTGGCCACAAC CGGTCCTGGTGCCTACAAAATTCCAGGGTTCAGGGATACTCCACAACGATTTGTGCTAT CAATGTATAAGGGAAGCGATTTCAGGCATTTGAGAACCATTCATTCATCTAAGGGAGTTG GTGAACCACCATTTTTCCTTGGTGCAAGTGTCCACTTTGCCCTCAGAGATGCAATTGGAC ATGCAAGAAGACAGAATGGCATTGAATCGGGAAGTCAGGGCTTGAGATTCCGGGTTCCT TTGACTACTGAGAGAATCAGAGTTGATTGTGGTGATAAGCTTGCCAAACAGTCGTTTGTG
GCAGCCAAAGAGGGCGAGGAGGAATTCTTTATCGAGGGATGA SEQID NO:24 Nucleotide sequence DbUro1 (Uric acid oxidase)
ATGGCTTACCTACAGGATTGTACATACGGTAAGAACAATGTTAGATTTTTGAAGGTCAAG AGGGACCCTATTAACCCTAAAATCCATCAGGTCATGGAGGCTAGTGTTAGGGTCATGCT TACGGGTGCTTTTGATGTCTCCTACACAAAGGCAGATAACAGCGTTATTATTCCAACCGA TACCATCAAAAATACCATTTTGGTTGAAGCCAAGCAAACAGATGTCTTCCCAATTGAGAG ATTTGCCGCTCACCTTGTGAAGCATTTCTTTGGTAAGTACAGTTGGATTGCAGGTATCAC TGTTCATATTGAGCAAGCCAAATGGTCTAAGTATTCTGTCGATGGAAAGCTCCAGCCACA TTCTTTTGTCAAGAATGGTGATGAAGTCAGGGTTTGTGAATTGGTTTCTAAGAAGAATGG TGACTTTGTTCTTACAGGCGGCGTGCAGGGGTTGACCGTTTTGAAGTCCTCTGGCTCTA TGTTCCATGGCTACAATGTTTGTGATTACACCACTTTGAAGCCTGTTAATGAGAGAATCTT GTCAACAGATGTAGACTGCAAGTACAAGTTTGACAGTGCCAAGATTGGTTCTGTCGATAA CATATTCACTTTAGCCGATTCTGGACTGTTTGATAAGGTCTTCCAATCTGCTCTTAAAATC ACTTTGGACAGATTTGCTCTCGAGAACTCGGCCTCTGTTCAAGCTACCATGTATAACATG GGTACCGATATTGTCAACGCTAACCCATATGTTTACAATGTCTCTTATGCCTTGCCAAAC AAGCACTACATTCTGTTTGACTTCTCCTGGAAGGGCTTGAAGAACGAAAACGAGATGTTC TACCCATCACCACATCCTAACGGTTTGATAAAGTGTACTGTTGGAAGGGAGCCTATTGCA
AAGTTATAA SEQ ID NO:25 Nucleotide sequence DbUro2 (5-hydroxyisourate hydrolase)
ATGTCTTCAAGGCCACCAATTACTTGCCATATTCTAGACACCACGTGTGGCAAACCCGCC GAAAATGTTAAGTGTGAGATAAGCTATATACCCTCAAATGGTATTACATCACCATCAGAA GTGAAACCATTTGGTTATGCGTATACGAATCAGGATGGAAGAATTGGATCTTGGAATGCT GCAAATAGTACAGAGACTTTCATTAATGCCGAAAATAATCAATGGACTAAATTAGTTAGTG GAACTTATCGAATTAGGTATCACACCAAGGACTACTTTTTGAAAAGAGATGGAACGACAT TTTTCCCCTTTATAGATATATGGTTTGAAGTCCCTGCTATCCCAGAGAAGCATTATCATGT
ACCATTGCTATTGAGCAATTATGGTTATTCTACATACAGAGGTTCATGA SEQ ID NO:26 Nucleotide sequence DbUro3 (2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase)
ATGAAATTACCAGATCCTCAAGCATTATCTCAAATTCTGAGATCAGAGCAGGTAACAGTT ATAGATACGCTATTTGAACATAACGATAAATTCGCGGACTTCATAATTAAAAAGGTCCTTA GCCATAATGAACGATATGGTTCATATAGAGAATTTATAAAGGCGGTGAGAATACAACTCA TTCAACTTGCAGATAATTATGAAAAAAGTATGACAGGAGACCTAGGTGATATGGTAAGAT CGGTTATTAGTGCACATCCTAGGCTAGGAATACAGCAGGCGTCGGCTCTTTCTGTCTCTT CTGCAAGAGAGCAAAAATCGCTACAATCAGGAAAGCCGGAGCTTGAGAGACAATTATTA GCATTAAATCAAGAATACGAACATTGCTTCCCCGGCTTGAGGTTCGTGGTATTTGTAAAT GGGCGGAGTCGCCAAGAGATCACCAAAATCATGCGGAAACGAATCACTCGTGATGACTA TAATCAGGAGGTGCGTGATGCATTCAGTGCAATGTGCGATATTGCACTGGATCGGATAA
AAAAGGAGAACAGCAAGCTGTAA SEQID NO:27 Sequence sequence EcMocA (Molybdenum cofactor cytidylyltransferase)
ATGTCCGCCATTGATTGCATTATTACTGCTGCTGGTTTGTCCTCTAGAATGGGTCAATGG AAAATGATGTTGCCATGGGAACAAGGTACTATCTTGGATACCTCTATTAAGAACGCCTTG CAATTCTGCTCCAGAATTATCTTGGTTACTGGTTACAGAGGTAACGAATTGCACGAAAGA TACGCCAATCAATCCAACATTACCATCATTCACAATCCAGATTACGCCCAAGGTTTGTTG ACTTCTGTTAAGGCTGCTGTTCCAGCTGTTCAAACTGAACATTGCTTTTTGACTCATGGT GATATGCCAACTTTGACCATCGATATCTTCAGAAAGATCTGGTCCTTGAGAAACGATGGT GCTATTTTGCCATTGCATAATGGTATTCCAGGTCACCCAATTTTGGTTTCTAAACCATGTT TGATGCAGGCCATTCAAAGACCAAACGTTACCAATATGAGACAGGCTTTGTTGATGGGT GATCACTACTCTGTTGAAATTGAAAACGCCGAAATCATCTTGGACATTGATACTCCAGAT
GACTTCATTACCGCCAAAGAAAGATACACCGAAATCTAA SEQ ID NO:28 Amino acid sequence HPODL_02673 (GTP 3',8-cyclase)
MISSLLLRRLHSTGTSQHLPRLERLRRMPVRHLKEFLTDTYGRKHDYLRISITERCNLRCVYC MPEQGVDLSPPEHMLTTEEIVKLATLFAQHGVRKVRLTGGEPTVRKDIVELVAKLNQITGIEEI CMTSNGLALHRKLPDLFKNGLTSLNLSLDTLINGKFLLITRRNGLSAVMRSLRTALELDIPKVKI NVVVMKNLNEDEILDFVELSKNDKVEVRFIEYMPFDGNKWSTNKLVSYEDILSNIKVRHPNIQ RLPHKHGDTAKVYQIPGFKGKVGFITSMTSDFCSTCTRLRITSDGNLKVCLFDNTEVSLRDML
RAGYSDDKLMQRIGEAVKNKKEKHAGIDVLGDQPNRPMILIGG SEQ ID NO:29 Amino acid sequence HPODL_02674 (Cyclic pyranopterin monophosphate synthase)
MVAIHEKEDTHRCAIAEGSIKFSNPESMKLLLSESNKKGDVISIARIAGIIAVKKTAELIPLCHPIS ITGIKVDLIHDEKENCIKVNCEVHCNGKTGVEMEALTGATISLLTVYDMCKAVDKMMTISDCR
VVKKSGGKSGDIDLSTIFK SEQ ID NO:30 Amino acid sequence HPODL_00195 (Molybdopterin synthase catalytic subunit)
MSIFVDITDKPLDSAEVLNYVRHPQAGAIVYFGGTTRNTFEGKEVVSLAYEAHPRLAIKTLESI AHEAKAKFQSVHKIAIVHRTGVVPVATESVMIAVSSTHRKEGWLCGEWVLEKVKERAEIWKI EKYADGDSVYKENDVSNVLSRT
SEQ ID NO:31 Amino acid sequence HPODL_01640 (Molybdopterin synthase sulfur carrier subunit)
MVAVAIEYFGPAKTYTNGVAHERVELTEPATLNTLIQHVGRSYSSEFAQYIVSSCGVVVNEDY
VETERIGIEFFGKNIALQSGDVVGIIPPVSSG SEQ ID NO:32 Amino acid sequence HPODL_03424 (Molybdopterin adenylyltransferase)
MTVGILVVSESVSRGLSTDKVVDALKQHLDGFELKAHKVVPDKKEDIQAAVVDWVKQDFKLI LTAGGTGFTKTDITPEAIEPLLDKKAPGLVHAMLSFSLQITPFAMLARPVAGVRGESLIITLPGS PKGATENFQAIKGVIGHALSQLGIESSRLLHKESGSGHHHHHHHHHHHGHLAKHELVDSVVA RHRVSPYPTISVDEAYSRIRENTPAPEVIELSILDPRLVGSVVAENVTAQMDVPNFRASIVDGY AMISSDGPGVYPVVSVSHASKNDQKELVAGQIARITTGAPLPEGADAVVMVEETRLVETTED GSEEKLVEILAKNVKTGENIRAIGSDTRKDSLVLAKGSRISPGGGEIGALASVGVNKIKVYRKP VIGLLSTGNELQDVQTEKLLHYGEIYDSNRPTLVSIVQNCGYELVDLGIASDTKESLIKIIKDAV DVKKIDCLITTGGVSMGELDLLKPTIENELHGTIHFGRVRMKPGKPTTFATIGKSTVVFALPGN PASSSVCYHLFVLPCLLKWQGLEPSVGOKLPTEPIVKVKLAEDLKLDPQRPEYQRVSICQSD
MALVANSTGFQRSSNIGSFKKANGLVCLPAASDFGKSVIEAGTVVDAILIDQIYV SEQ ID NO:33 Amino acid sequence HPODL_00948 (Molybdopterin-synthase adenylyltransferase)
MSLSLNEYLRYGRGQLIVPEFGLQGQISLKNSRVLVVGAGGLGCPALQYLVGAGFGTVGIVDH DTVDISNLHRQILHTSETVEMLKCESAKLQLAKLNPLVQINTHPVALSPDNSFGIFEQYDIILDC TDTPATRYLINDTAVLLGLTVISGSGLKTEGQLSILNFNNTGPCYRCFYPTPPPPSSVTACSD GGVLGPVIGIMGVMMALEAIKVVSGYYLREDVEFQPFLSLYSGYGPLQSLRTFKMRRRSPKC RVCNAGTREITRHVIETELDYAVWCGKMDYNVLEKDERVSVEQLSAQRAPYLVDVRAKEQY SIVHLPNSINIPLNALKHMDTLDVPKETMIYVICRFGNDSQLAVKHLKSIGYENCVDVIGGLTQ
WSRGQIDPNFPIY SEQ ID NO:34 Amino acid sequence HPODL_02128 (Cysteine desulfurase)
MYRFRIGAGVRRFSVSALRRTSSSGVKQAMPTSGAQPPQGSSISLKSATRDPSEYGTRPIYL DMQATTPTDPRVLDKMLEFYTGLYGNPHSSTHAYGWETDKAVEEARANVAAVIGADPKEIIF TSGATECNNMALKGVARFYGKSKKHIITTQTEHKCVLDSCRHLQDEGFEVTYLPVNSDGLID LELLEKSIRKDTCLVSVMAVNNEIGVIQPLEDIGRICRSHKVFFHTDAAQAYGKIPIDVNKCNID LMSISSHKIYGPMGVGATYVRRRPRVRLDPIINGGGQERGLRSGTLAPPLVCGFGEAARLMV QEYDADQAHIKALSTKLMDALLSMEHTQLNGSRIHRYPGCVNVSFAYIEGESLLMALKDIALS SGSACTSASLEPSYVLHALGADDALAHSSIRFGIGRFTTEAEVDYVIQALTERVKFLRELSPL
WEMVNEGIDLNSIEWAGH SEQ ID NO:35 Amino acid sequence CrMOT1 (molybdate transporter)
MALQNAWONTKERARETWAQLTWSEVSGSLGDLGTFLPLLIGLVQKVHLDLGTTLTITGLYN ISGWOQFRIPMCVQPMKTIAAVALAGGAAGLDLPQLLHAGLFVAGCVGLLGASQAIDLFNWLV PPPVIRGVQLAVGVKLAMKGVDMALRLHGGPSSGWRPWLGTEGLVVGAVALAAMIATTLPP RAARRGTLEAADEGGLGPRPTDTAFEPLLRRLPACCGGGDRAPQVEGAAVSAERAGLLAH AEGGERSGNLDDGTEAGVGAAAGGGGCGGGGGGGRIPSALIAVVVGLAMAVLHRPGLVW ELRLGPTLPRLLRPSWPDFKTGALRGGLPQLPLTTLNSVIAVTQLANALFGDKPEAERRRWR PSAVALSVALLNGAGVWLGAMPCCHGAGGLAAQYKFGARTGHAPILLGCIKAALGLLFGGSL VVLLEAFPQPLLGALLTVSGIELASVVRHTRSPRGYTFALLTAVAILALDNTGTGFLVGLVGVA
AVAAYEGAVAAAAARWPRVFARGGRA SEQ ID NO:36 Amino acid sequence HPODL_02384 (nitrate reductase NiaD)
MDSVVTEVTYGLEIKKIKEITELPFPVRQDSPLSEVLPTDLKTKDNFVARDPDLLRLTGSHPFN SEPPLAKLYDSGFLTPVSLHFVRNHGPVPYVPDENILDWEVSIEGMVETPYKIKLSDIMDQFDI YTTPVTMVCAGNRRKEQNMVKKGTGFNWGAAGTSTSLWTGCMLGDVIGKARPSKRARYI WMEGADNPANGAYGTCVRLSWAMDPERCIMMAYKQNGEWLHPDHGKPLRVVIPGVIGGR SVKWLRKLVVSDRPSENWYHYFDNRVLPTMVTPEMAKSDDRWWKDERYAIYDLNLQTIICK PENQQVIKISDDEYEIAGFGYNGGGIRIGRIEISLDKGKTWKLTEIDYPEDRYREAGYFRLFGG LVNVCDRMSCLCWCFWKLKVPLSELATSKDILVRGMDERMVVQPRTMYWNVTSMLNNWW YRVAIIREGDALRFEHPVVANKPGGWMDRVKAEGGDILDNNWGEVDDTVKQAERKPRVDE DIEMMCNPEKMDVIIKYSEFEAHKDSETEPWFAVKGHVFDGSSYLEDHPGGAQSILMVSGE DATDDFIAIHSSYAKKLLPPMHLGRLEEVSSVTKVKSVEENVKREVLLDPRKWHKITLAEKEII SSDSRIFKFDLEHPEQLIGLPTGKHLFLRLKDSSGKYVMRAYTPKSSNSLRGRLEILIKVYFPN REYPNGGIMTNLIENLQVGNQIEVKGPVGEFEYVKCGHCSFNNKPYQMKHFVMISGGSGITP TYQVLQAIFSDPEDTTSVQLFFGNKKVDDILLREELDCLQIKHPEQFKVDYSLSDLHHLPENW
SGLKGRLTFNILDSYVQGKNMGEYMLLVCGPPGMNGVVENWCKARNLDKQYVVYF SEQ ID NO:37 Amino acid sequence HPODL_02386 (nitrite reductase NirD)
MTCSVPPLPEDITPPAAKKKLVIVGLGMVGLSFLEKLLLNDSKLNEYSILVYGEEPYLAYNRVG LTEYFQHREFKNLLLSPEEFYQLRGEKWNYAIDEKVIDIDRQARTITTNKGNRASYDVLVLCT GSTAILPTDLLPPPTRKSYREMGCFVYRTIDDLYSMIDYCQGAKKQRAIVVGGGLLGLEAAKA LYDMESFEDITIVHRSHWLLSQQMDQKGGSLLTSKVKELGITCRTGTTVSELLFDEDQNLTG VKYDNGEIEECSLLCYTIGIKPRDELTSCGLNAGSRGGFKVNNMLQTSDENIYAIGECASWN NMTFGLIAPGYEMADILAFNLTQGKLHQPKEFSEPSMGTRLKLMGVDVASFGDFFADRNGP KWLPRGYEKEVRGLVFEDPIDGTYTKLLFTKDGKYMLGGILVGDTSNYTKFSAMIRKPLPKSP SELLIGKAGEDDMEKLSDETQICSCHNITKGKLVEAVKNGCSSLADLKKCTKAGTACGGCEP TVKVIFETEVKKLGGKVSNNLCVHFDYSRADLFSLIMVKNFRSFRRVMEELGNNPSSSGCEI CKPTIGSILSTLYNRHLLKKEVHGLQDTNDRYLGNIQRNGTFSVVPRMSAGEVTPEKLVSIGQ IAKKYGVYTKITGAQRLDLFGVKKSDLPKIWKDLNEAGFESGQAYGKSLRNVKSCVGSTWCR YGIGDSVGLAVRLEERYKGIRSPHKMKGGVSGCVRDCAEFHSKDFGLCAVKDGFNIYVGGN GGMKPAHAQLLATNVRPDEVIPILDRYLMFYITTADRLQRTARWLENLDGGIEYLKDVIIRDKL GICKDLEAQMRQLVAGYYDEWAKAVSEEKDNPIFKQFVNTSENQDTVEIVKERGQPRPAMW PEKAANQKFNEIKWSSVSWQEVCESSDLPLAEAGSSATVLVGDTQIALFRTSENELYCSQN MCGHKRAFVLNQGLLSEDGDKNCYISCPMHKRNFYLKSGACKNDEALSIATFEVKEENGKV
YAKLPPTTELDEVLGTSKWKVTSQETEAKQVRKITTEKNLVDKAISFDW SEQ ID NO:38 Amino acid sequence HPODL_02387 (high affinity nitrate transporter NrtB)
MRLSTLWEPPTVNPRNLKATSIPIFNLWNVYGRNFFFAWFGFFVCFLSWFAFPPLLHGMLKK DLKLTAVDISNNNICGLTGTLLGRFILGPLNDKYGPRITLVGVLVAGAIPTAFVPLVTNVAGLHA IRFFISFLGSSFICCSQFCAVFFDNNIIGTANAVSAGWGNAGGGVAFFVMPAISNALEKRGYS LHHSWSYSFVIGPFLILMITAIVIFVFGSDCPRGRWSLRGDILGINMDNMLVKSVSVTRHFSKD GELTSVCVEPVNAIDEVVVEPNQDQEIFEVADIINEDEIEDPTLKDVVKICFSPRTMLVGLCYM CSFGTELAVESIISNLFGQKMTKWSTSKAGAWGSMLGLLNVVTRPAGGIISDFLYQRFKTTKA KKFWMIFTGLMQGIFLIWVGLVPELSIAGLIVSVSFLCLWFEMGNGANYACVPVVNRHHSGIV
SGVTGAMGNLGGILFSLVFRYTIANGVNNYFKAFWIIGIVCTVVNLACVLIPIREERPRKAEI SEQ ID NO:39 Amino acid sequence EcMobA (Molybdenum cofactor guanylyltransferase)
MNLMTTITGVVLAGGKARRMGGVDKGLLELNGKPLWQHVADALMTQLSHVVVNANRHQEI YQASGLKVIEDSLADYPGPLAGMLSVMQQEAGEWFLFCPCDTPYIPPDLAARLNHQRKDAP VVWVHDGERDHPTIALVNRAIEPLLLEYLQAGERRVMVFMRLAGGHAVDFSDHKDAFVNVN
TPEELARWQEKR SEQ ID NO:40 Amino acid sequence EcMobB (Molybdopterin-guanine dinucleotide biosynthesis adapter protein)
MAGKTMIPLLAFAAWSGTGKTTLLKKLIPALCARGIRPGLIKHTHHDMDVDKPGKDSYELRKA GAAQTIVASQQRWALMTETPDEEELDLQFLASRMDTSKLDLILVEGFKHEEIAKIVLFRDGAG
HRPEELVIDRHVIAVASDVPLNLDVALLDINDVEGLADFVVEWMQKQNG SEQ ID NO:41 Amino acid sequence KoNasA (assimilatory nitrate reductase, subunit A)
MTETRTTCPYCGVGCGVIASRAPHGQVSVRGDEQHPANFGRLCVKGAALGETVGLEGRML FPEVDGERATWPQALAAAGSRLREIIDRHGPQAVAFYASGQLLTEDYYAANKLMKGFIGAAN IDTNSRLCMSSAVTGYKRALGADVVPCSYEDVENSDLVVLVGSNAAWAHPVLYQRLAQAKR DNPQMRVVVIDPRRTATCDIADRHLALAPGSDGGLFVGLLNAIAASGAISDDFNDAQRALTIA QDWDLDKVAQFCGLPRQQIADFYREFIAAPRAITLY TMGINQSASGSDKCNAIINVHLACGKY GRPGCGPFSLTGQPNAMGGREVGGLATMLAAHMNFEPDDLRRLARFWGSERLAQTPGLT GVELFAAIGRGEVKAVWIMGTNPVVSLPDSHAVSEALARCPLVIISDVVADTDTGRFAHIRFP ALAWGEKSGTVTNSERRISRQRAFMPPPGEARADWWIVARVAEALGFGSAFAWQHPHEVF SEHAALSGYENDGQRAFDIGGLADLSREAWDALEPVRWPVSRSEAAWSVHKGWHRDGKL RMVPVAPQPTRATTDAFYPLILNSGRIRDQWHTMTRTGAVPRLMQHINEPVVEVAPADAQR YHLLEGELARVRSPKGVMVAKVTIGDGQRPGSLFVPMHWNNQFARQGRVNNLLAAVTDPH SGQPESKQTAVAIATWLPAWKGELFSRQPVPLPASLHWRRRAAQGIIHLSLAGDTRSRDWL VEWCQRQGWQMQVAEGGKVWNLLAWRAGELMLGWWSDASEPAIDADWIHAAFRVPPQN AARRHALLSGRKGGVEMPRGRIICSCFSVGERAIGEAIAGGCRTPGALGGKLKCGTNCGSCI
PELKALLAAKLAQA SEQ ID NO:42 Amino acid sequence KoNasC (assimilatory nitrate reductase, subunit C)
MTRQLVIIGNGMAATPGEALVERDARRFSITIIGDEPRQAYNRIQLSPVLGAEKTAGATRLLPA EWYSQHNVTVRAGETVTAVDMAARTLQTTAGELGWDELVFATGSLPFLPPLPGITLPHVFAF RTLADVEGILAIDGPAVVIGGGVLGVEAAAALRRHGDSVTLLHRGSWLMEQQTDAFVGEQL QMLLAERGIGCVMESRIAAIDEHQVLLEDGRAFAASRVVLATGVQPNKRLAERSGLACGRGI LVDRRLATAQPGVSALGECCEIDGQTWGLVAPCLRQAEVLADRLCAIPGEDFRWQDSGTRL KVTDIELFSAGELRADEQDDVYTSWDPLDRHYRRLLLRDGKLRGVLLLGDCSSAAPLTALLG
RHGPRRQSGFSTHLQRCSRALWDKLR SEQ ID NO:43 Amino acid sequence RsBisC (biotin sulfoxide reductase)
MGVEPFAHDPAPSELIHSVPACGSPERRVMRPMVREGWLADRQHSDRRGRGRERFLPVS WDAALDLVAGEIRRVSADHGNAAIFAGSYGWTSCGRFHHASTLLKRMLNLVGGFTGHVDTY SIAAGPVILRHTLGDDRACGGQANTLDSIAEHSQTLVVFGAMSPRTAQSEAGGIGAHHLETY LRRIVERGVRVILVSPLKDDLPDWVAAEWWPIRPNTDTALMLGLAGEIVRSGRQDSDFLARC TSGSELYLAYLRGEGDGRPKDAEWASTITGLPAEAIRALAGDLPRTRSMLTVSWSLQRAHH GEQPFWAALGLAAVIGQIGRPGGGVGYGYGSLGGVGAPFTIGKSPAMSQLSKPINSFIPVAR ISDMLLNPGGPYSYEGEDRRYPDIRLVYWSGGNPFHHHQDLNRLSEAWTRPETIIVQDPMF TATAKRADIVLPASTSIERNDLAGNKRSDFILAMGQAIAPLGEARSDFDIFNALSGKLGVAAAF NEGRDEMGWIRHLYEESRNHAQRHHHFEMPDFETFWAQGHAPCPVQRDHTYLAAFREDP GAHPLDTESGLIVLGSATLARLGYADCGPHPAWIEPAEWLGKAQAGELHLISHQPKGRLHSQ LETAEASLAGKREGRDEVMLHPDDASVRGIADGQTVRLWNARGACLATAQVTDSVAAGVAI LPTGAWFTPAEAEGPELSGNPNVLTLDIGSSAFGQGCSAHTCLVRIEAHAGDAGDAVRIYDA
HLAAILPT SEQ ID NO:44 Amino acid sequence RcFdsA (Formate dehydrogenase, alpha subunit)
MKDLIIPPLDWTQDMGTPKREGAPVHLTIDGVEVTVPAGTSVLRAAAEAGISIPKLCATDNVE PVGSCRLCMVEIEGMRGTPTSCTTPVAPGMRVHTQTPQLQKLRRGVMELYISDHPLDCLTC AANGDCELQDMAGAVGLREVRYQAKDTHFARRDATGPNPRYIPKDNSNPYFSYDPAKCIVC MRCVRACEEVQGTFALTVMGRGFDARISPAAPDFLSSDCVSCGACVQACPTATLVEKSVER IGTPERKVVTTCAYCGVGCSFEAHMLGDQLVRMVPWKGGAANRGHSCVKGRFAYGYATH QDRILKPMIRDKITDPWREVNWTEALDFTATRLRALRDSHGADALGVITSSRCTNEETYLVQK LARAVFGTNNTDTCARVCHSPTGYGLKQTFGTSAGTQDFDSVEETDLALVIGANPTDGHPV FASRLRKRLRAGAKLIVVDPRRIDLLNTPHRGEAWHLQLKPGTNVAVMTAMAHVIVTEQIFDK RFIGDRCDWDEWADYAEFVANPEYAPEAVESLTGVPAGLLRQAARAYAAAPNAAIYYGLGV TEHSQGSTTVIAIANLAMMTGNIGRPGVGVNPLRGQNNVQGSCDMGSFPHEFPGYRHVSD DATRGLFERTWGVTLSSEPGLRIPNMLDAAVEGRFKALYVQGEDILQSDPDTRHVSAGLAA MDLVIVHDLFLNETANYAHVFLPGSTFLEKDGTFTNAERRINRVRRVMAPKAGFADWEVTQ MLANALGAGWHYTHPSEIMAEIAATTPGFAAVTYEMLDARGSVQWPCNEKAPEGSPIMHVE GFVRGKGRFIRTAYLPTDEKTGPRFPLLLTTGRILSQYNVGAQTRRTENTVWHGEDRLEIHP TDAETRGIRDGDWVRLASRAGETTLRATVTDRVSPGVVYTTFHHPDTQANVVTTDTSDWAT
NCPEYKVTAVQVAASNGPSDWQQDYAAQAAAARRIEAAE SEQ ID NO:45 Amino acid sequence RcFdsB (Formate dehydrogenase, beta subunit)
MKIWLPCDAAAKACGAEAVLAALRLEAEKRGGALDIARNGSRGMIWLEPLLEVETPAGRIGF GPMTPADVPALFDALESHPKALGLVEEIPFFKRQTRLTFARCGRIEPLSLAQFAAAEGWAGL RKALKMTPAEVVEEVLASGLRGRGGAGFPTGIKWRTVAAAQADQKYIVCNVDEGDSGSFAD RMLIEGDPFCLVEGMAIAGHAVGATRGYVYIRSEYPDAIAVMRAAIAMAKPFLAEAGFEMEV RVGAGAYVCGEETSLLNSLEGKRGTVRAKPPLPALKGLFGKPTVVNNLLSLAAVPWIIAHGA KAYESFGMDRSRGTIPLQIGGNVKRGGLFETGFGITLGELVEDICGGTASGRPVKAVQVGGP LGAYHPVSDYHLPFCYEQFAGQGGLVGHAGLVVHDDTADMLKLARFAMEFCAIESCGTCTP CRIGAVRGVEVIDRIAAGDASAMPLLDDLCQTMKLGSLCALGGFTPYPVQSAIRHFPADFPC
AREAAE SEQ ID NO:46 Amino acid sequence RcFdsG (Formate dehydrogenase, gamma subunit)
MTDTARLRAILAAHRGREGALLPILHDVQAAFGFIPEDAYAPIAADLGLTRAEVAGVVGFYHD FRKAPAGRHVIKLCRAEACQAMGMDAVQARLESALGLRLGDSSEAVTLEAVYCLGLCACAP
AAMVDDRLVGRLDAAAVAGIVAELGA SEQ ID NO:47 Amino acid sequence RcFdsD (Formate dehydrogenase, delta subunit)
MSDDKIIRMANQIAAFFAVQPGDRAGPVAAHISENWSAPMRAALLAHVAAQSPGLDPLVIAA
APQIRPVPA SEQ ID NO:48 Amino acid sequence RcFdhD (Sulfur carrier protein FdhD)
MSLPAGAVTVPLPGGARAVLAEEVPVALVFDGVTQAVMMASPVDLEDFLLGFALTEGMIAD RAELLRHEVVRQPQGIELRGWLAAPAGQRFAARRRAMAGPVGCGLCGLDSLAAVLRPLPR APRGGAPPPLADGALAALRAGQSLQDAVRSVHAAGFWDGAQMRALREDVGRHNALDKLA GALAGQGIDAAAGALVLTSRLSVDLVQKAAMIGARVLIAPSAPTALAVAEAQAAGLALIARGP
DGPTLYTETEAE SEQ ID NO:49 Amino acid sequence DbMOCOS (Molybdenum cofactor sulfurase)
MTVSEFYPLPVSEIREKYYPNLANQTYLDHAGTTVYSSLTLDKIHEVLSKTLLANPHSLSSASR DTASLVEETRYKILSIFHADPAEYDIVFSLNATHAIKIAASLIQDAAESSFNYYYNINCHTSLIGL RTLAAKYATFDDISSFEPVEDKDGNHPALNFVSWTGQSNFNGQKFPLGWCKEFRRRLDHC YTLYDASALSTSDPPDLSDANSSPDFVVMSFYKIFGMPDIGALILRRSTAKQLVEKRRYFGGG TIDALTIEEPFCRRSKQLHQSLEDGTIPVHAILELSVAIDSHYQIFGSFNSIRLHTDEIRKYAICKL KQLKYGNTGRRMLQIYDWPGAKHGPIIAFSLLSPAGDPIGYYGFGKLASARNISLRTGTLCNI GGIQKFLDRTNEDIRQDYEKGHKCGDILDIIDGKPTELVVKSLTVYPIKSCPGYRIPEGRKWKL TKHGFEFDRSFVLLDLLTQKPLLLKNNPRMALLDCRVDPEKHMLYVRDKRGGNKLVWVSTNIR RYKTKQMGDFIAISERKMVKFFSDVMSIGCTLAGFVTEKQMQNKTAFLLVNERSMRQVSKD DSLISRFRANIVVDSAHPYIEDKLSVLTDMDSGVVLKKRCKCDRCYMITVSDKGSRDPSLLVE LSKERKQKGKVYFGVNIDVENVGYRYMRVGDRIVGEE
SEQ ID NO:50 Amino acid sequence DbXDH (Xanthine dehydrogenase/oxidase)
MAPIAVEPSPYDGTANNDLKDIQFTDSIRFYLNDKLQVVKNPDPEERLIDYIRNEADLRGTKEA CSESGCNACSVTIASISYTDTDYPERPQVHYRSVNSCVTPLILADGKQVITVEGVGSSKNPHP VQERIAKFHGSQCGFCTPGFVMSLYALLREKNGHVSVAEIDEALESNLCRCTGYMPIYDAAY SFAYDSDNYNREKIRPFLKKKDTSFETGSDLYGGSVCALGTKCCRYKSGKEKADEECDKSA SNSDMEIDMNKIFTPNGLPLKPYNPAADLPFPLKLSRISPKPICYGNERKVWFRPVTKEGQFLQI YRIYPDAKIVAGASEVQIEVKFKAANYKVNIYAGDVKELKGWSYKKGKGLTIGGDIPLIELESIC GDLAKRLGRTAAGQTYNAIEEQLKVFASKAVRNVATPAGNIVTASPIADLNPIFVACGAIITAE KLTEDGKLEKTHIDMRDNFFTGYRRHKLPTSSLITEIFIPDTADNEYIHCYKQCKRKDDDISIVT ACLRMELDDEGNVLDSTLVYGGMAPITKNSPKAEKTIKGKNIYNSSFNEECCKCLSEDDYKM PYGVPGGAASYRRSLTLSFFYKFWQYVLATAPIPKANVATIQCRDAILDVDSLSEVTRVQKH GYREMNTPGHKTGIIGKPIVHVNAIKQATGEAQYTNDIPPLHRELFGVQVMSEKAHAKILSVD WSEALEVESVVGYVDINDLPNKEANLWGNLPFGKEPFFADGEVFFVGQAIGVILASSKERAY EASRKVRVVYDELPRIISVEDGVRQKSFFPDRREVKLGDWESAFKNSKYYLENTARLSAQEH FYFEVONCLVIPQEGGELKVYSSTQNPTETQLCAAQVTGVPANRVICRVKRLGGGFGGKET RSIQLSSLAAVAARKFNRPVRLELNRSEDMKTSGERHPFLVKYRASLDEDLKFTGLDMVLYA NAGWSMDLTRGVIDRSVLHASNAYYIPNARVCGIPVKTNIASNTAYRTFGAQAGFYAIESVVT EFAEKLGVDPEEIRRRNYLKPNCGEVFPYKQVVGEDITISNVVDENLKECNYKKMKQEINEFN KHSKWIKRGIAQIPAVFGVSFGVLFLNQAGALVHIYNDGSCLISTGGVEIGQGISTVMRMIAAE ELGVPFDKIFLSETSTECVPNTSSTAASSGSDLNGMALKDACMKLNKRLKPVKDAITKEKGD KWTWEELITKAYLDRVSLSATGFYKTPEIGFEWGDENPKPAFFYHTQGSAVSVVEVDTLTGD WSCLESHIKMDCGRPLNKAIIYGQIEGAFIQGMGYFTMEQSLWLSRTGGLATTGPGAYKIPG FRDTPQRFVLSMYKGSDFRHLRTIHSSKGVGEPPFFLGASVHFALRDAIGHARRQNGIESGS
QGLRFRVPLTTERIRVDCGDKLAKQSFVAAKEGEEEFFIEG SEQ ID NO:51 Amino acid sequence DbUro1 (Uric acid oxidase)
MAYLQDCTYGKNNVRFLKVKRDPINPKIHQVMEASVRVMLTGAFDVSYTKADNSVIIPTDTIK NTILVEAKQTDVFPIERFAAHLVKHFFGKYSWIAGITVHIEQAKWSKYSVDGKLQPHSFVKNG DEVRVCELVSKKNGDFVLTGGVQGLTVLKSSGSMFHGYNVCDYTTLKPVNERILSTDVDCK YKFDSAKIGSVDNIFTLADSGLFDKVFQSALKITLDRFALENSASVQATMYNMGTDIVNANPY
VYNVSYALPNKHYILFDFSWKGLKNENEMFYPSPHPNGLIKCTVGREPIAKL SEQ ID NO:52 Amino acid sequence DbUro2 (5-hydroxyisourate hydrolase)
MSSRPPITCHILDTTCGKPAENVKCEISYIPSNGITSPSEVKPFGYAYTNQDGRIGSWNAANS TETFINAENNQWTKLVSGTYRIRYHTKDYFLKRDGTTFFPFIDIWFEVPAIPEKHYHVPLLLSN
YGYSTYRGS SEQID NO:53 Amino acid sequence DbUro3 (2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase)
MKLPDPQALSQILRSEQVTVIDTLFEHNDKFADFIIKKVLSHNERYGSYREFIKAVRIQLIQLAD NYEKSMTGDLGDMVRSVISAHPRLGIQQASALSVSSAREQKSLQSGKPELERQLLALNGEY
EHCFPGLRFVVFVNGRSRQEITKIMRKRITRDDYNQEVRDAFSAMCDIALDRIKKENSKL SEQ ID NO:54 Amino acid sequence EcMocA (Molybdenum cofactor cytidylyltransferase)
MSAIDCIITAAGLSSRMGQWKMMLPWEQGTILDTSIKNALQFCSRIILVTGYRGNELHER YANQSNITIIHNPDYAQGLLTSVKAAVPAVQTEHCFLTHGDMPTLTIDIFRKIWSLRNDG AILPLHNGIPGHPILVSKPCLMQAIGRPNVTNMRQALLMGDHYSVEIENAEIILDIDTPD
DFITAKERYTEI In case of any inconsistency between the above SEQ ID Nos 1-54 and the SEQ ID Nos 1-54 as disclosed in the sequence listing, the above SEQ ID Nos 1-54 are preferred. Alternatively, the SEQ ID Nos 1-54 as disclosed in the sequence listing may be used. The following Examples illustrate the different embodiments of the invention. Unless stated otherwise all recombinant DNA techniques can be carried out according to standard protocols as described in e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA.
