WO2025083312A1 - Recombinant saccharomyces cerevisiae for the production of serotonin - Google Patents

Abstract

The present invention relates to a recombinant Saccharomyces cerevisiae cell comprising the nucleotide sequences that encode for a L-tryptophan decarboxylase enzyme, a tryptamine 5-hydroxylase enzyme and a modified ARO4 K229L enzyme. Said cell is capable of producing serotonin from a simple carbon source, such as glucose, and from ammonia as a nitrogen source.

Description

DESCRIPTION

Recombinant Saccharomyces cerevisiae for serotonin production

The present invention relates to a recombinant Saccharomyces cerevisiae cell comprising nucleotide sequences encoding an L-tryptophan decarboxylase enzyme, a tryptamine 5-hydroxylase enzyme, and a modified ARO4 K229L enzyme, wherein said cell has the capacity to produce serotonin. Therefore, the present invention falls within the field of biotechnology, in particular, in the use of recombinant microorganisms in industrial processes.

BACKGROUND OF THE INVENTION

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter for the proper functioning of the nervous system. It also plays a role in the immune system and the microbiota-gut-brain axis. It is also the precursor molecule to melatonin, another hormone that regulates several processes related to circadian rhythms and acts as an antioxidant. Other serotonin-derived molecules, such as feruloyl serotonin and 4-coumaryl serotonin, have also been shown to have antioxidant activity, which could be useful in the cosmetics industry.

Serotonin production today is primarily achieved through complex chemical synthesis using toxic solvents and catalysis. Therefore, in recent years, new strategies based on genetically modified microorganisms for serotonin production have been developed.

WO2017167866A1 describes recombinant microbial host cells comprising biosynthetic pathways, and their use, for the production of melatonin and related products such as serotonin.

WO2013127915A1 describes recombinant microbial cells and methods for producing melatonin and related compounds using such cells. More specifically, the recombinant microbial cell may comprise exogenous genes encoding one or more of an L-tryptophan hydroxylase, a 5-hydroxy-L-tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O- methyltransferase; an L-tryptophan decarboxylase and a tryptamine-5-hydroxylase, and means for providing tetrahydrobiopterin (THB).

WO2019173797A1 describes genetically modified microbial cells for biosynthesizing tryptamine and tryptamine derivatives. The microbes of the disclosure can be engineered to contain plasmids and stable gene integrations that contain sufficient genetic information for the conversion of an anthranilate or indole to a tryptamine.

Document WO2022248635A2 describes a method for the production of tryptamine derivatives, using a genetically modified microbial cell.

However, there is a need to provide new modified microorganisms for the efficient production of serotonin as an alternative to its production by chemical synthesis.

DESCRIPTION OF THE INVENTION

The present invention relates to a Saccharomyces cerevisiae cell genetically modified by multiple integration into the genome of nucleotide sequences encoding an L-tryptophan decarboxylase (TDC), a tryptamine 5-hydroxylase enzyme (T5H) and a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase enzyme of Saccharomyces cerevisiae with a point mutation, particularly the substitution of K (lysine) with L (leucine) at position 229 in the amino acid sequence of ARO4 (ARO K229L), wherein the modified Saccharomyces cerevisiae cell is capable of producing serotonin (5-HT) from glucose.

The development of the present invention enables the production of serotonin using the yeast S. cerevisiae as a cell factory, allowing the generation of these types of molecules in a more environmentally sustainable manner, as required for the chemical industry's transition to a "green" industry. Saccharomyces cerevisiae is considered a GRAS (Generally Recognized As Safe) organism, which means it is safe and makes it advantageous to use it as a cell factory.

Initially, the inventors developed recombinant Saccharomyces cerevisiae strains by multiple integration into the genome of 2 exogenous genes within from the same cassette to allow the conversion of L-tryptophan to serotonin. The genes are the Clostridium sporogenes L-tryptophan decarboxylase (CsTDC), which generates the decarboxylation of L-tryptophan to tryptamine, and the Oryza sativa T5H hydroxylase gene (OsT5H), which produces the hydroxylation of tryptamine to serotonin. The S. cerevisiae strain with the TDC + T5H modifications was able to produce 22 mg/L of serotonin in a medium containing only glucose and ammonium, compared to the control strain (without modifications in its genome) that produced 0.042 mg/ml of serotonin in a medium containing only glucose and ammonium (Table 4).

