WO2020209718A1

WO2020209718A1 - Yeast with engineered molybdenum co-factor biosynthesis

Info

Publication number: WO2020209718A1
Application number: PCT/NL2020/050241
Authority: WO
Inventors: Jean-Marc Georges Daran; Thomas PERLI; Jacobus Thomas Pronk
Original assignee: Technische Universiteit Delft
Priority date: 2019-04-09
Filing date: 2020-04-09
Publication date: 2020-10-15
Also published as: NL2022905B1

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 dinucleotide, sulfurated molybdenum co-factor and Mo-molybdopterin cytosine dinucleotide. 5 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; 10 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- factor and/or a Molybdenum co-factor cytidylyltransferase to form Molybdopterin cytosine 15 dinucleotide.

Description

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 ( BI01 and BI06) of the synthetic pathway, but this co-enzyme that is essential for carboxylation reactions (e.g. pyruvate carboxylase Pyd and Pyc2, urea carboxylase Dur1 ,2 or acetyl-CoA carboxylase Acd) 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 (Nid) 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 (MoO₄₂-)· 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.

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. 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, 95% "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:

- a gene 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, 96, 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, 96, 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, lie, 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 MOD 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: list of main molybdoenzymes with relative form of Moco.

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 NO: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

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:9;

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 10

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 11

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 14; and/or

a gene 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:

a gene encoding a formate dehydrogenase that allows said yeast to use formate as co-substrate;

a gene 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

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 16;

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 17;

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 18;

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO: 19;

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:20;

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:21 ;

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:23;

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:24;

a gene having at least 50, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% sequence identity with SEQ ID NO:25; and/or

a gene 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 I _or class II 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.

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 OD₆₆₀ of 1. A volume of 10 pi of normalized cell suspension was spotted on either SMA or SMNo plates. Strains were spotted on the 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 IMD019,

IMD020, IMD021 , IMD022, IMD023 and IMD027, 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 IMX1777 strain construction. Each expression module was amplified by PCR and 80 bp long unique homology arms were added to each fragment extremity. Fragments were co-transformed together with pUDR 119, a plasmid carrying a gRNA targeting the SGA1 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 60 bp long unique homology arms were added to each fragment extremity. Fragments were co-transformed together with pUDR119, a plasmid carrying a gRNA targeting the SGA1 locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.

Figure 6: schematic representation of IMX1779 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 pUDR1 19, a plasmid carrying a gRNA targeting the SG.A1 locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.

Figure 7: schematic representation of IMX1780 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 pUDR1 19, a plasmid carrying a gRNA targeting the SGA1 locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.

Figure 8: schematic representation of IMX1781 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 pUDR1 19, a plasmid carrying a gRNA targeting the SGA1 locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.

Figure 9: schematic representation of IMX1782 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 togetherwith pUDR119, a plasmid carrying a gRNA targeting the SGA1 locus (A). Correct assembly of each junction was verified by diagnostic PCR (B). Primers used for each PCR are shown.

Figure 10: copy number estimation of Chromosome IX for the S. cerevisiae strains adapted for growth on nitrate as sole nitrogen source. The SGA1 locus, where the different pathways where integrated, is located on ChriX at position 178004-179653. All adapted strains show an increase copy number at that locus, up to three copies for IMS0816 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 ( OD₆₆₀) 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.⁶⁷ Error bars represent the standard deviation. (n=2).

Figure 12: growth curve of IMS0816 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 (DMo-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 DMo-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 IMS0817 on synthetic medium with 10 mM ammonium nitrate as nitrogen source. OD is shown in squares. Nitrate, ammonium, glucose and ethanol 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 660 nm ( OD₆₆₀) 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, UK). 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 {Tie!, Nederiands) 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=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 YPRcTAU3 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.

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:

P0A738; Cyclic pyranopterin monophosphate synthase), MoeB (P12282; Molybdopterin- synthase adenylyltransferase), IscS (P0A6B7; Cysteine desulfurase), MoaD (P30748;

Molybdopterin synthase sulfur carrier subunit), MoaE (P30749; Molybdopterin synthase catalytic subunit), MogA (P0AF03; 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 ceils available at the Short Read Archive database under accession number - SRX365635 and SRX365636 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 ofE. coli Moco related proteins versus O. parapolymorpha transcriptome.

