WO2020234215A1 - Biotin prototrophy - Google Patents

Abstract

The present invention relates to a biological cell that can grow in the absence of biotin under aerobic conditions, comprising a BIO1 gene (i.e. CfBIO1), and preferably a BIO6, BIO2, BIO3, and BIO4 gene. The present disclosure additionally relates to a biological cell that can grow in the absence of biotin under aerobic and anaerobic conditions, comprising the genes fabD, bioC, fabB, fabG, fabZ, fabI, bioH, bioF, adpP, acpS, and preferably the genes BIO2, BIO3, and BIO4. The biological cell according to the disclosure may be a prokaryotic cell or an eukaryotic cell, preferably a yeast cell, such as a Saccharomycotina yeast, most preferably Saccharomyces cerevisiae.

Description

BIOTIN PROTOTROPHY

Technical field

The present disclosure relates to biological cells that can grow in the absence of biotin under aerobic and/or anaerobic conditions, i.e. biotin prototroph biological cells, in particular of a yeast such as Saccharomyces cerevisiae.

Background of the disclosure

A vitamin is an essential micronutrient that cannot be de novo synthesized by an organism and that is needed in small quantities for the proper functioning of cellular metabolic functions. Therefore, vitamins have to be acquired through diet. Biotin is also referred to as vitamin H or B7, essentially because animals cannot synthesize it.

Biotin is an indispensable cofactor responsible for carbon dioxide transfer in several carboxylase enzymes. In Saccharomyces cerevisiae, biotin is involved in four different reactions (Table A below).

Table A: Biotin dependent enzyme activities found in Saccharomyces cerevisiae.

Enzyme activity Protein Function References pyruvate carboxylase³ Pycl and Pyc2 anaplerotic formation of

oxaloacetate

urea amidolyase³ Durl and Dur2 releases ammonia from urea

aBiotin is covalently linked to these enzymes by the biotin-protein ligase Bpl1.

bOnly biotin-dependent protein that is not a carboxylase

However, in S. cerevisiae the term vitamin might not be totally appropriate. Although some strains as the reference strain S288C have a full biotin requirement as they miss two genes BI01 and BI06 (Goffeau et ai. 1996) that encode the first two steps of the biosynthetic pathway, other laboratory and wild strains have retained these genes and are able to grow in absence of biotin but at extremely low growth rate (Barbosa et ai. 2018, Hall and Dietrich 2007, Nijkamp et al. 2012). This is the case of the laboratory strain CEN.PK113-7D that is able to grow in medium devoid of biotin, however exhibiting a lower growth rate than in its presence. Evolutionary engineering of biotin independent growth of this strain led to the selection of mutants that grew as fast as the reference strain in presence of biotin (Bracher et al. 2017). This evolved phenotype was linked to a massive amplification of the BI01-BI06 genes cluster. Originally located on CHRI (Nijkamp et al. 2012), genome sequencing and karyotyping revealed that the BI01 - BI06 cluster was amplified over 40-times and was found on nearly all chromosomes (Nijkamp et al. 2012). Although successful, the genetic blue-print underlying this evolved phenotype cannot be easily transferred from one genetic background to another. Attempts to overexpress BI01 and BI01 - BI06 cluster did not yield strains able to grow at a growth rate similar to that of the same strain grown in presence of biotin suggesting still a limiting supply of de novo synthesised co-factor (Bracher et al. 2017, Hall and Dietrich 2007). Several hypothesis have been put forward, including the requirement of additional mutations in genes involved in transport of metabolic intermediates or requirement of codon optimisation of the BIO genes since they might have been inherited from horizontal gene transfer (Bracher et al. 2017, Hall and Dietrich 2007, Wu et al. 2005). However no further investigation has been yet performed and the only available option to implement full prototrophy seems to be restricted to an evolutionary engineering approach that is time consuming and by definition random.

Although the genetic make-up for the biosynthesis of the biotin fused rings structure is well characterised in S. cerevisiae (Phalip et al. 1999, Zhang et al. 1994), the origin of the pimeloyl moiety that contributes to the valeryl arm of the co-factor remains elusive. Together with alanine and pyridoxal phosphate, pimeloyl-CoA can be converted in 8-amino-7-oxononanoate (also referred to as KAPA) by Bio6 (KAPA synthase) (Wu et al. 2005). Further upon action of the BI03, BI04 and BI02 encoded enzymes, KAPA is sequentially converted to biotin (Figure 1). The lack of knowledge on the origin of the pimeloyl moiety has so far hindered further targeted molecular improvement of biotin synthesis. Contrasting with the situation in

Saccharomyces, several biotin biosynthetic pathways in model systems, Bacillus subtilis and Escherichia coli have been elucidated and no fewer than three pathways have been identified. In E.coli, biotin synthesis is produced by the so-called BioC-BioH pathway that diverts malonyl-coA from fatty acid synthesis. To do so, BioC masks the ra-carboxyl group of a malonyl-thioester by methylating it which allows recognition of this uncommon substrate by the fatty acid enzymatic machinery (Lin et al. 2010) (Figure 2). In two successive rounds of fatty acid synthesis the malonyl-thioester methyl ester is elongated in pimeloyl-ACP methyl ester. The second specific activity catalysed by BioH reverses the methylation introduced by BioC freeing pimeloyl-ACP that enters the biotin biosynthetic pathway (Cao et al. 2017). In Bacillus subtilis two distinct pathways have been identified. The first route is characterised by Biol that encodes a P450 dependent C-C bond cleaving oxygenase (Stok and De Voss 2000) which can release pimeloyl-ACP from long chain (C_M-C_{I S}) acyl-ACP molecules (Cryle and De Voss 2004). For instance cleavage of palmitoyl-ACP would generate nonanoate (Cg) in addition of pimeloyl-ACP (Cryle and Schlichting 2008). The second pathway depends on the activity of BioW that encodes a pimeloyl-CoA synthetase which activates free pimelic acid with Co-enzyme A (Bower et al. 1996, Ploux et al. 1992). However the origin of pimelic acid is still unknown, a recent study confirmed the occurrence of free pimelic acid in B. subtilis and ¹³C-NMR results would indicate that it derives from fatty acid synthesis (Manandhar and Cronan 2017). From these two options only BioW dependent pathway is essential. The deletion of biol resulted in growth reduction but was overall dispensable (Manandhar and Cronan 2017).

All these pathways share a tight link to fatty acid metabolism and use pimeloyl-ACP, a metabolite that is unlikely to be found freely available in cytoplasm of S. cerevisiae. Indeed S. cerevisiae as other fungi have a type I fatty acid synthetase, which comprises two large multi functional proteins (Fas1 and Fas2) that contain all of the necessary catalytic sites for the synthesis of fatty acid that eventually release acyl-CoA and not acyl-ACP (Lomakin et al.

2007, Schweizer et al. 1986, Wieland et al. 1979). This raises the question of the origin of pimeloyl-CoA in eukaryotic metabolism.

The rapid and controlled development of biotin prototrophic strains should held significant technological advantages. It would enable design of cheap easy to handle media since they would retain longer shelf life characteristics and would not require storage at low temperature to prevent biotin thermal degradation. This concept has been productively applied for heterologous proteins production with a Komagataella phaffii (formerly known as Pichia pastoris) expressing the S. cerevisiae BI01 and BI06 genes (Gasser et al. 2010). Additionally it could be anticipated that these media should further limit fermentation contamination to obligatory biotin auxotrophs.

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 have devised two methods to restore biotin prototrophy in S.

cerevisiae. In the first approach, the inventors alleviated metabolic bottleneck of biotin biosynthesis linked to BI01. To this end, the inventors screened a set of Saccharomycotina yeasts for biotin prototrophy, identified the orthologous ScBIOI gene and expressed it constitutively in S. cerevisiae. Expression of the Cyberlindnera fabianii BI01 gene was able to transfer the biotin prototrophy phenotype in a range of S. cerevisiae strains. It was found that biotin prototrophy phenotype is particularly restored (same growth rate in the presence or absence of biotin) if one, preferably all of the genes BI06, BI02, BI03, BI04 (already present in S. cerevisiae) are co-expressed with CfBIOI. The S. cerevisiae strains expressing CfBIOI exhibited a growth not less than 85% relative to growth rate in presence of biotin. Although expression of CfBIOI restored growth under aerobic conditions, it could not restore biotin prototrophy to the such extent under anaerobic conditions.