Subsequently, a modification was introduced into an enzyme involved in the aromatic amino acid synthesis pathway (tryptophan, phenylalanine, and tyrosine), thus increasing serotonin production directly from a glucose source. This modification consisted of multiple overexpression of the Saccharomyces cerevisiae ARO4 gene with a point mutation disrupting catabolic repression in aromatic amino acid metabolism, the substitution of K (lysine) for L (leucine) at position 229 of ARO4 [TDC + T5H + ARO4K229L]. The Saccharomyces cerevisiae strain TDC + T5H + ARO4K229L showed a significant increase in serotonin production, reaching a serotonin concentration of approximately 120 mg/L from glucose as the carbon source and ammonium as the nitrogen source. This represents a more than 5-fold increase in serotonin production compared to the strain with only the TDC + T5H modifications (Table 4). Therefore, the development of the present invention allows the production of serotonin from a simple carbon source by modified microorganisms suitable for use in the food industry.

Thus, in one aspect, the present invention relates to a recombinant Saccharomyces cerevisiae cell, hereinafter “cell of the invention”, comprising:

(i) a nucleotide sequence encoding an L-tryptophan decarboxylase enzyme or a functionally equivalent fragment thereof, hereinafter “nucleotide sequence (i) of the invention”, wherein the L-tryptophan decarboxylase enzyme comprises an amino acid sequence with at least 80% identity with the amino acid sequence SEQ ID NO: 1;

(i) a nucleotide sequence encoding a tryptamine 5-hydroxylase enzyme or a functionally equivalent fragment thereof, hereinafter referred to as hereinafter “nucleotide sequence (i) of the invention”, wherein the tryptamine 5-hydroxylase enzyme comprises an amino acid sequence with at least 80% identity with the amino acid sequence SEQ ID NO: 2; and

(iii) a nucleotide sequence encoding an ARO4 enzyme or a functionally equivalent fragment thereof, hereinafter “nucleotide sequence (iii) of the invention”, wherein the ARO4 enzyme comprises an amino acid sequence with at least 80% identity with the amino acid sequence SEQ ID NO: 3, and wherein said amino acid sequence comprises the substitution of K (lysine) for L (leucine) at position 229 (ARO4 K229L).

In the present invention, the term "recombinant Saccharomyces cerevisiae cell" refers to that cell of a wine yeast, particularly of the species Saccharomyces cerevisiae, which has been genetically modified, thus distinguishing itself from the "mother", "parental" or "wild type" strain. The cell of the invention has inserted into its genome the nucleotide sequences (i), (ii) and (iii) of the invention, the expression of which provides the cell with the enzymes that allow it to overproduce serotonin from a simple carbon source (e.g. glucose) and ammonium. Thus, the cell of the invention expresses the enzymes encoded by the nucleotide sequences (i), (iii) and (iii) of the invention. In the context of the present invention, the terms "recombinant Saccharomyces cerevisiae" and "transgenic Saccharomyces cerevisiae" are equivalent terms and have the same meaning.

In a preferred embodiment, the cell of the invention corresponds to the recombinant Saccharomyces cerevisiae BY4743 strain comprising the nucleotide sequences (i), (ii) and (iii). The Saccharomyces cerevisiae BY4743 strain is publicly available in public collections of recognized depository institutions and accessible without restrictions. In the present invention, the terms "cell" and "strain" are equivalent and are used interchangeably.

The amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 correspond to the amino acid sequences of the enzymes L-tryptophan decarboxylase from Clostridium sporogenes (CsTDC), tryptamine 5-hydroxylase from Oryza sativa (OsT5H) and 3-deoxy-D-arabino-heptulosonate-7-phosphate from Saccharomyces cerevisiae, respectively (ARO4). In the present invention, the terms “L-tryptophan decarboxylase” and “TDC”, used interchangeably, refer to an enzyme that catalyzes the decarboxylation of L-tryptophan to produce tryptamine as a product.