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 pUD697, 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^-1 Bacto tryptone, 5 g L^-1 Bacto yeast extract, 5 g L^-1 NaCI) supplemented with 100 mg L^-1 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 pUDP002 (Addgene plasmid number #103872) and pUD697 using Bsal and ligating with T4 ligase (Golden Gate cloning). Similarly, pUDP094 (gRNA_{HPOD __02674}), pUDP095 (gRNA_{HPODL_00948}), pUDP096 (gRNA_{HPODL_00195}), pUDP097 (gRNA_{HPOD __03424}), pUDP100 (gRNA_{HPODL_02128}) and pUDP101 (gRNA_HPOD__₀₁₆₄₀) 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 OpYNR 1, gene encoding the O.

parapolymorpha Moco dependent nitrate reductase gene, was also constructed. For this, plasmid pUD703 containing the gRNA sequence targeting the OpYNR 1 gene was de novo synthesized at GeneArt (Themo Fisher Scientific) and used in a Bsal golden gate assembly with pUDP002 as done for the other pUDP plasmids, yielding pUDP099.

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^-1 Bacto tryptone, 5 g L^-1 Bacto yeast extract, 5 g L^-1 NaCI) supplemented with 150 mg L^-1 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 L^-1, Bacto yeast extract, 20 g L^-1 Bacto peptone, 20 g L^-1 glucose and 20 g L^-1 agar) supplemented with hygromycin B at a final concentration of 200 mg L^-1. 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, OpYNR 1, 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 INDELs.

With the exception HPODL_02128, a ScNFSI 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, OpYNR 1, HPODL_01640 were renamed IMD019, IMD020, IMD021 , IMD022, IMD023, IMD025 and IMD027 respectively (Figure 2). The ability of the O. parapolymorpha DL-1 (CBS 11895) and its derived strains IMD019, IMD020, IMD021 , IMD022, 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^-1 glucose, 3 g L^-1 KH₂PO₄, 0.5 g L^-1 MgSO₄, 7 H₂O, 5g L^-1 (NH₄₂ SO₄, 1 m L^-1 of a trace element solution and of a vitamin solution a standard vitamin solution and on synthetic medium with nitrate (SMNo) in which (NH₄)₂SO₄ was substituted with 5 g L^-1 K₂SO₄ and 4.3 g L^-1 NaNO₃.

While the reference strain DL-1 was able to grow on plate containing nitrate as sole nitrogen source, the strain IMD025 that carries a disruptive mutation in OpYNR 1 (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)₂ 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 Sc NFS1 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 OpYNTI (HPODL_02387), nitrate reductase OpYNR 1 (HPODL_02384) and nitrite reductase OpYNH (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 Phusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific).

Primer pairs were used to amplify ScPYK 1t, ScTPi 1t, ScFBA1t, ScPDC1t, ScGPM1t respectively and used in a Golden Gate reaction together with the pMC entry vector to yield pGGKp040, pGGKpG42, pGGKpQ46, pGGKp048 respectively.

Moreover, other promoters and terminators compatible with the yeast toolkit were synthesized (GeneArt). Promoters regions of glycolytic genes were selected to be 800 bp long while terminators 300 bp long. Plasmids carrying ScFBAlp, ScTP11p, ScGPM 1p were renamed pGGKp1Q4, pGGKp114, pGGKpT18 respectively.

The entry vector pGGK017 was constructed by combining YTK plasmids, pYTK002,

PYTK047, pYTK072, pYTK074, pYTK082 and pYTK083 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, OpYNR 1 (HPODL_02384), OpYNH (HPODL_02386), OpYNTI (HPODL_02387) were directly PCR-amplified from the O. parapolymorpha DL-1 genome. Genomic DNA of O. parapolymorpha was isolated using the Qiagen 100/G Kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations. Instead, the high affinity molybdate importer CrMOTI 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, CrMOTI, OpYNH and OpYNR 1 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 pYTK096, pTK010 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 pYTK096, pTK011 and pYTK053 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUD1191.

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 pYTK096, pTK012 and pYTK054 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 pYTK096, pTK014 and pYTK056 in a Golden gate assembly reaction that yielded plasmid pUDI194.

CrMOTI 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.

OpYNTI (TIPODL_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.

OpYNR 1 (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^-1 Bacto tryptone, 5 g L^-1 Bacto yeast extract, 5 g L^-1 NaCI) supplemented with 150 mg L^-1 kanamycin at 37 °C. Samples were mixed with glycerol (30% v/v) and stored at -80 °C.

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 pYTK009 plasmid as a template and primers. The ScENOI terminator was amplified using the pYTK051 plasmid as template and primers. The plasmid backbone was amplified using pYTK096 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 Biolabs, Ipswich, MA). that yielded plasmid pUDI 189.

HPODL_02128 was amplified using primers. The ScHHFI promoter was amplified using the pYTK015 plasmid as a template and primers. The ScFBA I terminator was amplified using the pGGKp046 as template and primers. The plasmid backbone was amplified using pYTK096 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 pUDI 197.