To circumvent this, a second strategy was designed based on the E. coli malonyl-thioester methyl ester pathway, that derived fatty acid biosynthesis towards synthesis of pimeloyl-ACP. To do so, the pathway including EcfabD, EcbioC, EcfabB, EcfabG, EcfabZ, Ecfabl, EcbioH, EcbioF, EcacpP, EcacpS that encode for the [acyl-carrier-protein] S-malonyltransferase, [malonyl-acyl-carrier-protein] O-methyltransferase, beta-ketoacyl-[acyl-carrier-protein] synthase, 3-oxoacyl-[acyl-carrier-protein] reductase, 3-hydroxy-acyl-[acyl-carrier-protein] dehydratase, Enoyl-[acyl-carrier-protein] reductase, pimeloyl-acyl carrier protein methyl ester esterase, 8-amino-7-oxononanoate synthase and acyl-carrier-protein, holo-[acyl-carrier- protein] synthase respectively was expressed in S. cerevisiae and conferred growth in absence of biotin in presence as well as in absence of oxygen.

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 minimises 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 element is 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 cell, i.e. a biological cell, comprising a recombinant biotin pathway gene set that allows said cell to grow in the absence of biotin under aerobic and/or anaerobic conditions, as disclosed herein. Preferably, said cell is naturally devoid of such a gene set that allows said cell to grow in the absence of biotin. The cell according to the present disclosure is preferably a prokaryotic or eukaryotic cell, more preferably a yeast cell, even more preferably a Saccharomycotina yeast, or an ascomycete yeast, preferably Saccharomyces cerevisiae.

In a first embodiment (biotin pathway 1), the cell according to the present disclosure comprises a gene, particularly a BI01 gene ( CfBIOI ), having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:1. Preferably, the cell is not a Cyberlindnera fabianii cell.

Additionally or alternatively, the cell may comprise one or more of

- a gene, particularly a BI02 gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 12;

- a gene, particularly a BI03 gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 13;

- a gene, particularly a BI04 gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 14; and/or

- a gene, particularly a BI06 gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 15;

In a second embodiment (biotin pathway 2), the cell according to the present disclosure may comprise one or more of

- a gene, particularly a f abD gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:2;

- a gene, particularly a bioC gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:3;

- a gene, particularly a fabB gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:4;

- a gene, particularly a fabG gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:5;

- a gene, particularly a fabZ gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:6; - a gene, particularly a fabl gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID N0:7;

- a gene, particularly a bioH gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95,

96, 97, 98, 99, 100% sequence identity with SEQ ID N0:8;

- a gene, particularly a bioF gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95,

96, 97, 98, 99, 100% sequence identity with SEQ ID N0:9;

- a gene, in particular an acpP gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 10; and/or

- a gene, in particular an acpS gene, having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 11 , wherein preferably the cell is not an Escherichia coli cell.

Additionally or alternatively, the cell may comprise one or more of

- a BI02 gene having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:12;

- a BI03 gene having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 13; and/or

- a BI04 gene having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:14.

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.

The cell according to the present disclosure, comprising biotin pathway 1 or biotin pathway 2, was found to be able to grow on medium comprising less than 5, 4, 3, 2, 1 mg biotin per kg medium, wherein a cell comprising biotin pathway 1 according to the present disclosure is able to grow on such medium under aerobic conditions, and a cell comprising biotin pathway 2 according to the present disclosure is able to grow on such medium under aerobic conditions and anaerobic conditions.

For example, such medium devoid of biotin, may comprise ammonium sulfate (15 mM), monopotassium phosphate (6.6 mM), dipotassium phosphate (0.5 mM), sodium chloride (1.7 mM), calcium chloride (0.7 mM), magnesium chloride (2 mM), boric acid (0.5 pg/ml), copper chloride (0.04 pg/ml), potassium iodide (0.1 pg/ml), zinc chloride (0.19 pg/ml), calcium pantothenate (2 pg/ml), thiamine (2 pg/ml), pyridoxine (2 pg/ml), inositol (20 pg/ml), and glucose (2%).

The cell according to the present disclosure (having biotin pathway 1 or biotin pathway 2) may be able to grow at a specific growth rate in the absence of biotin (for example on the medium devoid of biotin as described above) of at least 25, 50, 75, 80, 85, 90, 95, 100% relative to specific growth rate in presence of biotin (for example on the medium as described above but wherein biotin is added to a final concentration of 2 pg/liter).

Specific growth rate may be determined under optimal conditions (e.g. 30-35 degrees Celsius and aerobic/anaerobic) as follows

Nt = No * ( 1 + if

where:

Nt: The amount at time t

No: The amount at time 0

r: specific growth rate

t: Time passed

In the present disclosure, anaerobic conditions mean that oxygen is absent, or present in a maximum concentration of at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 1000, 5000, 10000, 50000 ppm, e.g. in the medium and/or direct surroundings thereof. Conversely, aerobic conditions mean that oxygen is present, e.g. in a minimum concentration of at least 1000, 10000, 100000, 150000, 180000, 190000, 200000, and/or at most 300000, 250000, 220000 ppm, e.g. in the medium and/or direct surroundings thereof.

It will be clear to the skilled person, e.g. further to the first embodiment (biotin pathway 1) as described herein, that the cell according to the present disclosure may comprise a gene, particularly a BI01 gene, encoding a ( BI01 ) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:16. Preferably, the cell is not a Cyberlindnera fabianii cell. Additionally or alternatively, the cell may comprise one or more of

- a gene, particularly a BI02 gene, encoding a ( BI02 ) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:27;

- a gene, particularly a BI03 gene, encoding a ( BI03 ) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:28;

- a gene, particularly a BI04 gene, encoding a ( BI04 ) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:29; and/or

- a gene, particularly a BI06 gene, encoding a (BIOS) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:30;

It will also be clear to the skilled person, e.g. further to the second embodiment (biotin pathway 2) as described herein, that the cell according to the present disclosure may comprise one or more of

- a gene, particularly a fabD gene, encoding a (FabD) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 17;

- a gene, particularly a bioC gene, encoding a (BioC) protein having at least 50, 60, 70, 80,

85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 18;

- a gene, particularly a fabB gene, encoding a (FabB) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO: 19;

- a gene, particularly a fabG gene, encoding a (FabG) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:20;

- a gene, particularly a fabZ gene, encoding a (FabZ) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:21 ;

- a gene, particularly a fabl gene, encoding a (Fabl) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:22;

- a gene, particularly a bioH gene, encoding a (BioH) protein having at least 50, 60, 70, 80,

85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:23;

- a gene, particularly a bioF gene, encoding a (BoF) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:24;

- a gene, in particular an acpP gene, encoding a (AcpP) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:25;

and/or

- a gene, in particular an acpS gene, encoding a (AcpS) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:26, wherein preferably the cell is not an Escherichia coli cell. Additionally or alternatively, the cell may comprise one or more of

- a gene, particularly a BI02 gene, encoding a ( BI02 ) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:27;

- a gene, particularly a BI03 gene, encoding a ( BI03 ) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:28; and/or

- a gene, particularly a BI04 gene, encoding a ( BI04 ) protein having at least 50, 60, 70, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity with SEQ ID NO:29.

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.

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 aci d/nucleotide sequence comprising a nucleic acid according to the present disclosure 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.

Clauses

1. Cell comprising a BI01 gene having at least 70% sequence identity with SEQ ID NO: 1 , wherein the cell is not a Cyberlindnera fabianii cell.

2. Cell according to clause 1 , wherein the cell further comprises

- a BI06 gene having at least 70% sequence identity with SEQ ID NO: 15;

- a BI02 gene having at least 70% sequence identity with SEQ ID NO: 12;

- a BI03 gene having at least 70% sequence identity with SEQ ID NO: 13; and/or

- a BI04 gene having at least 70% sequence identity with SEQ ID NO: 14.

3. Cell comprising

- a fabD gene having at least 70% sequence identity with SEQ ID NO:2;

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

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

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

- a fabZ gene having at least 70% sequence identity with SEQ ID NO:6; - a fabl gene having at least 70% sequence identity with SEQ ID N0:7;

- a bioH gene having at least 70% sequence identity with SEQ ID N0:8;

(- a bioF gene having at least 70% sequence identity with SEQ ID N0:9;)

- an acpP gene having at least 70% sequence identity with SEQ ID NO: 10; and/or

- an acpS gene having at least 70% sequence identity with SEQ ID NO: 11 ,

wherein the cell is not an Escherichia coli cell.

4. Cell according to clause 3, wherein the cell further comprises

- a BI02 gene having at least 70% sequence identity with SEQ ID NO: 12;

- a BI03 gene having at least 70% sequence identity with SEQ ID NO: 13; and/or

- a BI04 gene having at least 70% sequence identity with SEQ ID NO: 14.

5. Cell according to any one of the previous clauses, wherein the cell is a prokaryotic cell, or an eukaryotic cell.

6. Cell according to any one of the previous clauses, wherein the cell is a yeast cell, preferably a Saccharomycotina yeast, more preferably an ascomycete yeast, most preferably Saccharomyces cerevisiae.