Amino acid sequence of Clostridium sporogenes L-tryptophan decarboxylase (CsTDC) SEQ ID NO: 1 :

MKFWRKYTQQEMDEKITESLEKTLNYDNTKTIGIPGTKLDDTVFYDDHSFVKHSPYLR TFIQNPNHIGCHTYDKADILFGGTFDIERELIQLLAIDVLNGNDEEFDGYVTQGGTEANI QAMWVYRNYFKKERKAKHEEIAIITSADTHYSAYKGSDLLNIDIIKVPVDFYSRKIQENT LDSIVKEAKEIGKKYFIVISNMGTTMFGSVDDPDLYANIFDKYNLEYKIHVDGAFGGFIY PIDNKECKTDFSNKNVSSITLDGHKMLQAPYGTGIFVSRKNLIHNTLTKEATYIENLDVT LSGSRSGSNAVAI WMVLASYGPYGWM EKI N KLRN RTKWLCKQLN DM Rl KYYKEDSM NIVTIEEQYVNKEIAEKYFLVPEVHNPTNNWYKIWMEHVELDILNSLVYDLRKFNKEHL KAM

In the present invention, the terms “tryptamine 5-hydroxylase” and “T5H”, used interchangeably, refer to an enzyme that catalyzes the hydroxylation of tryptamine to produce serotonin (5-hydroxytryptamine) as a product.

Amino acid sequence of tryptamine 5-hydroxylase from Oryza sativa fOsT5H SEQ ID NO: 2:

MELTMASTMSLALLVLSAAYVLVALRRSRSSSSKPRRLPPSPPGWPVIGHLHLMSGM PHHALAELARTMRAPLFRMRLGSVPAVVISKPDLARAALTTNDAALASRPHLLSGQFL SFGCSDVTFAPAGPYHRMARRVVVSELLSARRVATYGAVRVKELRRLLAHLTKNTSP AKPVDLSECFLNLANDVLCRVAFGRRFPHGEGDKLGAVLAEAQDLFAGFTIGDFFPEL EPVASTVTGLRRRLKKCLADLREACDVIVDEHISGNRQRIPGDRDEDFVDVLLRVQKS PDLEVPLTDDNLKALVLDMFVAGTDTTFATLEWVMTELVRHPRILKKAQEEVRRVVGD SGRVEESHLGELHYMRAIIKETFRLHPAVPLLVPRESVAPCTLGGYDIPARTRVFINTF AMGRDPEIWDNPLEYSPERFESAGGGGEIDLKDPDYKLLPFGGGRRGCPGYTFALAT VQVSLASLLYHFEWALPAGVRAEDVNLDETFGLATRKKEPLFVAVRKSDAYEFKGEEL SEV

In the present invention the terms “3-deoxy-D-arabino-heptulosonate-7-phosphate synthase” or “DAHP synthase”, used interchangeably, refer to an enzyme which catalyzes the conversion of phosphoenolpyruvate and erythrose 4-phosphate to 3-deoxy-D-arabinoheptulosonate-7-phosphate. The DAHP synthase enzyme catalyzes the first step in the biosynthesis of aromatic amino acids such as L-tryptophan. In the present invention, the enzyme “ARO4” refers to 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase from Saccharmyces cerevisiae.

Amino acid sequence of the ARO4 enzyme from Saccharomyces cerevisiae SEQ ID NO: 3:

MSESPM FAANGM PKVNQGAEEDVRI LGYDPLASPALLQVQI PATPTSLETAKRGRRE AIDIITGKDDRVLVIVGPCSIHDLEAAQEYALRLKKLSDELKGDLSIIMRAYLEKPRTTVG WKGLINDPDVNNTFNINKGLQSARQLFVNLTNIGLPIGSEMLDTISPQYLADLVSFGAIG ARTTESQLHRELASGLSFPVGFKNGTDGTLNVAVDACQAAAHSHHFMGVTKHGVAAI TTTKGNEHCFVILRGGKKGTNYDAKSVAEAKAQLPAGSNGLMIDYSHGNSNKDFRNQ PKVNDVVCEQIANGENAITGVMIESNINEGNQGIPAEGKAGLKYGVSITDACIGWETTE DVLRKLAAAVRQRREVNKK

The cell of the present invention comprises a nucleotide sequence encoding the ARO4 enzyme with a substitution of K (lysine) for L (leucine) at position 229 (referring to sequence SEQ ID NO: 3). The substitution of K (lysine) for L (leucine) at position 229 in the sequence of the S. cerevisiae ARO4 enzyme confers said enzyme resistance to inhibition mediated by L-tryptophan. This results in an increase in the production of L-tryptophan which is subsequently metabolized by the TDC and T5H enzymes thus producing serotonin. In the present invention the term “ARO4 K229L” refers to the S. cerevisiae ARO4 enzyme comprising the substitution of K (lysine) for L (leucine) at position 229.