EXPERIMENTAL PART Identification of the Molybdopterin cofactor biosynthetic genes in O. parapolymorpha In contrast to S. cerevisiae, other yeasts species such as Candida nitratophila, Pichia anomala, Pichia angusta and Ogataea parapolymorpha can grow on nitrate as sole nitrogen source and carry a functional Mo-dependent nitrate reductase enzyme, suggesting they are also able to synthesize Moco. The protein sequences of the E. coli Molybdopterin co-factor biosynthetic pathway enzymes MoaA (P30745; GTP 3',8-cyclase), MoaC (Uniprot ID: POA738; Cyclic pyranopterin monophosphate synthase), MoeB (P12282; Molybdopterin- synthase adenylyltransferase), IscS (POA6B7; Cysteine desulfurase), MoaD (P30748; Molybdopterin synthase sulfur carrier subunit), MoaE (P30749; Molybdopterin synthase catalytic subunit), MogA (POAF03; Molybdopterin adenylyltransferase) and MoeA (P12281; Molybdopterin molybdenumtransferase) were used as query in a tBLASTn (BLOSUM62 scoring matrix, gap costs of 11 for existence and 1 for extension) against a database comprising RNA sequencing data of O. parapolymorpha obtained for glucose and methanol grown cells availiable at the Short Read Archive dalabase under accession number - SRX365835 and SRKX385838 respectively. The identified coding sequences were manually annotated in the O. parapolymorpha genome sequence (PRJNA60503) and checked for the presence of alternative, in frame, start codon upstream of the annotated region.
For each of the eight E. coli gene an orthologous gene could be identified (Table 3). The MoaA ortholog, HPODL_02673, was found to be wrongly annotated since it was missing a mitochondrial localisation peptide at the 3’ end. In eukaryote the GTP 3',8-cyclase encoded by cnxA in Aspergillus niger or cnx2 in Arabidopsis thaliana, orthologous to MoaA were known to be localized in the mitochondria.
Table 3: tBLASTn analysis of E. coli Moco related proteins versus O. parapolymorpha transcriptome.
“Query protein Protein annotation ~~ Fungi/plant Gene name of first {Uniprot ID) s ortholog hitin O. gene name parapolymorpha (E value) TE coliMoaA {(P30745) GTP 3 8-cyclase 0 omxA2 HPODL_o02678 E. coli MoaC (POA738) Cyclic pyranopterin monophosphate ~~ cnxB/3 rob 02674 synthase (1e) E. coliMoeB (P12282} Molybdopterin-synthase cnxF/5 HPODL_00948 adenylyltransferase (7e°%
E. colilscS (POA6B7) Cysteine desulfurase NFS1 HPODL_02128 Ge" E. coli MoaD (P30748) Molybdopterin synthase sulfur carrier 6nxG/7 HPODL_01640 subunit (1.4) E. coli MoaE (P30748) Molybdopterin synthase catalytic onxH/6 HPODL 00195 subunit (eis E. coli MogA (POAF03) Molybdopterin adenylyltransferase cnXE/ (E) HPODL_03424 (ae) E. coli MoeA{P12281) Molybdopterin onxE/1 (G) HPODL_03424 molybdenumtransferase (ies To confirm the involvement of the Ogataea parapolymorpha genes (HPODL_02673, HPODL_02674, HPODL_00948, HPODL_02128, HPODL_ 01640, HPODL_00195, HPODL_03424) in Moco biosynthesis, single knock-out strains were constructed.
The sequences of gRNAs to reprogram SpyCas9 CRISPR endonuclease were manually selected to target the first part of each gene in order to increase the chances of loss of function due to frame-shift mutation.
Plasmids pUD897, pUD698, pUD699, pUD700, pUD701,pUD704, pUD705, containing the gRNA sequence targeting HPODL_02673, HPODL_02674, HPODL 00948, HPODL 00195, HPODL_03424, HPODL 02128, HPODL_01640 respectively, were de novo synthesized at GeneArt (Regensburg, Germany) (Thermo Fisher Scientific, Waltham, MA) and propagated in chemically competent Escherichia coli XL1-blue cells according to the supplier's instructions (Agilent Technologies, Santa Clara, CA) by chemical transformation.
Transformants were selected and propagated on Lysogeny Broth (LB) (10 g L™ Bacto tryptone, 5 g L™' Bacto yeast extract, 5 g L™ NaCl) supplemented with 100 mg L™' Ampicillin or Kanamycin and incubated at 37 °. Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma Aldrich, St.
Louis, MO) according to the supplier's instruction.
The plasmid pUDP093 expressing HPODL_02673 gRNA, was constructed in a one-pot reaction by digesting pUDPQ02 (Addgene plasmid number #103872) and pUD697 using Bsal and ligating with T4 ligase (Golden Gate cloning). Similarly, pPUDPO94 (gRNAueonL_o2674), pPUDP095 (gRNAupop. 00948), PUDPO96 (GRNAHRoDL 00195), PUDPO97 (gRNAkpoDL_03424), pUDP100 (gRNAuponr_o2128) and pUDP101 (gRNAkpop 91849) were constructed by Golden Gate cloning of pUDP002 and pUD698, pUD699, pUD700, pUD701, pUD704, pUD705 respectively.
Correct assembly of pUDP plasmids was verified by restriction analysis with Sspl and Pdml (Thermo Fisher Scientific). Next to the pUDP094-095-096-097-098-100-101 plasmids to delete genes of Moco biosynthesis pathway, a pUDP099 plasmid to delete Op YNR1, gene encoding the O.
parapolymorpha Moco dependent nitrate reductase gene, was also constructed. For this, plasmid pUD703 containing the gRNA sequence targeting the OpYNR1 gene was de novo synthesized at GeneArt (Themo Fisher Scientific) and used in a Bsal golden gate assembly with pUDPG02 as done for the other pUDP plasmids, yielding pUDPO99.
All gRNA expressing plasmids assembly were transformed to chemically competent Escherichia coli XL1-blue cells according to the supplier's instructions (Agilent Technologies, Santa Clara, CA) by the chemical E. coli transformation. Transformants were selected and propagated in Lysogeny Broth (10 g L™* Bacto tryptone, 5 g L™ Bacto yeast extract, 5g L 7 NaCl) supplemented with 150 mg L7t ampicillin at 37 °C and 200 rpm in an Innova 4000 shaker (Eppendorf). Samples to be stored at -80 °C were mixed with glycerol (30% v/v). Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma Aldrich) according to the supplier's instruction.
The resulting pUDP plasmids were then individually transformed by electroporation in O.
parapolymorpha DL-1 strain (CBS 11895) following the protocol previously described by Juergens, H. et al. FEMS Yeast Res. 18, (2018). Transformants were selected on YPD plates (10 g L7, Bacto yeast extract, 20 g L™" Bacto peptone, 20 g L glucose and 20 g L' agar) supplemented with hygromycin B at a final concentration of 200 mg L!. For each transformation, three colonies were selected. The targeted CRISPR-Cas9 edited loci were amplified by PCR. Editing at HPODL_02673, HPODL_02674, HPODL_00948, HPODL_00195, HPODL_03424, OpYNR1, HPODL_01640 was checked using specific primer pairs and the resulting fragments were Sanger sequenced (Baseclear, Leiden, The Netherlands) to check for the presence of INDELSs.
With the exception HPODL_02128, a ScNFS1 ortholog gene that did not yield any successful transformants since the open reading frame is known to be essential, sequencing results showed that in each case, the non-homologous end joining DNA repair mechanism introduced one or two nucleotides at the cut locus, 3 bp upstream of the PAM sequence (Figure 2). Mutants exhibiting a gene disruption were restreaked on YPD plates to lose the gRNA plasmid. Single colony isolates devoid of plasmid were then stocked at -80 °C and used for further experiments. Mutants exhibiting disruption in HPODL_02673, HPODL_02674, HPODL_00948, HPODL_00195, HPODL_03424, OpYNR1, HPODL_01640 were renamed IMDO19, IMDO020, IMD021, IMD022, IMD023, IMD025 and IMD027 respectively (Figure 2).
The ability of the O. parapolymorpha DL-1 (CBS 11895) and its derived strains IMDO019, IMDO20, IMDO021, IMDO022, IMD023, IMD025 and IMD027 to grow on nitrate was subsequently tested on SM medium with sodium nitrate as sole nitrogen source. The strains were spotted on synthetic medium (SMA) which contained 20 g L™ glucose, 3 g L™! KH2PO,4,
0.5 gL" MgSO,, 7 H20, 5g Lt (NHs)2SO,, 1 mL L™ of a trace element solution and of a vitamin solution a standard vitamin solution and on synthetic medium with nitrate (SMNo) in which (NH4)2SO, was substituted with 5 g L™* K2SOa and 4.3 g Lt NaNO:.
While the reference strain DL-1 was able to grow on plate containing nitrate as sole nitrogen source, the strain IMDO025 that carries a disruptive mutation in OpYNR1 (HPODL_02384), gene that encodes the nitrate reductase was as expected not able to grow that well on nitrate containing medium, while in the meantime both DL-1 and IMD025 were able to grow on NH4(SO4) plates.
The nitrate reductase is a Moco-dependent enzyme, therefore a strain unable to synthesise Moco would be also unable to assimilate nitrate. All the strains carrying a frameshift mutation in gene HPODL_02673 (IMD019), HPODL_02674 (IMD020), HPODL_02948 (IMD021), HPODL_0195 (IMD022), HPODL_03424 (IMD023) and HPODL_01640 (IMD027) showed impaired growth on SMNo comparable to the control strain IMD025 (Figure 3). In this way, the inventors were able to map and functionally link the O. parapolymorpha genes HPODL_02673, HPODL_02674, HPODL_00948, HPODL_00195, HPODL_03424, HPODL_02384, HPODL_01640 to Moco biosynthesis. The impossibility to delete the gene HPODL_02128, the ScNFS1 ortholog gene, confirmed that it was important since any disruption of HPODL_02128 would have been detrimental.
Engineering Moco and nitrate pathway in S. cerevisiae. Construction of individual expression modules of the Moco biosynthesis genes and nitrate utilization pathways.
Engineering the O. parapolymorpha Moco biosynthesic pathway in S. cerevisiae may require the transfer of six genes (HPODL_02673, HPODL_02674, HPODL_00948, HPODL_00195, HPODL_03424, HPODL_01640). Additionally to confirm the synthesis and functionality of the Moco pathway in S. cerevisiae, the O. parapolymorpha Moco dependent nitrate assimilation pathway including a high affinity nitrate transporter OpYNT1 (HPODL_02387), nitrate reductase OpYNR 1 (HPODL_02384) and nitrite reductase OpYN/1 (HPODL_02386) was introduced. Addition of a high affinity Molybdate transporter in S. cerevisiae could also be beneficial as S. cerevisiae may be unable of importing Molybdate with high affinities.
Construction of promoter and terminator dna parts for assembly of expression modules In order to assemble plasmids with promoter-gene-terminator expression modules, new promoters and terminator parts that are compatible with the Golden Gate based yeast toolkit
(Lee et al ACS Synth. Biol. 4, 975-986, 2015) (YTK) were cloned. For this purpose, terminator parts from S. cerevisiae were amplified with primers having the yeast toolkit compatible ends and CEN.PK113-7D genomic DNA as template. PCR products were used in a Golden Gate assembly, together with the entry vector pMC (GeneArt) using BsmBI and T4 DNA ligase. All PCR were performed using Fhusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific).
Primer pairs were used to amplify SoPYK TE ScTPITE, ScFBATL SCPDCTE ScGPMTE respectively and used in a Golden Gate reaction together with the DMO entry vector to yield pGGKPpO40, pGGREpL42, pGGRpU48, pGGKPL48 respectively.
Moreover, other promoters and terminators compatible with the veast toolkit were synthesized {SensArt). Promoters regions of glycolytic genes were selected to be 800 bp long while terminators 300 bp long. Plasmids carrying ScFBA Tp, ScTPitp, ScGRPM 1p were renamed pGERp104, pGGKp 114, pGGKp 118 respeciivaly.
The entry vector pGGKOTT was constructed by combining YTK plasmids, pYTK002, pYTKO47, pYTK072, pYTKO74, pYTKO082 and pYTKO83 in a Golden gate assembly reaction using Bsal and T4 DNA ligase. Assembly of expression modules The open reading frame HPODL_02673, HPODL_02674, HPODL_00948, HPODL_00195, HPODL_03424, HPODL_01640, OpYNR1 (HPODL_02384), OpYN/1 (HPODL_02386), OpYNT1 (HPODL_02387) were directly PCR-amplified from the O. parapolymorpha DL-1 genome. Genomic DNA of O. parapolymorghs was isolated using the Qiagen 100/G Kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations, Instead, the high affinity molybdate importer CrMOT1 from Chlamydomonas reinhardtii was codon optimized for expression in S. cerevisiae and gene synthesized (GeneArt). All PCR were performed using Phusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific).
The expression cassettes of HPODL 02674, HPODL_00195, HPODL_01640, HPODL_03424, CrMOT1, OpYNI1 and OpYNR1 were constructed in vitro by Golden Gate cloning.
HPODL_02674 was amplified using primers to add the yeast toolkit part 3 compatible ends. The PCR product was gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research, Irvine, CA) and combined with pYTKO986, pTKO10 and pYTK052 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI190.
HPODL_00195 was amplified using primers to add the yeast toolkit part 3 compatible ends.
The PCR product was gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and combined with pYTKO098, pTK011 and pYTK053 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI191.
HPODL_01640 was amplified using primers to add the yeast toolkit part 3 compatible ends. The PCR product was was gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and combined with pYTKO96, pTK012 and pYTKO054 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI192.
HPODL_03424 was amplified using primers to add the yeast toolkit part 3 compatible ends. The PCR product was gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and combined with pYTKO986, pTK014 and pY'TKO56 in a Golden gate assembly reaction that yielded plasmid pUDI194.
CrMOT1 coding sequence was codon optimized and ordered as synthetic dsDNA (plasmid name) and used as a template for a PCR reaction with primers to add the yeast toolkit part 3 compatible ends. The PCR product was gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and combined with pYTK096, pGGKp104 and pGGKp038 in a Golden gate assembly reaction that yielded plasmid pUDI195. OpYNT1 (HPODL_02387) was amplified using primers to add the yeast toolkit part 3 compatible ends. The PCR product was gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and combined with pYTK096, pYTK013 and pGGKp045 in a Golden gate assembly reaction that yielded plasmid pUDI198. OpYNR1 (HPODL_02384) was amplified using primers to add the yeast toolkit part 3 compatible ends. The PCR product was gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and combined with pYTK096, pYTK017 and pGGKp048 in a Golden gate assembly reaction that yielded plasmid pUDI199. Golden gate assembly mixes were transformed to chemically competent Escherichia coli XL1-blue cells according to the supplier's instructions (Agilent Technologies) by the chemical E. coli transformation. Transformants were selected and propagated on LB Broth (10 g L™ Bacto tryptone, 5 g L™ Bacto yeast extract, 5 g L7 NaCl) supplemented with 150 mg L™ kanamycin at 37 °C. Samples were mixed with glycerol (30% v/v) and stored at -80 °C. Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma Aldrich) according to the supplier's instruction. The expression cassettes for HPODL_02673 and HPODL_02128 were constructed using in vitro Gibson assembly. HPODL_02673 was amplified using primers. The ScTDH3 promoter was amplified using the pYTKOO9 plasmid as a template and primers. The ScENO1 terminator was amplified using the pYTKO51 plasmid as template and primers. The plasmid backbone was amplified using pYTKO096 as template and primers. All four PCR products were then gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and combined in equimolar amounts in an in vitro Gibson assembly reaction with NEBuilder® HiFi DNA Assembly Master Mix (New England Biclabs, Ipswich, MA). that yielded plasmid pUDI189.
HPODL_02128 was amplified using primers. The ScHHF1 promoter was amplified using the pYTKO15 plasmid as a template and primers. The ScFBA 1 terminator was amplified using the pGGKp046 as template and primers. The plasmid backbone was amplified using pYTKO096 as template and primers. All four PCR products were then gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and combined in equimolar amounts in a Gibson assembly reaction with NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs) that yielded plasmid pUDI197.
Gibson assembly mixes were transformed to chemically competent E. coli XL1-blue cells according to the supplier's instructions (Agilent Technologies) by the chemical E. coli transformation. Transformants were selected and propagated on LB Broth (10 g L™' Bacto tryptone, 5 g L™! Bacto yeast extract, 5 g L™! NaCl) supplemented with 150 mg L7? kanamycin at 37 °C. Samples were mixed with glycerol (30% v/v) and stored at -80 °C. Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma Aldrich) according to the supplier's instruction.
The expression cassettes for HPODL_00948 and OpYN/1 (HPODL_02386) were constructed using in vivo assembly in S. cerevisiae. HPODL_00948 was amplified using primers. The ScGPM1 promoter was amplified using the pGGKp116 plasmid as a template and primers. The ScPYK1 terminator was amplified using the pGGKO40 plasmid as template and primers. The plasmid backbone was amplified using pGGKd017 as template and primers. All four PCR products were gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and co-transformed in equimolar amounts in S. cerevisiae CEN.PK113-5D (MATa ura3-52) using the LiAc protocol (Gietz Methods Enzymol. 350, 87-96 (2002). Transformants were selected SMA. Plasmid pUDE796 was then purified using the yeast miniprep kit Il (Zymo research).
OpYNI1 (HPODL_02386) was amplified using primers. The ScTP/1 promoter was amplified using the pGGKp114 plasmid as a template and primers. The ScTP/1 terminator was amplified using the pGGKp042 plasmid as template and primers. The plasmid backbone was amplified using pGGKd017 as template and primers. All four PCR products were then gel purified and co-transformed in equimolar amounts in S. cerevisiae CEN.PK113-5 (MATa ura3-52) using the LiAc protocol. Transformants were selected on SMA plate. Plasmid pUDE797 was then purified using the yeast miniprep kit Il (Zymo research).
The in vivo assembled pasmids pUDE796 and pUDE797 were transformed to chemically competent £. coli XL1-blue celis according to the supplier's instructions (Agilent Technologies) using the chemical transformation protocol. Transformants were selected and propagated on LB Broth (10 g Lt Bacto tryptone, 5 g L™ Bacto yeast extract, 5 g L™! NaCl) supplemented with 150 mg L™ ampicillin at 37 °C. Samples were mixed with glycerol (30% v/v) and stored at -80 °C. Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma Aldrich) according to the supplier's instruction.
Assembly of Moco and nitrate pathway at the SGA1 locus in S. cerevisiae. Fragments necessary for CRISPR-Cas9 assisted in vivo assembly were obtained by PCR from plasmid template DNA using High fidelity Phusion polymerase (Termo Fisher Scientifc) according to supplier's instructions. The amplified fragments were stocked in TE buffer (10 mM Tris, pH8, 1 mM EDTA). Fragments amplified from plasmid templates were subjected to gel extraction to prevent false-positive transformants that might arise from contamination with linearized template plasmid. To construct S. cerevisiae strains with different pathways configurations, an expression module could be amplified with different primer pairs. The strain IMX1777 that expresses the Moco pathway was constructed as follows: The HPODL_02673 expression module was amplified using a primers pair and pUDI189 as template. The HPODL_02674 expression module was amplified using primers pairs and pUDI190 as template. The HPODL_00195 expression module was amplified using primers pairs and pUDI191 as template. The HPODL_01640 expression module was amplified using primers and pUDI192 as template. The HPODL_03424 expression module was amplified using primers pairs and pUDI194 as template. The HPODL_00948 expression module was amplified using primers pairs and pUDE796 as template. The HPODL_02128 expression module was amplified using primers pairs and pUDI197 as template. To integrate the different compatible module at the SGA1 locus, 200 ng of pUDR119 a plasmid expressing the gRNA targeting the SGA 7 gene was transformed in the strain IMX585 (MATa ura3-52 can1A::Spycas9). 100 fmol of each expression module were co-transformed using the LiAc protocol (Figure 4A). For selection of yeast strains harbouring an acetamidase marker carried by pUDR119, SMA was modified; (NH4)2SO4 was replaced by 0.6 g Lt acetamide as nitrogen source and 6.6 g L7 K2SO4 to compensate for sulfate (SM-Ac). Selection of the amdSYM marker was performed as previously described. Transformants were selected on SM-Ac plates. The strain IMX1778 that expresses the Moco pathway and the high affinity molybdate transporter was constructed has follows: The HPODL_02673 expression module was amplified using primers pair and pUDI189 as template. The HPODL_02674 expression module was amplified using primers pairs and pUDI190 as template. The HPODL_00195 expression module was amplified using primers pairs and pUDI191 as template. The HPODL_01640 expression module was amplified using primers pairs and pUDI192 as template. The HPODL_03424 expression module was amplified using primers pairs and pUDI194 as template. The HPODL_00948 expression module was amplified using primers pairs and pUDE796 as template. The HPODL_02128 expression module was amplified using primers pairs and pUDI197 as template. The CrMOT 1 expression module was amplified using primers pairs and pUDI195 as template. To integrate the different compatible module at the SGA1 locus, 200 ng of pUDR119 a plasmid expressing the gRNA targeting the SGA 7 gene was transformed in the strain IMX585 (MATa ura3-52 can1A4::Spycas9). 100 fmol of each expression module were co-transformed using the LiAc protocol (Figure 5A). Transformants were selected on SM-Ac plates.
The strain IMX1779 that expresses the high affinity molybdate transporter was constructed as follows: The CrMOT1 expression module was amplified using primers pairs and pUDI195 as template. To integrate the module at the SGA 17 locus, 200 ng of pUDR119 a plasmid expressing the gRNA targeting the SGA 7 gene was transformed in the strain IMX585. 100 fmol of the expression module was co-transformed using the LiAc protocol (Figure 6A). Transformants were selected on SM-Ac plates. The strain IMX1780 that expresses the nitrate assimilation pathway was constructed as follows: The OpYNT1 (HPODL_02387) expression module was amplified using primers pair and pUDI198 as template. The OpYNR1 (HPODL_02384) expression module was amplified using primers pairs and pUDI199 as template. The OpYN/1 (HPODL_023886) expression module was amplified using primers pairs and pUDE797 as template. To integrate the different compatible module at the SGA locus, 200 ng of pUDR118 a plasmid expressing the gRNA targeting the SGA 7 gene was transformed in the strain IMX585 (MATa ura3-52 can14::Spcas9). 100 fmol of each expression module were co-transformed using the LiAc protocol (Figure 7A). Transformants were selected on SM-Ac plates. The strain IMX1781 that expresses the Moco pathway, the high affinity molybdate transporter and the nitrate assimilation pathway was constructed has follows: The HPODL_02673 expression module was amplified using primers pair and pUDI189 as template. The HPODL_02674 expression module was amplified using primers pairs and pUDI1980 as template. The HPODL_00195 expression module was amplified using primers pairs and pUDI191 as template. The HPODL_01640 expression module was amplified using primers pairs and pUDI192 as template. The HPODL_03424 expression module was amplified using primers pairs and pUDI194 as template. The HPODL_00948 expression module was amplified using primers pairs and pUDE796 as template. The HPODL_02128 expression module was amplified using primers pairs and pUDI197 as template. The CrMOT1 expression module was amplified using primers pairs and pUDI195 as template. The OpYNT1 (HPODL_02387) expression module was amplified using primers pair and pUDI198 as template. The OpYNR1 (HPODL_02384) expression module was amplified using primers pairs and pUDI199 as template. The OpYN/71 (HPODL_023886) expression module was amplified using primers pairs and pUDE797 as template. To integrate the different compatible module at the SGA locus, 200 ng of pUDR 11952 a plasmid expressing the gRNA targeting the SGA 7 gene was transformed in the strain IMX585 (MATa ura3-52 can1A::Spcas9). 100 fmol of each expression module were co-transformed using the LiAc protocol (Figure 8A). Transformants were selected on SM-Ac plates.
The strain IMX1782 that expresses the Moco pathway and the nitrate assimilation pathway was constructed has follows: The HPODL_02673 expression module was amplified using primers pair and pUDI189 as template. The HPODL_02674 expression module was amplified using primers pairs and pUDI190 as template. The HPODL_00195 expression module was amplified using primers pairs and pUDI191 as template. The HPODL_01640 expression module was amplified using primers pairs and pUDI192 as template. The HPODL_03424 expression module was amplified using primers pairs and pUDI194 as template. The HPODL_00948 expression module was amplified using primers pairs and pUDE796 as template. The HPODL_02128 expression module was amplified using primers pairs and pUDI197 as template. The YNT7 (OpHPODL_02387) expression module was amplified using primers pair and pUDI198 as template. The OpYNR 1 (OpHPODL_02384) expression module was amplified using primers pairs and pUDI199 as template. The OpYN!1 (OpHPODL_02386) expression module was amplified using primers pairs and pUDE797 as template. To integrate the different compatible module at the SGA7 locus, 200 ng of pUDR119 a plasmid expressing the gRNA targeting the SGA 7 gene was transformed in the strain IMX585. 100 fmol of each expression module were co-transformed using the LiAc protocol (Figure 9A). Transformants were selected on SM. In order to verify the correct assembly of each pathway, single colonies were picked from the transformation plate and genomic DNA was extracted as previously described (Löoke Biotechniques 50, 325-328 (2011). DreamTagq polymerase (Thermo Scientific) was used to amplify each junction using primers that bind upstream and downstream the 60 bp homology flanks (SHR). For IMX1777, primers pair were used to verify the junction 3’ SGA1 SHR, SHR1, SHR2, SHRS3, SHR4, SHR5, SHR8, SHR7, 5’ SGA1 SHR respectively (Figure 4B). For IMX1778, primers pair were used to verify the junction 3' SGA7 SHR, SHR1, SHR2, SHR3, SHR4, SHR5, SHR6, SHR7, SHR8, 5° SGA 7 SHR respectively (Figure 5B).
For IMX1778, primers pair were used to verify the junction 3° SGA1 SHR, 5’ SGA1 SHR respectively (Figure 6B). For IMX1780, primers pair were used to verify the junction 3’ SGA1 SHR, SHR10, SHR11, 5 SGA1 SHR respectively (Figure 7B).
For IMX1781, primers pair were used to verify the junction 3' SGA? SHR, SHR1, SHR2, SHR3, SHR4, SHR5, SHR6, SHR7, SHR8, SHR9, SHR10, SHR11, 5 SGA7 SHR respectively (Figure 8B).
Following genotyping of the transformants, correctly assembled isolates were grown in 20 ml YPD in a 50 ml vented Greiner tube at 30 °C overnight by inoculating a single colony. The next day, 1 Jl was transferred to a new tube containing the same amount of medium and the sample was grown overnight. The day after, each liquid culture was restreaked to single colony by plating on YPD agar plates. Plates were incubated at 30 °C overnight and the next day single colonies were patched on both YPD and SMAc to assess which ones have lost the gRNA plasmid and was therefore not able to assimilate acetamide anymore. One clone for each strain that had lost the plasmid was then grown in YPD and 30 %v/v glycerol was added prior stocking samples at -80 °C. Each engineered S. cerevisiae strain was then inoculated in triplicate in 5 ml SMNs (a modified version of SMA in which ammonium sulfate was replaced by 50 mmol Lt KNO: and 38 mmol L* potassium sulfate) using 50 ml vented Greiner tubes and incubated in a shaker incubator at 30°C 200 rpm. After 14 days, only strains IMX1781 and IMX1782 that share the combined expression of the Moco and nitrate utilization pathways initiated growth, reaching high cell densities. From these liquid cultures, single colony isolates were re-streaked three times on SMNs plates and stocked (1 single colony isolate from each adaptation replicate), resulting in 6 nitrate adapted strains. The adapted strains IMS0815, IMS0816, IMS0819 were derived from IMX1781 and IMS0817, IMS0818 and IMS0821 were derived from IMX1782. Whole genome sequencing of IMS0815, IMS0816, IMS0819, IMS0817, IMS0818 and IMS0821 was performed. Genomic DNA of adapted S. cerevisiae strains, IMS0815, IMS0816. IMS0817, IMS0818, IMS0819, IMS0821 was isolated with a Qiagen Blood and Cell Culture DNA kit with 100/G genomic tips (Qiagen) according to the manufacturer's instructions. Genomic DNA was sequenced on a HiSeq2500 sequencer (Humina, San Diego, CA) with 150 bp paired-end reads using PCR-free library preparation by Novogene Bioinformatics Technology Co. Lid {Yuen Long, Hong Kong). Data mapping was performed against the CEN.PK113-7D genome® where an extra contig containing the integration cassette was previously added. Data processing and chromosome copy number variation determinations were done as previously described.