7. Cell according to any one of the previous clauses, wherein the cell is able to grow on medium comprising less than 5, 4, 3, 2, 1 , 0 mg biotin per kg medium.

8. Cell according to any one of the previous clauses, wherein the cell is able to grow under aerobic and/or anaerobic conditions, or used for growing under aerobic and/or anaerobic conditions.

9. Cell according to any one of the previous clauses, wherein the cell is able to grow at a growth rate in the absence of biotin of at least 25, 50, 75, 80, 85, 90, 95, 100% relative to growth rate in presence of biotin.

Enzymatic activity

Brief description of the figures

Figure 1: a biotin biosynthetic pathway

Figure 2: also showing a fatty acid enzymatic machinery

Sequences referred to:

In case of any inconsistency between the above SEQ ID Nos and the SEQ ID Nos as disclosed in the sequence listing, the above SEQ ID Nos are preferred. Alternatively, the SEQ ID Nos 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

Characterization of biotin requirement in Saccharomycotina yeasts

A total of 35 Saccharomycotina yeasts (Table 1) were tested for growth in absence of biotin. Along these yeast species, Saccharomyces cerevisiae strains CEN.PK1 13-7D and IMX585 were grown in synthetic medium (SM) containing 3.0 g L^~1 KH2PO4, 5.0 g L^~1 (NhU^SCU, 0.5 g L^_1 MgSCU, 7 H2O, 1 ml_ L ¹ trace element solution, and 1 ml_ L^~1 vitamin solution (0.05 g L·¹ D-(+)-biotin, 1.0 g L·¹ D-calcium pantothenate, 1.0 g L¹ nicotinic acid, 25 g L·¹ myo-inositol, 1.0 g L^-1 thiamine hydrochloride, 1.0 g L·¹ pyridoxol hydrochloride, 0.2 g L·¹ 4-aminobenzoic acid) (Verduyn et al., 1992). The pH was adjusted to 6 with 2 M KOH prior to autoclaving at 120 °C for 20 min. Vitamin solutions (Verduyn et al., 1992) were sterilized by filtration and added to the sterile medium. Concentrated sugar solutions were autoclaved at 110 °C for 20 min and added to the sterile medium to give a final concentration of 20 g L^-1 glucose (SMG). Biotin- free SM was prepared similarly but biotin was omitted from the vitamin solution (1.0 g L·¹ D-calcium pantothenate, 1.0 g L ¹ nicotinic acid, 25 g L ¹ myo-inositol, 1.0 g L ¹ thiamine hydrochloride, 1.0 g L ¹ pyridoxol hydrochloride, 0.2 g L ¹ 4-aminobenzoic acid). Similarly, after autoclaving concentrated glucose solutions at 1 10°C for 20 min, glucose was added to biotin-free SM to a final concentration of 20 g L ¹ (biotin-free SMG).

For maintenance, stock cultures of yeast strains were grown in YPD (10 g L^-1 yeast extract,

20 g L^-1 peptone, 20 g L^-1 glucose; pH adjusted to 6 with 2 M KOH prior to autoclaving) until late exponential phase, complemented with sterile glycerol to a final concentration of 30 % (v/v) and stored at -80 °C as 1 ml aliquots until further use.

Cultures for determination of biotin requirement of yeasts were performed as follows: 1 ml_ aliquot of a thawed stock culture was inoculated in 100 ml_ SMG in a 500 ml_ shake flask and incubated for 20 h at 30 °C. A second 100 ml_ SMG culture was started by inoculating 2 ml_ of the first shake flask culture. When the second culture reached mid-exponential phase (OD660 of 3-5) it was used to inoculate a third culture at an OD660 of 0.1 -0.3. Similarly a 1 ml_ aliquot of a thawed stock culture was inoculated in 100 ml_ biotin-free SMG in a 500 ml_ shake flask and incubated for 20 h at 30 °C. A second 100 ml_ biotin-free SMG culture was started by inoculating 2 ml_ of the first shake flask culture. If the second culture reached mid-exponential phase (OD660 of 3-5) it was used to inoculate a third culture at an OD660 of 0.1 -0.3. Shake flasks were incubated as biological duplicates at 30 °C and 200 rpm in an Innova incubator (Brunswick Scientific, Edison, NJ).

Growth studies were monitored by following cultures OD660 of an appropriate dilution of the third shake flask measured with a Jenway 7200 Spectrophotometer (Cole-Palmer, Stone, United Kingdom). Specific growth rates were calculated from a minimum of six data points during exponential growth covering 3 - 4 doublings of ODeeo.

Table 1 | List of Saccharomycotina yeasts tested for biotin prototrophy. Specific growth rates on biotin-free SMG of strains considered as biotin prototrophic are indicated.

* http://www.westerdijkinstitute.nl/

Out of the 35 strains tested in biotin-free SMG, six strains, which belong to the species Yarrowia lipolytica, Pichia kudriavzevii (also reported as Candida krusei and Issatchenkia oriental is), Wickerhamomyces ciferrii, Cyberlindnera fabianii (syn. Candida fabianii), Lachancea kluyveri and Torulaspora delbrueckii exhibited a specific growth rate higher than 0.25 h ¹.which represents less than 40 % reduction of the growth rate compared to rates measured in presence of biotin (SMG). In the same conditions, the S. cerevisiae strains CEN.PK113-7D and IMX585 were exhibiting a specific growth rate of 0.39+0.02 h ¹ on SMG but were not able to grow on biotin-free SMG (Table 2).

Table 2 | Specific growth rates of Saccharomycotina yeast on SMG and biotin-free SMG

Number of * = number of biological replicates

Identification of ScBIOI orthologs in Y. lipolytica, P. kudriavzevii, W. ciferrii, C. fabianii, L. kluyveri and T. delbrueckii.

Previous work describing adaptive evolution of the S. cerevisiae CEN.PK113-7D strain for biotin prototrophy showed that evolved strains carried a massive amplification of the ScBIOI gene. Although the reaction catalysed by the ScBiol remains unknown it was hypothesized that the first step of the biotin pathway represented a metabolic bottleneck for growth in the absence of biotin (Bracher et ai. 2017a). Biotin prototrophic yeasts as identified previously were hypothesized to encode and express ScBIOI orthologs with exceeding functionality. Although genome sequence information were available for all six species, the type and the quality of the data and most importantly the absence of proper genome assembly precluded direct search using pro Basic Local Alignment Search Tool (BLAST)

(https://blast.ncbi.nlm.nih.gov/Blast.cgi).

The identification of the putative ScBIOI orthologs in the whole genome sequence of the six biotin prototrophic yeasts was performed using the basic local alignment search tool tBLASTn that uses a protein sequence as query against translation of a DNA database. To this end, ScBiol amino acid sequence was queried against translation of whole genome shotgun data of single yeast species. In a reciprocal analysis the yeast specific best hits (Table 3) were aligned using the basic local alignment search tool tBLASTn against S. cerevisiae

CEN.PK113-7D DNA sequence to verify the similarity to the ScBIOI sequence. Table 3 | Yeast specific best hits for basic local alignment search tool tBLASTn querying ScBiol protein sequence.

The identified putative Biol amino acid sequences were pair-wise aligned using Clustal W (Scoring matrix BLOSUM 62) to determine amino acid sequence similarities. The sequences of L/cBid and TcfBiol showed the highest similarities to the ScBiol sequence (43-44 %). On the other end of the spectrum relative to the S. cerevisiae Biol V7Bio1 had the lowest similarty (15-20 %). Out of this set of Biol orthologs C Bid and l l/cBio1 exhibited the highest similarity (62 %) (Table 4).

Table 4 | Similarities of putative Biol of Saccharomycotina yeasts.

Functional characterization of putative ScBIOI orthologs in S. cerevisiae IMX585

In order to investigate if the expression of the putative ScBIOI orthologs was able to initiate growth of the S. cerevisiae strain CEN.PK113-7D in absence of biotin, each gene was expressed under a constitutive promoter and integrated into the genome of S. cerevisiae IMX585 at the locus SGA 1.

CONSTRUCTION OF FUNCTIONAL DNA YEAST TOOL KIT TYPE PLASMIDS

DNA fragments were amplified by PCR amplification with Phusion Hot Start II High Fidelity Polymerase (Thermo Fisher Scientific, Waltham, MA) and desalted or PAGE-purified oligonucleotide primers (Sigma-Aldrich, St. Louis, MO) performed according to the manufacturers’ instructions. For diagnostic PCR analysis DreamTaq polymerase (Thermo Fisher Scientific) was used according to manufacturers’ recommendations. Oligonucleotides and primers used in this study are listed in Table 5. PCR products were separated by electrophoresis on 1 % (w/v) agarose gels in TAE buffer (Thermo Fisher Scientific) and, if required, purified with a Zymoclean Gel DNA Recovery kit (Zymo Research, Irvine, CA) or GenElute PCR Clean-Up kit (Sigma-Aldrich).