The cell of the present invention may also be referred to as “Saccharomyces cerevisiae TDC+T5H+ARO4K229L”, “Saccharomyces cerevisiae CsTDC+OsT5H+ARO4K229L” or “Saccharomyces cerevisiae SER+ARO4K229L strain”, interchangeably.

In a preferred embodiment, the cell of the invention comprises: the nucleotide sequence (i) of the invention encoding an L-tryptophan decarboxylase enzyme or a functionally equivalent fragment thereof, wherein the L-tryptophan decarboxylase enzyme comprises a amino acid sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence SEQ ID NO: 1; the nucleotide sequence (i) of the invention encoding a tryptamine 5-hydroxylase enzyme or a functionally equivalent fragment thereof, wherein the tryptamine 5-hydroxylase enzyme comprises an amino acid sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence SEQ ID NO: 2; and the nucleotide sequence (iii) of the invention encoding an ARO4 enzyme or a functionally equivalent fragment thereof, wherein the ARO4 enzyme comprises an amino acid sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence SEQ ID NO: 3, and wherein said amino acid sequence comprises the substitution of K (lysine) for L (leucine) at position 229 (ARO4K229L); preferably where the % identity value of the amino acid sequences of the enzymes encoded by the nucleotide sequences (i), (ii) and (iii) (with their respective SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3), is the same % value.

In the present invention, "identity" or "sequence identity" refers to the degree of similarity between two nucleotide or amino acid sequences obtained through optimal alignment of the two sequences. A degree of identity expressed as a percentage will be obtained depending on the number of common residues between the aligned sequences. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the art, such as BLAST. BLAST programs, such as BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain on the website of the National Center for Biotechnology Information (NCBI).

In another preferred embodiment, the cell of the invention comprises: the nucleotide sequence (i) of the invention that codes for an L-tryptophan decarboxylase enzyme or a functionally equivalent fragment thereof. same, wherein the enzyme L-tryptophan decarboxylase comprises the amino acid sequence SEQ ID NO: 1; the nucleotide sequence (i) of the invention that codes for a tryptamine 5-hydroxylase enzyme or a functionally equivalent fragment thereof, wherein the tryptamine 5-hydroxylase enzyme comprises the amino acid sequence SEQ ID NO: 2; and the nucleotide sequence (iii) of the invention that codes for an ARO4 enzyme or a functionally equivalent fragment thereof, wherein the ARO4 enzyme comprises the amino acid sequence SEQ ID NO: 3, and wherein said amino acid sequence comprises the substitution of K (lysine) for L (leucine) at position 229 (ARO4 K229L).

In another preferred embodiment, the cell of the invention comprises: the nucleotide sequence (i) of the invention encoding an L-tryptophan decarboxylase enzyme or a functionally equivalent fragment thereof, wherein the L-tryptophan decarboxylase enzyme consists of the amino acid sequence SEQ ID NO: 1; the nucleotide sequence (ii) of the invention encoding a tryptamine 5-hydroxylase enzyme or a functionally equivalent fragment thereof, wherein the tryptamine 5-hydroxylase enzyme consists of the amino acid sequence SEQ ID NO: 2; and the nucleotide sequence (iii) of the invention encoding an ARO4 enzyme or a functionally equivalent fragment thereof, wherein the ARO4 enzyme consists of the amino acid sequence SEQ ID NO: 3, and wherein said amino acid sequence comprises the substitution of K (lysine) for L (leucine) at position 229 (ARO4 K229L).