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^-1 Bacto tryptone, 5 g L^-1 Bacto yeast extract, 5 g L^-1 NaCI) supplemented with 150 mg L^-1 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 OpYNI1 (HPODL_02386) were constructed using in vivo assembly in S. cerevisiae.

HPODL_00948 was amplified using primers. The ScGPMI promoter was amplified using the pGGKp116 plasmid as a template and primers. The ScPYKI terminator was amplified using the pGGK040 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.PK1 13-5D ( 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 II (Zymo research).

OpYNH (HPODL_02386) was amplified using primers. The ScTPH promoter was amplified using the pGGKpl 14 plasmid as a template and primers. The ScTPH 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 II (Zymo research). The in vivo assembled pasmids pUDE796 and pUDE797 were transformed to chemically competent E. coli XL1-blue cells according to the supplier’s instructions (Agilent

Technologies) using the chemical transformation protocol. Transformants were selected and propagated on LB Broth (10 g L^-1 Bacto tryptone, 5 g L^-1 Bacto yeast extract, 5 g L^-1 NaCI) supplemented with 150 mg L^-1 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 pUD1191 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 SGA1 gene was transformed in the strain IMX585 ( MATa ura3-52

can1D::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 L^-1 acetamide as nitrogen source and 6.6 g L^-1 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 pUD1191 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 CrMOTI 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 SGA1 gene was transformed in the strain IMX585 ( MATa ura3-52 can 1D::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 CrMOTI expression module was amplified using primers pairs and pUDI195 as template. To integrate the module at the SGA1 locus, 200 ng of pUDR119 a plasmid expressing the gRNA targeting the SGA1 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 Op YNT1 (HPODL_02387) expression module was amplified using primers pair and pUDI198 as template. The OpYNR 1 (HPODL_02384) expression module was amplified using primers pairs and pUDI199 as template. The OpYNH (HPODL_02386) expression module was amplified using primers pairs and pUDE797 as template. To integrate the different compatible module at the SGA1 locus, 200 ng of pUDR119 a plasmid expressing the gRNA targeting the SGA1 gene was transformed in the strain IMX585 ( MATa ura3-52 can1D::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 pUDI190 as template. The HPODL_00195 expression module was amplified using primers pairs and pUD1191 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 CrMOTI expression module was amplified using primers pairs and pUDI195 as template. The OpYNTI

(HPODL_02387) expression module was amplified using primers pair and pUDI198 as template. The OpYNR 1 (HPODL_02384) expression module was amplified using primers pairs and pUDI199 as template. The OpYNI1 (HPODL_02386) expression module was amplified using primers pairs and pUDE797 as template. To integrate the different compatible module at the SGA1 locus, 200 ng of pUDR119⁶² a plasmid expressing the gRNA targeting the SGA1 gene was transformed in the strain IMX585 ( MATa ura3-52 can1D::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 pUD1191 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 YNT1 (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 OpYNH

(OpHPODL_02386) expression module was amplified using primers pairs and pUDE797 as template. To integrate the different compatible module at the SGA1 locus, 200 ng of pUDR119 a plasmid expressing the gRNA targeting the SGA1 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 (Looke Biotechniques 50, 325-328 (2011). DreamTaq 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, SHR3, SHR4, SHR5, SHR6, SHR7, 5’ SGA 1 SHR respectively (Figure 4B).

For IMX1778, primers pair were used to verify the junction 3’ SGA1 SHR, SHR1 , SHR2, SHR3, SHR4, SHR5, SHR6, SHR7, SHR8, 5’ SGA 1 SHR respectively (Figure 5B).

For IMX1779, 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’ SGA1 SHR, SHR1 , SHR2, SHR3, SHR4, SHR5, SHR6, SHR7, SHR8, SHR9, SHR10, SHR1 1 , 5’ SGA1 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 pi 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 L- KNO₃ and 38 mmol L^-1 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 HiSeq250Q sequencer ( Illumina, San Diego, CA) with 150 bp paired-end reads using PCR-free library preparation by Novogene Bioinformatics Technology Co., Ltd (Yuen Long, Hong Kong). Data mapping was performed against the CEN.PK1 13-7D genome⁶⁵ 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 number 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 ( OD₆₆₀) was standardized to 0.2. The flasks were then incubated 30 °C and 200 rpm. The growth was monitored by OD₆₆₀ 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 h^-1 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 CrMOTI from Chlamydomonas reinhardtii was not essential at a molybdate concentration of 1.6 mM. 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 mM 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^-1. After twelve subsequent transfers to a new culture, the IMS0816 population grew at a growth rate of 0.14 h^-1 (Figure 12BC). Conversely, the strain

IMS0817(without CrMOTI ) did not show good growth even after extended incubation of 700 h-¹ (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^-1 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 YPRcTau3 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 OpYNTI (HPODL_02387) and nitrite reductase OpYNH (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 Phusion® 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 pYTK096, pTK009 and pYTK051 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 pYTK096, pTKOIO and pYTK052 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 pYTK096, pTK011 and pYTK053 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 pYTK054 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI216.