After confirming the absence of introns, the coding sequences of putative BI01 sequences from Y. lipolytica W29, P. kudriavzevii CBS 5147, I/I/ ciferrii CBS 111, C. fabianii CBS 5481 , L. kiuyveri CBS 3082 and T. delbrueckii CBS 813 were obtained by PCR with primer combinations 14925/14926, 14892/14893, 15104/15105, 13963/13964, 13291/13039 as well as 13290/13038 and 12991/12992 respectively using genomic DNA of the respective yeast as a template, which was isolated using the YeaStar Genomic DNA kit (Zymo Research). In case of S. cerevisiae CEN.PK113-7D BI01, the plasmid pUDE450 (Bracher et al. 2017b) was isolated from E. coli cultures using the SIGMA GenElute Plasmid kit (Sigma-Aldrich) and used as a template for PCR with primer pair 11614/11615. The BI01 containing DNA fragments from Y. lypolytica, T. delbrueckii, L kluyverii and CEN.PK113-7D coding sequences were in vitro assembled with entry vector pUD565 (syn pMC) from GeneArt (Thermo Fisher Scientific) using BsmBI-T4 ligase directed Golden Gate cloning resulting in Yeast Tool Kit type 3 plasmids (Lee et al. 2015) pGGkp243, pGGKp169, pGGKp178 and pGGKp080 respectively. Similarly, the ScBIOI terminator was PCR amplified using plasmid pUDE450 (Bracher et al. 2017) as template and primer pairs 11618/11619 respectively. The ScBIOI terminator fragment was in vitro assembled in pUD565 using BsmBI-T4 DNA ligase directed Golden Gate cloning yielding the Yeast T ool Kit type 4 plasmid pGGKp078.

In order to remove a Bsal site in the coding sequence of LkBIOI, the open reading frame was PCR amplified from L kluyveri CBS 3082 genomic DNA using two primer pairs 13291/13039 and 13290/13038 with overhangs allowing for BsmBI-T4 ligase directed Golden Gate cloning of the two DNA fragments into entry vector pUD565, leaving a sequence without Bsal site behind and resulting in the Yeast Tool Kit type 3 plasmid pGGKp178.

Subsequently, 5 pL of the assembly mix were chemically transformed into E. coli and the transformed cells plated on selective LB medium (5.0 g L ¹ yeast extract, 10 g L ¹ trypton, 5.0 g L ¹ NaCI) supplemented with 170 pg mL ¹ chloramphenicol (LB chloramphenicol) (Inoue’, Nojima and Okayama 1990) at 37 °C. Four to eight colonies were selected and plasmid DNA isolated using the Sigma GenElute Plasmid kit (Sigma-Aldrich). The Yeast Tool Kit type plasmids pggkp080, pggkp169 and pggkp178 were confirmed by diagnostic PCR with primer pair 12616/4892, 12616/13287 and 12616/13290 respectively. Yeast Tool Kit type plasmid pggkp078 was confirmed by diagnostic PCR with primer pair 12616/10235. Yeast Tool Kit type plasmid pggkp243 was confirmed by restriction analysis with restriction enzymes pvuii and drai (thermo fisher scientific) according to manufacturer’s recommendations. The Yeast Tool Kit type plasmids were stored in transformed E. coli cultures at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase liquid LB chloramphenicol cultures.

The promoter ScPYKIp was synthesized by GeneArt (Thermo Fisher Scientific) and is stored as Yeast Tool Kit type 2 plasmid pGGkpl 17 in transformed E. coli cultures at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase liquid LB chloramphenicol cultures.

CONSTRUCTION OF TRANSCRIPTIONAL MODULES

The control S. cerevisiae BI01 transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd017, pGGKp117, pGGKp080, pGGKp078 yielding plasmid pUDE718. The plasmid pGGkd017 was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from pYTK002, pYTK047, PYTK072, pYTK074, pYTK082 and pYTK083.

The T. delbrueckii BI01 transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGKp1 17, pGGKp169, pGGKp078 yielding plasmid pUD788. The entry plasmids pGGkd015 was obtained by Bsal-T4 DNA ligase directed Golden Gate cloning with Yeast Tool Kit type plasmids pYTK002, pYTK047, pYTK067 and pYTK095.

The L kluyverii BI01 transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGKp117, pGGKp178, pGGKp078 yielding plasmid pUD789.

The Y. lipolytica BI01 transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragment with compatible overhangs from plasmids pGGkd015, pGGKp1 17, pGGKp243, pGGKp078 yielding plasmid pUD989.

The transcriptional modules of P. kudriavzevii, W. ciferrii and C. fabianii putative BI01 genes were constructed by Gibson assembly (Gibson et al. 2009) (New England Biolabs, Ipswich) using pUDE718 as plasmid backbone including the ScPYKIp and ScBIOIt. The BI01 genes from P. kudriavzevii CBS 5147, W. ciferrii CBS 1 11 , C. fabianii CBS 5481 were amplified by primer pairs 14892/14893, 15104/15105, 13963/13964 respectively using genomic DNA of the respective yeast as a template which was isolated using the YeaStar Genomic DNA kit (Zymo Research). After electrophoresis and gel purification the DNA fragments were Gibson assembled with linearized pUDE718 backbone using primer pair 7428/14891 yielding plasmids pUD988, pUD990 and pUD790 respectively.

Following Golden Gate or Gibson Assembly reaction, a volume of 5 pl_ of the assembly mix was chemically transformed in chemically competent E. coli using the appropriate protocol as described previously (INOUE’, NOJIMA AND OKAYAMA 1990). The transformed cells were propagated on selective LB medium (5 g L^-1 yeast extract, 10 g L^-1 trypton, 5 g L^-1 NaCI) supplemented with 100 pg mL^-1 ampicillin at 37 °C (LB ampicillin). Four to eight colonies were selected and plasmid dna isolated using the Sigma GenElute Plasmid kit (Sigma-Aldrich).

The transcriptional module plasmids were first confirmed by diagnostic PCR with the forward primer 10320 and a gene-specific reverse primer as follows: 13287 for TdBIOI, 13293 for LkBI01, 14928 for YIBI01, 4892 for ScBIO I, 14909 for PkBIO I, 14907 for WcBIOI and 14162 for CfBIOI. The transcriptional module plasmids were stored in transformed E. coli cultures at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase liquid LB ampicillin cultures. INTEGRATION OF TRANSCRIPTIONAL MODULES AT SCSGA1 LOCUS IN IMX585

The transcriptional modules were PCR amplified by using primer pair 12086/12108 adding specific sequences for homologous recombination into the SGA1 locus in S. cerevisiae directed by CRISPR/Cas9 (Mans et al. 2015). The transcriptional module was amplified from plasmid pUD788 for TdBIOI, from plasmid pUD789 for LkBIOI, from plasmid pUD989 for YIBI01, from plasmid pUDE718 for ScBIOI, from plasmid pUD988 for PkBIOI, from plasmid pUD990 for WcBIOI and from plasmid pUD790 for CfBIOI. DNA fragments were amplified by PCR amplification with Phusion Hot Start II High Fidelity Polymerase (Thermo Fisher