The amino acid sequence of the ARO4 enzyme comprising the substitution of K (lysine) for L (leucine) at position 229 (ARO4 K229L) consists of the amino acid sequence SEQ ID NO: 7. Amino acid sequence of the Saccharomyces cerevisiae ARO4 enzyme comprising the substitution of K (lysine) for L (leucine) at position 229, SEQ ID NO: 7:

MSESPM FAANGM PKVNQGAEEDVRI LGYDPLASPALLQVQI PATPTSLETAKRGRRE

AIDIITGKDDRVLVIVGPCSIHDLEAAQEYALRLKKLSDELKGDLSIIMRAYLEKPRTTVG

WKGLINDPDVNNTFNINKGLQSARQLFVNLTNIGLPIGSEMLDTISPQYLADLVSFGAIG

ARTTESQLHRELASGLSFPVGFKNGTDGTLNVAVDACQAAAHSHHFMGVTLHGVAAI

TTTKGNEHCFVILRGGKKGTNYDAKSVAEAKAQLPAGSNGLMIDYSHGNSNKDFRNQ PKVNDVVCEQIANGENAITGVMIESNINEGNQGIPAEGKAGLKYGVSITDACIGWETTE DVLRKLAAAVRQRREVNKK

In another preferred embodiment of the cell of the invention: the nucleotide sequence (i) of the invention encoding the enzyme L-tryptophan decarboxylase comprises a nucleotide sequence with at least 80% identity with the nucleotide sequence SEQ ID NO: 4; the nucleotide sequence (ii) of the invention encoding the enzyme tryptamine 5-hydroxylase comprises a nucleotide sequence with at least 80% identity with the nucleotide sequence SEQ ID NO: 5; and the nucleotide sequence (iii) of the invention encoding the enzyme ARO4 K229L comprises a nucleotide sequence with at least 80% identity with the nucleotide sequence SEQ ID NO: 6.

Nucleotide sequence encoding the enzyme L-tryptophan decarboxylase of Clostridium sporogenes SEQ ID NO: 4:

ATGAAATTTTGGAGGAAATATACACAACAAGAGATGGATGAGAAAATTACAGAAAG

CTTAGAGAAAACCCTTAACTACGATAACACCAAAACAATAGGTATCCCAGGAACTA

AATTGGATGATACCGTCTTTTATGATGACCATAGTTTTGTGAAGCATTCACCCTATT

TGAGAACGTTCATACAGAATCCAAATCATATTGGCTGTCATACCTACGATAAAGCC

GACATTCTGTTTGGAGGTACTTTCGACATTGAGAGAGAGTTGATTCAACTTCTAGC

GATAGATGTCCTAAATGGCAACGATGAAGAATTTGACGGTATGTTACTCAAGGT

GGTACAGAAGCCAATATTCAAGCAATGTGGGTTTATAGAAACTACTTCAAGAAGGA GAGAAAAGCTAAACACGAAGAAATTGCCATTATTACTTCTGCGGATACACACTATT CTGCATACAAAGGTAGTGATTTACTAAACATAGACATCATCAAAGTACCAGTAGAC TTTTACTCTAGAAAGATCCAGGAAAATACATTGGATTCAATCGTTAAGGAAGCAAA GGAAATTGGTAAGAAATATTTTATCGTCATTAGCAATATGGGCACTACGATGTTTG GGTCAGTAGATGATCCAGATCTTTATGCCAATATCTTTGACAAATACAACTTGGAG TATAAGATTCATGTTGATGGTGCTTTTGGCGGATTTATATATCCGATTGACAACAAA GAATGCAAAACTGATTTCTCCAATAAGAATGTTTCCTCCATAACTCTGGATGGTCA CAAGATGTTACAAGCTCCTTACGGAACTGGGATATTCGTGAGTCGTAAGAATTTGA TTCATAATACCTTAACGAAAGAAGCTACCTATATTGAGAACTTGGACGTCACTTTAT CTGGTTCAAGATCTGGATCAAATGCTGTTGCAATTTGGATGGTCTTAGCTTCGTAT GGTCCTTATGGTTGGATGGAGAAGATTAACAAATTGAGAAACAGAACGAAATGGTT GTGTAAGCAACTGAATGATATGCGTATAAAATACTATAAGGAAGATTCTATGAATAT CGTAACAATCGAAGAACAGTACGTGAACAAGGAAATCGCTGAAAAGTACTTCTTAG TTCCTGAAGTTCATAACCCCACAAATAATTGGTATAAAATAGTGGTTATGGAACAT GTAGAACTAGATATACTGAATTCGCTTGTGTACGACTTGAGGAAATTCAATAAGGA ACACTTAAAAGCAATGTAA

In the present invention, all amino acid or nucleotide sequences that have a sequence identity of at least 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 1 or with the sequence SEQ ID NO: 4, respectively, are considered functionally equivalent variants of the Clostridium sporogenes L-tryptophan decarboxylase enzyme and gene (CsTDC), that is, although they comprise different amino acid or nucleotide sequences, they have L-tryptophan decarboxylase activity to catalyze the decarboxylation of L-tryptophan, producing tryptamine as a product.