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 Op YNT1 (HPODL_02387) expression module was amplified using primers pair and pUDI198 as template. The OpYNI1 (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 Phusion® High-Fidelity 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, pTK011 and pYTK053 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI219.

Golden gate assembly mix was transformed to chemically competent Escherichia coli X L1- 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^-1 Bacto tryptone, 5 g L^-1 Bacto yeast extract, 5 g L^-1 NaCI) supplemented with 150 mg L^-1 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.

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, RcFdsG, 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 Phusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific).

The expression cassettes for RcFdsG, RcFdsD, RcFdhD were constructed in vitro by Golden Gate cloning.

The plasmid carrying the RcFdsG gene pUD961 was combined with pYTK096, pTK013 and pYTK055 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 pYTK056 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 pYTK096, pTK015 and pGGKp045 in a Golden gate assembly reaction using Bsal and T4 DNA ligase that yielded plasmid pUDI224.

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 ScTDH3 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 NEBuilder® HiFi DNA

Assembly Master Mix (New England Biolabs, 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 SkADHI promoter, RcFdsA gene and ScFBAI 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.

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 SkADHI promoter was amplified using primers pairs and pGGKp062 as template. The RcFdsA coding sequence was amplified using primers pairs and pUD959 as template. The ScFBAI 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.

Claims

1. Saccharomycotina yeast that is naturally devoid of a molybdenum cofactor (Moco) pathway gene set comprising a recombinant Moco pathway gene set that allows said

Saccharomycotina yeast to produce Molybdenum co-factor, wherein the Moco pathway gene set comprises

- a gene having at least 70% sequence identity with SEQ ID NO:1 and encoding a GTP 3',8- cyclase;

- a gene having at least 70% sequence identity with SEQ ID NO:2 and encoding a Cyclic pyranopterin monophosphate synthase;

- a gene having at least 70% sequence identity with SEQ ID NO:3 and encoding a

Molybdopterin synthase catalytic subunit;

- a gene having at least 70% sequence identity with SEQ ID NO:4 and encoding a

Molybdopterin synthase sulfur carrier subunit;

- a gene having at least 70% sequence identity with SEQ ID NO:5 and encoding a

Molybdopterin adenylyltransferase; and/or

- a gene having at least 70% sequence identity with SEQ ID NO:6 and encoding a

Molybdopterin-synthase adenylyltransferase and/or a Molybdopterin molybdenumtransferase.

2. Saccharomycotina yeast according to claim 1 , wherein the Moco pathway gene set further comprises

- a gene having at least 70% sequence identity with SEQ ID NO: 12 and encoding a

Molybdenum co-factor guanylyl transferase; and/or

- a gene having at least 70% sequence identity with SEQ ID NO: 13 and encoding a

Molybdenum-guanine dinucleotide biosynthesis adapter protein,

which allows said Saccharomycotina yeast to produce sulfurylated Molybdenum co-factor and/or Molybdopterin cytosine dinucleotide.

3. Saccharomycotina yeast according to any one of the previous claims, wherein the Saccharomycotina yeast further comprises a gene having at least 70% sequence identity with SEQ ID NO:7 and encoding a Cysteine desulfurase.

4. Saccharomycotina yeast according to any one of the previous claims, wherein the Saccharomycotina yeast further comprises a gene having at least 70% sequence identity with SEQ ID NO:8 and encoding a molybdate transporter allowing the Saccharomycotina yeast to import Molybdate with higher affinity than without a molybdate transporter.

5. Saccharomycotina yeast according to any one of the previous claims, further comprising a Moco dependent nitrate assimilation pathway gene set.

6. Saccharomycotina yeast according to claim 5, 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.

7. Saccharomycotina yeast according to any one of claims 5-6, wherein the Moco dependent nitrate assimilation pathway gene set allows said Saccharomycotina yeast to grow on nitrate as sole nitrogen source.

8. Saccharomycotina yeast according to any one of the previous claims, 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.

9. Saccharomycotina yeast according to any one of the previous claims, wherein the Saccharomycotina yeast further comprises a gene encoding a xanthine oxidase.

10. Saccharomycotina yeast according to any one of the previous claims, 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.

11. Saccharomycotina yeast according to any one of the previous claims, wherein the yeast is an ascomycete yeast, preferably chosen from the group consisting of

Saccharomyces cerevisiae and Yarrowia lipolytica.