Scientific) and PAGE-purified oligonucleotide primers (Sigma-Aldrich) performed according to the manufacturers’ instructions. In case of plasmids pUDE718, pUD988, pUD990 and pUD790 which carry a yeast replication origin the PCR mix was digested with Dpnl (Thermo Fisher Scientific) before gel DNA purification (Zymo Research) in order to remove putative contamination with yeast replicative plasmids in the subsequent transformation. Targeting at the ScSGA I in IMX585 was directed by CRISPR-Cas9 and a target-specific gRNA

expressing plasmid (Mans et al. 2015). Each transcriptional module was co-transformed with plasmid pUDR119 (Papapetridis et al. 2018) expressing the gRNA to target Cas9 activity to the ScSGA I locus in strain IMX585 using the lithium-acetate transformation protocol (Daniel Gietz and Woods 2002). The transformed cells were plated on selective synthetic medium with acetamide as nitrogen source (SMG acetamide) (20 g L ¹ glucose, 1.2 g L ¹ acetamide, 3.0 g L^-1 KH2PO4, 6.6 g L^-1 K₂S0₄, 0.5 g L ¹, MgS0₄-7 H₂0, 1 ml_ L^-1 trace element solution, and 1 ml_ L^-1 vitamin solution) and incubated for 3 days at 30 °C. Genomic DNA of transformants was isolated using the SDS/LiAc protocol (Looke, Kristjuhan and Kristjuhan). The desired genotype was confirmed by diagnostic PCR using primer pair 11898/11899 and a gene-specific primer pair with 11898 as forward primer and the following reverse primers: 13287 for TdBIOI, 13293 for LkBIOI, 14928 for YIBI01, 4892 for ScBIOI, 14909 for PkBIOI, 14907 for WcBIOI and 14162 for CfBIOI together with DreamTaq polymerase (Thermo Fisher Scientific) according to the manufacturer’s recommendations. The correct clone was inoculated in 20 ml_ non-selective YPD for plasmid removal and incubated for 24 h at 30 °C. The cells were plated on solid YPD agar in order to obtain single colony isolates. One isolate was restreaked on both SMG acetamide and YPD. When no growth was observed on SMG acetamide the respective clone was once again confirmed by diagnostic PCR with gene- specific primers. Furthermore, the genetic modification at the ScSGAI locus was verified by Sanger sequencing using primers 11898/11899 to PCR amplify the modified locus and further using primers 11898, 11915 and 10235 for sequencing. The strain with the transcriptional module coding for TdBIOI was stocked as IMX1857, LkBIOI as IMX1858, YIBI01 as

IMX1862, ScBIOI as IMX1511 , PkBIOI as IMX1861, WcBIOI as IMX1863 and CfBIOI AS IMX1859. THE STRAINS WERE STORED at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase shake flask cultures in biotin-free SMG.

PHYSIOLOGICAL CHARACTERIZATION OF S. CEREVISIAE STRAINS EXPRESSING SCBI01 ORTHOLOGS

Firstly, a 1 ml_ aliquot of a thawed stock culture of strains IMX1857, IMX1858, IMX1862, IMX1511 , IMX1861, IMX1863 and IMX1859 was inoculated in 100 mL SMG in a 500 mL shake flask and incubated for 20 h at 30 °C. A second 100 mL SMG culture was started by inoculating 2 L of the first shake flask culture. When the second culture reached mid exponential phase (OD₆₆o of 3-5) it was used to inoculate the third culture at an OD₆₆o of 0.1- 0.3. For biotin-free growth studies all strains were incubated in biotin-free SMG. Shake flasks were incubated at 30 °C and 200 rpm in an Innova incubator (Brunswick Scientific). Strain IMX585 and CEN.PK1 13-7D, which consistently failed to grow on biotin-free SMG in the third culture, were used as a negative control in all growth experiments. Growth studies were monitored by following cultures OD₆₆o of an appropriate dilution of the third shake flask measured with a Jenway 7200 Spectrophotometer (Cole-Palmer). Specific growth rates were calculated from a minimum of six data points during exponential growth covering 3 - 4 doublings of ODeeo_.

S. cerevisiae strains expressing TdBIOI, LkBI01, YIBI01, ScBI01, PkBI01 and WcBI01 failed to grow in the third culture of biotin-free SMG but grew in SMG (growth was not monitored). However, strain IMX1859 expressing CfBIOI showed in the third biotin-free SMG culture growth at a specific growth rate of m = 0.40±0.02 h ¹ and on SMG a specific growth rate of 0.34±0.02 h ¹ , which is not significantly different (two-tailed t-test; p<0.05). Strain IMX585 was grown on SMG supplemented with biotin with a specific growth rate of 0.39±0.02 h ¹ , which did not differ significantly from IMX1859 in absence of biotin (two-tailed t-test; p<0.05) (Table 5).

Table 5| Specific growth rates of IMX585 and IMX1859 in biotin-free SMG and SMG.

Furthermore, strain IMX1859 was physiologically characterized under anaerobic conditions. A 1 mL aliquot of a thawed stock culture was inoculated in SMG and after 20 h of incubation under aerobic conditions at 30 °C transferred to a second aerobic SMG culture as previously described. A biomass sample of approx. 2 mL of the second culture was inoculated in 40 mL SMG supplemented with 420 mg L ¹ Tween 80 and 10 mg L^-1 ergosterol dissolved in ethanol. This culture was incubated at 30 °C on a IKA®KS 130 basic rotary shaker (IKA, Staufen, DE) at 240 rpm in a Bactron anaerobic chamber (Sheldon Manufacturing, Cornelius, OR).

Subsequently, when the third culture reached mid-exponential phase (OD₆₆o of 3-5) it was used to inoculate the fourth culture at an OD₆₆o of 0.1 -0.3. For biotin-free growth studies all strains were incubated in biotin-free SMG. Growth studies were monitored by following cultures OD₆₆o of an appropriate dilution of the fourth shake flask measured with a Biochrom Ultrospec 10 cell density meter (Biochrom, Berlin, DE). Specific growth rates were calculated from a minimum of four data points during exponential growth._. Strain IMX585 grown in SMG without anaerobic growth factors was used as control in all experiments. Strain IMX1859 failed to grow under anaerobic conditions in the fourth culture in biotin-free SMG

supplemented with anaerobic growth factors, while growth was observed when biotin was supplied.

Deletion of native ScBIOI gene in CfBIOI expressing IMX585

To further characterize the effect of the expressed BI01 orthologue from C. fabianii on the growth of S. cerevisiae in absence of biotin the deletion of the native ScBIOI locus in strain IMX1859 was directed by CRISPR/Cas9. In order to target ScBIOI a specific gRNA expressing plasmid pUDR244 was constructed by in vitro Gibson assembly. The linearized pROS11 plasmid, obtained by PCR with 6005/6006 was assembled together with a PCR amplified fragment using primer 14139 and pROS11 as a template as described before (Mans et al. 2015). The assembly mix was chemically transformed into E. coli and incubated on selective LB ampicillin agar plates. Four colonies were selected and propagated in 4 mL liquid LB ampicillin and subsequently the plasmid DNA was isolated using the Sigma

GenElute Plasmid kit (Sigma-Aldrich). The correct assembly of plasmid pUDR244 was confirmed by PCR with primers 3841/14167/5941. Plasmid pUDR244 was co-transformed with annealed repair oligo-nucleotides 12223/12224 in strain IMX1859 using the lithium- acetate transformation protocol (Daniel Gietz and Woods 2002). The transformed cells were plated on selective SMG acetamide and incubated for 3 days at 30 °C. Genomic DNA of the transformants was isolated using the SDS/LiAc protocol (Looke, Kristjuhan and Kristjuhan) and the desired genotype confirmed by diagnostic PCR using primer pair 7469/10873. The correct clone was inoculated in 20 mL non-selective YPD for plasmid removal and incubated for 24 h at 30 °C. The cells were plated on solid YPD agar in order to obtain single colony isolates. One isolate was restreaked on both SMG acetamide and YPD. When no growth was observed on SMG acetamide the respective clone was once again confirmed by diagnostic PCR with primer pair 7469/10873 and stored as IMX1860 at -80 °C after addition of 30 %

(v/v) glycerol to a stationary-phase shake flask culture. PHYSIOLOGICAL CHARACTERIZATION OF S. CEREVISIAE BI01 DELETION STRAIN

EXPRESSING CFBI01

The physiological characterization of strain IMX1860 with ScBIOI deletion and expression of CfBIOI in SMG and biotin-free SMG under aerobic conditions was performed as previously described for strains expressing ScBIOI orthologs from Y. lipolytica (IMX1862), P.

kudriavzevii (I MX1861), I/I/ ciferrii (IMX1863), C. fabianii (IMX1859), L kluyveri (IMX1858) and T. delbrueckii (IMX1857) The specific growth rate of strain IMX1860 in biotin-free SMG was determined as 0.37±0.00 IT¹ which was not significantly differing from specific growth rate of IMX1859 in biotin-free SMG determined as 0.40±0.02 IT¹ (two-tailed t-test; p<0.05). Hence, the native ScBIOI did not have an influence on the phenotype of modified IMX585 in absence of biotin.

Functional characterization of ScBIOI ortholog from C. fabianii in S. cerevisiae strains S288c, PE-2, Ethanol Red and CBS 8066

Integration of transcriptional modules at ScSGAI locus in S288c, Ethanol Red,

CBS8066 and PE-2

To verify whether the results obtained in the laboratory CEN.PK strain lineage could be extrapolated to other genetic backgrounds, CfBIOI was expressed in the S. cerevisiae strains CBS 8066, PE-2 used in the Brazilian bio-ethanol programme and Ethanol Red from an ethanol producing strain from Lesaffre. As previously described the transcriptional module harbouring CfBIOI was integrated at the ScSGA I locus. In contrast to the S. cerevisiae strain IMX585 previously described the strains CBS 8066, PE-2 and Ethanol Red do not express Cas9. To achieve integration of the CfBIOI module at the SGA 1 locus of these strains the plasmid pUDP145 that carried the ScSGAI targeting gRNA and a Cas9 expression module was constructed. Assembly of pUDP145 was performed in vitro by Bsal -T4 DNA ligase directed Golden Gate cloning with the gRNA entry plasmid pUDP002 (Juergens et al. 2018) with a de novo synthesized DNA fragment (GeneArt Thermo Fisher Scientific) encoding a gRNA targeting the ScSGA I locus in the three S. cerevisiae strains.