Sequence data analysis of IMS0815, IMS0816. IMS0817, IMS0818, IMS0819, IMS0821 confirmed that the various engineered pathways were correctly integrated. However all isolates independently of the pathway configuration showed a copy humber increase of chromosome IX, on which the engineered pathways were integrated (Figure 10). Additionally, sequence analysis of IMS0821 revealed an absence of sequencing reads covering mitochondrial DNA.
This strongly suggests that this isolate lost mitochondrial DNA and therefore is expected the be respiratory deficient. IMS0821 was not used any further. Physiological characterization of strains expressing Moco biosynthesis and nitrate utilisation pathways grown on nitrate as sole N-source. Next, growth of the adapted strains in synthetic media with KNO3 (SMNs) as a sole nitrogen source was characterized. The strains were grown in 500 mL shake flask with 100 mL of medium. Thawed aliquots of frozen stock cultures were inoculated in a shake flask containing SMNs and incubated overnight. This pre-culture was used to inoculate biological replicates 500 mL shake flasks containing 100 mL SMNs. The initial optical density at 660 nm (ODsss) was standardized to 0.2. The flasks were then incubated 30 °C and 200 rpm. The growth was monitored by ODesss measurement throughout the growth using a 7200 Jenway Spectrometer (Jenway, Stone, UK). At each time point, in addition of optical density samples, 2 ml of the culture were centrifuged with a benchtop centrifuge, supernatant were collected and stored at -20 °C for subsequent analysis. Specific growth rates were calculated from at least five time points in the exponential growth phase of each culture. Analysis of nitrate, nitrite and ammonium concentration in culture supernatants was performed using the HACH (Tiel, Nederlands) cuvette test kits LCK339, LCK341 and LCK304 respectively, following manufacturer instructions. Glucose and ethanol concentrations were measured.
Strains IMS0815, IMS0818 and IMS0819 showed a bi-phasic grow curve with two different exponential phases. In contrast the strains IMS0816 and IMS0817 showed a typical exponential growth profile. All strains grew faster than 0.1 ht on SMNs. IMS0816 and IMS0817 exhibited the highest specific growth rate of all strains reaching 0.14 and 0.17 respectively. Fast growth of IMS0817 showed that the molybdate transporter CrMOT1 from Chlamydomonas reinhardtii was not essential at a molybdate concentration of 1.6 pM. Analysis of nitrate, nitrite and ammonium concentrations confirmed that growth of IMS0816 and IMS0817 was concomitant with nitrate consumption. Upon glucose and ethanol depletion trace of nitrite and ammonium ions accumulated in culture supernatants (Figure 11).
To test the role of the high affinity Molybdate importer, the regular trace element that contains
1.6 uM Molybdate was modified. The molybdate concentration was lowered by 100-fold to 16 nM. In this new condition, only IMS0816 (CrMOT1) exhibited growth after an adaptation period of about 300 h™'. After twelve subsequent transfers to a new culture, the IMS0816 population grew at a growth rate of 0.14 n° (Figure 12BC). Conversely, the strain IMS0817 (without CrMOT1) did not show good growth even after extended incubation of 700 hr (Figure 12A).
Ammonium nitrate is a nitrogen source commonly used in industrial fermentation, it is also predominantly used in agriculture as a high-nitrogen fertilizer, and it is found in plant biomass feedstock that may serve as substrate for microbial fermentation as in second generation bioethanol processes. To test the ability of the strain IMS0817 to co-consume nitrate and ammonium, the strain was grown in a modified SMNs medium (SMNA) in which potassium nitrate was substituted with 10 mM of ammonium nitrate as sole nitrogen source. In this condition, IMS0817 was able to grow with a maximum specific growth rate of 0.34 h” and consume both nitrogen sources simultaneously demonstrating absence of repression of ammonium on the heterologous nitrate assimilation pathway (Figure 13).
Engineering bis-MGD and bis-MGD-dependent nitrate assimilation in S. cerevisiae. Cloning of the gRNA carrying plasmid pUDR514 targeting the YPRcTau3 locus A new gRNA carrying plasmid targeting the YPRcTaus locus (targeting sequence was constructed as previously described (Mans et Al., 2015) by Gibson assembly of a 2micron fragment with two copies of the same gRNA and a linearized pROS13 backbone yielding plasmid pUDR514.
Construction of individual expression modules of the bis-MGD biosynthesis genes and bis- MGD-dependent nitrate utilization pathways. Engineering the bis-MGD biosynthesic pathway in S. cerevisiae may require the transfer of two additional genes (EcMobA, EcMobB) in the Moco background strain IMX1778.
Additionally, to confirm the synthesis and functionality of the bis-MGD pathway in S. cerevisiae, the bis-MGD dependent nitrate reductase from Klebsiella oxycotica (KoNasA, KoNasC) was introduced together with the O. parapolymorpha high affinity nitrate transporter OpYNT1 (HPODL_02387) and nitrite reductase OpYN/1 (HPODL_02386).
Assembly of expression modules
The bis-MGD-dependent nitrate reductase (KoNasA, KoNasC) and Moco modifying genes (EcMobA, EcMobB) were codon optimized for expression in S. cerevisiae, flanked by yeast toolkit compatible ends and gene synthesized (GeneArt). All PCR were performed using Fhusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific).
The expression cassettes were constructed in vitro by Golden Gate cloning. The plasmid carrying the EcMobA gene pUD951 was combined with pYTKO96, pTKO09 and pYTKO51 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI212. The plasmid carrying the EcMobB gene pUD952 was combined with pYTK0S6, pTK010 and pYTKO52 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI213.
The plasmid carrying the KoNasA gene pUD954 was combined with pYTKO96, pTKO11 and pYTKO53 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI215.
The plasmid carrying the KoNasC gene pUD955 was combined with pYTK096, pTK012 and pYTKO54 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI216.
Golden gate assembly mixes were transformed to chemically competent Escherichia coli XL1-blue cells according to the supplier's instructions (Agilent Technologies) by the chemical E. coli transformation. Transformants were selected and propagated on LB Broth (10 g L™ Bacto tryptone, 5 g L™! Bacto yeast extract, 5 g L7 NaCl) supplemented with 150 mg L™ kanamycin at 37 °C. Samples were mixed with glycerol (30% v/v) and stored at -80 °C. Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma Aldrich) according to the supplier's instruction.
Assembly of bis-MGD and bis-MGD-dependent nitrate pathway at the YPRcTau3 locus in S. cerevisiae.
Fragments necessary for CRISPR-Cas9 assisted in vivo assembly were obtained by PCR from plasmid template DNA using High fidelity Phusion polymerase (Termo Fisher Scientifc) according to supplier's instructions. The amplified fragments were stocked in TE buffer (10 mM Tris, pH8, 1 mM EDTA). Fragments amplified from plasmid templates were subjected to gel extraction to prevent false-positive transformants that might arise from contamination with linearized template plasmid. For selection of yeast strains harbouring an KanMX marker carried by pUDR514, SMA was supplemented with 200 mg/L G418 (SM-G418).
The strain IMX2130 that expresses the Moco modifying genes and bis-MGD-dependent nitrate assimilation pathway was constructed as follows: The EcMobA expression module was amplified using primers pairs and pUDI212 as template. The EcMobB expression module was amplified using primers pairs and pUDI213 as template. The KoNasA expression module was amplified using primers pairs and pUDI215 as template. The KoNasC expression module was amplified using primers pairs and pUDI216 as template. The OpYNT1 (HPODL_02387) expression module was amplified using primers pair and pUDI198 as template. The OpYN/1 (HPODL_02386) expression module was amplified using primers pairs and pUDE797 as template. To integrate the different compatible module at the YPRcTau3 locus, 200 ng of pUDR514 a plasmid expressing the gRNA targeting the YPRcTau3 locus was transformed in the strain IMX1778 (Moco chassis strain with high affinity Molybdenum importer). 100 fmol of each expression module were co-transformed using the LiAc protocol (Figure 14A). Transformants were selected on SM-G418 plates.
Engineering bis-MGD and bis-MGD-dependent biotin sulfoxide reductase in S.
cerevisiae. Construction of individual expression modules of the bis-MGD biosynthesis genes and bis- MGD-dependent nitrate utilization pathways.
Engineering of the bis-MGD-dependent biotin sulfoxide reductase in S. cerevisiae may require the transfer of three additional genes in the Moco background strain IMX1778. The E. coli genes EcMobA, EcMobB were introduced to allow bis-MGD biosynthesis while the biotin sulfoxide gene from Rhodobacter sphaeroides (RsBisC) was introduced to allow reduction of biotin sulfoxide to biotin.
Construction of expression module for the bis-MGD-dependent biotin sulfoxide reductase. The bis-MGD-dependent biotin sulfoxide gene (RsBisC) and Moco modifying genes (EcMobA, EcMobB) were codon optimized for expression in S. cerevisiae, flanked by yeast toolkit compatible ends and gene synthesized (GeneArt). All PCR were performed using Fhusion® High-Fideiity DNA polymerase (Thermo Fisher Scientific).
The expression cassette for RsBisC was constructed in vitro by Golden Gate cloning. The plasmid carrying the RsBisC gene pUD958 was combined with pYTK096, pTKO11 and pYTKO53 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI218.
Golden gate assembly mix was transformed to chemically competent Escherichia coli XL1- blue cells according to the supplier's instructions (Agilent Technologies) by the chemical E.
coli transformation. Transformants were selected and propagated on LB Broth (10 g L™* Bacto tryptone, 5 g L™! Bacto yeast extract, 5 g L7’ NaCl) supplemented with 150 mg L™* kanamycin at 37 °C. Samples were mixed with glycerol (30% v/v) and stored at -80 °C. Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma Aldrich) according to the supplier's instruction. Assembly of bis-MGD and bis-MGD-dependent biotin sulfoxide reductase at the YPRcTau3 locus in S. cerevisiae.
Fragments necessary for CRISPR-Cas9 assisted in vivo assembly were obtained by PCR from plasmid template DNA using High fidelity Phusion polymerase (Termo Fisher Scientifc) according to supplier's instructions. The amplified fragments were stocked in TE buffer (10 mM Tris, pH8, 1 mM EDTA). Fragments amplified from plasmid templates were subjected to gel extraction to prevent false-positive transformants that might arise from contamination with linearized template plasmid. For selection of yeast strains harbouring an KanMX marker carried by pUDR514, SMA was supplemented with 200 mg/L G418 (SM-G418).
The strain IMX2133 that expresses the Moco modifying genes and bis-MGD-dependent biotin sulfoxide protein was constructed as follows: The EcMobA expression module was amplified using primers pairs and pUDI212 as template. The EcMobB expression module was amplified using primers pairs and pUDI213 as template. The RcBisC expression module was amplified using primers pairs and pUDI219 as template. To integrate the different compatible module at the YPRcTau3 locus, 200 ng of pUDR514 a plasmid expressing the gRNA targeting the YPRcTau3 locus was transformed in the strain IMX1778 (Moco chassis strain with high affinity Molybdenum importer). 100 fmol of each expression module were co-transformed using the LiAc protocol (Figure 14A).
Transformants were selected on SM-G418 plates.
Engineering bis-MGD and bis-MGD-dependent formate dehydrogenase in S. cerevisiae.
Construction of individual expression modules of the bis-MGD-dependent formate dehydrogenase genes. Engineering the bis-MGD-dependent formate dehydrogenase in S. cerevisiae may require the transfer of seven additional genes in the Moco background strain IMX1778. The E. coli genes EcMobA, EcMobB were introduced to allow bis-MGD biosynthesis while the formate dehydrogenase genes from Rhodobacter capsulatus (RcFdsA, RcFdsB, FdsG, FdsD) were introduced together with the formate dehydrogenase-specific chaperone and sulfur carrier protein (RcFdhD) to allow formation of NADH and CO2 from formate. Assembly of expression modules The bis-MGD-dependent dehydrogenase genes (RcFdsA, RcFdsB, ReFdsG, RcFdsD) and the formate dehydrogenase-specific chaperone (RcFdhD) gene were codon optimized for expression in S. cerevisiae, flanked by yeast toolkit compatible ends and gene synthesized (GeneArt). All PCR were performed using Fhusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific).
The expression cassettes for RcFdsG, RcFdsD, RcFdhD were constructed jn vitro by Golden Gate cloning. The plasmid carrying the RcFdsG gene pUD961 was combined with pYTK096, pTK013 and pYTKO55 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI222.
The plasmid carrying the RcFdsD gene pUD962 was combined with pYTK096, pTK014 and pYTKO56 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI223.
The plasmid carrying the RcFdhD gene pUD963 was combined with pYTKO096, pTK015 and pGGKp045 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI224.
Golden gate assembly mixes were transformed to chemically competent Escherichia coli XL1-blue cells according to the supplier's instructions (Agilent Technologies) by the chemical E. coli transformation. Transformants were selected and propagated on LB Broth (10 g L™ Bacto tryptone, 5 g Lt Bacto yeast extract, 5 g L7t NaCl) supplemented with 150 mg Lt kanamycin at 37 °C. Samples were mixed with glycerol (30% v/v) and stored at -80 °C.
The expression cassettes for RcFdsB were constructed in vitro by Gibson assembly.
RcFdsB was amplified using pUD960 as a template and primers. The SKFBA1 promoter was amplified using the pGGKp065 plasmid as a template and primers. The Sc TDH3 terminator was amplified using the pGGK106 plasmid as template and primers. The plasmid backbone was amplified using pGGKd017 as template and primers. All four PCR products were then gel purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) and combined in equimolar amounts in an in vitro Gibson assembly reaction with NEBuiider® HiFi DNA Assembly Master Mix (New England Biclabs, Ipswich, MA). that yielded plasmid pUDE869.
Plasmids were isolated from E. coli with the GenElute Plasmid Miniprep Kit (Sigma Aldrich) according to the supplier's instruction. RcFdsA could not be successfully assembled in an expression unit so SKADH 1 promoter, RcFdsA gene and ScFBA 1 terminator were used as independent fragments for the in vivo assembly of the pathway. Assembly of bis-MGD and bis-MGD-dependent formate dehydrogenase at the YPRcTau3 locus in S. cerevisiae.
Fragments necessary for CRISPR-Cas9 assisted in vivo assembly were obtained by PCR from plasmid template DNA using High fidelity Phusion polymerase (Termo Fisher Scientifc) according to supplier's instructions. The amplified fragments were stocked in TE buffer (10 mM Tris, pH8, 1 mM EDTA). Fragments amplified from plasmid templates were subjected to gel extraction to prevent false-positive transformants that might arise from contamination with linearized template plasmid. For selection of yeast strains harbouring an KanMX marker carried by pUDR514, SMA was supplemented with 200 mg/L G418 (SM-G418). The strain IMX2156 that expresses the Moco modifying genes, bis-MGD-dependent formate dehydrogenase and formate dehydrogenase-specific chaperone protein was constructed as follows: The EcMobA expression module was amplified using primers pairs and pUDI212 as template. The EcMobB expression module was amplified using primers pairs and pUDI213 as template. The SKADH 1 promoter was amplified using primers pairs and pGGKp062 as template. The RcFdsA coding sequence was amplified using primers pairs and pUD959 as template. The ScFBA1 terminator was amplified using primers pairs and pGGKp105 as template. The RcFdsB expression module was amplified using primers pairs and pUDE869 as template. The RcFdsG expression module was amplified using primers pair and pUDI222 as template. The RcFdsD expression module was amplified using primers pairs and pUDI223 as template. The RcFdhD expression module was amplified using primers pairs and pUDI224 as template. To integrate the different compatible module at the YPRcTau3 locus, 200 ng of PUDR514 a plasmid expressing the gRNA targeting the YPRcTau3 locus was transformed in the strain IMX1778 (Moco chassis strain with high affinity Molybdenum importer). 100 fmol of each expression module were co-transformed using the LiAc protocol (Figure 14A). Transformants were selected on SM-G418 plates.
SEQLTXT
SEQUENCE LISTING <110> Technische Universiteit Delft <120> Yeast with engineered Molybdenum co-factor biosynthesis <130> P33965NL09 <160> 54 <170> PatentIn version 3.5 <210> 1 <211> 1083 <212> DNA <213> Ogataea parapolymorpha <400> 1 atgatatcct cattactgct gaggcggctc cattcaacgg gtacttcaca gcacctgcca 60 cggcttgaaa gactgaggcg tatgcctgta agacacttga aagagtttct gacagacacg 120 tatgggcgca agcacgacta tcttcgcatc tccatcaccg aacgatgtaa tctccgctgc 180 gtttactgta tgcctgagca aggcgttgac ttgtcgccac cagagcacat gcttacaaca 240 gaggagatag tcaaactggc cactcttttt gcacagcacg gagtacggaa ggtcagactg 300 actggcggag agcccacggt cagaaaagat atcgttgagc ttgtggccaa gctcaatcaa 360 attacaggca ttgaagagat ctgtatgaca tctaatggtc tagcactcca tcgaaaatta 420 ccagatcttt tcaagaatgg gctgacatcg ctcaatttga gtcttgatac tcttatcaat 480 ggtaagtttc tcttaataac gcgacgcaac ggactcagcg cagtaatgag aagtttgaga 540 acagcacttg aattggacat tcccaaggtg aaaatcaatg tggtagtaat gaagaatctg 600 aacgaagatg agatactgga tttcgttgaa ctcagcaaaa atgataaagt cgaggttcga 660 tttatagagt acatgccttt cgatggaaac aaatggtcta cgaataagct ggtctcatac 720 gaggacatac tttccaatat caaagtgaga catccgaata tccaaagact cccccataaa 780 catggtgaca cggccaaagt ctaccaaatt cccggattca aaggaaaagt aggttttata 840 acgtccatga cttcggactt ttgcagtact tgcacacgtt tgaggattac ttctgacggt 900 aacctgaaag tttgcttgtt cgataacaca gaagtatctc tgcgagatat gcttcgtgct 960 ggatacagcg atgataaatt aatgcaacgc atcggtgaag cagtgaaaaa taagaaggag 1020 Pagina 1
SEQLTXT aaacatgcag ggattgacgt attaggagat caacccaata ggccgatgat tttaattgga 1080 gga 1083 <210> 2 <211> 450 <212> DNA <213> Ogataea parapolymorpha <400> 2 atggttgcaa ttcatgaaaa agaagacacc catagatgtg ctattgcaga gggatcaatc 60 aagttcagca acccagaatc gatgaaattg ctgctatcag aaagcaacaa gaagggagat 120 gtgatttcaa tcgcaagaat cgcagggata attgctgtga agaagaccgc agagttgatt 180 cctctctgtc atccaatcag catcactgga atcaaggttg atctgattca cgacgagaaa 240 gagaactgca tcaaagtaaa ctgtgaggtg cactgtaatg ggaaaaccgg cgtcgaaatg 300 gaagcattaa cgggtgcgac gatttcgttg cttaccgttt atgatatgtg caaagctgtc 360 gacaaaatga tgacgattag cgattgtcga gttgttaaga agtcaggagg taaaagcggt 420 gacatagatc tatcaaccat cttcaaatag 450 <210> 3 <211> 447 <212> DNA <213> Ogataea parapolymorpha <400> 3 atgtccatct ttgtagatat tactgataag ccgctggatt cggctgaggt gcttaattat 60 gttcggcatc ctcaagcggg agcaattgtg tattttggag gcacaacaag aaatacgttt 120 gaaggcaagg aggttgtatc tctcgcatac gaggcgcatc caaggttggc gatcaaaact 180 ctcgagtcca tcgcccacga ggcgaaagcc aaatttcaga gtgttcataa gatagcaatt 240 gtgcaccgca caggcgtggt tccagtagct acagagtccg ttatgattgc cgtcagttca 300 acacatcgaa aagaagggtg gctttgtgga gagtgggtat tggagaaagt caaggaaagg 360 gcagaaatct ggaagatcga aaagtacgcg gacggagata gtgtctacaa agagaatgac 420 gtttctaacg tgcttagtcg cacctaa 447 <210> 4
Pagina 2
SEQLTXT <211> 288 <212> DNA <213> Ogataea parapolymorpha <400> 4 atggtcgcag ttgctatcga atatttcggg cccgcaaaaa catatacaaa cggcgtggcg 60 cacgagagag tagagctgac agagccggca acgcttaaca cgctaattca gcacgtcggt 120 cgctcgtact cgtccgaatt cgctcaatat atcgtctcca gctgtggagt cgtggtcaat 180 gaggactacg tggaaaccga gcgcataggc atcgaattct tcggtaaaaa cattgctctc 240 cagtcaggcg acgtggtagg catcattccg ccagtttcaa gtggataa 288 <210> 5 <211> 1878 <212> DNA <213> Ogataea parapolymorpha <400> 5 atgactgttg gtatcttggt tgtatcagaa tcggtttcca ggggtttatc gaccgacaag 60 gttgttgacg cattgaaaca acaccttgat ggctttgaat tgaaggctca caaggttgtg 120 ccagacaaga aggaagatat ccaggctgca gtggtggact gggtgaaaca ggatttcaag 180 cttatcttga ccgctggcgg aaccggcttc accaagactg acatcacacc agaggccatt 240 gagccattgt tggacaaaaa ggcgcctgga ctggttcatg ctatgctgtc cttttctctt 300 caaatcaccc cttttgccat gcttgctcgg ccggtagctg gtgttcgcgg agaatctttg 360 attatcacgc tgcccggctc gccaaaggga gccacggaaa atttccaggc aatcaaaggg 420 gtcattgggc acgcactttc gcaactgggg attgaaagct caagattgct gcacaaggag 480 tccggttcag gccatcatca tcatcatcat catcaccatc atcatggaca ccttgccaaa 540 cacgaattgg tcgattcggt cgttgcgaga catcgagtct cgccctatcc cacgatctct 600 gttgatgaag catactcaag gatcagagag aacacgccag ctccagaagt aatcgagctg 660 agcattttgg atcctcgtct agtgggcagc gtggttgctg aaaatgttac tgcccagatg 720 gacgttccaa attttcgcgc aagtattgtg gacggatatg cgatgatcag ctctgatgga 780 ccgggtgttt accctgttgt tagtgtttcg catgcttcca aaaatgacca gaaagagctg 840 gttgcaggac aaatcgcgag aatcaccacg ggggcgccgc ttcctgaggg agccgacgcc 900 Pagina 3 gtagtgatgg tcgaggaaac acgactagtt Gasnccacts aggacgggag cgaggagaag 960 cttgttgaga tactggcgaa aaacgtcaag acaggggaga acatcagggc cattggttct 1020 gacaccagga aggacagttt ggtgctcgcg aaaggatcaa gaatctcgcc aggaggagga 1080 gaaatagggg cgttggcgag cgtgggggtc aacaaaatta aagtgtaccg caaaccggtg 1140 attgggctgc tatctactgg aaatgaactg caggacgtgc aaaccgaaaa attgttacat 1200 tatggagaaa tatacgacag caacagaccg acgctagtgt ccatagtgca gaattgtggg 1260 tacgagttag tcgacctcgg aatagcctca gataccaagg agtctcttat caaaattata 1320 aaggacgctg tggacgtcaa aaaaattgat tgtttaatta ccactggagg agtgtccatg 1380 ggagagcttg acctgctaaa accgacgatc gaaaatgaac ttcacggtac aatccatttt 1440 ggacgtgtca gaatgaagcc tggcaagcca acaacttttg caacaatcgg caaatccacc 1500 gttgtctttg ctctacccgg taatcctgcc tcttcttctg tttgctacca tctatttgtc 1560 ctaccttgtt tgttgaaatg gcaaggactt gagccatcag ttggccaaaa attgcctaca 1620 gagccaattg ttaaggtcaa gcttgcagag gacttgaaac tggacccaca acgtcctgaa 1680 taccagcggg tcagcatttg tcagtcagac atggctctag ttgcgaactc tacagggttc 1740 Caaagaagca gcaacatcgg atcttttaag aaggccaacg gacttgtgtg tcttcctgct 1800 gccagtgact tcggaaaatc ggtaatcgaa gcagggacgg tggtggacgc cattctcatc 1860 gaccagatct atgtgtga 1878 <210> 6 <211> 1170 <212> DNA <213> Ogataea parapolymorpha <400> 6 atgtctttgt ccttaaatga gtaccttcgc tatgggcgac agctcatagt gcccgagttt 60 ggtctgcagg gccagatctc gctgaagaat tctcgtgtac tggtcgttgg agccggtggt 120 cttgggtgtc cagctttgca gtatcttgtt ggagctggtt ttggcacggt aggaatcgtg 180 gatcacgata ccgtcgatat ctcgaacctt catagacaaa tcttgcacac atcagaaaca 240 gtggagatgt tgaaatgcga gtctgcaaag ctccagcttg ccaaactcaa tcctttagtg 300 caaatcaata ctcatccggt ggctctcagt ccggataaca gctttggaat ttttgaacag 360 Pagina 4