Nucleotide sequence encoding the enzyme tryptamine 5-hydroxylase from Oryza sativa SEQ ID NO: 5:

ATGGAATTGACAATGGCTTCTACAATGTCACTGGCTCTGTTAGTTTTATCCGCAGC ATATGTCTTAGTTGCATTGAGAAGATCTAGAAGTAGCTCTTCGAAGCCGCGTAGAT TGCCACCATCTCCACCAGGTTGGCCAGTTATTGGGCATTTGCATTTGATGTCAGG CATGCCTCATCATGCTTTAGCTGAACTAGCGAGGACAATGAGAGCTCCACTATTCA GAATGAGGTTGGGTTCTGTTCCTGCAGTTGTGATTAGTAAACCCGATTTAGCGAG GGCAGCATTAACGACAAATGACGCAGCCTTGGCTTCACGTCCCCATCTTCTTAGT GGTCAGTTCCTATCCTTCGGATGCTCGGATGTAACATTCGCTCCAGCTGGTCCAT ACCACAGAATGGCTAGAAGAGTTGTGGTGTCTGAGCTATTAAGCGCCAGACGTGT TGCCACTTATGGAGCGGTTAGAGTAAAGGAGCTAAGGAGATTATTGGCTCATTTG ACTAAGAATACTAGTCCCGCAAAACCCGTAGACTTATCCGAATGCTTTCTGAACTT GGCAAATGATGTCCTATGTAGAGTGGCATTTGGGAGAAGGTTTCCTCATGGTGAA GGAGATAAATTGGGAGCAGTTTTAGCTGAAGCCCAAGATTTGTTTGCTGGCTTTAC GATCGGCGATTTCTTTCCGGAATTAGAGCCTGTGGCCTCAACTGTTACTGGCTTG AGGAGAAGGTTGAAGAAGTGCCTAGCTGACTTAAGAGGCTTGTGACGTTATAG TCGATGAACACATTAGCGGTAATAGGCAAAGGATACCAGGCGATAGAGATGAGGA CTTCGTAGATGTGCTGTTGCGTGTGCAGAAATCCCCTGATTTAGAAGTACCTCTGA CTGATGATAACCTTAAGGCCTTGGTACTAGACATGTTCGTAGCTGGCACAGATACA ACCTTTGCTACCTTGGAATGGGTTATGACCGAACTTGTCAGACATCCGAGAATTTT GAAGAAAGCCCAAGAAGAAGTGAGAAGAGTTGTAGGTGATAGCGGTAGAGTTGAA GAATCACACTTAGGAGAGCTGCACTACATGAGAGCTATCATCAAAGAAACCTTCA GACTGCATCCTGCAGTCCCTTTACTTGTACCTAGAGAATCAGTCGCTCCTTGTACT TTGGGTGGTTATGACATTCCAGCAAGAACTCGTGTTTTCATTAACACGTTCGCTAT GGGTAGAGATCCAGAGATATGGGATAATCCATTGGAATATTCACCGGAGAGGTTT GAGTCTGCAGGAGGAGGTGGGGAAATAGACCTTAAAGATCCAGACTACAAATTGT TGCCTTTTGGTGGTGGTAGACGTGGCTGTCCAGGTTACACATTTGCGTTAGCGAC TGTCCAAGTTAGTCTTGCCTCCCTTCTTTATCACTTTGAATGGGCCTTACCAGCCG GAGTTAGAGCCGAAGATGTCAACTTGGACGAAACCTTTGGTCTAGCAACCCGTAA GAAAGAGCCCCTATTTGTTGCGGTGAGAAAATCTGATGCCTATGAATTTAAAGGG GAAGAATTATCGGAAGTCTAA

In the present invention, all amino acid or nucleotide sequences that have a sequence identity of at least 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO:2 or with the sequence SEQ ID NO:5, respectively, are considered functionally equivalent variants of the tryptamine 5-hydroxylase enzyme and gene of Oryza sativa, that is, although they comprise different amino acid or nucleotide sequences, they have tryptamine 5-hydroxylase activity to catalyze the hydroxylation of tryptamine, producing serotonin as a product.