In contrast to CBS8066, PE-2 and Ethanol Red the S. cerevisiae strains S288c misses both the ScBIOI and ScBI06 gene (Goffeau et al. 1996). Consequently, the plasmid pUDP145 was co-transformed with the CfBIOI transcriptional module which was PCR amplified using primer pair 12086/14663 from pUD790 and an additional transcriptional module harbouring ScBI06 which was PCR amplified using primer pair 14661/14662 and plasmid pUDE448 (Bracher et ai. 2017c) as template. S. cerevisiae strains S288c and PE-2 were transformed using the lithium-acetate transformation protocol (Daniel Gietz and Woods 2002). Strains CBS 8066 and Ethanol Red were transformed by electroporation using 50 pl_ of competent cells and up to 5 mI_ DNA as previously described (Thompson et al. 1998). Chemically and electro-chemically transformed cells were incubated in 0.5 ml_ YPD for 1 h, after which they were resuspended in 100 pl_ of sterile demi-water and plated on selective YPD supplemented with 200 mg L¹ hygromycin (YPD hygromycin). After 3 days of incubation at 30 °C, genomic DNA of transformed colonies was isolated using the SDS/LiAc protocol (Looke, Kristjuhan and Kristjuhan) and the desired genotype confirmed by diagnostic PCR using primer pair 11898/11899 and using a CfBIO 7-specific PCR with primer pair 11898/14162. In case of transformation into strain S288c an additional diagnostic PCR with primer pair 8737/11899 was conducted. The correct clones were inoculated in 20 ml_ non-selective YPD for plasmid removal and incubated for 24 h at 30 °C. The cells were plated on solid YPD agar in order to obtain single colony isolates. One isolate was restreaked on both YPD hygromycin and YPD. When no growth was observed on YPD hygromycin the respective clone was once again confirmed by diagnostic PCR with gene-specific primers. The strain with the CfBIOI and ScBI06 transcriptional module in S288c was stocked as IMX2103. CBS8066 expressing CfBIOI was stocked as IMX2104, Ethanol Red expressing CfBIOI was stocked as IMX2101 and PE-2 expressing CfBIOI was stocked as IMX2090. THE STRAINS WERE STORED at - 80 °C after addition of 30 % (v/v) glycerol to stationary-phase shake flask cultures.

PHYSIOLOGICAL CHARACTERIZATION OF CFBIOI EXPRESSING S288C, ETHANOL RED, CBS8066 AND PE-2

A 1 mL aliquot of a thawed stock culture of strains IMX2103, IMX2104, IMX2101 and

IMX2090 was inoculated in 100 mL SMG in a 500 mL shake flask and incubated for 20 h at 30 °C. A second 100 mL SMG culture was started by inoculating 2 mL of the first shake flask culture. When the second culture reached mid-exponential phase (OD₆₆o of 3-5) it was used to inoculate a third culture at an OD₆₆o of 0.1 -0.3. For biotin-free growth studies all strains were incubated in biotin-free SMG. Shake flasks were incubated at 30 °C and 200 rpm in an Innova incubator (Brunswick Scientific). Strains S288c, Ethanol Red, CBS 8066 and PE-2 which consistently failed to grow in the third biotin-free SMG culture, were used as a negative control. Growth studies were monitored by following cultures OD₆₆o of an appropriate dilution of the third shake flask measured with a Jenway 7200 Spectrophotometer (Cole-Palmer). Specific growth rates were calculated from a minimum of six data points during exponential growth covering 3 - 4 doublings of OOb_do_.

S. cerevisiae strains IMX2103, IMX2104, IMX2101 and IMX2090 expressing CfBIOI grew during the third culture of biotin-free SMG and SMG (Table 6). For strain IMX2104 and IMX2103 the specific growth rate in biotin-free SMG and SMG was significantly different (two- tailed t-test; p<0.05). However, all strains expressing CfBIOI exhibit in biotin-free conditions a growth rate that was at least higher than 85 % of the rate measured in SMG. Table 6 | Specific growth rates of S. cerevisiae strains on SMG and biotin-free SMG

Functional characterization of a S. cerevisiae strain expressing the E. coli 8-amino-7- oxonanoate biosynthetic pathway.

Despite enabling fast growth of S. cerevisiae strain, expression of CfBIOI could not restore growth in absence of biotin under anaerobic conditions. Strain S. cerevisiae IMX585 that exhibits strict biotin growth dependency was equipped with the E. coli pathway for biosynthesis of 8-amino-7-oxonanoate that includes the genes EcfabD, EcbioC, EcfabB,

EcfabG, EcfabZ, Ecfabl, EcbioH, EcbioF, EcacpP and EcacpS (Table 7) to enable growth in absence of biotin under anaerobic conditions.

Table 7 | E. coli 8-amino-7-oxonanoate synthesis genes and corresponding protein annotations.

CONSTRUCTION OF FUNCTIONAL DNA YEAST TOOL KIT TYPE PLASMIDS

DNA fragments were amplified by PCR amplification with Phusion Hot Start II High Fidelity Polymerase (Thermo Fisher Scientific) and desalted or PAGE-purified oligonucleotide primers (Sigma-Aldrich) performed according to the manufacturers’ recommendations. For diagnostic PCR analysis DreamTaq polymerase (Thermo Fisher Scientific) according to manufacturers’ guidelines was used. PCR products were separated by electrophoresis on 1 % (w/v) agarose gels in TAE buffer (Thermo Fisher Scientific) and, if required, purified with a Zymoclean Gel DNA Recovery kit (Zymo Research) or GenElute PCR Clean-Up kit (Sigma-Aldrich).

The coding sequences of the genes EcfabD, EcbioC, EcfabB, EcfabG, EcfabZ, Ecfabl, EcbioH, EcbioF, EcacpP and EcacpS were codon optimized and synthesized by GeneArt (Thermo Fisher Scientific). The plasmids harbouring the coding sequences together with 5’ and 3’ flanking YTK type 3 Bsal sites (Lee et al. 2015) WERE PROPAGATED in chemically transformed E. coli cultures in liquid LB chloramphenicol medium grown at 37 °C on a rotary shaker and the plasmid DNA isolated using THE SIGMA GenElute Plasmid kit (Sigma-Aldrich). The Yeast Tool Kit type plasmids were stored at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase LB chloramphenicol liquid cultures.

The promoter sequences ScEN02p, ScPFK2p, ScPGUp were obtained by PCR with primer combinations 9739/9740, 10614/10615 and 9630/9631 respectively using genomic DNA of CEN.PK113-7D as a template which was isolated using the YeaStar Genomic DNA kit (Zymo Research). The promoter sequence SePDCIp was obtained by PCR with primer pair 9729/9730 using genomic DNA of S. eubayanus CBS12357 which was isolated using the YeaStar Genomic DNA kit (Zymo Research). The promoter sequences were in vitro assembled in pUD565 using BsmBI-T4 DNA ligase directed Golden Gate cloning yielding the Yeast Tool Kit type 2 plasmids pGGkp028, pGGKp031 , pGGKp033 and pGGKp074 respectively. The correct assembly of plasmids pGGKp028, pGGkp031 and pGGkp033 was confirmed by restriction analysis with enzyme Pvull (Thermo Fisher Scientific) according to manufacturer’s recommendations. Plasmid pGGkp074 was confirmed by diagnostic PCR using primer pairs 2012/2397 and 4707/2398. The Yeast Tool Kit type plasmids were propagated in E. coli grown in liquid LB chloramphenicol at 37 °C and stored in E. coli cultures at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase LB chloramphenicol cultures.