SEQLTXT tatgatatca ttttggactg cacggacact cctgccaccc ggtatttgat caatgatact 420 gccgtgctac ttggtttgac ggttatttcc ggatctggtc tcaagaccga gggtcagtta 480 tccattctca atttcaacaa caccggtccc tgttatcgtt gtttctatcc aacaccgcca 540 cctcctagct ctgtgactgc ttgcagcgac ggaggagttc ttggcccagt tatcgggatt 600 atgggtgtga tgatggcact ggaagccatt aaggtggtta gtggctacta cttgagggag 660 gatgtggagt ttcaaccttt tttatcttta tacagcggtt atggcccgtt gcaaagctta 720 cggacgttca aaatgcgcag acgatcgccc aagtgccgag tgtgcaatgc cggaacgcga 780 gaaataacaa ggcacgtgat agaaacagag ctcgactatg ctgtgtggtg cggtaagatg 840 gactataatg ttcttgagaa ggatgaaaga gtatctgtgg agcagctttc tgcgcagaga 900 gcgccctatc tggtcgatgt gcgagccaaa gaacagtatt ccattgtcca tctgccaaat 960 tcgattaata taccactcaa cgcactaaaa cacatggaca ctctggatgt gccaaaggaa 1020 actatgatat acgttatttg ccgatttgga aatgactcgc agcttgcagt gaaacatttg 1080 aagagcatcg gatatgagaa ctgtgtggat gtcataggcg ggctaacgca atggtctcgg 1140 cagatagacc caaacttccc tatttactaa 1170 <210> 7 <211> 1374 <212> DNA <213> Ogataea parapolymorpha <400> 7 atgtacaggt tcaggatcgg agctggcgtg agacgctttt ccgtctctgc gctgagaagg 60 acatcgtcga gtggcgtcaa acaggcaatg cccacgtctg gggcccagcc gcctcaggga 120 tcatcaatat ctctcaagtc cgcaacaaga gatccttcgg agtatggcac aagaccgata 180 tacttggata tgcaggcaac aacgccgaca gacccacgtg tgttggacaa aatgctggaa 240 ttttatacgg ggctttatgg caacccgcac tcgtctaccc acgcctatgg atgggagacc 300 gataaggctg ttgaggaggc gcgcgcaaac gtcgctgccg tgatcggcgc agacccaaag 360 gagattattt tcacttcggg cgccaccgaa tgtaacaaca tggctctgaa gggcgtggct 420 cggttctacg gaaagagcaa gaagcacatc atcaccaccc agaccgagca caagtgcgtt 480 Pagina 5 ctggactcct gcagacatct gcaggatgag cocttoanns tgacatattt gccggtcaac 540 tcggacggtc tgatcgattt ggagcttttg gagaagtcta tcagaaaaga tacctgtttg 600 gtgtctgtta tggctgtgaa taacgagata ggagtgatcc agcctttgga ggatattggc 660 cgcatctgtc gctcgcacaa ggtgttcttc cacaccgacg ccgcccaggc gtacggcaag 720 ataccgatcg acgtcaacaa gtgcaacatt gacctgatgt cgatctcgtc acacaagata 780 tatggaccta tgggtgttgg tgccacgtac gtgcgaagac ggcctagagt cagactggac 840 ccaatcatca acggcggagg ccaagagaga ggtctgcggt ccggaacttt ggctcctccg 900 cttgtttgtg gctttggaga ggccgcaaga ctgatggtgc aagagtacga tgcagaccag 960 gcccacatca aggccctaag cacgaagctg atggatgccc tgttgtctat ggagcacaca 1020 caattgaacg gatcccgcat ccatagatac cctggctgcg ttaacgtttc ctttgcgtac 1080 atcgagggag agtctctttt gatggcattg aaggacattg ctctttcgtc aggatccgcc 1140 tgcacgtccg cgtctcttga gccttcgtac gttttgcatg ctctgggcgc cgacgacgcc 1200 cttgcacact catcgatccg gttcggcatc ggcagattca ccaccgaggc cgaggtcgac 1260 tacgtgatcc aggccctgac cgagagagtc aagttcttgc gggagctgag tccgctgtgg 1320 gagatggtca acgagggaat cgacttgaat tcgatcgaat gggcaggaca ttga 1374 <210> 8 <211> 1560 <212> DNA <213> Ogataea parapolymorpha <400> 8 atggccttgc aaaatgcttg gcaaaacaca aaagaaagag ctagagaaac ttgggctcaa 60 ttgacttggt ctgaagtttc tggttctttg ggtgatttgg gtactttttt gcctttgttg 120 atcggtttgg ttcaaaaggt tcatttggac ttgggtacta ctttgactat tactggcttg 180 tacaacatca tttccggttg gcaattcaga attccaatgt gtgttcaacc catgaagact 240 attgctgctg ttgctttggc tggtggtgct gctggtttgg atttgccaca attattgcat 300 gcaggtttgt ttgttgctgg ttgtgttggt ttgttgggtg cttctcaagc tattgatttg 360 ttcaattggt tggttccacc accagttatt agaggtgttc aattagctgt tggtgttaag 420 ttggctatga agggtgttga tatggctttg agattgcatg gtggtccatc ttcaggttgg 480 Pagina 6
SEQLTXT aggccatggt tgggtactga aggtttagtt gttggtgctg ttgcattagc tgctatgatt 540 gctactactt taccaccaag agctgctaga agaggtactt tggaagctgc tgatgaaggt 600 ggtttgggtc caagaccaac tgatactgca tttgaaccat tgctaagaag attgccagct 660 tgttgcggtg gtggtgatag agcaccacaa gttgaaggtg ctgcagtttc tgctgaaaga 720 gctggtttgt tagctcatgc cgaaggtggt gaaagatctg gtaatttgga tgatggaact 780 gaagctggtg ttggtgcagc tgctggtggc ggtggttgtg gtggtggcgg aggtggcggt 840 agaattccat ctgctttaat tgcagttgtt gttggtctag ctatggctgt tttacacaga 900 ccaggtttgg tttgggaatt gagattaggt ccaactttgc caagactgtt aagaccatct 960 tggccagatt ttaagactgg tgctttgaga ggtggtttac cacaattgcc attgactacc 1020 ttgaactccg ttattgctgt tactcaattg gctaatgctt tgttcggtga taagcctgaa 1080 gctgaaagac gtagatggcg tccatctgca gttgctttat ccgttgcttt gttgaatggt 1140 gccggtgttt ggttaggtgc tatgccatgt tgtcatggtg ctggcggttt agctgctcag 1200 tacaaatttg gtgctagaac tggtcatgct ccaattttgt taggttgtat taaggctgct 1260 ttgggtttgt tgtttggtgg ttcattggtt gttttgttgg aagcttttcc acagccatta 1320 ttgggtgcat tattgaccgt ttctggtatt gaattggcct ctgttgttag acatacaaga 1380 tctccaagag gttacacttt cgctttgtta acagcagttg ctattttggc tttggataac 1440 actggtactg gttttttggt tggcttggtt ggtgttgctg ccgttgcagc ttatgaaggt 1500 gccgttgctg ctgctgccgc tagatggcca agagtttttg ctagaggtgg tagagcttaa 1560 <210> 9 <211> 2580 <212> DNA <213> Ogataea parapolymorpha <400> 9 atggactctg ttgtcactga ggtgacctat ggtctggaga ttaagaaaat caaggagatc 60 acggagctgc cttttccagt caggcaagac tctcctttaa gcgaggtgct tcctacagat 120 ctgaagacca aagataattt cgtcgctaga gatcctgacc ttcttagact tactggttca 180 cacccattca attctgagcc accactggca aagctctacg attcggggtt tctcactcca 240 Pagina 7 gtgagtcttc attttgtgag aaatcacggc cccpttectt acgttcctga tgaaaatatt 300 ttggactggg aagtgtcgat tgaaggcatg gttgaaacgc cttataagat caaactgtca 360 gacataatgg accagttcga tatctataca accccagtta ctatggtctg tgctggaaac 420 agaagaaagg agcagaacat ggtgaaaaag gggaccggtt tcaattgggg agcagctgga 480 acatctactt ctctttggac cggatgcatg cttggagatg tgataggcaa agccagacca 540 tcaaagcggg caagatacat atggatggag ggtgcagata acccggcaaa tggcgcatac 600 ggcacctgtg tccgcctaag ctgggctatg gaccctgaac gatgcatcat gatggcatac 660 aaacaaaacg gcgagtggtt gcatcctgac catggaaagc cccttcgagt agtcatcccc 720 ggtgtcattg gtggacgatc agtcaaatgg ctaagaaagt tagtggtgag cgaccggccg 780 tctgaaaatt ggtatcatta ttttgataat cgggttcttc cgaccatggt gacgccagaa 840 atggccaaaa gtgatgacag gtggtggaaa gacgagcgat atgccatcta tgatctgaac 900 ttgcaaacta tcatttgcaa gcccgaaaat cagcaggtta tcaagatttc agacgacgag 960 tacgaaattg caggttttgg ctacaatgga ggtgggatca gaataggccg aatcgaaatc 1020 agtcttgaca aggggaagac ctggaagctg acagagatcg actatccgga agacagatat 1080 agggaggcag gttatttcag attgtttggc ggacttgtga acgtttgcga cagaatgagc 1140 tgtctgtgct ggtgtttctg gaagctcaag gttcctcttt ctgaattagc aacgtcaaaa 1200 gatattctcg tccgtggcat ggatgagcgt atggtggtcc agccgcgcac aatgtactgg 1260 aatgtaacgt ccatgttgaa caattggtgg tatcgagtcg ccattatccg cgagggtgac 1320 gctcttcgat ttgagcatcc agtggtggcc aacaagcctg gcggttggat ggatagggtc 1380 aaggcagaag gtggagatat tttagataat aattggggag aagtggacga tacggtcaag 1440 caggctgaaa ggaagccgcg tgttgatgag gatatcgaga tgatgtgcaa cccggagaaa 1500 atggacgtca ttatcaaata ctcagagttt gaagcacaca aggacagcga gacagaacca 1560 tggtttgctg tcaagggcca cgtatttgat ggtagctcgt atttggaaga ccatccagga 1620 ggtgcccagt cgatcttgat ggtgagcggc gaagacgcta ccgacgattt cattgcaatt 1680 cactcatctt atgccaaaaa gctgctccct cctatgcact tgggaagact cgaagaggtc 1740 agctctgtta caaaagtaaa gtctgtcgag gaaaatgtca agcgagaagt tttgctcgat 1800 Pagina 8 ccgcgaaaat ggcacaagat aacacttgcg il ttatctcttc cgactcgaga 1860 atattcaagt tcgaccttga gcatccagaa cagcttatcg gtcttccaac gggtaaacac 1920 ctgtttctga ggctaaaaga ttcatctggc aagtatgtga tgagggcata cacccctaaa 1980 tcgagcaatt ctttgcgggg tcgtctagag atattgataa aggtttattt cccaaatagg 2040 gaatacccca acggcggaat tatgacaaat cttatcgaaa acctccaagt gggaaaccag 2100 atcgaggtca aaggacctgt cggcgagttt gagtatgtca agtgcgggca ctgcagtttc 2160 aataacaagc cttatcaaat gaagcatttt gttatgatct cgggaggatc gggcatcacc 2220 ccaacttacc aggttctgca agctattttc agcgatcctg aagatacaac aagcgtgcag 2280 ttgttttttg gtaataagaa agttgacgat atcctgcttc gagaagagct tgactgtcta 2340 cagataaaac atccagaaca attcaaagtt gattactcgc tatcagatct gcatcatcta 2400 ccggagaatt ggagcggatt gaaaggcagg ttaacattca atattctgga cagctacgtt 2460 cagggaaaaa atatgggaga gtatatgcta ctggtatgtg gaccgccagg aatgaacggt 2520 gtggtcgaaa actggtgcaa agcgcgcaat ttggataaac agtatgtagt gtacttctga 2580 <210> 10 <211> 3135 <212> DNA <213> Ogataea parapolymorpha <400> 10 atgacttgtt ctgttcctcc cttgccagag gacatcactc ctccggccgc taagaagaag 60 cttgtcattg tgggcctggg catggtcggc ctgtcatttc tcgaaaaatt acttttgaac 120 gactcaaaac taaatgagta tagtatactg gtttatgggg aggagccata tcttgcttat 180 aaccgcgttg gtctcaccga gtatttccag caccgtgagt tcaaaaatct tcttctgtcg 240 ccagaggagt tctaccagct acgcggcgaa aaatggaact acgccattga tgagaaggtg 300 attgacatag atcggcaagc tcggactatc accacgaaca aaggaaacag ggcgtcttac 360 gatgtgcttg ttctttgcac aggctctacg gccatccttc ctaccgatct gctaccacct 420 cctactagaa aaagctaccg tgaaatggga tgctttgtct acagaaccat cgacgatctc 480 tattcaatga tagattattg ccagggtgca aagaaacaga gagccattgt cgttggtggt 540 gggcttctcg gtctagaagc agccaaagct ctctacgaca tggagtcgtt tgaggatatc 600 Pagina 9
SEQLTXT accattgtcc ataggtccca ttggctgctt tctcaacaaa tggaccagaa gggtggttct 660 ctacttacca gcaaggtgaa agagctgggg attacttgtc gaactggaac aactgtgtca 720 gagctgcttt ttgacgaaga ccaaaatctg acaggagtga aatacgataa cggtgaaatt 780 gaagaatgct cccttctttg ctacactatt ggtatcaaac caagagatga gcttacaagc 840 tgtggcctga atgcgggctc aaggggcgga tttaaagtga ataacatgtt gcaaacttcg 900 gatgaaaata tctatgcgat tggagagtgt gcttcttgga ataacatgac ttttggactg 960 attgctcccg gatatgaaat ggctgatata ctggccttca accttactca gggaaaactc 1020 caccagccaa aggagttttc tgaacctagt atgggcacca ggctcaagct gatgggtgtg 1080 gatgttgcat cttttggcga tttttttgca gacagaaatg ggcctaaatg gcttcctcgc 1140 ggatatgaga aagaagttcg tggccttgtg tttgaggatc caatagatgg aacttacaca 1200 aagttgcttt tcacaaagga tggcaagtac atgcttggag gaattctggt tggcgataca 1260 agcaactaca ccaagttctc cgcgatgata aggaaaccat tacccaagtc accatccgag 1320 cttctgattg gaaaagcggg agaagatgac atggagaagc tttcagatga gacccaaata 1380 tgctcctgtc acaatatcac caagggaaaa cttgtggaag ctgtgaaaaa tggttgcagc 1440 agtctcgcag atctgaaaaa gtgcactaag gcgggaacag cttgcggggg ttgcgagcct 1500 acagtgaagg tcatttttga aacagaggtt aaaaagttgg gtggcaaggt ctccaacaac 1560 ctatgcgtgc acttcgacta ttcaagagca gacttgttct cactgattat ggtcaaaaat 1620 ttccggtctt ttagaagggt catggaggag ctgggaaaca accccagctc gtcgggatgt 1680 gaaatttgca agcctaccat tggctccatt ctctcaacac tatacaaccg acacctcttg 1740 aaaaaggagg ttcatggtct gcaagacaca aatgaccgat accttggaaa catacagcga 1800 aacggcacct tctcggttgt tcctcggatg tcagccggcg aggtcactcc ggaaaagctt 1860 gtttctattg ggcaaattgc taagaagtac ggtgtctaca caaagatcac aggggcacaa 1920 agactggatt tgtttggcgt caagaagagc gatctgccga aaatatggaa agatctcaac 1980 gaagctggct tcgagagtgg acaggcttac ggcaagagtt tgagaaacgt caagtcctgc 2040 gttggatcaa catggtgtag atacggaatt ggagactctg ttggtttggc tgtcaggttg 2100 gaagaacggt acaaaggcat acgctcgcct cataaaatga aggggggcgt gtctgggtgt 2160 Pagina 10
SEQLTXT gttagagact gtgccgagtt ccactcaaag gactttggtc tctgcgctgt gaaggatgga 2220 ttcaacatat atgttggtgg aaatggtgga atgaagccag cgcacgcgca gcttcttgct 2280 accaatgtgc gtcctgacga ggtgatccct attcttgaca gatacctaat gttttacatt 2340 accacggcag atagactgca gcggacagct cgatggcttg aaaaccttga tggtggaatc 2400 gaatatttga aggacgttat catccgcgat aaattgggga tttgcaagga tctggaagcc 2460 caaatgcgcc agcttgtagc cggttactac gatgagtggg ccaaggccgt ttctgaagag 2520 aaagacaacc caattttcaa gcaatttgtg aatacatctg agaaccaaga tactgtggag 2580 atagtcaaag aaagaggcca accgagaccg gcaatgtggc cggagaaagc ggccaaccag 2640 aaattcaatg agatcaagtg gtcttctgtg agctggcagg aagtctgtga gagctcagat 2700 ttgcctctag cagaggcagg gtcctcggca acggttttgg ttggggacac ccaaattgct 2760 ctgttcagaa ctagcgaaaa cgagctctat tgctctcaga acatgtgcgg acataaacgt 2820 gcctttgtgc taaatcaggg acttctttcg gaagatggtg acaaaaactg ctacatatcg 2880 tgtccgatgc ataaacgaaa tttttacctc aagtccggtg cctgcaaaaa cgatgaagcg 2940 ctatctatcg cgacgtttga ggttaaggag gagaatggca aagtgtatgc caaacttccg 3000 cccacaacag agctggatga ggtcctggga acttctaaat ggaaggtgac aagtcaggaa 3060 acagaggcta aacaggtgcg caagataaca acggagaaaa atttagttga caaagcaata 3120 tcgttcgact ggtaa 3135 <210> 11 <211> 1527 <212> DNA <213> Ogataea parapolymorpha <400> 11 atgcgacttt ctaccttatg ggaaccgcca acggtgaatc caagaaacct gaaagcgacc 60 tcgataccaa tttttaacct gtggaacgtc tatggaagaa acttcttttt cgcgtggttt 120 gggttttttg tgtgctttct ttcctggttt gcttttccgc ctcttcttca tggtatgcta 180 aagaaggacc taaagctcac cgcagtggat atatccaata ataacatatg tggactgacc 240 ggaactttac taggcagatt tattttgggg ccccttaacg acaagtatgg ccctcggatt 300 Pagina 11 actttggtag gcgtgctggt tgcaggagca ttceeactg catttgttcc tttggttaca 360 aatgttgcag gtctacatgc catccgtttc tttatcagct tcctaggctc ctcgtttatt 420 tgctgctccc aattctgcgc tgtatttttt gataacaata ttataggaac agcaaatgcc 480 gtctctgccg gttggggaaa tgctggaggg ggtgtggcat tctttgtcat gcctgccatt 540 tcaaatgcat tagaaaaaag aggttactct ttacaccatt cgtggagcta ctcttttgtg 600 attgggccgt tcttgattct gatgatcaca gcaattgtga tttttgtatt tggcagcgac 660 tgcccgagag gcagatggtc ccttcgtgga gatatccttg gaatcaacat ggataatatg 720 ctcgtgaagt ctgtctctgt cacaagacac ttctctaagg atggagaact cacttctgtc 780 tgtgttgagc ctgttaacgc aatcgatgag gttgtggttg agccaaatca ggaccaggaa 840 atttttgaag tcgcagacat cataaatgag gacgaaatca tcgaagaccc aactctcaaa 900 gacgtggtca agatctgttt ttccccacgc acaatgctgg tcggactttg ctacatgtgc 960 tcgtttggta ctgagcttgc agtggagtct attatatcca acctattcgg gcaaaagatg 1020 acaaagtgga gcacctctaa agctggagca tggggctcaa tgcttggact tctcaacgtg 1080 gtgacaagac cagccggagg aattatctcc gatttcctat accaaagatt caagaccacc 1140 aaggctaaaa agttctggat gatattcact ggcttgatgc agggaatttt tttgatctgg 1200 gttggactgg ttccggaatt gtccatcgcg ggactcatag tgtcggtttc gtttttgtgt 1260 ctttggtttg agatgggaaa cggtgcaaat tatgcctgtg ttccggttgt gaatagacac 1320 cacagtggta ttgtgagcgg agttacggga gcaatgggta acctaggagg cattctgttt 1380 agtttagtgt tcaggtacac tatagcaaat ggagtgaaca actacttcaa ggcgttctgg 1440 atcataggaa ttgtttgcac tgttgtgaat ttggcctgtg tgcttattcc aattagggag 1500 gagaggccta ggaaagcgga aatttga 1527 <210> 12 <211> 585 <212> DNA <213> Ogataea parapolymorpha <400> 12 atgaatttga tgactaccat taccggtgtt gttttggctg gtggtaaagc tagaagaatg 60 ggtggtgttg ataagggttt gttggaattg aatggtaagc cattgtggca acacgttgct 120 Pagina 12
SEQLTXT gatgctttaa tgactcaatt gtcccacgtt gttgttaacg ctaacagaca tcaagaaatc 180 taccaagctt ctggtttgaa ggtcatcgaa gattctttgg ctgattatcc aggtccattg 240 gctggtatgt tgtctgttat gcaacaagag gctggtgaat ggtttttgtt ttgtccatgt 300 gatacccctt acatcccacc agatttggct gctagattga atcatcaaag aaaggatgct 360 ccagttgttt gggttcatga tggtgaaaga gatcatccaa ctattgcctt ggttaacaga 420 gctattgaac ccttgttgtt ggagtacttg caagctggtg agagaagagt tatggttttt 480 atgagattag ctggtggtca tgccgttgat ttttctgatc ataaggatgc ctttgtcaac 540 gttaacactc ctgaagaatt ggctagatgg caagaaaaga gatga 585 <210> 13 <211> 528 <212> DNA <213> Ogataea parapolymorpha <400> 13 atggctggta aaactatgat tcctttgttg gcttttgctg cttggtctgg tacaggtaaa 60 acaactttgt tgaagaagtt gattccagct ttgtgcgcta gaggtattag accaggttta 120 atcaaacata cccaccacga tatggatgtt gataagccag gtaaagactc ctacgaattg 180 agaaaagctg gtgctgctca aactatcgtt gcttcacaac aaagatgggc tttgatgact 240 gaaactccag atgaagaaga attggacttg caattcttgg cctctagaat ggatacttcc 300 aagttggatt tgatcttggt cgaaggtttt aagcacgaag aaattgccaa gatcgtcttg 360 tttagagatg gtgctggtca tagaccagaa gagttggtta ttgatagaca tgttattgcc 420 gttgcctctg atgttccatt gaatttggat gttgctctgt tggatatcaa cgacgttgaa 480 ggtttggctg attttgttgt tgaatggatg caaaagcaga acggctaa 528 <210> 14 <211> 2601 <212> DNA <213> Ogataea parapolymorpha <400> 14 atgactgaaa ctagaactac ttgtccatac tgtggtgttg gttgtggtgt tattgcttct 60 agagcaccac atggtcaagt ttctgttaga ggtgatgaac aacatccagc taatttcggt 120 Pagina 13
SEQLTXT agattgtgtg ttaagggtgc tgctttgggt gaaactgttg gtttggaagg tagaatgttg 180 ttccctgaag ttgatggtga aagagctact tggccacaag ctttggctgc tgctggttct 240 agattgagag aaattattga tagacatggt ccacaagctg ttgcttttta tgcttctggt 300 caactgttga ctgaagatta ttacgctgct aacaagttga tgaagggttt tattggtgct 360 gccaacattg ataccaactc tagattatgt atgtcctctg ctgttactgg ttacaaacgt 420 gctttaggtg ctgatgttgt accatgttct tacgaagatg ttgagaactc cgatttggtt 480 gttttggttg gttctaatgc tgcttgggct catccagtct tgtatcaaag attggctcaa 540 gctaaaaggg acaacccaca aatgagagtt gttgttattg atcctagaag aaccgctacc 600 tgtgatattg cagatagaca tttggctttg gctccaggtt ctgatggtgg tttgtttgtt 660 ggtttattga acgctattgc tgcctctggt gctatttctg atgattttaa tgatgcccaa 720 agggccttga ctattgctca agattgggac ttagataagg ttgctcaatt ttgtggtttg 780 ccaagacaac aaattgccga tttctacaga gaattcattg ctgctccaag agctattacc 840 ttgtacacta tgggtatcaa tcaatccgct tctggttctg ataagtgcaa cgccattatt 900 aacgttcatt tggcctgtgg taaatacggt agaccaggtt gtggtccatt ttctttgact 960 ggtcaaccta atgctatggg tggtagagaa gttggtggtt tagctactat gttggctgct 1020 catatgaatt tcgaaccaga tgacttgaga agattggcaa gattttgggg ttctgaaaga 1080 ttagcacaaa ctccaggttt aactggtgtt gaattatttg ctgctatcgg tagaggtgaa 1140 gttaaggctg tttggattat gggtactaat ccagttgttt ccttgccaga ttctcatgct 1200 gtttctgaag ctttagctag atgtccattg gttatcatct cagatgttgt tgctgatact 1260 gatactggta gattcgccca tattagattt ccagctttgg catggggtga aaaatctggt 1320 actgttacca attccgaaag aagaatctct agacaaaggg cttttatgcc accaccaggt 1380 gaagctagag ctgattggtg gatagttgct agagttgctg aagccttagg ttttggttct 1440 gcttttgctt ggcaacatcc acatgaagtt ttttctgaac atgctgcatt gtccggttac 1500 gaaaatgacg gtcaaagggc atttgatata ggtggtttgg ctgatttgtc aagagaagct 1560 tgggatgctt tggaaccagt tagatggcca gtttctagat ctgaagctgc ttggtctgtt 1620 cacaaaggtt ggcatagaga tggtaaattg agaatggttc cagttgctcc acaacctact 1680 Pagina 14
SEQLTXT agagctacta ctgatgcttt ttacccactg attttgaact ccggtagaat cagagatcaa 1740 tggcatacaa tgactagaac tggtgctgtt ccaagattga tgcaacatat taacgaacca 1800 gttgttgaag ttgctcctgc tgatgctcaa agatatcatt tgttagaagg tgaattggcc 1860 agagtcagat ctccaaaagg tgttatggtt gctaaggtta ctattggtga tggtcaaaga 1920 cctggttctt tgttcgttcc tatgcactgg aacaatcaat ttgctagaca aggtagagtc 1980 aacaacttat tggctgcagt tactgatcca cattcaggtc aaccagaatc taaacaaact 2040 gctgttgcta ttgctacttg gttgccagct tggaagggtg aattattctc aagacaacca 2100 gttccattac cagcttcatt gcattggaga agaagggctg ctcaaggtat tattcatttg 2160 tctttagctg gtgacaccag atcaagagat tggttagttg aatggtgtca aagacaagga 2220 tggcaaatgc aagttgcaga aggtggtaaa gtttggaatt tgttagcttg gagagctggt 2280 gaattgatgt taggttggtg gtctgatgct tctgaaccag ctattgatgc tgattggatt 2340 catgctgctt ttagagttcc acctcaaaat gctgctagaa ggcatgcttt gttatctggt 2400 agaaaaggtg gtgtagaaat gccaagaggt agaattatct gttcctgttt ctctgttggt 2460 gaacgtgcaa ttggtgaagc cattgctggt ggttgtagaa caccaggtgc attaggtggt 2520 aagttgaaat gtggtactaa ttgcggttct tgcatcccag aattgaaagc tttattggca 2580 gctaaattgg cccaagccta a 2601 <210> 15 <211> 1197 <212> DNA <213> Ogataea parapolymorpha <400> 15 atgactagac aattggttat tatcggtaac ggtatggctg ctactccagg tgaagctttg 60 gttgaaagag atgctagaag attctccatt accattatcg gtgatgaacc tagacaagcc 120 tacaacagaa ttcaattgtc tccagttttg ggtgctgaaa aaactgctgg tgctactaga 180 ttattgccag ctgaatggta ctctcaacat aacgttacag ttagagctgg tgaaactgtt 240 actgctgttg atatggctgc aagaacattg caaactaccg ctggtgaatt aggttgggat 300 gaattggttt ttgctactgg ttctttgcca tttttgccac cattgccagg tattactttg 360 Pagina 15 ccacatgttt ttgctttcag aaccttggct gate gaon gtattttggc tattgatggt 420 ccagctgttg ttattggtgg tggtgtttta ggtgttgaag ctgctgctgc tttgagaagg 480 catggtgatt ctgttacttt gttgcataga ggttcttggt tgatggaaca acaaactgat 540 gctttcgttg gtgaacaatt gcaaatgttg ttggctgaaa gaggtattgg ttgcgttatg 600 gaatctagaa ttgctgccat tgatgaacac caagtcttgt tggaagatgg tagagctttt 660 gctgcttcta gagttgtttt ggctacaggt gttcaaccta acaaaagatt ggcagaaaga 720 tctggtttgg cttgtggtag aggtatttta gttgatagaa gattggctac tgctcaacca 780 ggtgtttctg ctttgggtga atgttgtgaa atagatggtc aaacctgggg tttagttgct 840 ccatgtttga gacaagctga agttttggct gatagattgt gtgctattcc tggtgaagat 900 ttcagatggc aagattctgg tactagattg aaggttactg acatcgaatt attctctgcc 960 ggtgaattga gagctgatga acaagatgat gtttacactt cttgggatcc attggataga 1020 cattacagaa ggttgttgtt gagagatggt aaattgagag gtgtcttgtt gttgggtgac 1080 tgttcttctg ctgctccatt gactgctttg ttgggtagac atggtccaag aaggcaatct 1140 ggtttttcta cacacttgca aagatgttct agagccttgt gggataagtt gagatga 1197 <210> 16 <211> 2235 <212> DNA <213> Ogataea parapolymorpha <400> 16 atgggtgttg aaccatttgc tcatgatcca gctccatctg aattgattca ttctgttcca 60 gcttgtggtt ctcctgagag aagagttatg aggccaatgg ttagagaagg ttggttggct 120 gatagacaac attctgatag aagaggtaga ggtagggaaa gatttttgcc agtttcttgg 180 gatgctgctt tggatttggt tgctggtgaa attagaagag tttctgctga tcatggtaac 240 gctgctattt ttgctggttc ttatggttgg acttcttgcg gtagatttca tcatgcttct 300 accttgttga agaggatgtt gaacttggtt ggtggtttta ctggtcatgt tgatacctat 360 tctattgctg ctggtccagt tattttgaga catactttgg gtgatgatag agcatgtggt 420 ggtcaagcta ataccttgga ttcaattgct gaacactctc aaaccttggt tgtttttggt 480 gctatgtctc caagaactgc tcaatctgaa gctggtggta ttggtgctca tcatttggaa 540 Pagina 16
SEQLTXT acttacttga gaagaatcgt cgaaagaggt gttagagtca ttttggtttc tccattgaag 600 gatgatttgc cagattgggt tgctgcagaa tggtggccaa ttagaccaaa tactgatacc 660 gctttgatgt tgggtttagc cggtgaaata gttagatctg gtagacaaga ctctgatttc 720 ttggctagat gtacttctgg ttctgaatta tacttggcct acttgagagg tgaaggtgat 780 ggtagaccaa aagatgctga atgggcttct actattactg gtttgccagc tgaagctatt 840 agagctttgg ctggtgattt gcctagaact agatctatgt tgactgtctc ttggtcttta 900 caaagggctc atcatggtga acaaccattt tgggctgctt taggtttagc tgctgttatt 960 ggtcaaattg gtagacctgg tggtggtgtt ggttatggtt acggttcttt aggtggtgta 1020 ggtgctcctt ttactattgg taaatctcca gctatgtccc aattgtctaa gccaatcaat 1080 tccttcattc cagttgccag aatctccgat atgttgttga atccaggtgg tccatattct 1140 tacgaaggtg aagatagaag atacccagat atcagattgg tgtattggtc tggtggtaat 1200 ccattccatc atcaccaaga tttgaacaga ttgtctgaag cttggactag accagaaacc 1260 attatagttc aagatccaat gttcactgct accgctaaaa gagctgatat agttttacca 1320 gcctccacct ctattgaaag aaatgatttg gcaggtaaca agaggtccga tttcattttg 1380 gctatgggtc aagccattgc tccattgggt gaagctagat ctgatttcga tattttcaac 1440 gccttgtctg gtaaattggg tgttgctgct gcttttaatg aaggtagaga tgagatgggt 1500 tggatcagac acttgtatga agaatctaga aaccacgctc aaagacatca tcactttgaa 1560 atgccagatt tcgaaacttt ttgggctcaa ggtcatgctc catgtccagt tcaaagagat 1620 catacctatt tggctgcttt cagagaagat ccaggtgcac atccattgga tactgaatct 1680 ggtttgatag ttttgggttc tgctacttta gccagattag gttatgctga ttgtggtcca 1740 catccagctt ggattgaacc agctgaatgg ttgggtaaag ctcaagctgg tgaattgcat 1800 ttgatttctc atcaacctaa gggcagattg cactctcaat tggaaactgc tgaagcttct 1860 ttagctggta aacgtgaagg tcgtgatgaa gttatgttgc atccagatga tgcttccgtt 1920 agaggtattg cagatggtca aactgttaga ttgtggaatg ctagaggtgc ttgtttggct 1980 actgctcaag ttactgattc agttgcagct ggtgttgcta ttttgccaac tggtgcttgg 2040 tttactccag cagaagctga aggtccagaa ttgtcaggta atccaaatgt tttgaccttg 2100 Pagina 17