The promoter sequences SkFBA lp, SkPDCIp, SkADHIp and SkTDH3p were obtained by PCR with primer combinations 9640/9641 , 9731/9732 and 9737/9738 and 9751/9752 respectively using genomic DNA of S. kudriavzevii CBS 8840 as a template which was isolated using the YeaStar Genomic DNA kit (Zymo Research). The promoter sequences were in vitro assembled in pUD564 which was derived as pMK from GeneArt (Thermo Fisher Scientific) using BsmBI-T4 DNA ligase directed Golden Gate cloning yielding the Yeast Tool Kit type 2 plasmids pGGkp062, pGGKp063, pGGkp064 and pGGKp065 respectively. After assembly reaction 5 pL of the assembly mix were chemically transformed in chemically competent E. coli and the transformed cells were plated on selective LB supplemented with 50 pg mL^-1 kanamycin (LB kanamycin) (Inoue’, Nojima and Okayama 1990). Four to eight colonies were selected and plasmid DNA isolated using the Sigma GenElute Plasmid kit (Sigma-Aldrich). The plasmids were confirmed by restriction analysis with Pvull and Bsp1 191 (pGGKp062), Xbal (pGGKp063) and Hindi 11 (pGGKp064) (Thermo Fisher Scientific) according to manufacturer’s recommendations. The Yeast Tool Kit type plasmids were stored in transformed E. coli cultures at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase LB kanamycin liquid cultures.

The terminator sequences ScADHIt, ScTEF2t, ScPYKIt, ScFBA 1t, ScPDCIt, ScGPMIt, ScTPIt, ScPGUt and ScTDH3t were obtained by PCR with primer combinations 10769/10770, 10884/10885, 10886/10887, 10773/10774, 10757/10758, 10759/10760, 10765/10766, 10771/10772, 10761/10762 respectively using genomic DNA of CEN.PK113-7D as template. The promoter sequences were in vitro assembled in pUD565 using BsmBI-T4 DNA ligase directed Golden Gate cloning yielding the Yeast Tool Kit type 4 plasmids pGGKp037, pGGKp038, pGGKp040, pGGKp046, pGGKp045, pGGKp048, pGGKp042, pGGKp044 and pGGKp041 respectively. After assembly reaction 5 pL of the assembly mix were transformed in chemically competent E. coli and the transformed cells were plated on selective LB chloramphenicol (Inoue’, Nojima and Okayama 1990). Four to eight colonies were selected and plasmid DNA isolated using the Sigma GenElute Plasmid kit (Sigma-Aldrich). The plasmids harbouring the terminator sequences were confirmed by restriction analysis with enzyme Sspl (Thermo Fisher Scientific) according to manufacturer’s recommendations the Yeast Tool Kit type plasmids were stored in transformed E. coli cultures at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase LB chloramphenicol liquid cultures.

The promoter sequences ScPYKIp and ScHXK2p as well as the terminator sequence ScPFK2t were synthesized by GeneArt (Thermo Fisher Scientific) and are harboured by Yeast Tool Kit type 2 plasmids pGGkp117, pGGKp096 and pGGkp103. The Yeast Tool Kit type plasmids were propagated in chemically transformed E. coli cultures in liquid LB chloramphenicol medium grown at 37 °C on a rotary shaker and stored at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase liquid cultures.

CONSTRUCTION OF TRANSCRIPTIONAL MODULES

The E. coli fabD transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGKp062, pUD671 , pGGKp037 yielding plasmid pUD978.

The E. coli bioC transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGKp063, pUD663, pGGKp038 yielding plasmid pUD979.

The E. coli fabB transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGKp064, pUD664, pGGKp040 yielding plasmid pUD980.

The E. coli fabG transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGKp065, pUD665, pGGKp046 yielding plasmid pUD981.

The E. coli fabZ transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGKp074, pUD666, pGGKp045 yielding plasmid pUD982.

The E. coli fabl transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGKp028, pUD667, pGGKp103 yielding plasmid pUD983.

The E. coli bioH transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGkp1 17, pUD668, pGGKp044 yielding plasmid pUD984.

The E. coli bioF transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGkp031 , pUD669, pGGKp042 yielding plasmid pUD985.

The E. coli acpP transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGkp033, pUD661 , pGGKp048 yielding plasmid pUD986.

The E. coli acpS transcriptional module was constructed by Bsal-T4 DNA ligase directed Golden Gate cloning combining DNA fragments with compatible overhangs from plasmids pGGkd015, pGGkp096, pUD662, pGGKp041 yielding plasmid pUD987. After assembly reaction 5 mI_ of the assembly mix were transformed in chemically competent E. coli and the transformed cells were plated on selective LB ampicillin medium (Inoue’, Nojima and Okayama 1990). Four to eight colonies were selected and plasmid DNA isolated using the Sigma GenElute Plasmid kit (Sigma-Aldrich). The transcriptional module plasmids were first confirmed by diagnostic PCR primer combinations as follows: 13483/12761 for EcfabD, 10320/10325 for EcbioC, 13483/12745 for EcfabB, 13483/12751 for EcfabG, 13483/12759 for EcfabZ, 13483/12763 for Ecfabl, 10320/10325 for EcbioH, 13483/13283 for EcbioF, 10320/10325 for EcacpP and 13483/12749 for EcacpS. The transcriptional module plasmids were stored in transformed E. coli cultures at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase Lb ampicillin liquid cultures.

INTEGRATION OF TRANSCRIPTIONAL MODULES AT SCSGA1 LOCUS IN IMX585

The transcriptional modules were PCR amplified by using the following primer pairs adding homologous sequences to enable in vivo assembly of the transcriptional modules into the Sc SGA 1 locus of S. cerevisiae : 12655/12665 for EcfabD, 12656/12666 for EcbioC,

12657/12667 for EcfabB, 12658/12668 for EcfabG, 12659/12669 for EcfabZ, 12660/14000 for Ecfabl, 12455/12450 for EcbioH, 14448/13718 for EcbioF, 12663/13748 for EcacpP and 12664/12674 for EcacpS. The transcriptional module was amplified from plasmid pUD978 for EcfabD, pUD979 for EcbioC, pUD980 for EcfabB, pUD981 for EcfabG, pUD982 for EcfabZ, pUD983 for Ecfabl, pUD984 for EcbioH, pUD985 for EcbioF, pUD986 for EcacpP and pUD987 for EcacpS. The linear DNA fragments were separated by electrophoresis on 1 % (w/v) agarose gels n and purified with a Gel DNA Recovery Kit (Zymo Research). ScSGA I gene editing in IMX585 was directed by CRISPR-Cas9 (Mans et al. 2015). The

transcriptional modules carrying EcfabD, EcbioC, EcfabB, EcfabG, EcfabZ, Ecfabl, EcbioH, EcbioF, EcacpP and EcacpS were co-transformed with the plasmid pUDR119 (Papapetridis et al. 2018) into IMX585 using the lithium-acetate transformation protocol (Daniel Gietz and Woods 2002). The transformed cells were plated on selective SMG acetamide and incubated for 3 days at 30 ° C. Genomic DNA of colonies was isolated using the SDS/LiAc protocol (Looke, Kristjuhan and Kristjuhan) and the desired genotype confirmed by diagnostic PCR using following primer combinations 11898/12761, 12762/13545, 13284/12745, 12746/12751 , 12752/12759, 12760/12763, 12764/13281 , 13280/13283, 1719/12747 and 12750/11899. The correct clone was inoculated in 20 mL non-selective YPD for plasmid removal and incubated for 24 h at 30 °C. The cells were plated on solid YPD agar to obtain single colony isolates. One isolate was restreaked on both SMG acetamide and YPD. When no growth was observed on SMG acetamide the respective clone was once again confirmed by diagnostic PCR with above-mentioned primer combinations. Additionally, the strain IMX2035 was genome sequenced using lllumina (San Diego, CA) sequencing technology. The sequencing reads (add SRA file) were mapped onto the CEN.PK113-7D reference genome assembly (add reference). Analysis of the recombined ScSGAI locus did not reveal the presence of mutations. The strain with in vivo assembled transcriptional modules coding for 8-amino-7- oxonanoate synthesis into ScSGAI WAS STOCKED AS IMX2035 at -80 °C after addition of 30 % (v/v) glycerol to stationary-phase shake flask cultures.

PHYSIOLOGICAL CHARACTERIZATION OF S. CEREVISIAE STRAIN EXPRESSING E.

COLI 8-AMINO-7-OXONANOATE BIOSYNTHESIS

A 1 ml_ aliquot of a thawed stock culture of strain IMX2035 was inoculated in 100 ml_ SMG in a 500 ml_ shake flask and incubated for 20 h at 30 °C. A second 100 ml_ SMG culture was started by inoculating 2 ml_ of the first culture. When the second culture reached mid exponential phase (OD₆₆o of 3-5) it was used to inoculate a third culture at an OD₆₆o of 0.1- 0.3. For biotin-free growth studies all cultures were incubated in biotin-free SMG. Shake flasks were incubated at 30 °C and 200 rpm in an Innova incubator (Brunswick Scientific). Strain IMX585, which consistently failed to grow on biotin-free SMG in the third culture, was used as a control in all growth experiments. Growth studies were monitored by following cultures OD₆₆o of an appropriate dilution of the third shake flask measured with a Jenway 7200 Spectrophotometer (Cole-Palmer). Specific growth rates were calculated from a minimum of six data points during exponential growth covering 3 - 4 doublings of ODeeo. S. cerevisiae strain IMX2035 expressing EcfabD, EcbioC, EcfabB, EcfabG, EcfabZ, Ecfabl, EcbioH, EcbioF, EcacpP and EcacpS showed growth in SMG, but also on biotin-free SMG. The strain IMX2035 exhibited a specific growth rate of 0.26±0.01 h ¹ in biotin-free SMG under aerobic conditions. The growth rate represented a ca. 30 % reduction relative to that of IMX2035 grown in SMG. Subsequently, strain IMX2035 was physiologically characterized under anaerobic conditions. The first and second culture were grown aerobically as described previously. The second culture was used to inoculate a third culture with 40 mL SMG supplemented with 420 mg L ¹ Tween 80 and 10 mg L ¹ ergosterol dissolved in ethanol which was incubated at 30 °C° on a IKA®KS 130 basic rotary shaker at 240 rpm in a Bactron anaerobic chamber (Sheldon Manufacturing). Subsequently, mid-exponential (OD₆₆o of 3-5) cells were transferred to the fourth shake flask culture at an OD₆₆o of 0.1 -0.3. For biotin-free growth studies all strains were incubated in biotin-free SMG. Growth studies were monitored by following cultures OD₆₆o of an appropriate dilution of the fourth shake flask measured with a Biochrom Ultrospec 10 cell density meter (Biochrom). Specific growth rates were calculated from a minimum of four data points during exponential growth._. Strain IMX585 grown in SMG without anaerobic growth factors was used as negative control in all experiments. Strain IMX2035 grew under anaerobic conditions with a specific growth rate of 0.16±0.00 h ¹ in the fourth culture in biotin-free SMG and at a specific growth rate of 0.16±0.00 h ¹ in the fourth culture in SMG both supplemented with anaerobic growth factors (Table 7).

Table 7 | Specific growth rates of strain IMX2035 under aerobic and anaerobic conditions on SMG and biotin-free SMG

Deletion of native ScBIOI gene in IMX2035

To further characterize the influence of the expressed biotin biosynthetic pathway from E. coli on the growth of S. cerevisiae in absence of biotin the deletion of the native ScBIOI locus in strain IMX2035 was directed by CRISPR/Cas9. In order to target ScBIOI a specific gRNA expressing plasmid pUDR244 was constructed by in vitro Gibson assembly. Therefore linearized pROS11 plasmid, obtained by PCR with primer pair 6005/6006 was assembled together with a PCR amplified fragment using primer 14139 and pROS1 1 as a template as described before (Mans et ai. 2015). The assembly mix was chemically transformed into E. coli and incubated on selective LB ampicillin agar plates. Four colonies were selected and propagated in 4 mL liquid LB ampicillin and subsequently the plasmid DNA was isolated using the Sigma GenElute Plasmid kit (Sigma-Aldrich). The correct assembly of plasmid pUDR244 was confirmed by PCR with primers 3841/14167/5941. Plasmid pUDR244 was co

transformed with annealed repair oligo-nucleotides 12223/12224 into strain IMX2035. The transformation was performed using the lithium-acetate transformation protocol (Daniel Gietz and Woods 2002) after which the transformed cells were plated on selective SMG acetamide and incubated for 3 days at 30 °C. Genomic DNA of colonies was isolated using the

SDS/LiAc protocol (Looke, Kristjuhan and Kristjuhan) and the desired genotype confirmed by diagnostic PCR using primer pair 7469/10873 and DreamTaq polymerase (Thermo Fisher Scientific) according to the manufacturer’s guidelines. The correct clone was inoculated in 20 mL non-selective YPD for plasmid removal and incubated for 24 h at 30 °C. The cells were plated on solid YPD agar in order to obtain single colony isolates. One isolate was restreaked on both SMG acetamide and YPD. When no growth was observed on SMG acetamide the respective clone was once again confirmed by diagnostic PCR with primer pair 7469/10873 and stocked as IMX2122 at -80 °C after addition of 30 % (v/v) glycerol to a stationary-phase shake flask culture. PHYSIOLOGICAL CHARACTERIZATION OF SCBI01 DELETION STRAIN EXPRESSING

E. COLI 8-AMINO-7-OXONANOATE BIOSYNTHETIC PATHWAY

The physiological characterization of strain IMX2122 with ScBIOI deletion and expression of E. coli 8-amino-7-oxonanoate biosynthetic pathway was performed in aerobic and anaerobic batch cultivations in 2-L bioreactors with 0.8 L working volume. All cultures were grown on biotin-free SMG supplemented in case of anaerobic cultivations with sterile solutions of anaerobic growth factors ergosterol (10 mg L ¹) and Tween 80 (420 mg L ¹), as well as with 0.2 g L^-1 sterile antifoam C (Sigma-Aldrich). In case of aerobic batch cultivations biotin-free SMG was supplemented with 0.2 g L^-1 sterile pluronic PE6100 (BASF). Anaerobic conditions were maintained by sparging of a gas mixture of N2/CO2 (90/10 %, < 10 ppm oxygen) at a rate of 0.5 L min^-1 and culture pH was maintained at 5 by automatic addition of 2 M KOH. All cultures were grown at a stirrer speed of 800 rpm and at a temperature of 30 °C. Oxygen diffusion in the bioreactors was minimized by equipping them with Norprene tubing and Viton O-rings, and evaporation was minimized by cooling of outlet gas to 4 °C. Aerobic conditions were maintained by sparging with air at a rate of 0.5 L min^-1. For bioreactor inocula, 1 ml_ aliquot of a thawed stock culture of strain IMX2122 was inoculated in 100 ml_ biotin-free SMG in a 500 ml_ shake flask and incubated for 20 h at 30 °C. A second 100 ml_ biotin-free SMG culture was started by inoculating 2 ml_ of the first shake flask culture. Shake flasks were incubated at 30 °C and 200 rpm in an Innova incubator (Brunswick Scientific). When the second culture reached mid-exponential phase (OD660 of 3-5) it was used to inoculate the bioreactors at an OD660 of 0.1-0.3. Growth in the bioreactor was monitored based on the CO2 concentration in the off gas. In case of aerobic batch cultivations specific growth rates were calculated from CO2 profile of the batch cultivation. When, in anaerobic conditions after first having reached the CO2 production peak, the CO2 percentage in the off gas decreased below more than 20 % of the previously measured value, a computer-controlled peristaltic pump automatically removed approximately 90 % of the culture volume, leaving approximately 10 % as an inoculum for the next batch. Specific growth rates under anaerobic conditions were determined from the CO2 profile after two empty-refill cycles in order to deplete anaerobic growth factors from the aerobic pre-cultures.

IMX2122 grew under aerobic conditions in biotin-free SMG with a specific growth rate of 0.25±0.00 h ¹. Under anaerobic conditions IMX2122 grew in biotin-free SMG supplemented with anaerobic growth factors at a specific growth rate of 0.21 ±0.00 IT¹ demonstrating that expression of the E. coli 8-amino-7-oxonanoate biosynthesis pathway could support biotin synthesis under anaerobic conditions. References

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Claims

1. Cell comprising a BI01 gene having at least 70% sequence identity with SEQ ID NO: 1 , wherein the cell is not a Cyberlindnera fabianii cell, and wherein the cell further comprises:

- a BI06 gene having at least 70% sequence identity with SEQ ID NO: 15;

- a BI02 gene having at least 70% sequence identity with SEQ ID NO: 12;

- a BI03 gene having at least 70% sequence identity with SEQ ID NO: 13; and

- a BI04 gene having at least 70% sequence identity with SEQ ID NO: 14.

2. Cell according to claim 1 , wherein the cell is a prokaryotic cell, or an eukaryotic cell.

3. Cell according to any one of the previous claims, wherein the cell is a yeast cell, preferably a Saccharomycotina yeast, more preferably an ascomycete yeast, most preferably Saccharomyces cerevisiae.

4. Cell according to any one of the previous claims, wherein the cell is able to grow on medium comprising less than 5, 4, 3, 2, 1 , 0 mg biotin per kg medium.

5. Cell according to any one of the previous claims, wherein the cell is able to grow under aerobic and/or anaerobic conditions.

6. Cell according to any one of the previous claims, wherein the cell is able to grow at a growth rate in the absence of biotin of at least 25, 50, 75, 80, 85, 90, 95, 100% relative to growth rate in presence of biotin.