SEQLTXT gacattggtt cctctgcttt tggtcaaggt tgttctgctc atacttgctt ggttagaatt 2160 gaagcacatg ctggtgatgc cggtgatgct gttagaatct atgatgctca tttggcagca 2220 atcttgccaa cttaa 2235 <210> 17 <211> 2877 <212> DNA <213> Ogataea parapolymorpha <400> 17 atgaaggatt tgattatccc accattggat tggactcaag atatgggtac tccaaaaaga 60 gaaggcgctc cagttcattt gactattgat ggtgttgaag ttactgttcc agctggtact 120 tctgttttga gagctgctgc tgaagctggt atttctattc caaaattgtg tgccaccgat 180 aacgttgaac cagttggttc ttgtagattg tgcatggttg aaatcgaagg tatgagaggt 240 actcctactt cttgtactac tccagttgct ccaggtatga gggttcatac tcaaactcca 300 caattgcaaa agttgagaag aggtgttatg gagttgtaca tctctgatca tccattggac 360 tgtttgactt gtgctgctaa tggtgattgt gaattgcaag acatggctgg tgctgttggt 420 ttgagagaag ttagatatca agctaaggat acccatttcg ctagaagaga tgctactggt 480 ccaaatccaa gatatatccc aaaggataac agcaacccat acttctctta tgatccagct 540 aagtgtattg tctgcatgag atgtgttaga gcttgcgaag aagttcaagg tacttttgct 600 ttgactgtta tgggtagagg tttcgatgct agaatttcac cagctgctcc agattttttg 660 tcctctgatt gtgtttcttg tggtgcttgt gttcaagctt gtccaactgc tactttggtt 720 gaaaagtccg ttgaaagaat tggtactcct gaaagaaagg ttgttactac ctgtgcttac 780 tgtggtgttg gttgttcttt tgaagctcat atgttgggtg atcagttggt tagaatggtt 840 ccttggaaag gtggtgctgc aaatagaggt cattcttgtg ttaagggtag attcgcttat 900 ggttacgcta ctcatcaaga cagaattttg aagccaatga tcagggataa gattaccgat 960 ccttggagag aagtaaattg gactgaagct ttggatttca ctgctactag attgagagct 1020 ttgagagatt ctcatggtgc tgatgctttg ggtgttatta cttcttctag atgcactaac 1080 gaggaaacct atttggttca aaaattggct agagccgttt tcggtacaaa caacactgat 1140 Pagina 18 acttgtgcta gagtttgtca ttctccaaca vette cagtt tgaagcaaac ttttggtaca 1200 tctgctggta ctcaagattt cgattctgtt gaagaaaccg atttggcctt ggttattggt 1260 gctaatccaa cagatggtca tccagttttt gcctccagat tgagaaaaag attaagagct 1320 ggtgccaagt tgatcgttgt tgatccaaga agaattgact tgttgaacac tccacataga 1380 ggtgaagctt ggcacttgca attgaaacca ggtactaatg ttgcagttat gactgctatg 1440 gctcatgtta tcgttaccga acaaattttc gacaagagat tcatcggtga tagatgcgat 1500 tgggatgaat gggctgatta tgctgaattt gttgctaacc cagaatatgc tccagaagct 1560 gttgaatctt tgactggtgt tccagcaggt ttgttgagac aagctgctag agcttatgct 1620 gctgctccaa atgctgctat ctattatggt ttaggtgtca ccgaacattc tcaaggttct 1680 actactgtta ttgccattgc taacttggct atgatgactg gtaatattgg tagaccaggt 1740 gttggtgtta atcctttaag aggtcaaaac aacgtccaag gttcttgtga tatgggttct 1800 tttccacatg aattcccagg ttacagacac gtttctgatg atgctacaag aggtttgttt 1860 gaaagaacat ggggtgttac cttatcttct gaaccaggtt tgagaatccc aaacatgttg 1920 gatgctgctg ttgaaggtag attcaaagcc ttgtatgttc agggtgaaga tatcctacaa 1980 tctgatccag ataccagaca tgtttcagct ggtttggctg ctatggattt ggttatagtt 2040 cacgacttgt tcttgaacga aactgctaat tacgcccatg tttttttgcc aggttctacc 2100 tttttggaaa aggatggtac tttcaccaat gccgaaagaa gaatcaacag agttagaaga 2160 gttatggctc caaaagcagg ttttgctgat tgggaagtta ctcaaatgtt ggctaatgct 2220 ttaggtgctg gttggcatta tactcatcca tctgaaatta tggctgaaat tgctgcaact 2280 actcctggtt ttgccgctgt tacttacgaa atgttagatg ctagaggttc tgttcaatgg 2340 ccatgtaacg aaaaagcacc tgaaggttct ccaatcatgc acgttgaagg ttttgttaga 2400 ggtaagggca gattcattag aactgcttat ttgccaactg acgaaaaaac cggtccaaga 2460 tttcctttgt tgttgaccac tggtagaatc ttgtctcagt ataatgttgg tgctcaaact 2520 agaagaaccg aaaacactgt ttggcatggt gaggatagat tggaaattca tccaactgat 2580 gctgaaacca gaggtattag agatggtgac tgggttagat tggcttctag agccggtgaa 2640 actactttaa gagctactgt tactgataga gtttctccag gtgtagttta cactactttc 2700 Pagina 19 catcatcctg atactcaagc caacgttgtt acancagata cttctgattg ggctactaat 2760 tgtccagagt acaaagttac tgctgttcaa gttgctgctt ctaatggtcc atctgattgg 2820 caacaagatt acgctgctca agctgccgct gctagaagaa tagaagctgc agaatga 2877 <210> 18 <211> 1503 <212> DNA <213> Ogataea parapolymorpha <400> 18 atgaagattt ggttgccatg tgatgctgct gctaaagctt gtggtgctga agctgttttg 60 gctgctttga gattggaagc tgaaaaaaga ggtggcgctt tggatattgc tagaaatggt 120 tctagaggta tgatctggtt ggaacctttg ttggaagttg aaactccagc tggtagaatt 180 ggttttggtc caatgacacc agctgatgtt ccagctttgt ttgatgcttt ggaatctcat 240 ccaaaggcct tgggtttagt tgaagaaatt ccattcttca agagacagac cagattgact 300 tttgctagat gtggtagaat cgagccattg tctttggctc aatttgctgc tgcagaaggt 360 tgggctggtt tgagaaaagc tttgaaaatg actccagccg aagttgttga agaagttttg 420 gcttctggtt taagaggtag aggtggtgct ggttttccaa ctggtattaa gtggcgtact 480 gttgctgcag ctcaagctga tcaaaagtac attgtctgta acgttgatga aggtgactct 540 ggttcttttg ctgatagaat gttaatcgaa ggtgacccat tctgtttggt tgaaggtatg 600 gctattgctg gtcatgctgt tggtgctact agaggttatg tttacatcag atcagaatac 660 ccagatgcca ttgctgttat gagagctgct attgctatgg ctaaaccatt tttggctgaa 720 gccggttttg aaatggaagt tagagttggt gccggtgctt atgtttgtgg tgaagaaact 780 tctctgttga actctttgga aggtaagaga ggtactgtta gagctaaacc accattgcca 840 gctttgaagg gtttgtttgg taaacctact gtcgtgaaca acttgttgtc tttagctgct 900 gttccatgga ttattgctca tggtgcaaaa gcttacgaat cctttggtat ggatagatcc 960 agaggtacta ttccattgca aattggtggt aatgtaaaga gaggtggttt gttcgaaact 1020 ggtttcggta ttactttggg tgaattggtc gaagatattt gtggtggtac tgcttctggt 1080 agaccagtta aggctgttca agttggtggt ccattgggtg cttatcatcc agtttctgat 1140 taccatttgc cattctgcta cgaacaattc gctggtcaag gtggtctagt tggtcatgca 1200 Pagina 20
SEQLTXT ggtttggttg ttcatgatga tacagctgat atgttgaagt tggctagatt cgctatggaa 1260 ttctgcgcta ttgaatcttg tggtacttgt accccatgta gaataggtgc agttagaggt 1320 gttgaagtca ttgatagaat tgctgctggt gatgcttcag ctatgccatt attggatgat 1380 ttgtgtcaga ctatgaagtt gggttctttg tgtgctttag gtggttttac tccatatcca 1440 gttcaatccg ctattagaca ttttccagca gattttccat gtgctagaga agctgctgaa 1500 tga 1503 <210> 19 <211> 453 <212> DNA <213> Ogataea parapolymorpha <400> 19 atgactgata ctgctagatt gagagctatt ttggctgctc atagaggtag agaaggtgct 60 ttgttgccaa tattgcatga tgttcaagct gcctttggtt tcattccaga agatgcttac 120 gctccaattg ctgctgattt gggtttgact agagctgaag ttgctggtgt tgttggtttt 180 taccatgatt ttagaaaagc tccagctggt agacacgtta ttaagttgtg tagagccgaa 240 gcttgtcaag ctatgggtat ggatgctgtt caagcaagat tggaatctgc tttgggttta 300 agattgggtg attcttctga agctgttacc ttggaagctg tttactgttt aggtttgtgt 360 gcttgtgctc cagcagctat ggttgatgat agattggttg gtagattgga tgctgctgct 420 gttgctggta tagttgctga attgggtgct taa 453 <210> 20 <211> 216 <212> DNA <213> Ogataea parapolymorpha <400> 20 atgtctgatg ataagattat cagaatggcc aatcaaattg ctgctttctt tgctgttcaa 60 ccaggtgata gagctggtcc agttgctgct catatttctg aaaattggtc tgctccaatg 120 agagctgctt tgttggctca tgttgctgca caatctccag gtttggatcc attggttatt 180 gctgctgctc cacaaattag accagttcca gcttaa 216 Pagina 21
SEQLTXT <210> 21 <211> 768 <212> DNA <213> Ogataea parapolymorpha <400> 21 atgtctttgc cagctggtgc tgttactgtt ccattaccag gtggtgctag agctgttttg 60 gctgaagaag ttccagttgc tttggttttt gatggtgtta ctcaagctgt tatgatggct 120 tctcctgttg atttggaaga tttcttgttg ggtttcgctt tgaccgaagg tatgattgct 180 gatagagctg aattattgag acacgaagtt gtcagacaac cacaaggtat tgaattgaga 240 ggttggttgg ctgctccagc aggtcaaaga tttgctgcta gacgtagagc tatggctggt 300 ccagttggtt gcggtttgtg tggtttggat tctttggctg ctgttttaag accattgcca 360 agagcaccaa gaggtggtgc cccaccacca ttggctgatg gtgctttagc tgctttgaga 420 gctggtcaat ctttacaaga tgctgttaga tctgttcatg ctgctggttt ttgggatggt 480 gctcaaatga gagctttaag agaagatgtt ggtagacata acgccttgga taagttggct 540 ggtgcattgg ctggtcaagg tatagatgca gctgctggtg ccttggtttt gacttctaga 600 ttgtctgtag atttggttca aaaggctgct atgattggtg ccagagtttt gattgctcca 660 tctgctccaa ctgctttggc tgttgctgaa gctcaagctg caggtttggc tttaattgct 720 agaggtccag atggtccaac attatacact gaaactgaag ccgaatga 768 <210> 22 <211> 2013 <212> DNA <213> Ogataea parapolymorpha <400> 22 atgactgttt ctgagttcta cccgctacca gtttcagaga taagagagaa gtactacccc 60 aatttggcca atcaaacata tctagatcat gctggaacaa ctgtgtactc atcactcaca 120 ttagataaaa tccatgaggt attatccaaa acgcttcttg caaatccgca ctccctatct 180 tcagcatcac gcgatactgc cagtcttgtc gaggaaacca gatacaagat tttaagcata 240 tttcacgccg atccggcgga atatgacatt gtcttttctc taaatgcaac acacgcaata 300 aagattgctg cctcccttat ccaggatgcc gcagaaagtt cattcaacta ttattacaac 360 attaactgtc acacttcgtt gattgggttg cgaactttag ccgccaaata cgcaactttt 420 Pagina 22
SEQLTXT gacgatatat ccagttttga gccagttgaa gacaaagatg gcaaccatcc agcacttaac 480 tttgtctcgt ggacggggca gtcgaatttc aacggccaga agtttcctct tggctggtgt 540 aaggagttta gaaggcgatt ggatcattgc tacacacttt atgatgcctc tgcattgtca 600 acatccgatc ctcccgactt aagtgatgca aacagctcgc cagactttgt ggtgatgtca 660 ttctacaaga tttttgggat gccagatata ggagcgctca ttttaagacg ttcaactgca 720 aaacagctcg ttgaaaaaag aagatacttt ggtggaggca caattgatgc tctcacgatt 780 gaggagccgt tttgccgccg gagtaagcaa ttgcatcaaa gcctagaaga cggaacgatt 840 ccggtacatg caattttgga attgtccgtg gcgattgatt cgcattatca gattttcggg 900 tcttttaaca gcattcgttt gcacacggat gaaatcagaa aatacgccat ttgcaagttg 960 aagcagctga aatatggcaa cactgggcgc agaatgctgc agatttatga ttggccaggt 1020 gccaagcacg gccctattat tgcattttcg cttttatcgc cagcaggaga cccgattggg 1080 tattatggct ttgggaagct tgcctcagct cgaaatattt cgctaagaac gggaacattg 1140 tgcaatatag gcggaattca gaagtttctc gatcgcacaa atgaggatat aaggcaagac 1200 tacgaaaaag ggcacaagtg cggtgatata ttggatataa tcgatggaaa accaaccgaa 1260 cttgtagtta aaagcttgac ggtatatcca ataaaatcgt gtccaggtta tcgaataccg 1320 gaaggccgaa aatggaaatt gaccaagcac ggtttcgagt tcgatcgcag ttttgtgctt 1380 ctcgatttgc tgacgcaaaa gcctcttttg ctaaagaata atcccagaat ggcactttta 1440 gattgtcgtg tggatccaga aaagcatatg ctctatgtga gagataaaag aggcggtaat 1500 aagctttggg tcagtaccaa catccggagg tacaaaacca agcagatggg agatttcata 1560 gcgatttctg aaagaaagat ggtcaaattt ttcagtgatg tcatgagcat aggctgcact 1620 ttggctgggt ttgttactga gaagcagatg cagaataaaa ctgctttctt actagtaaat 1680 gagagaagta tgcgacaggt ttcgaaagat gattctctca tttcaaggtt cagagccaac 1740 attgttgtcg attcggcaca tccttacatt gaggacaagt tgtccgttct aacagacatg 1800 gacagcggag ttgtgctcaa gaaaagatgc aaatgtgaca gatgctacat gattacagtg 1860 tctgataagg ggtcacgtga tccgtcgttg cttgttgagc tttcaaagga gcgaaaacag 1920 aagggaaaag tctattttgg ggtgaatatc gatgttgaga atgttggtta caggtacatg 1980 Pagina 23
SEQLTXT agagttgggg atcgcattgt tggagaggag tga 2013 <210> 23 <211> 4266 <212> DNA <213> Ogataea parapolymorpha <400> 23 atggcaccaa tagcggtcga acccagccca tatgatggca cagcaaacaa tgatcttaaa 60 gatattcaat tcacagatag cataagattt tacctcaacg ataaactcca ggtggtgaaa 120 aatccagatc cggaggagcg actcattgac tacatcagaa atgaggctga tctcagaggc 180 acaaaagaag catgttcgga atcgggatgt aatgcttgct ctgttaccat tgcatccatt 240 tcatacacag acacggatta tccagagcgc ccacaggttc actatcggtc cgtaaattca 300 tgtgttactc ctttgatact tgcagatggc aagcaagtga tcacagttga aggtgttggc 360 tcgtctaaaa acccacatcc agttcaggag agaattgcta aattccacgg ttcacagtgt 420 ggattttgca cacctggttt tgtgatgtct ttgtatgcgt tgctaaggga gaaaaatggc 480 catgtgtcgg ttgcagaaat cgatgaagca ttggaatcga atttgtgcag atgcacgggc 540 tacatgccaa tctatgatgc ggcatattcg tttgcgtacg attctgataa ttacaacagg 600 gagaagatca gacctttcct caagaagaag gacacgagtt ttgagactgg aagtgacttg 660 tatggcggtt cagtgtgtgc gttaggaacc aaatgctgca gatacaagtc tggaaaagag 720 aaagctgacg aagaatgtga taagtctgca tccaattcgg acatggagat cgatatgaac 780 aaaatattca caccaaacgg tctgccattg aagccataca atccggcagc agatttgccg 840 ttccctctta aactttccag gattagccct aagccaattt gctacggaaa tgagcgaaag 900 gtgtggtttc gccccgtgac taaagaacaa tttctacaga tatacagaat ctatccggat 960 gcgaaaattg tcgccggagc atcggaggtt cagatcgagg tgaagttcaa ggctgccaat 1020 tataaggtga atatttatgc tggggacgtc aaggaactca aaggatggtc atacaagaag 1080 gggaagggtt taacaattgg aggtgatatt ccattgatcg agttagaatc tatatgcggt 1140 gatttagcaa aacggttggg tagaactgca gctggacaga catacaacgc aattgaggag 1200 cagttgaagg tctttgcttc taaagctgtt cgtaatgtgg ccactccggc aggtaatatc 1260 Pagina 24 gtgacagcat cgccaattgc ggatctaaat ccaatetttg tggcttgtgg tgcaatcata 1320 acggcggaaa aactgactga ggatggtaaa ctagaaaaga cgcacattga tatgcgtgac 1380 aacttcttta ctgggtacag gcggcacaag ttacctacat catcgttgat taccgagatc 1440 ttcattccag atactgcaga taacgaatac atccactgct ataagcagtg taagcggaaa 1500 gatgacgata tctccattgt cactgcctgc ttgagaatgg agctagatga cgaaggaaat 1560 gtgctcgatt caacattagt ctacggtggt atggctccta taaccaagaa ctcaccaaag 1620 gccgaaaaaa ctatcaaggg gaagaatatt tacaactcat catttaacga ggaatgctgt 1680 aagtgcctta gtgaagatga ttataagatg ccgtacggcg tccctggcgg tgctgcctcg 1740 tatagaagat ccctaacatt gtccttcttc tacaaatttt ggcagtacgt tttggccaca 1800 gcgccaattc ccaaggctaa cgttgccaca atccagtgta gggatgccat tctggatgtt 1860 gattcgttga gtgaagtgac aagggtccaa aagcatggct acagagaaat gaacactcca 1920 gggcacaaga ctggaatcat tggcaagcct atcgttcatg tgaatgcaat aaaacaggca 1980 acaggcgaag ctcagtatac caatgatatt ccaccattgc accgcgaact atttggtgtg 2040 caggtgatgt ctgaaaaggc gcatgctaag attctctccg tggactggag tgaggccttg 2100 gaagtggaat cagttgttgg atacgtggac attaacgact tgcctaacaa ggaggccaat 2160 ctttggggca atttaccctt tggaaaggaa cctttctttg cagatggtga ggtgttcttt 2220 gttggccagg ctattggtgt tatccttgcc tcaagtaaag agagggccta tgaggcatct 2280 agaaaagtta gagttgtgta cgatgagctt ccccgaatca tctccgttga ggacggtgtg 2340 cgacagaagt cgttctttcc agacaggaga gaagtcaagc ttggggattg ggaatctgca 2400 ttcaagaaca gtaaatatta tcttgagaac actgcgagat tatcggccca ggagcatttc 2460 tacttcgagg ttcagaactg cctcgttata ccacaggaag gtggtgagct caaggtgtat 2520 tcttccacac aaaatcctac cgagacccag ttgtgtgccg cacaggtcac gggcgttcct 2580 gcaaacagag tcatatgccg tgttaaaaga ctcggaggtg gatttggtgg taaggagacc 2640 agatctatcc aactttcgag tttggcggca gttgcagcca gaaagtttaa tcgtccagtc 2700 aggttggaac tcaacagaag tgaagacatg aaaacttccg gtgaaagaca tccatttttg 2760 gtaaagtacc gggcctcgtt ggacgaagat ttaaagttca ctggattgga catggttctt 2820 Pagina 25 tatgcgaatg ctggttggtc gatggatttg acteetssts tcattgacag atccgttctc 2880 catgcatcaa atgcttatta cattcctaat gctcgggttt gtggcatacc agtgaagacc 2940 aacattgcct ctaacacggc atatcggacg tttggtgccc aggctggatt ctatgcgatt 3000 gagtctgttg tgacggagtt tgccgagaag cttggtgtgg atccggagga aataagaaga 3060 aggaattact tgaagccaaa ttgtggtgag gtgttcccat acaagcaggt agttggcgaa 3120 gacatcacga tttccaacgt ggtggatgaa aacttgaagg agtgcaacta caagaagatg 3180 aagcaggaaa tcaatgagtt caacaagcat tcaaaatgga taaagcgtgg tatagcacaa 3240 attcccgcag tgtttggtgt ttcgttcggt gttcttttct tgaatcaggc tggtgcttta 3300 gtccacattt ataacgatgg ctcctgcttg atttcaaccg gtggtgttga gattggccag 3360 ggtattagta ccgtgatgag gatgattgca gctgaagaat tgggcgttcc gtttgacaag 3420 atattcttga gtgagacttc taccgaatgt gttcctaaca catcatctac tgcagcttct 3480 tccggatccg atttgaacgg aatggcgtta aaggatgcat gcatgaagct taacaagcgt 3540 ttaaagccgg tgaaggatgc aatcacgaag gaaaaaggtg acaaatggac gtgggaggag 3600 ttgatcacaa aggcctactt ggacagagtc tcattgagtg caactggatt ctacaagacg 3660 ccagaaatcg gttttgagtg gggagatgag aatcctaaac ccgccttttt ctatcacact 3720 cagggatccg ctgtatctgt agtcgaagta gatacgttaa cgggtgattg gagttgcttg 3780 gagtcacaca tcaagatgga ctgtggacgg ccgttaaata aagcaatcat atacggtcaa 3840 attgaaggag ctttcatcca gggaatgggc tactttacca tggaacagtc attatggtta 3900 agcaggacag gagggctggc cacaaccggt cctggtgcct acaaaattcc agggttcagg 3960 gatactccac aacgatttgt gctatcaatg tataagggaa gcgatttcag gcatttgaga 4020 accattcatt catctaaggg agttggtgaa ccaccatttt tccttggtgc aagtgtccac 4080 tttgccctca gagatgcaat tggacatgca agaagacaga atggcattga atcgggaagt 4140 cagggcttga gattccgggt tcctttgact actgagagaa tcagagttga ttgtggtgat 4200 aagcttgcca aacagtcgtt tgtggcagcc aaagagggcg aggaggaatt ctttatcgag 4260 ggatga 4266 <210> 24 Pagina 26
SEQLTXT <211> 909 <212> DNA <213> Ogataea parapolymorpha <400> 24 atggcttacc tacaggattg tacatacggt aagaacaatg ttagattttt gaaggtcaag 60 agggacccta ttaaccctaa aatccatcag gtcatggagg ctagtgttag ggtcatgctt 120 acgggtgctt ttgatgtctc ctacacaaag gcagataaca gcgttattat tccaaccgat 180 accatcaaaa ataccatttt ggttgaagcc aagcaaacag atgtcttccc aattgagaga 240 tttgccgctc accttgtgaa gcatttcttt ggtaagtaca gttggattgc aggtatcact 300 gttcatattg agcaagccaa atggtctaag tattctgtcg atggaaagct ccagccacat 360 tcttttgtca agaatggtga tgaagtcagg gtttgtgaat tggtttctaa gaagaatggt 420 gactttgttc ttacaggcgg cgtgcagggg ttgaccgttt tgaagtcctc tggctctatg 480 ttccatggct acaatgtttg tgattacacc actttgaagc ctgttaatga gagaatcttg 540 tcaacagatg tagactgcaa gtacaagttt gacagtgcca agattggttc tgtcgataac 600 atattcactt tagccgattc tggactgttt gataaggtct tccaatctgc tcttaaaatc 660 actttggaca gatttgctct cgagaactcg gcctctgttc aagctaccat gtataacatg 720 ggtaccgata ttgtcaacgc taacccatat gtttacaatg tctcttatgc cttgccaaac 780 aagcactaca ttctgtttga cttctcctgg aagggcttga agaacgaaaa cgagatgttc 840 tacccatcac cacatcctaa cggtttgata aagtgtactg ttggaaggga gcctattgca 900 aagttataa 909 <210> 25 <211> 411 <212> DNA <213> Ogataea parapolymorpha <400> 25 atgtcttcaa ggccaccaat tacttgccat attctagaca ccacgtgtgg caaacccgcc 60 gaaaatgtta agtgtgagat aagctatata ccctcaaatg gtattacatc accatcagaa 120 gtgaaaccat ttggttatgc gtatacgaat caggatggaa gaattggatc ttggaatgct 180 gcaaatagta cagagacttt cattaatgcc gaaaataatc aatggactaa attagttagt 240 Pagina 27
SEQLTXT ggaacttatc gaattaggta tcacaccaag gactactttt tgaaaagaga tggaacgaca 300 tttttcccct ttatagatat atggtttgaa gtccctgcta tcccagagaa gcattatcat 360 gtaccattgc tattgagcaa ttatggttat tctacataca gaggttcatg a 411 <210> 26 <211> 561 <212> DNA <213> Ogataea parapolymorpha <400> 26 atgaaattac cagatcctca agcattatct caaattctga gatcagagca ggtaacagtt 60 atagatacgc tatttgaaca taacgataaa ttcgcggact tcataattaa aaaggtcctt 120 agccataatg aacgatatgg ttcatataga gaatttataa aggcggtgag aatacaactc 180 attcaacttg cagataatta tgaaaaaagt atgacaggag acctaggtga tatggtaaga 240 tcggttatta gtgcacatcc taggctagga atacagcagg cgtcggctct ttctgtctct 300 tctgcaagag agcaaaaatc gctacaatca ggaaagccgg agcttgagag acaattatta 360 gcattaaatc aagaatacga acattgcttc cccggcttga ggttcgtggt atttgtaaat 420 gggcggagtc gccaagagat caccaaaatc atgcggaaac gaatcactcg tgatgactat 480 aatcaggagg tgcgtgatgc attcagtgca atgtgcgata ttgcactgga tcggataaaa 540 aaggagaaca gcaagctgta a 561 <210> 27 <211> 579 <212> DNA <213> Ogataea parapolymorpha <400> 27 atgtccgcca ttgattgcat tattactgct gctggtttgt cctctagaat gggtcaatgg 60 aaaatgatgt tgccatggga acaaggtact atcttggata cctctattaa gaacgccttg 120 caattctgct ccagaattat cttggttact ggttacagag gtaacgaatt gcacgaaaga 180 tacgccaatc aatccaacat taccatcatt cacaatccag attacgccca aggtttgttg 240 acttctgtta aggctgctgt tccagctgtt caaactgaac attgcttttt gactcatggt 300 gatatgccaa ctttgaccat cgatatcttc agaaagatct ggtccttgag aaacgatggt 360 Pagina 28
SEQLTXT gctattttgc cattgcataa tggtattcca ggtcacccaa ttttggtttc taaaccatgt 420 ttgatgcagg ccattcaaag accaaacgtt accaatatga gacaggcttt gttgatgggt 480 gatcactact ctgttgaaat tgaaaacgcc gaaatcatct tggacattga tactccagat 540 gacttcatta ccgccaaaga aagatacacc gaaatctaa 579 <210> 28 <211> 361 <212> PRT <213> Ogataea parapolymorpha <400> 28 Met Ile Ser Ser Leu Leu Leu Arg Arg Leu His Ser Thr Gly Thr Ser 1 5 10 15 Gln His Leu Pro Arg Leu Glu Arg Leu Arg Arg Met Pro Val Arg His
Leu Lys Glu Phe Leu Thr Asp Thr Tyr Gly Arg Lys His Asp Tyr Leu 40 45 Arg Ile Ser Ile Thr Glu Arg Cys Asn Leu Arg Cys Val Tyr Cys Met 50 55 60 Pro Glu Gln Gly Val Asp Leu Ser Pro Pro Glu His Met Leu Thr Thr 65 70 75 80 Glu Glu Ile Val Lys Leu Ala Thr Leu Phe Ala Gln His Gly Val Arg 85 90 95 Lys Val Arg Leu Thr Gly Gly Glu Pro Thr Val Arg Lys Asp Ile Val 100 105 110 Glu Leu Val Ala Lys Leu Asn Gln Ile Thr Gly Ile Glu Glu Ile Cys 115 120 125 Met Thr Ser Asn Gly Leu Ala Leu His Arg Lys Leu Pro Asp Leu Phe 130 135 140 Pagina 29
SEQLTXT Lys Asn Gly Leu Thr Ser Leu Asn Leu Ser Leu Asp Thr Leu Ile Asn 145 150 155 160 Gly Lys Phe Leu Leu Ile Thr Arg Arg Asn Gly Leu Ser Ala Val Met 165 170 175 Arg Ser Leu Arg Thr Ala Leu Glu Leu Asp Ile Pro Lys Val Lys Ile 180 185 190 Asn Val Val Val Met Lys Asn Leu Asn Glu Asp Glu Ile Leu Asp Phe 195 200 205 Val Glu Leu Ser Lys Asn Asp Lys Val Glu Val Arg Phe Ile Glu Tyr 210 215 220 Met Pro Phe Asp Gly Asn Lys Trp Ser Thr Asn Lys Leu Val Ser Tyr 225 230 235 240 Glu Asp Ile Leu Ser Asn Ile Lys Val Arg His Pro Asn Ile Gln Arg 245 250 255 Leu Pro His Lys His Gly Asp Thr Ala Lys Val Tyr Gln Ile Pro Gly 260 265 270 Phe Lys Gly Lys Val Gly Phe Ile Thr Ser Met Thr Ser Asp Phe Cys 275 280 285 Ser Thr Cys Thr Arg Leu Arg Ile Thr Ser Asp Gly Asn Leu Lys Val 290 295 300 Cys Leu Phe Asp Asn Thr Glu Val Ser Leu Arg Asp Met Leu Arg Ala 305 310 315 320 Gly Tyr Ser Asp Asp Lys Leu Met Gln Arg Ile Gly Glu Ala Val Lys 325 330 335 Asn Lys Lys Glu Lys His Ala Gly Ile Asp Val Leu Gly Asp Gln Pro 340 345 350 Pagina 30
SEQLTXT Asn Arg Pro Met Ile Leu Ile Gly Gly 355 360 <210> 29 <211> 149 <212> PRT <213> Ogataea parapolymorpha <400> 29 Met Val Ala Ile His Glu Lys Glu Asp Thr His Arg Cys Ala Ile Ala 1 5 10 15 Glu Gly Ser Ile Lys Phe Ser Asn Pro Glu Ser Met Lys Leu Leu Leu
Ser Glu Ser Asn Lys Lys Gly Asp Val Ile Ser Ile Ala Arg Ile Ala 40 45 Gly Ile Ile Ala Val Lys Lys Thr Ala Glu Leu Ile Pro Leu Cys His 50 55 60 Pro Ile Ser Ile Thr Gly Ile Lys Val Asp Leu Ile His Asp Glu Lys 65 70 75 80 Glu Asn Cys Ile Lys Val Asn Cys Glu Val His Cys Asn Gly Lys Thr 85 90 95 Gly Val Glu Met Glu Ala Leu Thr Gly Ala Thr Ile Ser Leu Leu Thr 100 105 110 Val Tyr Asp Met Cys Lys Ala Val Asp Lys Met Met Thr Ile Ser Asp 115 120 125 Cys Arg Val Val Lys Lys Ser Gly Gly Lys Ser Gly Asp Ile Asp Leu 130 135 140 Ser Thr Ile Phe Lys 145 <210> 30 Pagina 31
SEQLTXT <211> 148 <212> PRT <213> Ogataea parapolymorpha <400> 30 Met Ser Ile Phe Val Asp Ile Thr Asp Lys Pro Leu Asp Ser Ala Glu 1 5 10 15 Val Leu Asn Tyr Val Arg His Pro Gln Ala Gly Ala Ile Val Tyr Phe
Gly Gly Thr Thr Arg Asn Thr Phe Glu Gly Lys Glu Val Val Ser Leu 40 45 Ala Tyr Glu Ala His Pro Arg Leu Ala Ile Lys Thr Leu Glu Ser Ile 50 55 60 Ala His Glu Ala Lys Ala Lys Phe Gln Ser Val His Lys Ile Ala Ile 65 70 75 80 Val His Arg Thr Gly Val Val Pro Val Ala Thr Glu Ser Val Met Ile 85 90 95 Ala Val Ser Ser Thr His Arg Lys Glu Gly Trp Leu Cys Gly Glu Trp 100 105 110 Val Leu Glu Lys Val Lys Glu Arg Ala Glu Ile Trp Lys Ile Glu Lys 115 120 125 Tyr Ala Asp Gly Asp Ser Val Tyr Lys Glu Asn Asp Val Ser Asn Val 130 135 140 Leu Ser Arg Thr 145 <210> 31 <211> 95 <212> PRT <213> Ogataea parapolymorpha <400> 31 Pagina 32
SEQLTXT Met Val Ala Val Ala Ile Glu Tyr Phe Gly Pro Ala Lys Thr Tyr Thr 1 5 10 15 Asn Gly Val Ala His Glu Arg Val Glu Leu Thr Glu Pro Ala Thr Leu
Asn Thr Leu Ile Gln His Val Gly Arg Ser Tyr Ser Ser Glu Phe Ala 40 45 Gln Tyr Ile Val Ser Ser Cys Gly Val Val Val Asn Glu Asp Tyr Val 50 55 60
Glu Thr Glu Arg Ile Gly Ile Glu Phe Phe Gly Lys Asn Ile Ala Leu 65 70 75 80 Gln Ser Gly Asp Val Val Gly Ile Ile Pro Pro Val Ser Ser Gly
85 90 95 <210> 32 <211> 625 <212> PRT <213> Ogataea parapolymorpha <400> 32 Met Thr Val Gly Ile Leu Val Val Ser Glu Ser Val Ser Arg Gly Leu 1 5 10 15 Ser Thr Asp Lys Val Val Asp Ala Leu Lys Gln His Leu Asp Gly Phe
20 25 30 Glu Leu Lys Ala His Lys Val Val Pro Asp Lys Lys Glu Asp Ile Gln 35 40 45 Ala Ala Val Val Asp Trp Val Lys Gln Asp Phe Lys Leu Ile Leu Thr 50 55 60 Ala Gly Gly Thr Gly Phe Thr Lys Thr Asp Ile Thr Pro Glu Ala Ile 65 70 75 80 Pagina 33
SEQLTXT Glu Pro Leu Leu Asp Lys Lys Ala Pro Gly Leu Val His Ala Met Leu 85 90 95 Ser Phe Ser Leu Gln Ile Thr Pro Phe Ala Met Leu Ala Arg Pro Val 100 105 110 Ala Gly Val Arg Gly Glu Ser Leu Ile Ile Thr Leu Pro Gly Ser Pro 115 120 125 Lys Gly Ala Thr Glu Asn Phe Gln Ala Ile Lys Gly Val Ile Gly His 130 135 140 Ala Leu Ser Gln Leu Gly Ile Glu Ser Ser Arg Leu Leu His Lys Glu 145 150 155 160 Ser Gly Ser Gly His His His His His His His His His His His Gly 165 170 175 His Leu Ala Lys His Glu Leu Val Asp Ser Val Val Ala Arg His Arg 180 185 190 Val Ser Pro Tyr Pro Thr Ile Ser Val Asp Glu Ala Tyr Ser Arg Ile 195 200 205 Arg Glu Asn Thr Pro Ala Pro Glu Val Ile Glu Leu Ser Ile Leu Asp 210 215 220 Pro Arg Leu Val Gly Ser Val Val Ala Glu Asn Val Thr Ala Gln Met 225 230 235 240 Asp Val Pro Asn Phe Arg Ala Ser Ile Val Asp Gly Tyr Ala Met Ile 245 250 255 Ser Ser Asp Gly Pro Gly Val Tyr Pro Val Val Ser Val Ser His Ala 260 265 270 Ser Lys Asn Asp Gln Lys Glu Leu Val Ala Gly Gln Ile Ala Arg Ile 275 280 285 Pagina 34
SEQLTXT Thr Thr Gly Ala Pro Leu Pro Glu Gly Ala Asp Ala Val Val Met Val 290 295 300 Glu Glu Thr Arg Leu Val Glu Thr Thr Glu Asp Gly Ser Glu Glu Lys 305 310 315 320 Leu Val Glu Ile Leu Ala Lys Asn Val Lys Thr Gly Glu Asn Ile Arg 325 330 335 Ala Ile Gly Ser Asp Thr Arg Lys Asp Ser Leu Val Leu Ala Lys Gly 340 345 350 Ser Arg Ile Ser Pro Gly Gly Gly Glu Ile Gly Ala Leu Ala Ser Val 355 360 365 Gly Val Asn Lys Ile Lys Val Tyr Arg Lys Pro Val Ile Gly Leu Leu 370 375 380 Ser Thr Gly Asn Glu Leu Gln Asp Val Gln Thr Glu Lys Leu Leu His 385 390 395 400 Tyr Gly Glu Ile Tyr Asp Ser Asn Arg Pro Thr Leu Val Ser Ile Val 405 410 415 Gln Asn Cys Gly Tyr Glu Leu Val Asp Leu Gly Ile Ala Ser Asp Thr 420 425 430 Lys Glu Ser Leu Ile Lys Ile Ile Lys Asp Ala Val Asp Val Lys Lys 435 440 445 Ile Asp Cys Leu Ile Thr Thr Gly Gly Val Ser Met Gly Glu Leu Asp 450 455 460 Leu Leu Lys Pro Thr Ile Glu Asn Glu Leu His Gly Thr Ile His Phe 465 470 475 480 Gly Arg Val Arg Met Lys Pro Gly Lys Pro Thr Thr Phe Ala Thr Ile 485 490 495 Pagina 35
SEQLTXT
Gly Lys Ser Thr Val Val Phe Ala Leu Pro Gly Asn Pro Ala Ser Ser 500 505 519
Ser Val Cys Tyr His Leu Phe Val Leu Pro Cys Leu Leu Lys Trp Gln
515 520 525 Gly Leu Glu Pro Ser Val Gly Gln Lys Leu Pro Thr Glu Pro Ile Val 530 535 540
Lys Val Lys Leu Ala Glu Asp Leu Lys Leu Asp Pro Gln Arg Pro Glu
545 550 555 560
Tyr Gln Arg Val Ser Ile Cys Gln Ser Asp Met Ala Leu Val Ala Asn
565 570 575
Ser Thr Gly Phe Gln Arg Ser Ser Asn Ile Gly Ser Phe Lys Lys Ala 580 585 590
Asn Gly Leu Val Cys Leu Pro Ala Ala Ser Asp Phe Gly Lys Ser Val
595 600 605 Ile Glu Ala Gly Thr Val Val Asp Ala Ile Leu Ile Asp Gln Ile Tyr 610 615 620
Val
625
<210> 33
<211> 389
<212> PRT
<213> Ogataea parapolymorpha
<400> 33
Met Ser Leu Ser Leu Asn Glu Tyr Leu Arg Tyr Gly Arg Gln Leu Ile
1 5 10 15
Val Pro Glu Phe Gly Leu Gln Gly Gln Ile Ser Leu Lys Asn Ser Arg
Val Leu Val Val Gly Ala Gly Gly Leu Gly Cys Pro Ala Leu Gln Tyr
Pagina 36
SEQLTXT 40 45 Leu Val Gly Ala Gly Phe Gly Thr Val Gly Ile Val Asp His Asp Thr 50 55 60 Val Asp Ile Ser Asn Leu His Arg Gln Ile Leu His Thr Ser Glu Thr 65 70 75 80 Val Glu Met Leu Lys Cys Glu Ser Ala Lys Leu Gln Leu Ala Lys Leu 85 90 95 Asn Pro Leu Val Gln Ile Asn Thr His Pro Val Ala Leu Ser Pro Asp 100 105 110 Asn Ser Phe Gly Ile Phe Glu Gln Tyr Asp Ile Ile Leu Asp Cys Thr 115 120 125 Asp Thr Pro Ala Thr Arg Tyr Leu Ile Asn Asp Thr Ala Val Leu Leu 130 135 140 Gly Leu Thr Val Ile Ser Gly Ser Gly Leu Lys Thr Glu Gly Gln Leu 145 150 155 160 Ser Ile Leu Asn Phe Asn Asn Thr Gly Pro Cys Tyr Arg Cys Phe Tyr 165 170 175 Pro Thr Pro Pro Pro Pro Ser Ser Val Thr Ala Cys Ser Asp Gly Gly 180 185 190 Val Leu Gly Pro Val Ile Gly Ile Met Gly Val Met Met Ala Leu Glu 195 200 205 Ala Ile Lys Val Val Ser Gly Tyr Tyr Leu Arg Glu Asp Val Glu Phe 210 215 220 Gln Pro Phe Leu Ser Leu Tyr Ser Gly Tyr Gly Pro Leu Gln Ser Leu 225 230 235 240 Arg Thr Phe Lys Met Arg Arg Arg Ser Pro Lys Cys Arg Val Cys Asn Pagina 37
SEQLTXT 245 250 255 Ala Gly Thr Arg Glu Ile Thr Arg His Val Ile Glu Thr Glu Leu Asp 260 265 270 Tyr Ala Val Trp Cys Gly Lys Met Asp Tyr Asn Val Leu Glu Lys Asp 275 280 285 Glu Arg Val Ser Val Glu Gln Leu Ser Ala Gln Arg Ala Pro Tyr Leu 290 295 300 Val Asp Val Arg Ala Lys Glu Gln Tyr Ser Ile Val His Leu Pro Asn 305 310 315 320 Ser Ile Asn Ile Pro Leu Asn Ala Leu Lys His Met Asp Thr Leu Asp 325 330 335 Val Pro Lys Glu Thr Met Ile Tyr Val Ile Cys Arg Phe Gly Asn Asp 340 345 350 Ser Gln Leu Ala Val Lys His Leu Lys Ser Ile Gly Tyr Glu Asn Cys 355 360 365 Val Asp Val Ile Gly Gly Leu Thr Gln Trp Ser Arg Gln Ile Asp Pro 370 375 380 Asn Phe Pro Ile Tyr 385 <210> 34 <211> 457 <212> PRT <213> Ogataea parapolymorpha <400> 34 Met Tyr Arg Phe Arg Ile Gly Ala Gly Val Arg Arg Phe Ser Val Ser 1 5 10 15 Ala Leu Arg Arg Thr Ser Ser Ser Gly Val Lys Gln Ala Met Pro Thr
Pagina 38
SEQLTXT Ser Gly Ala Gln Pro Pro Gln Gly Ser Ser Ile Ser Leu Lys Ser Ala 40 45 Thr Arg Asp Pro Ser Glu Tyr Gly Thr Arg Pro Ile Tyr Leu Asp Met 50 55 60 Gln Ala Thr Thr Pro Thr Asp Pro Arg Val Leu Asp Lys Met Leu Glu 65 70 75 80 Phe Tyr Thr Gly Leu Tyr Gly Asn Pro His Ser Ser Thr His Ala Tyr 85 90 95 Gly Trp Glu Thr Asp Lys Ala Val Glu Glu Ala Arg Ala Asn Val Ala 100 105 110 Ala Val Ile Gly Ala Asp Pro Lys Glu Ile Ile Phe Thr Ser Gly Ala 115 120 125 Thr Glu Cys Asn Asn Met Ala Leu Lys Gly Val Ala Arg Phe Tyr Gly 130 135 140 Lys Ser Lys Lys His Ile Ile Thr Thr Gln Thr Glu His Lys Cys Val 145 150 155 160 Leu Asp Ser Cys Arg His Leu Gln Asp Glu Gly Phe Glu Val Thr Tyr 165 170 175 Leu Pro Val Asn Ser Asp Gly Leu Ile Asp Leu Glu Leu Leu Glu Lys 180 185 190 Ser Ile Arg Lys Asp Thr Cys Leu Val Ser Val Met Ala Val Asn Asn 195 200 205 Glu Ile Gly Val Ile Gln Pro Leu Glu Asp Ile Gly Arg Ile Cys Arg 210 215 220 Ser His Lys Val Phe Phe His Thr Asp Ala Ala Gln Ala Tyr Gly Lys 225 230 235 240 Pagina 39
SEQLTXT Ile Pro Ile Asp Val Asn Lys Cys Asn Ile Asp Leu Met Ser Ile Ser 245 250 255 Ser His Lys Ile Tyr Gly Pro Met Gly Val Gly Ala Thr Tyr Val Arg 260 265 270 Arg Arg Pro Arg Val Arg Leu Asp Pro Ile Ile Asn Gly Gly Gly Gln 275 280 285 Glu Arg Gly Leu Arg Ser Gly Thr Leu Ala Pro Pro Leu Val Cys Gly 290 295 300 Phe Gly Glu Ala Ala Arg Leu Met Val Gln Glu Tyr Asp Ala Asp Gln 305 310 315 320 Ala His Ile Lys Ala Leu Ser Thr Lys Leu Met Asp Ala Leu Leu Ser 325 330 335 Met Glu His Thr Gln Leu Asn Gly Ser Arg Ile His Arg Tyr Pro Gly 340 345 350 Cys Val Asn Val Ser Phe Ala Tyr Ile Glu Gly Glu Ser Leu Leu Met 355 360 365 Ala Leu Lys Asp Ile Ala Leu Ser Ser Gly Ser Ala Cys Thr Ser Ala 370 375 380 Ser Leu Glu Pro Ser Tyr Val Leu His Ala Leu Gly Ala Asp Asp Ala 385 390 395 400 Leu Ala His Ser Ser Ile Arg Phe Gly Ile Gly Arg Phe Thr Thr Glu 405 410 415 Ala Glu Val Asp Tyr Val Ile Gln Ala Leu Thr Glu Arg Val Lys Phe 420 425 430 Leu Arg Glu Leu Ser Pro Leu Trp Glu Met Val Asn Glu Gly Ile Asp 435 440 445 Pagina 40
SEQLTXT Leu Asn Ser Ile Glu Trp Ala Gly His 450 455 <210> 35 <211> 519 <212> PRT <213> Ogataea parapolymorpha <400> 35 Met Ala Leu Gln Asn Ala Trp Gln Asn Thr Lys Glu Arg Ala Arg Glu 1 5 10 15 Thr Trp Ala Gln Leu Thr Trp Ser Glu Val Ser Gly Ser Leu Gly Asp
Leu Gly Thr Phe Leu Pro Leu Leu Ile Gly Leu Val Gln Lys Val His 40 45 Leu Asp Leu Gly Thr Thr Leu Thr Ile Thr Gly Leu Tyr Asn Ile Ile 50 55 60 Ser Gly Trp Gln Phe Arg Ile Pro Met Cys Val Gln Pro Met Lys Thr 65 70 75 80 Ile Ala Ala Val Ala Leu Ala Gly Gly Ala Ala Gly Leu Asp Leu Pro 85 90 95 Gln Leu Leu His Ala Gly Leu Phe Val Ala Gly Cys Val Gly Leu Leu 100 105 110 Gly Ala Ser Gln Ala Ile Asp Leu Phe Asn Trp Leu Val Pro Pro Pro 115 120 125 Val Ile Arg Gly Val Gln Leu Ala Val Gly Val Lys Leu Ala Met Lys 130 135 140 Gly Val Asp Met Ala Leu Arg Leu His Gly Gly Pro Ser Ser Gly Trp 145 150 155 160 Pagina 41
SEQLTXT Arg Pro Trp Leu Gly Thr Glu Gly Leu Val Val Gly Ala Val Ala Leu 165 170 175 Ala Ala Met Ile Ala Thr Thr Leu Pro Pro Arg Ala Ala Arg Arg Gly 180 185 190 Thr Leu Glu Ala Ala Asp Glu Gly Gly Leu Gly Pro Arg Pro Thr Asp 195 200 205 Thr Ala Phe Glu Pro Leu Leu Arg Arg Leu Pro Ala Cys Cys Gly Gly 210 215 220 Gly Asp Arg Ala Pro Gln Val Glu Gly Ala Ala Val Ser Ala Glu Arg 225 230 235 240 Ala Gly Leu Leu Ala His Ala Glu Gly Gly Glu Arg Ser Gly Asn Leu 245 250 255 Asp Asp Gly Thr Glu Ala Gly Val Gly Ala Ala Ala Gly Gly Gly Gly 260 265 270 Cys Gly Gly Gly Gly Gly Gly Gly Arg Ile Pro Ser Ala Leu Ile Ala 275 280 285 Val Val Val Gly Leu Ala Met Ala Val Leu His Arg Pro Gly Leu Val 290 295 300 Trp Glu Leu Arg Leu Gly Pro Thr Leu Pro Arg Leu Leu Arg Pro Ser 305 310 315 320 Trp Pro Asp Phe Lys Thr Gly Ala Leu Arg Gly Gly Leu Pro Gln Leu 325 330 335 Pro Leu Thr Thr Leu Asn Ser Val Ile Ala Val Thr Gln Leu Ala Asn 340 345 350 Ala Leu Phe Gly Asp Lys Pro Glu Ala Glu Arg Arg Arg Trp Arg Pro 355 360 365 Pagina 42
SEQLTXT Ser Ala Val Ala Leu Ser Val Ala Leu Leu Asn Gly Ala Gly Val Trp 370 375 380 Leu Gly Ala Met Pro Cys Cys His Gly Ala Gly Gly Leu Ala Ala Gln 385 390 395 400 Tyr Lys Phe Gly Ala Arg Thr Gly His Ala Pro Ile Leu Leu Gly Cys 405 410 415 Ile Lys Ala Ala Leu Gly Leu Leu Phe Gly Gly Ser Leu Val Val Leu 420 425 430 Leu Glu Ala Phe Pro Gln Pro Leu Leu Gly Ala Leu Leu Thr Val Ser 435 440 445 Gly Ile Glu Leu Ala Ser Val Val Arg His Thr Arg Ser Pro Arg Gly 450 455 460 Tyr Thr Phe Ala Leu Leu Thr Ala Val Ala Ile Leu Ala Leu Asp Asn 465 470 475 480 Thr Gly Thr Gly Phe Leu Val Gly Leu Val Gly Val Ala Ala Val Ala 485 490 495 Ala Tyr Glu Gly Ala Val Ala Ala Ala Ala Ala Arg Trp Pro Arg Val 500 505 519 Phe Ala Arg Gly Gly Arg Ala 515 <210> 36 <211> 859 <212> PRT <213> Ogataea parapolymorpha <400> 36 Met Asp Ser Val Val Thr Glu Val Thr Tyr Gly Leu Glu Ile Lys Lys 1 5 10 15 Pagina 43
SEQLTXT Ile Lys Glu Ile Thr Glu Leu Pro Phe Pro Val Arg Gln Asp Ser Pro
Leu Ser Glu Val Leu Pro Thr Asp Leu Lys Thr Lys Asp Asn Phe Val 40 45 Ala Arg Asp Pro Asp Leu Leu Arg Leu Thr Gly Ser His Pro Phe Asn 50 55 60 Ser Glu Pro Pro Leu Ala Lys Leu Tyr Asp Ser Gly Phe Leu Thr Pro 65 70 75 80 Val Ser Leu His Phe Val Arg Asn His Gly Pro Val Pro Tyr Val Pro 85 90 95 Asp Glu Asn Ile Leu Asp Trp Glu Val Ser Ile Glu Gly Met Val Glu 100 105 110 Thr Pro Tyr Lys Ile Lys Leu Ser Asp Ile Met Asp Gln Phe Asp Ile 115 120 125 Tyr Thr Thr Pro Val Thr Met Val Cys Ala Gly Asn Arg Arg Lys Glu 130 135 140 Gln Asn Met Val Lys Lys Gly Thr Gly Phe Asn Trp Gly Ala Ala Gly 145 150 155 160 Thr Ser Thr Ser Leu Trp Thr Gly Cys Met Leu Gly Asp Val Ile Gly 165 170 175 Lys Ala Arg Pro Ser Lys Arg Ala Arg Tyr Ile Trp Met Glu Gly Ala 180 185 190 Asp Asn Pro Ala Asn Gly Ala Tyr Gly Thr Cys Val Arg Leu Ser Trp 195 200 205 Ala Met Asp Pro Glu Arg Cys Ile Met Met Ala Tyr Lys Gln Asn Gly 210 215 220 Pagina 44
SEQLTXT Glu Trp Leu His Pro Asp His Gly Lys Pro Leu Arg Val Val Ile Pro 225 230 235 240 Gly Val Ile Gly Gly Arg Ser Val Lys Trp Leu Arg Lys Leu Val Val 245 250 255 Ser Asp Arg Pro Ser Glu Asn Trp Tyr His Tyr Phe Asp Asn Arg Val 260 265 270 Leu Pro Thr Met Val Thr Pro Glu Met Ala Lys Ser Asp Asp Arg Trp 275 280 285 Trp Lys Asp Glu Arg Tyr Ala Ile Tyr Asp Leu Asn Leu Gln Thr Ile 290 295 300 Ile Cys Lys Pro Glu Asn Gln Gln Val Ile Lys Ile Ser Asp Asp Glu 305 310 315 320 Tyr Glu Ile Ala Gly Phe Gly Tyr Asn Gly Gly Gly Ile Arg Ile Gly 325 330 335 Arg Ile Glu Ile Ser Leu Asp Lys Gly Lys Thr Trp Lys Leu Thr Glu 340 345 350 Ile Asp Tyr Pro Glu Asp Arg Tyr Arg Glu Ala Gly Tyr Phe Arg Leu 355 360 365 Phe Gly Gly Leu Val Asn Val Cys Asp Arg Met Ser Cys Leu Cys Trp 370 375 380 Cys Phe Trp Lys Leu Lys Val Pro Leu Ser Glu Leu Ala Thr Ser Lys 385 390 395 400 Asp Ile Leu Val Arg Gly Met Asp Glu Arg Met Val Val Gln Pro Arg 405 410 415 Thr Met Tyr Trp Asn Val Thr Ser Met Leu Asn Asn Trp Trp Tyr Arg 420 425 430 Pagina 45
SEQLTXT Val Ala Ile Ile Arg Glu Gly Asp Ala Leu Arg Phe Glu His Pro Val 435 440 445 Val Ala Asn Lys Pro Gly Gly Trp Met Asp Arg Val Lys Ala Glu Gly 450 455 460 Gly Asp Ile Leu Asp Asn Asn Trp Gly Glu Val Asp Asp Thr Val Lys 465 470 475 480 Gln Ala Glu Arg Lys Pro Arg Val Asp Glu Asp Ile Glu Met Met Cys 485 490 495 Asn Pro Glu Lys Met Asp Val Ile Ile Lys Tyr Ser Glu Phe Glu Ala 500 505 519 His Lys Asp Ser Glu Thr Glu Pro Trp Phe Ala Val Lys Gly His Val 515 520 525 Phe Asp Gly Ser Ser Tyr Leu Glu Asp His Pro Gly Gly Ala Gln Ser 530 535 540 Ile Leu Met Val Ser Gly Glu Asp Ala Thr Asp Asp Phe Ile Ala Ile 545 550 555 560 His Ser Ser Tyr Ala Lys Lys Leu Leu Pro Pro Met His Leu Gly Arg 565 570 575 Leu Glu Glu Val Ser Ser Val Thr Lys Val Lys Ser Val Glu Glu Asn 580 585 590 Val Lys Arg Glu Val Leu Leu Asp Pro Arg Lys Trp His Lys Ile Thr 595 600 605 Leu Ala Glu Lys Glu Ile Ile Ser Ser Asp Ser Arg Ile Phe Lys Phe 610 615 620 Asp Leu Glu His Pro Glu Gln Leu Ile Gly Leu Pro Thr Gly Lys His 625 630 635 640 Pagina 46
SEQLTXT Leu Phe Leu Arg Leu Lys Asp Ser Ser Gly Lys Tyr Val Met Arg Ala 645 650 655 Tyr Thr Pro Lys Ser Ser Asn Ser Leu Arg Gly Arg Leu Glu Ile Leu 660 665 670 Ile Lys Val Tyr Phe Pro Asn Arg Glu Tyr Pro Asn Gly Gly Ile Met 675 680 685 Thr Asn Leu Ile Glu Asn Leu Gln Val Gly Asn Gln Ile Glu Val Lys 690 695 700 Gly Pro Val Gly Glu Phe Glu Tyr Val Lys Cys Gly His Cys Ser Phe 705 710 715 720 Asn Asn Lys Pro Tyr Gln Met Lys His Phe Val Met Ile Ser Gly Gly 725 730 735 Ser Gly Ile Thr Pro Thr Tyr Gln Val Leu Gln Ala Ile Phe Ser Asp 740 745 750 Pro Glu Asp Thr Thr Ser Val Gln Leu Phe Phe Gly Asn Lys Lys Val 755 760 765 Asp Asp Ile Leu Leu Arg Glu Glu Leu Asp Cys Leu Gln Ile Lys His 770 775 780 Pro Glu Gln Phe Lys Val Asp Tyr Ser Leu Ser Asp Leu His His Leu 785 790 795 800 Pro Glu Asn Trp Ser Gly Leu Lys Gly Arg Leu Thr Phe Asn Ile Leu 805 810 815 Asp Ser Tyr Val Gln Gly Lys Asn Met Gly Glu Tyr Met Leu Leu Val 820 825 830 Cys Gly Pro Pro Gly Met Asn Gly Val Val Glu Asn Trp Cys Lys Ala 835 840 845 Pagina 47
SEQLTXT Arg Asn Leu Asp Lys Gln Tyr Val Val Tyr Phe 850 855 <210> 37 <211> 1044 <212> PRT <213> Ogataea parapolymorpha <400> 37 Met Thr Cys Ser Val Pro Pro Leu Pro Glu Asp Ile Thr Pro Pro Ala 1 5 10 15 Ala Lys Lys Lys Leu Val Ile Val Gly Leu Gly Met Val Gly Leu Ser
Phe Leu Glu Lys Leu Leu Leu Asn Asp Ser Lys Leu Asn Glu Tyr Ser 40 45 Ile Leu Val Tyr Gly Glu Glu Pro Tyr Leu Ala Tyr Asn Arg Val Gly 50 55 60 Leu Thr Glu Tyr Phe Gln His Arg Glu Phe Lys Asn Leu Leu Leu Ser 65 70 75 80 Pro Glu Glu Phe Tyr Gln Leu Arg Gly Glu Lys Trp Asn Tyr Ala Ile 85 90 95 Asp Glu Lys Val Ile Asp Ile Asp Arg Gln Ala Arg Thr Ile Thr Thr 100 105 110 Asn Lys Gly Asn Arg Ala Ser Tyr Asp Val Leu Val Leu Cys Thr Gly 115 120 125 Ser Thr Ala Ile Leu Pro Thr Asp Leu Leu Pro Pro Pro Thr Arg Lys 130 135 140 Ser Tyr Arg Glu Met Gly Cys Phe Val Tyr Arg Thr Ile Asp Asp Leu 145 150 155 160 Tyr Ser Met Ile Asp Tyr Cys Gln Gly Ala Lys Lys Gln Arg Ala Ile Pagina 48
SEQLTXT 165 170 175 Val Val Gly Gly Gly Leu Leu Gly Leu Glu Ala Ala Lys Ala Leu Tyr 180 185 190 Asp Met Glu Ser Phe Glu Asp Ile Thr Ile Val His Arg Ser His Trp 195 200 205 Leu Leu Ser Gln Gln Met Asp Gln Lys Gly Gly Ser Leu Leu Thr Ser 210 215 220 Lys Val Lys Glu Leu Gly Ile Thr Cys Arg Thr Gly Thr Thr Val Ser 225 230 235 240 Glu Leu Leu Phe Asp Glu Asp Gln Asn Leu Thr Gly Val Lys Tyr Asp 245 250 255 Asn Gly Glu Ile Glu Glu Cys Ser Leu Leu Cys Tyr Thr Ile Gly Ile 260 265 270 Lys Pro Arg Asp Glu Leu Thr Ser Cys Gly Leu Asn Ala Gly Ser Arg 275 280 285 Gly Gly Phe Lys Val Asn Asn Met Leu Gln Thr Ser Asp Glu Asn Ile 290 295 300 Tyr Ala Ile Gly Glu Cys Ala Ser Trp Asn Asn Met Thr Phe Gly Leu 305 310 315 320 Ile Ala Pro Gly Tyr Glu Met Ala Asp Ile Leu Ala Phe Asn Leu Thr 325 330 335 Gln Gly Lys Leu His Gln Pro Lys Glu Phe Ser Glu Pro Ser Met Gly 340 345 350 Thr Arg Leu Lys Leu Met Gly Val Asp Val Ala Ser Phe Gly Asp Phe 355 360 365 Phe Ala Asp Arg Asn Gly Pro Lys Trp Leu Pro Arg Gly Tyr Glu Lys Pagina 49
SEQLTXT 370 375 380 Glu Val Arg Gly Leu Val Phe Glu Asp Pro Ile Asp Gly Thr Tyr Thr 385 390 395 400 Lys Leu Leu Phe Thr Lys Asp Gly Lys Tyr Met Leu Gly Gly Ile Leu 405 410 415 Val Gly Asp Thr Ser Asn Tyr Thr Lys Phe Ser Ala Met Ile Arg Lys 420 425 430 Pro Leu Pro Lys Ser Pro Ser Glu Leu Leu Ile Gly Lys Ala Gly Glu 435 440 445 Asp Asp Met Glu Lys Leu Ser Asp Glu Thr Gln Ile Cys Ser Cys His 450 455 460 Asn Ile Thr Lys Gly Lys Leu Val Glu Ala Val Lys Asn Gly Cys Ser 465 470 475 480 Ser Leu Ala Asp Leu Lys Lys Cys Thr Lys Ala Gly Thr Ala Cys Gly 485 490 495 Gly Cys Glu Pro Thr Val Lys Val Ile Phe Glu Thr Glu Val Lys Lys 500 505 519 Leu Gly Gly Lys Val Ser Asn Asn Leu Cys Val His Phe Asp Tyr Ser 515 520 525 Arg Ala Asp Leu Phe Ser Leu Ile Met Val Lys Asn Phe Arg Ser Phe 530 535 540 Arg Arg Val Met Glu Glu Leu Gly Asn Asn Pro Ser Ser Ser Gly Cys 545 550 555 560 Glu Ile Cys Lys Pro Thr Ile Gly Ser Ile Leu Ser Thr Leu Tyr Asn 565 570 575 Arg His Leu Leu Lys Lys Glu Val His Gly Leu Gln Asp Thr Asn Asp Pagina 50
SEQLTXT 580 585 590 Arg Tyr Leu Gly Asn Ile Gln Arg Asn Gly Thr Phe Ser Val Val Pro 595 600 605 Arg Met Ser Ala Gly Glu Val Thr Pro Glu Lys Leu Val Ser Ile Gly 610 615 620 Gln Ile Ala Lys Lys Tyr Gly Val Tyr Thr Lys Ile Thr Gly Ala Gln 625 630 635 640 Arg Leu Asp Leu Phe Gly Val Lys Lys Ser Asp Leu Pro Lys Ile Trp 645 650 655 Lys Asp Leu Asn Glu Ala Gly Phe Glu Ser Gly Gln Ala Tyr Gly Lys 660 665 670 Ser Leu Arg Asn Val Lys Ser Cys Val Gly Ser Thr Trp Cys Arg Tyr 675 680 685 Gly Ile Gly Asp Ser Val Gly Leu Ala Val Arg Leu Glu Glu Arg Tyr 690 695 700 Lys Gly Ile Arg Ser Pro His Lys Met Lys Gly Gly Val Ser Gly Cys 705 710 715 720 Val Arg Asp Cys Ala Glu Phe His Ser Lys Asp Phe Gly Leu Cys Ala 725 730 735 Val Lys Asp Gly Phe Asn Ile Tyr Val Gly Gly Asn Gly Gly Met Lys 740 745 750 Pro Ala His Ala Gln Leu Leu Ala Thr Asn Val Arg Pro Asp Glu Val 755 760 765 Ile Pro Ile Leu Asp Arg Tyr Leu Met Phe Tyr Ile Thr Thr Ala Asp 770 775 780 Arg Leu Gln Arg Thr Ala Arg Trp Leu Glu Asn Leu Asp Gly Gly Ile Pagina 51
SEQLTXT 785 790 795 800 Glu Tyr Leu Lys Asp Val Ile Ile Arg Asp Lys Leu Gly Ile Cys Lys 805 810 815 Asp Leu Glu Ala Gln Met Arg Gln Leu Val Ala Gly Tyr Tyr Asp Glu 820 825 830 Trp Ala Lys Ala Val Ser Glu Glu Lys Asp Asn Pro Ile Phe Lys Gln 835 840 845 Phe Val Asn Thr Ser Glu Asn Gln Asp Thr Val Glu Ile Val Lys Glu 850 855 860 Arg Gly Gln Pro Arg Pro Ala Met Trp Pro Glu Lys Ala Ala Asn Gln 865 870 875 880 Lys Phe Asn Glu Ile Lys Trp Ser Ser Val Ser Trp Gln Glu Val Cys 885 890 895 Glu Ser Ser Asp Leu Pro Leu Ala Glu Ala Gly Ser Ser Ala Thr Val 900 905 910 Leu Val Gly Asp Thr Gln Ile Ala Leu Phe Arg Thr Ser Glu Asn Glu 915 920 925 Leu Tyr Cys Ser Gln Asn Met Cys Gly His Lys Arg Ala Phe Val Leu 930 935 940 Asn Gln Gly Leu Leu Ser Glu Asp Gly Asp Lys Asn Cys Tyr Ile Ser 945 950 955 960 Cys Pro Met His Lys Arg Asn Phe Tyr Leu Lys Ser Gly Ala Cys Lys 965 970 975 Asn Asp Glu Ala Leu Ser Ile Ala Thr Phe Glu Val Lys Glu Glu Asn 980 985 990 Gly Lys Val Tyr Ala Lys Leu Pro Pro Thr Thr Glu Leu Asp Glu Val Pagina 52
SEQLTXT 995 1000 1005 Leu Gly Thr Ser Lys Trp Lys Val Thr Ser Gln Glu Thr Glu Ala 1010 1015 1020 Lys Gln Val Arg Lys Ile Thr Thr Glu Lys Asn Leu Val Asp Lys 1025 1030 1035 Ala Ile Ser Phe Asp Trp 1040 <210> 38 <211> 508 <212> PRT <213> Ogataea parapolymorpha <400> 38 Met Arg Leu Ser Thr Leu Trp Glu Pro Pro Thr Val Asn Pro Arg Asn 1 5 10 15 Leu Lys Ala Thr Ser Ile Pro Ile Phe Asn Leu Trp Asn Val Tyr Gly
Arg Asn Phe Phe Phe Ala Trp Phe Gly Phe Phe Val Cys Phe Leu Ser 40 45 Trp Phe Ala Phe Pro Pro Leu Leu His Gly Met Leu Lys Lys Asp Leu 50 55 60 Lys Leu Thr Ala Val Asp Ile Ser Asn Asn Asn Ile Cys Gly Leu Thr 65 70 75 80 Gly Thr Leu Leu Gly Arg Phe Ile Leu Gly Pro Leu Asn Asp Lys Tyr 85 90 95 Gly Pro Arg Ile Thr Leu Val Gly Val Leu Val Ala Gly Ala Ile Pro 100 105 110 Thr Ala Phe Val Pro Leu Val Thr Asn Val Ala Gly Leu His Ala Ile 115 120 125 Pagina 53
SEQLTXT Arg Phe Phe Ile Ser Phe Leu Gly Ser Ser Phe Ile Cys Cys Ser Gln 130 135 140 Phe Cys Ala Val Phe Phe Asp Asn Asn Ile Ile Gly Thr Ala Asn Ala 145 150 155 160 Val Ser Ala Gly Trp Gly Asn Ala Gly Gly Gly Val Ala Phe Phe Val 165 170 175 Met Pro Ala Ile Ser Asn Ala Leu Glu Lys Arg Gly Tyr Ser Leu His 180 185 190 His Ser Trp Ser Tyr Ser Phe Val Ile Gly Pro Phe Leu Ile Leu Met 195 200 205 Ile Thr Ala Ile Val Ile Phe Val Phe Gly Ser Asp Cys Pro Arg Gly 210 215 220 Arg Trp Ser Leu Arg Gly Asp Ile Leu Gly Ile Asn Met Asp Asn Met 225 230 235 240 Leu Val Lys Ser Val Ser Val Thr Arg His Phe Ser Lys Asp Gly Glu 245 250 255 Leu Thr Ser Val Cys Val Glu Pro Val Asn Ala Ile Asp Glu Val Val 260 265 270 Val Glu Pro Asn Gln Asp Gln Glu Ile Phe Glu Val Ala Asp Ile Ile 275 280 285 Asn Glu Asp Glu Ile Ile Glu Asp Pro Thr Leu Lys Asp Val Val Lys 290 295 300 Ile Cys Phe Ser Pro Arg Thr Met Leu Val Gly Leu Cys Tyr Met Cys 305 310 315 320 Ser Phe Gly Thr Glu Leu Ala Val Glu Ser Ile Ile Ser Asn Leu Phe 325 330 335 Pagina 54
SEQLTXT Gly Gln Lys Met Thr Lys Trp Ser Thr Ser Lys Ala Gly Ala Trp Gly 340 345 350 Ser Met Leu Gly Leu Leu Asn Val Val Thr Arg Pro Ala Gly Gly Ile 355 360 365 Ile Ser Asp Phe Leu Tyr Gln Arg Phe Lys Thr Thr Lys Ala Lys Lys 370 375 380 Phe Trp Met Ile Phe Thr Gly Leu Met Gln Gly Ile Phe Leu Ile Trp 385 390 395 400 Val Gly Leu Val Pro Glu Leu Ser Ile Ala Gly Leu Ile Val Ser Val 405 410 415 Ser Phe Leu Cys Leu Trp Phe Glu Met Gly Asn Gly Ala Asn Tyr Ala 420 425 430 Cys Val Pro Val Val Asn Arg His His Ser Gly Ile Val Ser Gly Val 435 440 445 Thr Gly Ala Met Gly Asn Leu Gly Gly Ile Leu Phe Ser Leu Val Phe 450 455 460 Arg Tyr Thr Ile Ala Asn Gly Val Asn Asn Tyr Phe Lys Ala Phe Trp 465 470 475 480 Ile Ile Gly Ile Val Cys Thr Val Val Asn Leu Ala Cys Val Leu Ile 485 490 495 Pro Ile Arg Glu Glu Arg Pro Arg Lys Ala Glu Ile 500 505 <210> 39 <211> 194 <212> PRT <213> Ogataea parapolymorpha <400> 39 Pagina 55
SEQLTXT Met Asn Leu Met Thr Thr Ile Thr Gly Val Val Leu Ala Gly Gly Lys 1 5 10 15 Ala Arg Arg Met Gly Gly Val Asp Lys Gly Leu Leu Glu Leu Asn Gly
Lys Pro Leu Trp Gln His Val Ala Asp Ala Leu Met Thr Gln Leu Ser 40 45 His Val Val Val Asn Ala Asn Arg His Gln Glu Ile Tyr Gln Ala Ser 50 55 60 Gly Leu Lys Val Ile Glu Asp Ser Leu Ala Asp Tyr Pro Gly Pro Leu 65 70 75 80 Ala Gly Met Leu Ser Val Met Gln Gln Glu Ala Gly Glu Trp Phe Leu 85 90 95 Phe Cys Pro Cys Asp Thr Pro Tyr Ile Pro Pro Asp Leu Ala Ala Arg 100 105 110 Leu Asn His Gln Arg Lys Asp Ala Pro Val Val Trp Val His Asp Gly 115 120 125 Glu Arg Asp His Pro Thr Ile Ala Leu Val Asn Arg Ala Ile Glu Pro 130 135 140 Leu Leu Leu Glu Tyr Leu Gln Ala Gly Glu Arg Arg Val Met Val Phe 145 150 155 160 Met Arg Leu Ala Gly Gly His Ala Val Asp Phe Ser Asp His Lys Asp 165 170 175 Ala Phe Val Asn Val Asn Thr Pro Glu Glu Leu Ala Arg Trp Gln Glu 180 185 190 Lys Arg Pagina 56
SEQLTXT <210> 40 <211> 175 <212> PRT <213> Ogataea parapolymorpha <400> 40 Met Ala Gly Lys Thr Met Ile Pro Leu Leu Ala Phe Ala Ala Trp Ser 1 5 10 15 Gly Thr Gly Lys Thr Thr Leu Leu Lys Lys Leu Ile Pro Ala Leu Cys
Ala Arg Gly Ile Arg Pro Gly Leu Ile Lys His Thr His His Asp Met 40 45 Asp Val Asp Lys Pro Gly Lys Asp Ser Tyr Glu Leu Arg Lys Ala Gly 50 55 60 Ala Ala Gln Thr Ile Val Ala Ser Gln Gln Arg Trp Ala Leu Met Thr 65 70 75 80 Glu Thr Pro Asp Glu Glu Glu Leu Asp Leu Gln Phe Leu Ala Ser Arg 85 90 95 Met Asp Thr Ser Lys Leu Asp Leu Ile Leu Val Glu Gly Phe Lys His 100 105 110 Glu Glu Ile Ala Lys Ile Val Leu Phe Arg Asp Gly Ala Gly His Arg 115 120 125 Pro Glu Glu Leu Val Ile Asp Arg His Val Ile Ala Val Ala Ser Asp 130 135 140 Val Pro Leu Asn Leu Asp Val Ala Leu Leu Asp Ile Asn Asp Val Glu 145 150 155 160 Gly Leu Ala Asp Phe Val Val Glu Trp Met Gln Lys Gln Asn Gly 165 170 175 Pagina 57
SEQLTXT <210> 41 <211> 866 <212> PRT <213> Ogataea parapolymorpha <400> 41 Met Thr Glu Thr Arg Thr Thr Cys Pro Tyr Cys Gly Val Gly Cys Gly 1 5 10 15 Val Ile Ala Ser Arg Ala Pro His Gly Gln Val Ser Val Arg Gly Asp
Glu Gln His Pro Ala Asn Phe Gly Arg Leu Cys Val Lys Gly Ala Ala 40 45 Leu Gly Glu Thr Val Gly Leu Glu Gly Arg Met Leu Phe Pro Glu Val 50 55 60 Asp Gly Glu Arg Ala Thr Trp Pro Gln Ala Leu Ala Ala Ala Gly Ser 65 70 75 80 Arg Leu Arg Glu Ile Ile Asp Arg His Gly Pro Gln Ala Val Ala Phe 85 90 95 Tyr Ala Ser Gly Gln Leu Leu Thr Glu Asp Tyr Tyr Ala Ala Asn Lys 100 105 110 Leu Met Lys Gly Phe Ile Gly Ala Ala Asn Ile Asp Thr Asn Ser Arg 115 120 125 Leu Cys Met Ser Ser Ala Val Thr Gly Tyr Lys Arg Ala Leu Gly Ala 130 135 140 Asp Val Val Pro Cys Ser Tyr Glu Asp Val Glu Asn Ser Asp Leu Val 145 150 155 160 Val Leu Val Gly Ser Asn Ala Ala Trp Ala His Pro Val Leu Tyr Gln 165 170 175 Arg Leu Ala Gln Ala Lys Arg Asp Asn Pro Gln Met Arg Val Val Val Pagina 58
SEQLTXT 180 185 190 Ile Asp Pro Arg Arg Thr Ala Thr Cys Asp Ile Ala Asp Arg His Leu 195 200 205 Ala Leu Ala Pro Gly Ser Asp Gly Gly Leu Phe Val Gly Leu Leu Asn 210 215 220 Ala Ile Ala Ala Ser Gly Ala Ile Ser Asp Asp Phe Asn Asp Ala Gln 225 230 235 240 Arg Ala Leu Thr Ile Ala Gln Asp Trp Asp Leu Asp Lys Val Ala Gln 245 250 255 Phe Cys Gly Leu Pro Arg Gln Gln Ile Ala Asp Phe Tyr Arg Glu Phe 260 265 270 Ile Ala Ala Pro Arg Ala Ile Thr Leu Tyr Thr Met Gly Ile Asn Gln 275 280 285 Ser Ala Ser Gly Ser Asp Lys Cys Asn Ala Ile Ile Asn Val His Leu 290 295 300 Ala Cys Gly Lys Tyr Gly Arg Pro Gly Cys Gly Pro Phe Ser Leu Thr 305 310 315 320 Gly Gln Pro Asn Ala Met Gly Gly Arg Glu Val Gly Gly Leu Ala Thr 325 330 335 Met Leu Ala Ala His Met Asn Phe Glu Pro Asp Asp Leu Arg Arg Leu 340 345 350 Ala Arg Phe Trp Gly Ser Glu Arg Leu Ala Gln Thr Pro Gly Leu Thr 355 360 365 Gly Val Glu Leu Phe Ala Ala Ile Gly Arg Gly Glu Val Lys Ala Val 370 375 380 Trp Ile Met Gly Thr Asn Pro Val Val Ser Leu Pro Asp Ser His Ala Pagina 59
SEQLTXT 385 390 395 400 Val Ser Glu Ala Leu Ala Arg Cys Pro Leu Val Ile Ile Ser Asp Val 405 410 415 Val Ala Asp Thr Asp Thr Gly Arg Phe Ala His Ile Arg Phe Pro Ala 420 425 430 Leu Ala Trp Gly Glu Lys Ser Gly Thr Val Thr Asn Ser Glu Arg Arg 435 440 445 Ile Ser Arg Gln Arg Ala Phe Met Pro Pro Pro Gly Glu Ala Arg Ala 450 455 460 Asp Trp Trp Ile Val Ala Arg Val Ala Glu Ala Leu Gly Phe Gly Ser 465 470 475 480 Ala Phe Ala Trp Gln His Pro His Glu Val Phe Ser Glu His Ala Ala 485 490 495 Leu Ser Gly Tyr Glu Asn Asp Gly Gln Arg Ala Phe Asp Ile Gly Gly 500 505 519 Leu Ala Asp Leu Ser Arg Glu Ala Trp Asp Ala Leu Glu Pro Val Arg 515 520 525 Trp Pro Val Ser Arg Ser Glu Ala Ala Trp Ser Val His Lys Gly Trp 530 535 540 His Arg Asp Gly Lys Leu Arg Met Val Pro Val Ala Pro Gln Pro Thr 545 550 555 560 Arg Ala Thr Thr Asp Ala Phe Tyr Pro Leu Ile Leu Asn Ser Gly Arg 565 570 575 Ile Arg Asp Gln Trp His Thr Met Thr Arg Thr Gly Ala Val Pro Arg 580 585 590 Leu Met Gln His Ile Asn Glu Pro Val Val Glu Val Ala Pro Ala Asp Pagina 60
SEQLTXT 595 600 605 Ala Gln Arg Tyr His Leu Leu Glu Gly Glu Leu Ala Arg Val Arg Ser 610 615 620 Pro Lys Gly Val Met Val Ala Lys Val Thr Ile Gly Asp Gly Gln Arg 625 630 635 640 Pro Gly Ser Leu Phe Val Pro Met His Trp Asn Asn Gln Phe Ala Arg 645 650 655 Gln Gly Arg Val Asn Asn Leu Leu Ala Ala Val Thr Asp Pro His Ser 660 665 670 Gly Gln Pro Glu Ser Lys Gln Thr Ala Val Ala Ile Ala Thr Trp Leu 675 680 685 Pro Ala Trp Lys Gly Glu Leu Phe Ser Arg Gln Pro Val Pro Leu Pro 690 695 700 Ala Ser Leu His Trp Arg Arg Arg Ala Ala Gln Gly Ile Ile His Leu 705 710 715 720 Ser Leu Ala Gly Asp Thr Arg Ser Arg Asp Trp Leu Val Glu Trp Cys 725 730 735 Gln Arg Gln Gly Trp Gln Met Gln Val Ala Glu Gly Gly Lys Val Trp 740 745 750 Asn Leu Leu Ala Trp Arg Ala Gly Glu Leu Met Leu Gly Trp Trp Ser 755 760 765 Asp Ala Ser Glu Pro Ala Ile Asp Ala Asp Trp Ile His Ala Ala Phe 770 775 780 Arg Val Pro Pro Gln Asn Ala Ala Arg Arg His Ala Leu Leu Ser Gly 785 790 795 800 Arg Lys Gly Gly Val Glu Met Pro Arg Gly Arg Ile Ile Cys Ser Cys Pagina 61
SEQLTXT 805 810 815 Phe Ser Val Gly Glu Arg Ala Ile Gly Glu Ala Ile Ala Gly Gly Cys 820 825 830 Arg Thr Pro Gly Ala Leu Gly Gly Lys Leu Lys Cys Gly Thr Asn Cys 835 840 845 Gly Ser Cys Ile Pro Glu Leu Lys Ala Leu Leu Ala Ala Lys Leu Ala 850 855 860 Gln Ala 865 <210> 42 <211> 398 <212> PRT <213> Ogataea parapolymorpha <400> 42 Met Thr Arg Gln Leu Val Ile Ile Gly Asn Gly Met Ala Ala Thr Pro 1 5 10 15 Gly Glu Ala Leu Val Glu Arg Asp Ala Arg Arg Phe Ser Ile Thr Ile
Ile Gly Asp Glu Pro Arg Gln Ala Tyr Asn Arg Ile Gln Leu Ser Pro 40 45 Val Leu Gly Ala Glu Lys Thr Ala Gly Ala Thr Arg Leu Leu Pro Ala 50 55 60 Glu Trp Tyr Ser Gln His Asn Val Thr Val Arg Ala Gly Glu Thr Val 65 70 75 80 Thr Ala Val Asp Met Ala Ala Arg Thr Leu Gln Thr Thr Ala Gly Glu 85 90 95 Leu Gly Trp Asp Glu Leu Val Phe Ala Thr Gly Ser Leu Pro Phe Leu 100 105 110 Pagina 62
SEQLTXT Pro Pro Leu Pro Gly Ile Thr Leu Pro His Val Phe Ala Phe Arg Thr 115 120 125 Leu Ala Asp Val Glu Gly Ile Leu Ala Ile Asp Gly Pro Ala Val Val 130 135 140 Ile Gly Gly Gly Val Leu Gly Val Glu Ala Ala Ala Ala Leu Arg Arg 145 150 155 160 His Gly Asp Ser Val Thr Leu Leu His Arg Gly Ser Trp Leu Met Glu 165 170 175 Gln Gln Thr Asp Ala Phe Val Gly Glu Gln Leu Gln Met Leu Leu Ala 180 185 190 Glu Arg Gly Ile Gly Cys Val Met Glu Ser Arg Ile Ala Ala Ile Asp 195 200 205 Glu His Gln Val Leu Leu Glu Asp Gly Arg Ala Phe Ala Ala Ser Arg 210 215 220 Val Val Leu Ala Thr Gly Val Gln Pro Asn Lys Arg Leu Ala Glu Arg 225 230 235 240 Ser Gly Leu Ala Cys Gly Arg Gly Ile Leu Val Asp Arg Arg Leu Ala 245 250 255 Thr Ala Gln Pro Gly Val Ser Ala Leu Gly Glu Cys Cys Glu Ile Asp 260 265 270 Gly Gln Thr Trp Gly Leu Val Ala Pro Cys Leu Arg Gln Ala Glu Val 275 280 285 Leu Ala Asp Arg Leu Cys Ala Ile Pro Gly Glu Asp Phe Arg Trp Gln 290 295 300 Asp Ser Gly Thr Arg Leu Lys Val Thr Asp Ile Glu Leu Phe Ser Ala 305 310 315 320 Pagina 63
SEQLTXT Gly Glu Leu Arg Ala Asp Glu Gln Asp Asp Val Tyr Thr Ser Trp Asp 325 330 335 Pro Leu Asp Arg His Tyr Arg Arg Leu Leu Leu Arg Asp Gly Lys Leu 340 345 350 Arg Gly Val Leu Leu Leu Gly Asp Cys Ser Ser Ala Ala Pro Leu Thr 355 360 365 Ala Leu Leu Gly Arg His Gly Pro Arg Arg Gln Ser Gly Phe Ser Thr 370 375 380 His Leu Gln Arg Cys Ser Arg Ala Leu Trp Asp Lys Leu Arg 385 390 395 <210> 43 <211> 744 <212> PRT <213> Ogataea parapolymorpha <400> 43 Met Gly Val Glu Pro Phe Ala His Asp Pro Ala Pro Ser Glu Leu Ile 1 5 10 15 His Ser Val Pro Ala Cys Gly Ser Pro Glu Arg Arg Val Met Arg Pro
Met Val Arg Glu Gly Trp Leu Ala Asp Arg Gln His Ser Asp Arg Arg 40 45 Gly Arg Gly Arg Glu Arg Phe Leu Pro Val Ser Trp Asp Ala Ala Leu 50 55 60 Asp Leu Val Ala Gly Glu Ile Arg Arg Val Ser Ala Asp His Gly Asn 65 70 75 80 Ala Ala Ile Phe Ala Gly Ser Tyr Gly Trp Thr Ser Cys Gly Arg Phe 85 90 95 Pagina 64
SEQLTXT His His Ala Ser Thr Leu Leu Lys Arg Met Leu Asn Leu Val Gly Gly 100 105 110 Phe Thr Gly His Val Asp Thr Tyr Ser Ile Ala Ala Gly Pro Val Ile 115 120 125 Leu Arg His Thr Leu Gly Asp Asp Arg Ala Cys Gly Gly Gln Ala Asn 130 135 140 Thr Leu Asp Ser Ile Ala Glu His Ser Gln Thr Leu Val Val Phe Gly 145 150 155 160 Ala Met Ser Pro Arg Thr Ala Gln Ser Glu Ala Gly Gly Ile Gly Ala 165 170 175 His His Leu Glu Thr Tyr Leu Arg Arg Ile Val Glu Arg Gly Val Arg 180 185 190 Val Ile Leu Val Ser Pro Leu Lys Asp Asp Leu Pro Asp Trp Val Ala 195 200 205 Ala Glu Trp Trp Pro Ile Arg Pro Asn Thr Asp Thr Ala Leu Met Leu 210 215 220 Gly Leu Ala Gly Glu Ile Val Arg Ser Gly Arg Gln Asp Ser Asp Phe 225 230 235 240 Leu Ala Arg Cys Thr Ser Gly Ser Glu Leu Tyr Leu Ala Tyr Leu Arg 245 250 255 Gly Glu Gly Asp Gly Arg Pro Lys Asp Ala Glu Trp Ala Ser Thr Ile 260 265 270 Thr Gly Leu Pro Ala Glu Ala Ile Arg Ala Leu Ala Gly Asp Leu Pro 275 280 285 Arg Thr Arg Ser Met Leu Thr Val Ser Trp Ser Leu Gln Arg Ala His 290 295 300 Pagina 65
SEQLTXT His Gly Glu Gln Pro Phe Trp Ala Ala Leu Gly Leu Ala Ala Val Ile 305 310 315 320 Gly Gln Ile Gly Arg Pro Gly Gly Gly Val Gly Tyr Gly Tyr Gly Ser 325 330 335 Leu Gly Gly Val Gly Ala Pro Phe Thr Ile Gly Lys Ser Pro Ala Met 340 345 350 Ser Gln Leu Ser Lys Pro Ile Asn Ser Phe Ile Pro Val Ala Arg Ile 355 360 365 Ser Asp Met Leu Leu Asn Pro Gly Gly Pro Tyr Ser Tyr Glu Gly Glu 370 375 380 Asp Arg Arg Tyr Pro Asp Ile Arg Leu Val Tyr Trp Ser Gly Gly Asn 385 390 395 400 Pro Phe His His His Gln Asp Leu Asn Arg Leu Ser Glu Ala Trp Thr 405 410 415 Arg Pro Glu Thr Ile Ile Val Gln Asp Pro Met Phe Thr Ala Thr Ala 420 425 430 Lys Arg Ala Asp Ile Val Leu Pro Ala Ser Thr Ser Ile Glu Arg Asn 435 440 445 Asp Leu Ala Gly Asn Lys Arg Ser Asp Phe Ile Leu Ala Met Gly Gln 450 455 460 Ala Ile Ala Pro Leu Gly Glu Ala Arg Ser Asp Phe Asp Ile Phe Asn 465 470 475 480 Ala Leu Ser Gly Lys Leu Gly Val Ala Ala Ala Phe Asn Glu Gly Arg 485 490 495 Asp Glu Met Gly Trp Ile Arg His Leu Tyr Glu Glu Ser Arg Asn His 500 505 510 Pagina 66
SEQLTXT Ala Gln Arg His His His Phe Glu Met Pro Asp Phe Glu Thr Phe Trp 515 520 525 Ala Gln Gly His Ala Pro Cys Pro Val Gln Arg Asp His Thr Tyr Leu 530 535 540 Ala Ala Phe Arg Glu Asp Pro Gly Ala His Pro Leu Asp Thr Glu Ser 545 550 555 560 Gly Leu Ile Val Leu Gly Ser Ala Thr Leu Ala Arg Leu Gly Tyr Ala 565 570 575 Asp Cys Gly Pro His Pro Ala Trp Ile Glu Pro Ala Glu Trp Leu Gly 580 585 590 Lys Ala Gln Ala Gly Glu Leu His Leu Ile Ser His Gln Pro Lys Gly 595 600 605 Arg Leu His Ser Gln Leu Glu Thr Ala Glu Ala Ser Leu Ala Gly Lys 610 615 620 Arg Glu Gly Arg Asp Glu Val Met Leu His Pro Asp Asp Ala Ser Val 625 630 635 640 Arg Gly Ile Ala Asp Gly Gln Thr Val Arg Leu Trp Asn Ala Arg Gly 645 650 655 Ala Cys Leu Ala Thr Ala Gln Val Thr Asp Ser Val Ala Ala Gly Val 660 665 670 Ala Ile Leu Pro Thr Gly Ala Trp Phe Thr Pro Ala Glu Ala Glu Gly 675 680 685 Pro Glu Leu Ser Gly Asn Pro Asn Val Leu Thr Leu Asp Ile Gly Ser 690 695 700 Ser Ala Phe Gly Gln Gly Cys Ser Ala His Thr Cys Leu Val Arg Ile 705 710 715 720 Pagina 67
SEQLTXT Glu Ala His Ala Gly Asp Ala Gly Asp Ala Val Arg Ile Tyr Asp Ala 725 730 735 His Leu Ala Ala Ile Leu Pro Thr 740 <210> 44 <211> 958 <212> PRT <213> Ogataea parapolymorpha <400> 44 Met Lys Asp Leu Ile Ile Pro Pro Leu Asp Trp Thr Gln Asp Met Gly 1 5 10 15 Thr Pro Lys Arg Glu Gly Ala Pro Val His Leu Thr Ile Asp Gly Val
Glu Val Thr Val Pro Ala Gly Thr Ser Val Leu Arg Ala Ala Ala Glu 40 45 Ala Gly Ile Ser Ile Pro Lys Leu Cys Ala Thr Asp Asn Val Glu Pro 50 55 60 Val Gly Ser Cys Arg Leu Cys Met Val Glu Ile Glu Gly Met Arg Gly 65 70 75 80 Thr Pro Thr Ser Cys Thr Thr Pro Val Ala Pro Gly Met Arg Val His 85 90 95 Thr Gln Thr Pro Gln Leu Gln Lys Leu Arg Arg Gly Val Met Glu Leu 100 105 110 Tyr Ile Ser Asp His Pro Leu Asp Cys Leu Thr Cys Ala Ala Asn Gly 115 120 125 Asp Cys Glu Leu Gln Asp Met Ala Gly Ala Val Gly Leu Arg Glu Val 130 135 140 Pagina 68
SEQLTXT Arg Tyr Gln Ala Lys Asp Thr His Phe Ala Arg Arg Asp Ala Thr Gly 145 150 155 160 Pro Asn Pro Arg Tyr Ile Pro Lys Asp Asn Ser Asn Pro Tyr Phe Ser 165 170 175 Tyr Asp Pro Ala Lys Cys Ile Val Cys Met Arg Cys Val Arg Ala Cys 180 185 190 Glu Glu Val Gln Gly Thr Phe Ala Leu Thr Val Met Gly Arg Gly Phe 195 200 205 Asp Ala Arg Ile Ser Pro Ala Ala Pro Asp Phe Leu Ser Ser Asp Cys 210 215 220 Val Ser Cys Gly Ala Cys Val Gln Ala Cys Pro Thr Ala Thr Leu Val 225 230 235 240 Glu Lys Ser Val Glu Arg Ile Gly Thr Pro Glu Arg Lys Val Val Thr 245 250 255 Thr Cys Ala Tyr Cys Gly Val Gly Cys Ser Phe Glu Ala His Met Leu 260 265 270 Gly Asp Gln Leu Val Arg Met Val Pro Trp Lys Gly Gly Ala Ala Asn 275 280 285 Arg Gly His Ser Cys Val Lys Gly Arg Phe Ala Tyr Gly Tyr Ala Thr 290 295 300 His Gln Asp Arg Ile Leu Lys Pro Met Ile Arg Asp Lys Ile Thr Asp 305 310 315 320 Pro Trp Arg Glu Val Asn Trp Thr Glu Ala Leu Asp Phe Thr Ala Thr 325 330 335 Arg Leu Arg Ala Leu Arg Asp Ser His Gly Ala Asp Ala Leu Gly Val 340 345 350 Pagina 69
SEQLTXT Ile Thr Ser Ser Arg Cys Thr Asn Glu Glu Thr Tyr Leu Val Gln Lys 355 360 365 Leu Ala Arg Ala Val Phe Gly Thr Asn Asn Thr Asp Thr Cys Ala Arg 370 375 380 Val Cys His Ser Pro Thr Gly Tyr Gly Leu Lys Gln Thr Phe Gly Thr 385 390 395 400 Ser Ala Gly Thr Gln Asp Phe Asp Ser Val Glu Glu Thr Asp Leu Ala 405 410 415 Leu Val Ile Gly Ala Asn Pro Thr Asp Gly His Pro Val Phe Ala Ser 420 425 430 Arg Leu Arg Lys Arg Leu Arg Ala Gly Ala Lys Leu Ile Val Val Asp 435 440 445 Pro Arg Arg Ile Asp Leu Leu Asn Thr Pro His Arg Gly Glu Ala Trp 450 455 460 His Leu Gln Leu Lys Pro Gly Thr Asn Val Ala Val Met Thr Ala Met 465 470 475 480 Ala His Val Ile Val Thr Glu Gln Ile Phe Asp Lys Arg Phe Ile Gly 485 490 495 Asp Arg Cys Asp Trp Asp Glu Trp Ala Asp Tyr Ala Glu Phe Val Ala 500 505 519 Asn Pro Glu Tyr Ala Pro Glu Ala Val Glu Ser Leu Thr Gly Val Pro 515 520 525 Ala Gly Leu Leu Arg Gln Ala Ala Arg Ala Tyr Ala Ala Ala Pro Asn 530 535 540 Ala Ala Ile Tyr Tyr Gly Leu Gly Val Thr Glu His Ser Gln Gly Ser 545 550 555 560 Pagina 70
SEQLTXT Thr Thr Val Ile Ala Ile Ala Asn Leu Ala Met Met Thr Gly Asn Ile 565 570 575 Gly Arg Pro Gly Val Gly Val Asn Pro Leu Arg Gly Gln Asn Asn Val 580 585 590 Gln Gly Ser Cys Asp Met Gly Ser Phe Pro His Glu Phe Pro Gly Tyr 595 600 605 Arg His Val Ser Asp Asp Ala Thr Arg Gly Leu Phe Glu Arg Thr Trp 610 615 620 Gly Val Thr Leu Ser Ser Glu Pro Gly Leu Arg Ile Pro Asn Met Leu 625 630 635 640 Asp Ala Ala Val Glu Gly Arg Phe Lys Ala Leu Tyr Val Gln Gly Glu 645 650 655 Asp Ile Leu Gln Ser Asp Pro Asp Thr Arg His Val Ser Ala Gly Leu 660 665 670 Ala Ala Met Asp Leu Val Ile Val His Asp Leu Phe Leu Asn Glu Thr 675 680 685 Ala Asn Tyr Ala His Val Phe Leu Pro Gly Ser Thr Phe Leu Glu Lys 690 695 700 Asp Gly Thr Phe Thr Asn Ala Glu Arg Arg Ile Asn Arg Val Arg Arg 705 710 715 720 Val Met Ala Pro Lys Ala Gly Phe Ala Asp Trp Glu Val Thr Gln Met 725 730 735 Leu Ala Asn Ala Leu Gly Ala Gly Trp His Tyr Thr His Pro Ser Glu 740 745 750 Ile Met Ala Glu Ile Ala Ala Thr Thr Pro Gly Phe Ala Ala Val Thr 755 760 765 Pagina 71
SEQLTXT Tyr Glu Met Leu Asp Ala Arg Gly Ser Val Gln Trp Pro Cys Asn Glu 770 775 780 Lys Ala Pro Glu Gly Ser Pro Ile Met His Val Glu Gly Phe Val Arg 785 790 795 800 Gly Lys Gly Arg Phe Ile Arg Thr Ala Tyr Leu Pro Thr Asp Glu Lys 805 810 815 Thr Gly Pro Arg Phe Pro Leu Leu Leu Thr Thr Gly Arg Ile Leu Ser 820 825 830 Gln Tyr Asn Val Gly Ala Gln Thr Arg Arg Thr Glu Asn Thr Val Trp 835 840 845 His Gly Glu Asp Arg Leu Glu Ile His Pro Thr Asp Ala Glu Thr Arg 850 855 860 Gly Ile Arg Asp Gly Asp Trp Val Arg Leu Ala Ser Arg Ala Gly Glu 865 870 875 880 Thr Thr Leu Arg Ala Thr Val Thr Asp Arg Val Ser Pro Gly Val Val 885 890 895 Tyr Thr Thr Phe His His Pro Asp Thr Gln Ala Asn Val Val Thr Thr 900 905 910 Asp Thr Ser Asp Trp Ala Thr Asn Cys Pro Glu Tyr Lys Val Thr Ala 915 920 925 Val Gln Val Ala Ala Ser Asn Gly Pro Ser Asp Trp Gln Gln Asp Tyr 930 935 940 Ala Ala Gln Ala Ala Ala Ala Arg Arg Ile Glu Ala Ala Glu 945 950 955 <210> 45 <211> 500 <212> PRT <213> Ogataea parapolymorpha Pagina 72
SEQLTXT
<400> 45
Met Lys Ile Trp Leu Pro Cys Asp Ala Ala Ala Lys Ala Cys Gly Ala
1 5 10 15
Glu Ala Val Leu Ala Ala Leu Arg Leu Glu Ala Glu Lys Arg Gly Gly
Ala Leu Asp Ile Ala Arg Asn Gly Ser Arg Gly Met Ile Trp Leu Glu
40 45 Pro Leu Leu Glu Val Glu Thr Pro Ala Gly Arg Ile Gly Phe Gly Pro 50 55 60
Met Thr Pro Ala Asp Val Pro Ala Leu Phe Asp Ala Leu Glu Ser His
65 70 75 80
Pro Lys Ala Leu Gly Leu Val Glu Glu Ile Pro Phe Phe Lys Arg Gln
85 90 95
Thr Arg Leu Thr Phe Ala Arg Cys Gly Arg Ile Glu Pro Leu Ser Leu 100 105 110
Ala Gln Phe Ala Ala Ala Glu Gly Trp Ala Gly Leu Arg Lys Ala Leu
115 120 125 Lys Met Thr Pro Ala Glu Val Val Glu Glu Val Leu Ala Ser Gly Leu 130 135 140
Arg Gly Arg Gly Gly Ala Gly Phe Pro Thr Gly Ile Lys Trp Arg Thr
145 150 155 160
Val Ala Ala Ala Gln Ala Asp Gln Lys Tyr Ile Val Cys Asn Val Asp
165 170 175
Glu Gly Asp Ser Gly Ser Phe Ala Asp Arg Met Leu Ile Glu Gly Asp 180 185 190
Pro Phe Cys Leu Val Glu Gly Met Ala Ile Ala Gly His Ala Val Gly
Pagina 73
SEQLTXT 195 200 205 Ala Thr Arg Gly Tyr Val Tyr Ile Arg Ser Glu Tyr Pro Asp Ala Ile 210 215 220 Ala Val Met Arg Ala Ala Ile Ala Met Ala Lys Pro Phe Leu Ala Glu 225 230 235 240 Ala Gly Phe Glu Met Glu Val Arg Val Gly Ala Gly Ala Tyr Val Cys 245 250 255 Gly Glu Glu Thr Ser Leu Leu Asn Ser Leu Glu Gly Lys Arg Gly Thr 260 265 270 Val Arg Ala Lys Pro Pro Leu Pro Ala Leu Lys Gly Leu Phe Gly Lys 275 280 285 Pro Thr Val Val Asn Asn Leu Leu Ser Leu Ala Ala Val Pro Trp Ile 290 295 300 Ile Ala His Gly Ala Lys Ala Tyr Glu Ser Phe Gly Met Asp Arg Ser 305 310 315 320 Arg Gly Thr Ile Pro Leu Gln Ile Gly Gly Asn Val Lys Arg Gly Gly 325 330 335 Leu Phe Glu Thr Gly Phe Gly Ile Thr Leu Gly Glu Leu Val Glu Asp 340 345 350 Ile Cys Gly Gly Thr Ala Ser Gly Arg Pro Val Lys Ala Val Gln Val 355 360 365 Gly Gly Pro Leu Gly Ala Tyr His Pro Val Ser Asp Tyr His Leu Pro 370 375 380 Phe Cys Tyr Glu Gln Phe Ala Gly Gln Gly Gly Leu Val Gly His Ala 385 390 395 400 Gly Leu Val Val His Asp Asp Thr Ala Asp Met Leu Lys Leu Ala Arg Pagina 74
SEQLTXT 405 410 415 Phe Ala Met Glu Phe Cys Ala Ile Glu Ser Cys Gly Thr Cys Thr Pro 420 425 430 Cys Arg Ile Gly Ala Val Arg Gly Val Glu Val Ile Asp Arg Ile Ala 435 440 445 Ala Gly Asp Ala Ser Ala Met Pro Leu Leu Asp Asp Leu Cys Gln Thr 450 455 460 Met Lys Leu Gly Ser Leu Cys Ala Leu Gly Gly Phe Thr Pro Tyr Pro 465 470 475 480 Val Gln Ser Ala Ile Arg His Phe Pro Ala Asp Phe Pro Cys Ala Arg 485 490 495 Glu Ala Ala Glu 500 <210> 46 <211> 150 <212> PRT <213> Ogataea parapolymorpha <400> 46 Met Thr Asp Thr Ala Arg Leu Arg Ala Ile Leu Ala Ala His Arg Gly 1 5 10 15 Arg Glu Gly Ala Leu Leu Pro Ile Leu His Asp Val Gln Ala Ala Phe
Gly Phe Ile Pro Glu Asp Ala Tyr Ala Pro Ile Ala Ala Asp Leu Gly 40 45 Leu Thr Arg Ala Glu Val Ala Gly Val Val Gly Phe Tyr His Asp Phe 50 55 60 Arg Lys Ala Pro Ala Gly Arg His Val Ile Lys Leu Cys Arg Ala Glu 65 70 75 80 Pagina 75
SEQLTXT Ala Cys Gln Ala Met Gly Met Asp Ala Val Gln Ala Arg Leu Glu Ser 85 90 95 Ala Leu Gly Leu Arg Leu Gly Asp Ser Ser Glu Ala Val Thr Leu Glu 100 105 110 Ala Val Tyr Cys Leu Gly Leu Cys Ala Cys Ala Pro Ala Ala Met Val 115 120 125 Asp Asp Arg Leu Val Gly Arg Leu Asp Ala Ala Ala Val Ala Gly Ile 130 135 140 Val Ala Glu Leu Gly Ala 145 150 <210> 47 <211> 71 <212> PRT <213> Ogataea parapolymorpha <400> 47 Met Ser Asp Asp Lys Ile Ile Arg Met Ala Asn Gln Ile Ala Ala Phe 1 5 10 15 Phe Ala Val Gln Pro Gly Asp Arg Ala Gly Pro Val Ala Ala His Ile
Ser Glu Asn Trp Ser Ala Pro Met Arg Ala Ala Leu Leu Ala His Val 40 45 Ala Ala Gln Ser Pro Gly Leu Asp Pro Leu Val Ile Ala Ala Ala Pro 50 55 60 Gln Ile Arg Pro Val Pro Ala 65 70 <210> 48 <211> 255 <212> PRT Pagina 76
SEQLTXT <213> Ogataea parapolymorpha <400> 48 Met Ser Leu Pro Ala Gly Ala Val Thr Val Pro Leu Pro Gly Gly Ala 1 5 10 15 Arg Ala Val Leu Ala Glu Glu Val Pro Val Ala Leu Val Phe Asp Gly
Val Thr Gln Ala Val Met Met Ala Ser Pro Val Asp Leu Glu Asp Phe 40 45 Leu Leu Gly Phe Ala Leu Thr Glu Gly Met Ile Ala Asp Arg Ala Glu 50 55 60 Leu Leu Arg His Glu Val Val Arg Gln Pro Gln Gly Ile Glu Leu Arg 65 70 75 80 Gly Trp Leu Ala Ala Pro Ala Gly Gln Arg Phe Ala Ala Arg Arg Arg 85 90 95 Ala Met Ala Gly Pro Val Gly Cys Gly Leu Cys Gly Leu Asp Ser Leu 100 105 110 Ala Ala Val Leu Arg Pro Leu Pro Arg Ala Pro Arg Gly Gly Ala Pro 115 120 125 Pro Pro Leu Ala Asp Gly Ala Leu Ala Ala Leu Arg Ala Gly Gln Ser 130 135 140 Leu Gln Asp Ala Val Arg Ser Val His Ala Ala Gly Phe Trp Asp Gly 145 150 155 160 Ala Gln Met Arg Ala Leu Arg Glu Asp Val Gly Arg His Asn Ala Leu 165 170 175 Asp Lys Leu Ala Gly Ala Leu Ala Gly Gln Gly Ile Asp Ala Ala Ala 180 185 190 Pagina 77
SEQLTXT Gly Ala Leu Val Leu Thr Ser Arg Leu Ser Val Asp Leu Val Gln Lys 195 200 205 Ala Ala Met Ile Gly Ala Arg Val Leu Ile Ala Pro Ser Ala Pro Thr 210 215 220
Ala Leu Ala Val Ala Glu Ala Gln Ala Ala Gly Leu Ala Leu Ile Ala 225 230 235 240 Arg Gly Pro Asp Gly Pro Thr Leu Tyr Thr Glu Thr Glu Ala Glu
245 250 255 <210> 49 <211> 670 <212> PRT <213> Ogataea parapolymorpha <400> 49 Met Thr Val Ser Glu Phe Tyr Pro Leu Pro Val Ser Glu Ile Arg Glu 1 5 10 15 Lys Tyr Tyr Pro Asn Leu Ala Asn Gln Thr Tyr Leu Asp His Ala Gly
Thr Thr Val Tyr Ser Ser Leu Thr Leu Asp Lys Ile His Glu Val Leu 40 45 Ser Lys Thr Leu Leu Ala Asn Pro His Ser Leu Ser Ser Ala Ser Arg 50 55 60
Asp Thr Ala Ser Leu Val Glu Glu Thr Arg Tyr Lys Ile Leu Ser Ile 65 70 75 80 Phe His Ala Asp Pro Ala Glu Tyr Asp Ile Val Phe Ser Leu Asn Ala
85 90 95 Thr His Ala Ile Lys Ile Ala Ala Ser Leu Ile Gln Asp Ala Ala Glu
100 105 110 Ser Ser Phe Asn Tyr Tyr Tyr Asn Ile Asn Cys His Thr Ser Leu Ile Pagina 78
SEQLTXT 115 120 125 Gly Leu Arg Thr Leu Ala Ala Lys Tyr Ala Thr Phe Asp Asp Ile Ser 130 135 140 Ser Phe Glu Pro Val Glu Asp Lys Asp Gly Asn His Pro Ala Leu Asn 145 150 155 160 Phe Val Ser Trp Thr Gly Gln Ser Asn Phe Asn Gly Gln Lys Phe Pro 165 170 175 Leu Gly Trp Cys Lys Glu Phe Arg Arg Arg Leu Asp His Cys Tyr Thr 180 185 190 Leu Tyr Asp Ala Ser Ala Leu Ser Thr Ser Asp Pro Pro Asp Leu Ser 195 200 205 Asp Ala Asn Ser Ser Pro Asp Phe Val Val Met Ser Phe Tyr Lys Ile 210 215 220 Phe Gly Met Pro Asp Ile Gly Ala Leu Ile Leu Arg Arg Ser Thr Ala 225 230 235 240 Lys Gln Leu Val Glu Lys Arg Arg Tyr Phe Gly Gly Gly Thr Ile Asp 245 250 255 Ala Leu Thr Ile Glu Glu Pro Phe Cys Arg Arg Ser Lys Gln Leu His 260 265 270 Gln Ser Leu Glu Asp Gly Thr Ile Pro Val His Ala Ile Leu Glu Leu 275 280 285 Ser Val Ala Ile Asp Ser His Tyr Gln Ile Phe Gly Ser Phe Asn Ser 290 295 300 Ile Arg Leu His Thr Asp Glu Ile Arg Lys Tyr Ala Ile Cys Lys Leu 305 310 315 320 Lys Gln Leu Lys Tyr Gly Asn Thr Gly Arg Arg Met Leu Gln Ile Tyr Pagina 79
SEQLTXT 325 330 335 Asp Trp Pro Gly Ala Lys His Gly Pro Ile Ile Ala Phe Ser Leu Leu 340 345 350 Ser Pro Ala Gly Asp Pro Ile Gly Tyr Tyr Gly Phe Gly Lys Leu Ala 355 360 365 Ser Ala Arg Asn Ile Ser Leu Arg Thr Gly Thr Leu Cys Asn Ile Gly 370 375 380 Gly Ile Gln Lys Phe Leu Asp Arg Thr Asn Glu Asp Ile Arg Gln Asp 385 390 395 400 Tyr Glu Lys Gly His Lys Cys Gly Asp Ile Leu Asp Ile Ile Asp Gly 405 410 415 Lys Pro Thr Glu Leu Val Val Lys Ser Leu Thr Val Tyr Pro Ile Lys 420 425 430 Ser Cys Pro Gly Tyr Arg Ile Pro Glu Gly Arg Lys Trp Lys Leu Thr 435 440 445 Lys His Gly Phe Glu Phe Asp Arg Ser Phe Val Leu Leu Asp Leu Leu 450 455 460 Thr Gln Lys Pro Leu Leu Leu Lys Asn Asn Pro Arg Met Ala Leu Leu 465 470 475 480 Asp Cys Arg Val Asp Pro Glu Lys His Met Leu Tyr Val Arg Asp Lys 485 490 495 Arg Gly Gly Asn Lys Leu Trp Val Ser Thr Asn Ile Arg Arg Tyr Lys 500 505 519 Thr Lys Gln Met Gly Asp Phe Ile Ala Ile Ser Glu Arg Lys Met Val 515 520 525 Lys Phe Phe Ser Asp Val Met Ser Ile Gly Cys Thr Leu Ala Gly Phe Pagina 80
SEQLTXT 530 535 540 Val Thr Glu Lys Gln Met Gln Asn Lys Thr Ala Phe Leu Leu Val Asn 545 550 555 560 Glu Arg Ser Met Arg Gln Val Ser Lys Asp Asp Ser Leu Ile Ser Arg 565 570 575 Phe Arg Ala Asn Ile Val Val Asp Ser Ala His Pro Tyr Ile Glu Asp 580 585 590 Lys Leu Ser Val Leu Thr Asp Met Asp Ser Gly Val Val Leu Lys Lys 595 600 605 Arg Cys Lys Cys Asp Arg Cys Tyr Met Ile Thr Val Ser Asp Lys Gly 610 615 620 Ser Arg Asp Pro Ser Leu Leu Val Glu Leu Ser Lys Glu Arg Lys Gln 625 630 635 640 Lys Gly Lys Val Tyr Phe Gly Val Asn Ile Asp Val Glu Asn Val Gly 645 650 655 Tyr Arg Tyr Met Arg Val Gly Asp Arg Ile Val Gly Glu Glu 660 665 670 <210> 50 <211> 1421 <212> PRT <213> Ogataea parapolymorpha <400> 50 Met Ala Pro Ile Ala Val Glu Pro Ser Pro Tyr Asp Gly Thr Ala Asn 1 5 10 15 Asn Asp Leu Lys Asp Ile Gln Phe Thr Asp Ser Ile Arg Phe Tyr Leu
Asn Asp Lys Leu Gln Val Val Lys Asn Pro Asp Pro Glu Glu Arg Leu 40 45 Pagina 81
SEQLTXT Ile Asp Tyr Ile Arg Asn Glu Ala Asp Leu Arg Gly Thr Lys Glu Ala 50 55 60 Cys Ser Glu Ser Gly Cys Asn Ala Cys Ser Val Thr Ile Ala Ser Ile 65 70 75 80 Ser Tyr Thr Asp Thr Asp Tyr Pro Glu Arg Pro Gln Val His Tyr Arg 85 90 95 Ser Val Asn Ser Cys Val Thr Pro Leu Ile Leu Ala Asp Gly Lys Gln 100 105 110 Val Ile Thr Val Glu Gly Val Gly Ser Ser Lys Asn Pro His Pro Val 115 120 125 Gln Glu Arg Ile Ala Lys Phe His Gly Ser Gln Cys Gly Phe Cys Thr 130 135 140 Pro Gly Phe Val Met Ser Leu Tyr Ala Leu Leu Arg Glu Lys Asn Gly 145 150 155 160 His Val Ser Val Ala Glu Ile Asp Glu Ala Leu Glu Ser Asn Leu Cys 165 170 175 Arg Cys Thr Gly Tyr Met Pro Ile Tyr Asp Ala Ala Tyr Ser Phe Ala 180 185 190 Tyr Asp Ser Asp Asn Tyr Asn Arg Glu Lys Ile Arg Pro Phe Leu Lys 195 200 205 Lys Lys Asp Thr Ser Phe Glu Thr Gly Ser Asp Leu Tyr Gly Gly Ser 210 215 220 Val Cys Ala Leu Gly Thr Lys Cys Cys Arg Tyr Lys Ser Gly Lys Glu 225 230 235 240 Lys Ala Asp Glu Glu Cys Asp Lys Ser Ala Ser Asn Ser Asp Met Glu 245 250 255 Pagina 82
SEQLTXT Ile Asp Met Asn Lys Ile Phe Thr Pro Asn Gly Leu Pro Leu Lys Pro 260 265 270 Tyr Asn Pro Ala Ala Asp Leu Pro Phe Pro Leu Lys Leu Ser Arg Ile 275 280 285 Ser Pro Lys Pro Ile Cys Tyr Gly Asn Glu Arg Lys Val Trp Phe Arg 290 295 300 Pro Val Thr Lys Glu Gln Phe Leu Gln Ile Tyr Arg Ile Tyr Pro Asp 305 310 315 320 Ala Lys Ile Val Ala Gly Ala Ser Glu Val Gln Ile Glu Val Lys Phe 325 330 335 Lys Ala Ala Asn Tyr Lys Val Asn Ile Tyr Ala Gly Asp Val Lys Glu 340 345 350 Leu Lys Gly Trp Ser Tyr Lys Lys Gly Lys Gly Leu Thr Ile Gly Gly 355 360 365 Asp Ile Pro Leu Ile Glu Leu Glu Ser Ile Cys Gly Asp Leu Ala Lys 370 375 380 Arg Leu Gly Arg Thr Ala Ala Gly Gln Thr Tyr Asn Ala Ile Glu Glu 385 390 395 400 Gln Leu Lys Val Phe Ala Ser Lys Ala Val Arg Asn Val Ala Thr Pro 405 410 415 Ala Gly Asn Ile Val Thr Ala Ser Pro Ile Ala Asp Leu Asn Pro Ile 420 425 430 Phe Val Ala Cys Gly Ala Ile Ile Thr Ala Glu Lys Leu Thr Glu Asp 435 440 445 Gly Lys Leu Glu Lys Thr His Ile Asp Met Arg Asp Asn Phe Phe Thr 450 455 460 Pagina 83
SEQLTXT Gly Tyr Arg Arg His Lys Leu Pro Thr Ser Ser Leu Ile Thr Glu Ile 465 470 475 480 Phe Ile Pro Asp Thr Ala Asp Asn Glu Tyr Ile His Cys Tyr Lys Gln 485 490 495 Cys Lys Arg Lys Asp Asp Asp Ile Ser Ile Val Thr Ala Cys Leu Arg 500 505 519 Met Glu Leu Asp Asp Glu Gly Asn Val Leu Asp Ser Thr Leu Val Tyr 515 520 525 Gly Gly Met Ala Pro Ile Thr Lys Asn Ser Pro Lys Ala Glu Lys Thr 530 535 540 Ile Lys Gly Lys Asn Ile Tyr Asn Ser Ser Phe Asn Glu Glu Cys Cys 545 550 555 560 Lys Cys Leu Ser Glu Asp Asp Tyr Lys Met Pro Tyr Gly Val Pro Gly 565 570 575 Gly Ala Ala Ser Tyr Arg Arg Ser Leu Thr Leu Ser Phe Phe Tyr Lys 580 585 590 Phe Trp Gln Tyr Val Leu Ala Thr Ala Pro Ile Pro Lys Ala Asn Val 595 600 605 Ala Thr Ile Gln Cys Arg Asp Ala Ile Leu Asp Val Asp Ser Leu Ser 610 615 620 Glu Val Thr Arg Val Gln Lys His Gly Tyr Arg Glu Met Asn Thr Pro 625 630 635 640 Gly His Lys Thr Gly Ile Ile Gly Lys Pro Ile Val His Val Asn Ala 645 650 655 Ile Lys Gln Ala Thr Gly Glu Ala Gln Tyr Thr Asn Asp Ile Pro Pro 660 665 670 Pagina 84
SEQLTXT Leu His Arg Glu Leu Phe Gly Val Gln Val Met Ser Glu Lys Ala His 675 680 685 Ala Lys Ile Leu Ser Val Asp Trp Ser Glu Ala Leu Glu Val Glu Ser 690 695 700 Val Val Gly Tyr Val Asp Ile Asn Asp Leu Pro Asn Lys Glu Ala Asn 705 710 715 720 Leu Trp Gly Asn Leu Pro Phe Gly Lys Glu Pro Phe Phe Ala Asp Gly 725 730 735 Glu Val Phe Phe Val Gly Gln Ala Ile Gly Val Ile Leu Ala Ser Ser 740 745 750 Lys Glu Arg Ala Tyr Glu Ala Ser Arg Lys Val Arg Val Val Tyr Asp 755 760 765 Glu Leu Pro Arg Ile Ile Ser Val Glu Asp Gly Val Arg Gln Lys Ser 770 775 780 Phe Phe Pro Asp Arg Arg Glu Val Lys Leu Gly Asp Trp Glu Ser Ala 785 790 795 800 Phe Lys Asn Ser Lys Tyr Tyr Leu Glu Asn Thr Ala Arg Leu Ser Ala 805 810 815 Gln Glu His Phe Tyr Phe Glu Val Gln Asn Cys Leu Val Ile Pro Gln 820 825 830 Glu Gly Gly Glu Leu Lys Val Tyr Ser Ser Thr Gln Asn Pro Thr Glu 835 840 845 Thr Gln Leu Cys Ala Ala Gln Val Thr Gly Val Pro Ala Asn Arg Val 850 855 860 Ile Cys Arg Val Lys Arg Leu Gly Gly Gly Phe Gly Gly Lys Glu Thr 865 870 875 880 Pagina 85
SEQLTXT Arg Ser Ile Gln Leu Ser Ser Leu Ala Ala Val Ala Ala Arg Lys Phe 885 890 895 Asn Arg Pro Val Arg Leu Glu Leu Asn Arg Ser Glu Asp Met Lys Thr 900 905 910 Ser Gly Glu Arg His Pro Phe Leu Val Lys Tyr Arg Ala Ser Leu Asp 915 920 925
Glu Asp Leu Lys Phe Thr Gly Leu Asp Met Val Leu Tyr Ala Asn Ala 930 935 940
Gly Trp Ser Met Asp Leu Thr Arg Gly Val Ile Asp Arg Ser Val Leu
945 950 955 960
His Ala Ser Asn Ala Tyr Tyr Ile Pro Asn Ala Arg Val Cys Gly Ile
965 970 975 Pro Val Lys Thr Asn Ile Ala Ser Asn Thr Ala Tyr Arg Thr Phe Gly 980 985 990 Ala Gln Ala Gly Phe Tyr Ala Ile Glu Ser Val Val Thr Glu Phe Ala 995 1000 1005
Glu Lys Leu Gly Val Asp Pro Glu Glu Ile Arg Arg Arg Asn Tyr 1010 1015 1020
Leu Lys Pro Asn Cys Gly Glu Val Phe Pro Tyr Lys Gln Val Val 1025 1030 1035
Gly Glu Asp Ile Thr Ile Ser Asn Val Val Asp Glu Asn Leu Lys 1040 1045 1050
Glu Cys Asn Tyr Lys Lys Met Lys Gln Glu Ile Asn Glu Phe Asn 1055 1060 1065
Lys His Ser Lys Trp Ile Lys Arg Gly Ile Ala Gln Ile Pro Ala 1070 1075 1080
Pagina 86
SEQLTXT
Val Phe Gly Val Ser Phe Gly Val Leu Phe Leu Asn Gln Ala Gly 1085 1090 1095
Ala Leu Val His Ile Tyr Asn Asp Gly Ser Cys Leu Ile Ser Thr 1100 1105 1110
Gly Gly Val Glu Ile Gly Gln Gly Ile Ser Thr Val Met Arg Met 1115 1120 1125
Ile Ala Ala Glu Glu Leu Gly Val Pro Phe Asp Lys Ile Phe Leu 1130 1135 1140
Ser Glu Thr Ser Thr Glu Cys Val Pro Asn Thr Ser Ser Thr Ala 1145 1150 1155
Ala Ser Ser Gly Ser Asp Leu Asn Gly Met Ala Leu Lys Asp Ala 1160 1165 1170
Cys Met Lys Leu Asn Lys Arg Leu Lys Pro Val Lys Asp Ala Ile 1175 1180 1185
Thr Lys Glu Lys Gly Asp Lys Trp Thr Trp Glu Glu Leu Ile Thr 1190 1195 1200
Lys Ala Tyr Leu Asp Arg Val Ser Leu Ser Ala Thr Gly Phe Tyr 1205 1210 1215
Lys Thr Pro Glu Ile Gly Phe Glu Trp Gly Asp Glu Asn Pro Lys 1220 1225 1230
Pro Ala Phe Phe Tyr His Thr Gln Gly Ser Ala Val Ser Val Val 1235 1240 1245
Glu Val Asp Thr Leu Thr Gly Asp Trp Ser Cys Leu Glu Ser His 1250 1255 1260
Ile Lys Met Asp Cys Gly Arg Pro Leu Asn Lys Ala Ile Ile Tyr 1265 1270 1275
Pagina 87
SEQLTXT
Gly Gln Ile Glu Gly Ala Phe Ile Gln Gly Met Gly Tyr Phe Thr 1280 1285 1290
Met Glu Gln Ser Leu Trp Leu Ser Arg Thr Gly Gly Leu Ala Thr 1295 1300 1305
Thr Gly Pro Gly Ala Tyr Lys Ile Pro Gly Phe Arg Asp Thr Pro 1310 1315 1320
Gln Arg Phe Val Leu Ser Met Tyr Lys Gly Ser Asp Phe Arg His 1325 1330 1335
Leu Arg Thr Ile His Ser Ser Lys Gly Val Gly Glu Pro Pro Phe 1340 1345 1350
Phe Leu Gly Ala Ser Val His Phe Ala Leu Arg Asp Ala Ile Gly 1355 1360 1365
His Ala Arg Arg Gln Asn Gly Ile Glu Ser Gly Ser Gln Gly Leu 1370 1375 1380
Arg Phe Arg Val Pro Leu Thr Thr Glu Arg Ile Arg Val Asp Cys 1385 1390 1395
Gly Asp Lys Leu Ala Lys Gln Ser Phe Val Ala Ala Lys Glu Gly 1400 1405 1410
Glu Glu Glu Phe Phe Ile Glu Gly 1415 1420
<210> 51
<211> 302
<212> PRT
<213> Ogataea parapolymorpha
<400> 51
Met Ala Tyr Leu Gln Asp Cys Thr Tyr Gly Lys Asn Asn Val Arg Phe
1 5 10 15
Pagina 88
SEQLTXT Leu Lys Val Lys Arg Asp Pro Ile Asn Pro Lys Ile His Gln Val Met
Glu Ala Ser Val Arg Val Met Leu Thr Gly Ala Phe Asp Val Ser Tyr 40 45 Thr Lys Ala Asp Asn Ser Val Ile Ile Pro Thr Asp Thr Ile Lys Asn 50 55 60 Thr Ile Leu Val Glu Ala Lys Gln Thr Asp Val Phe Pro Ile Glu Arg 65 70 75 80 Phe Ala Ala His Leu Val Lys His Phe Phe Gly Lys Tyr Ser Trp Ile 85 90 95 Ala Gly Ile Thr Val His Ile Glu Gln Ala Lys Trp Ser Lys Tyr Ser 100 105 110 Val Asp Gly Lys Leu Gln Pro His Ser Phe Val Lys Asn Gly Asp Glu 115 120 125 Val Arg Val Cys Glu Leu Val Ser Lys Lys Asn Gly Asp Phe Val Leu 130 135 140 Thr Gly Gly Val Gln Gly Leu Thr Val Leu Lys Ser Ser Gly Ser Met 145 150 155 160 Phe His Gly Tyr Asn Val Cys Asp Tyr Thr Thr Leu Lys Pro Val Asn 165 170 175 Glu Arg Ile Leu Ser Thr Asp Val Asp Cys Lys Tyr Lys Phe Asp Ser 180 185 190 Ala Lys Ile Gly Ser Val Asp Asn Ile Phe Thr Leu Ala Asp Ser Gly 195 200 205 Leu Phe Asp Lys Val Phe Gln Ser Ala Leu Lys Ile Thr Leu Asp Arg 210 215 220 Pagina 89
SEQLTXT Phe Ala Leu Glu Asn Ser Ala Ser Val Gln Ala Thr Met Tyr Asn Met 225 230 235 240 Gly Thr Asp Ile Val Asn Ala Asn Pro Tyr Val Tyr Asn Val Ser Tyr 245 250 255 Ala Leu Pro Asn Lys His Tyr Ile Leu Phe Asp Phe Ser Trp Lys Gly 260 265 270 Leu Lys Asn Glu Asn Glu Met Phe Tyr Pro Ser Pro His Pro Asn Gly 275 280 285 Leu Ile Lys Cys Thr Val Gly Arg Glu Pro Ile Ala Lys Leu 290 295 300 <210> 52 <211> 136 <212> PRT <213> Ogataea parapolymorpha <400> 52 Met Ser Ser Arg Pro Pro Ile Thr Cys His Ile Leu Asp Thr Thr Cys 1 5 10 15 Gly Lys Pro Ala Glu Asn Val Lys Cys Glu Ile Ser Tyr Ile Pro Ser
Asn Gly Ile Thr Ser Pro Ser Glu Val Lys Pro Phe Gly Tyr Ala Tyr 40 45 Thr Asn Gln Asp Gly Arg Ile Gly Ser Trp Asn Ala Ala Asn Ser Thr 50 55 60 Glu Thr Phe Ile Asn Ala Glu Asn Asn Gln Trp Thr Lys Leu Val Ser 65 70 75 80 Gly Thr Tyr Arg Ile Arg Tyr His Thr Lys Asp Tyr Phe Leu Lys Arg 85 90 95 Pagina 90
SEQLTXT Asp Gly Thr Thr Phe Phe Pro Phe Ile Asp Ile Trp Phe Glu Val Pro 100 105 110 Ala Ile Pro Glu Lys His Tyr His Val Pro Leu Leu Leu Ser Asn Tyr 115 120 125 Gly Tyr Ser Thr Tyr Arg Gly Ser 130 135 <210> 53 <211> 186 <212> PRT <213> Ogataea parapolymorpha <400> 53 Met Lys Leu Pro Asp Pro Gln Ala Leu Ser Gln Ile Leu Arg Ser Glu 1 5 10 15 Gln Val Thr Val Ile Asp Thr Leu Phe Glu His Asn Asp Lys Phe Ala
Asp Phe Ile Ile Lys Lys Val Leu Ser His Asn Glu Arg Tyr Gly Ser 40 45 Tyr Arg Glu Phe Ile Lys Ala Val Arg Ile Gln Leu Ile Gln Leu Ala 50 55 60 Asp Asn Tyr Glu Lys Ser Met Thr Gly Asp Leu Gly Asp Met Val Arg 65 70 75 80 Ser Val Ile Ser Ala His Pro Arg Leu Gly Ile Gln Gln Ala Ser Ala 85 90 95 Leu Ser Val Ser Ser Ala Arg Glu Gln Lys Ser Leu Gln Ser Gly Lys 100 105 110 Pro Glu Leu Glu Arg Gln Leu Leu Ala Leu Asn Gln Glu Tyr Glu His 115 120 125 Cys Phe Pro Gly Leu Arg Phe Val Val Phe Val Asn Gly Arg Ser Arg Pagina 91
SEQLTXT 130 135 140 Gln Glu Ile Thr Lys Ile Met Arg Lys Arg Ile Thr Arg Asp Asp Tyr 145 150 155 160 Asn Gln Glu Val Arg Asp Ala Phe Ser Ala Met Cys Asp Ile Ala Leu 165 170 175 Asp Arg Ile Lys Lys Glu Asn Ser Lys Leu 180 185 <210> 54 <211> 192 <212> PRT <213> Ogataea parapolymorpha <400> 54 Met Ser Ala Ile Asp Cys Ile Ile Thr Ala Ala Gly Leu Ser Ser Arg 1 5 10 15 Met Gly Gln Trp Lys Met Met Leu Pro Trp Glu Gln Gly Thr Ile Leu
Asp Thr Ser Ile Lys Asn Ala Leu Gln Phe Cys Ser Arg Ile Ile Leu 40 45 Val Thr Gly Tyr Arg Gly Asn Glu Leu His Glu Arg Tyr Ala Asn Gln 50 55 60 Ser Asn Ile Thr Ile Ile His Asn Pro Asp Tyr Ala Gln Gly Leu Leu 65 70 75 80 Thr Ser Val Lys Ala Ala Val Pro Ala Val Gln Thr Glu His Cys Phe 85 90 95 Leu Thr His Gly Asp Met Pro Thr Leu Thr Ile Asp Ile Phe Arg Lys 100 105 110 Ile Trp Ser Leu Arg Asn Asp Gly Ala Ile Leu Pro Leu His Asn Gly 115 120 125 Pagina 92
SEQLTXT
Ile Pro Gly His Pro Ile Leu Val Ser Lys Pro Cys Leu Met Gln Ala 130 135 140 Ile Gln Arg Pro Asn Val Thr Asn Met Arg Gln Ala Leu Leu Met Gly 145 150 155 160 Asp His Tyr Ser Val Glu Ile Glu Asn Ala Glu Ile Ile Leu Asp Ile 165 170 175 Asp Thr Pro Asp Asp Phe Ile Thr Ala Lys Glu Arg Tyr Thr Glu Ile 180 185 190 Pagina 93

Claims (1)

CONCLUSIESCONCLUSIONS 1. Saccharomycotina gist die van nature geen Moco-pathway-genset heeft omvattende een recombinante Moco-pathway-genset omvat die het mogelijk maakt dat genoemde Saccharomycotina gist Molybdeen-co-factor produceert.Saccharomycotina yeast which does not naturally have a Moco pathway gene set comprising a recombinant Moco pathway gene set that allows said Saccharomycotina yeast to produce Molybdenum co-factor. 2. Saccharomycotina gist volgens conclusie 1, waarbij de Moco-pathway-genset omvat: - een gen met ten minste 70% sequentie-identiteit met SEQ ID NO: 1 en / of coderend voor een GTP 3',8-cyclase; -een gen met ten minste 70% sequentie-identiteit met SEQ ID NO: 2 en / of coderend voor een cyclisch pyranopterinemonofosfaatsynthase; - een gen met ten minste 70% sequentie-identiteit met SEQ ID NO: 3 en / of coderend voor een Molybdopterine synthase katalytische subeenheid; - een gen met ten minste 70% sequentie-identiteit met SEQ ID NO: 4 en / of coderend voor een Molybdopterine synthase zwaveldrager subeenheid; - een gen met ten minste 70% sequentie-identiteit met SEQ ID NO: 5 en / of coderend voor een Molybdopterine adenylyltransferase; en / of - een gen met ten minste 70% sequentie-identiteit met SEQ ID NO: 6 en / of coderend voor een Molybdopterine-synthase-adenylyltransferase en / of een Molybdopterine- molybdeentransferase.Saccharomycotina yeast according to claim 1, wherein the Moco pathway gene set comprises: a gene having at least 70% sequence identity with SEQ ID NO: 1 and / or coding for a GTP 3 ', 8 cyclase; a gene with at least 70% sequence identity to SEQ ID NO: 2 and / or encoding a cyclic pyranopterin monophosphate synthase; a gene with at least 70% sequence identity with SEQ ID NO: 3 and / or encoding a Molybdopterin synthase catalytic subunit; a gene with at least 70% sequence identity with SEQ ID NO: 4 and / or encoding a Molybdopterin synthase sulfur support subunit; a gene with at least 70% sequence identity with SEQ ID NO: 5 and / or encoding a Molybdopterin adenylyl transferase; and / or - a gene with at least 70% sequence identity to SEQ ID NO: 6 and / or encoding a Molybdopterin synthase adenylyl transferase and / or a Molybdopterin molybdenum transferase. 3. Saccharomycotina gist volgens één van de voorgaande conclusies, waarbij de Moco- pathway-genset verder omvat: - een gen met ten minste 70% sequentie-identiteit met SEQ ID NO: 12 en / of coderend voor een molybdeen co-factor guanylyltransferase; en / of - een gen met ten minste 70% sequentie-identiteit met SEQ ID NO: 13 en / of coderend voor een Molybdeen-guanine dinucleotide-biosynthese-adaptereiwit, welk de Saccharomycotina gist in staat stelt gesulfuryleerd Molybdeen co-factor en / of Molybdopterine cytosine dinucleotide te produceren.Saccharomycotina yeast according to any one of the preceding claims, wherein the Moco pathway gene set further comprises: - a gene having at least 70% sequence identity with SEQ ID NO: 12 and / or encoding a molybdenum cofactor guanylyl transferase; and / or - a gene with at least 70% sequence identity to SEQ ID NO: 13 and / or encoding a Molybdenum-guanine dinucleotide biosynthesis adapter protein, which enables the Saccharomycotina yeast to sulfurylated Molybdenum co-factor and / or Molybdopterin to produce cytosine dinucleotide. 4. Saccharomycotina-gist volgens één van de voorgaande conclusies, waarbij de Saccharomycotina gist verder een gen omvat met ten minste 70% sequentie-identiteit met SEQ ID NO: 7 en / of coderend voor een cysteine-desulfurase.Saccharomycotina yeast according to any preceding claim, wherein the Saccharomycotina yeast further comprises a gene having at least 70% sequence identity to SEQ ID NO: 7 and / or encoding a cysteine desulfurase. 5. Saccharomycotina-gist volgens één van de voorgaande conclusies, waarbij de Saccharomycotina gist verder een gen omvat met ten minste 70% sequentie-identiteit met SEQ ID NO: 8 en / of coderend voor een molybdaattransporter waardoor deSaccharomycotina yeast according to any preceding claim, wherein the Saccharomycotina yeast further comprises a gene having at least 70% sequence identity with SEQ ID NO: 8 and / or encoding a molybdate transporter whereby the Saccharomycotina gist molybdaat met hogere affiniteit kan importeren dan zonder een molybdaattransporter.Saccharomycotina yeast can import molybdate with higher affinity than without a molybdate transporter. 8. Saccharomycotina gist volgens één van de voorgaande conclusies, verder omvattende een moco-afhankelijke nitraatassimilatieroute-genset.Saccharomycotina yeast according to any one of the preceding claims, further comprising a moco-dependent nitrate assimilation pathway gene set. 7. Saccharomycotina gist volgens conclusie 6, waarbij de Moco-afhankelijke nitraliteitsassimilatieroute genset één of meer van een gen coderend voor nitraattransporter, een gen dat codeert voor nitraatreductase en een gen dat codeert voor nitrietreductase omvat.The saccharomycotina yeast of claim 6, wherein the Moco-dependent nitrality assimilation pathway gene set comprises one or more of a gene encoding nitrate transporter, a gene encoding nitrate reductase, and a gene encoding nitrite reductase. 8. Saccharomycotina-gist volgens één van de conclusies 6-7, waarbij de Moco-afhankelijke nitraliteitsassimilatieroute-genset het mogelijk maakt dat de Saccharomycotina gist op nitraat kan groeien als enige stikstofbron.The Saccharomycotina yeast of any one of claims 6-7, wherein the Moco-dependent nitrality assimilation pathway gene set allows the Saccharomycotina yeast to grow on nitrate as the sole nitrogen source. 9. Saccharomycotina-gist volgens één van de voorgaande conclusies, waarbij de Saccharomycotina gist verder een gen omvat dat codeert voor bis-MGD afhankelijke formiaat-dehydrogenase dat het mogelijk maakt dat de Saccharomycotina gist formiaat als cosubstraat kan gebruiken.Saccharomycotina yeast according to any of the preceding claims, wherein the Saccharomycotina yeast further comprises a gene encoding bis-MGD dependent formate dehydrogenase that allows the Saccharomycotina yeast to use formate as a co-substrate. 10. Saccharomycotina gist volgens één van de voorgaande conclusies, waarbij de Saccharomycotina gist verder een gen omvat dat codeert voor een xanthineoxidase.Saccharomycotina yeast according to any of the preceding claims, wherein the Saccharomycotina yeast further comprises a gene encoding a xanthine oxidase. 11. Saccharomycotina gist volgens één van de voorgaande conclusies, waarbij de Saccharomycotina gist verder een gen omvat dat codeert voor een biotinesulfoxide-reductase dat het mogelijk maakt dat de Saccharomycotina gist biotine uit peptidegebonden biotine kan verkrijgen.Saccharomycotina yeast according to any one of the preceding claims, wherein the Saccharomycotina yeast further comprises a gene encoding a biotin sulfoxide reductase that allows the Saccharomycotina yeast to obtain biotin from peptide-bound biotin. 12. Saccharomycotina gist volgens één van de voorgaande conclusies, waarbij de gist een ascomycetengist is, bij voorkeur gekozen uit de groep bestaande uit Saccharomyces cerevisiae en Yarrowia lipolytica.Saccharomycotina yeast according to any one of the preceding claims, wherein the yeast is an ascomycee yeast, preferably selected from the group consisting of Saccharomyces cerevisiae and Yarrowia lipolytica.
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