Nucleotide sequence encoding the ARO4 K229L enzyme of Saccharomyces cerevisiae SEQ ID NO: 6:

ATGAGTGAATCTCCAATGTTCGCTGCCAACGGCATGCCAAAGGTAAATCAAGGTG

CTGAAGAAGATGTCAGAATTTTAGGTTACGACCCATTAGCTTCTCCAGCTCTCCTT CAAGTGCAAATCCCAGCCACACCAACTTCTTTGGAAACTGCCAAGAGAGGTAGAA GAGAAGCTATAGATATTATTACCGGTAAAGACGACAGAGTTCTTGTCATTGTCGGT CCTTGTTCCATCCATGATCTAGAAGCCGCTCAAGAATACGCTTTGAGATTAAAGAA ATTGTCAGATGAATTAAAAGGTGATTTATCCATCATTATGAGAGCATACTTGGAGA AGCCAAGAACAACCGTCGGCTGGAAAGGTCTAATTAATGACCCTGATGTTAACAA CACTTTCAACATCAACAAGGGTTTGCAATCCGCTAGACAATTGTTTGTCAACTTGA CAAATATCGGTTTGCCAATTGGTTCTGAAATGCTTGATACCATTTCTCCTCAATACT TGGCTGATTTGGTCTCCTTCGGTGCCATTGGTGCCAGAACCACCGAATCTCAACT GCACAGAGAATTGGCCTCCGGTTGTCTTTCCCAGTTGGTTTCAAGAACGGTACC GATGGTACCTTAAATGTTGCTGTGGATGCTTGTCAAGCCGCTGCTCATTCTCACCA TTTCATGGGTGTTACTTTGCATGGTGTTGCTGCTATCACCACTACTAAGGGTAACG AACACTGCTTCGTTATTCTAAGAGGTGGTAAAAAGGGTACCAACTACGACGCTAA GTCCGTTGCAGAAGCTAAGGCTCAATTGCCTGCCGGTTCCAACGGTCTAATGATT GACTACTCTCACGGTAACTCCAATAAGGATTTCAGAAACCAACCAAAGGTCAATGA CGTTGTTTGTGAGCAAATCGCTAACGGTGAAAACGCCATTACCGGTGTCATGATT GAATCAAACATCAACGAAGGTAACCAAGGCATCCCAGCCGAAGGTAAAGCCGGCT TGAAATATGGTGTTTCCATCACTGATGCTTGTATAGGTTGGGAAACTACTGAAGAC GTCTTGAGGAAATTGGCTGCTGCTGTCAGACAAAGAAGAGAAGTTAACAAGAAAT AG

In the present invention, all amino acid or nucleotide sequences that have a sequence identity of at least 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 3 where said sequence comprises the substitution of K (lysine) for L (leucine) at position 229 (alternatively the amino acid sequence SEQ ID NO: 7), or with the sequence SEQ ID NO: 6, respectively, are considered functionally equivalent variants of the ARO4 K229L gene and enzyme of Saccharomyces cerevisiae, that is, although they comprise different nucleotide or amino acid sequences, they have 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase activity to catalyze the first step in the biosynthesis of aromatic amino acids.

The nucleotide sequence SEQ ID NO: 6 preserves the TTG triplet (coding for the amino acid leucine, L at position 229 in the amino acid sequence) at nucleotide position 685. It is routine practice for one skilled in the art to test whether different sequences are considered functionally equivalent.

In the present invention, the term "vahante" refers to a polypeptide or protein (or, where appropriate, a polynucleotide) that has the same activity as a reference polypeptide or protein (or polynucleotide), but that comprises an alteration in its sequence, that is, a substitution, insertion and/or deletion, at one or more (e.g., vahas) positions. By way of example, a substitution means the replacement of the amino acid (or a nucleotide) occupying a position with a different amino acid (or a nucleotide); a deletion means the deletion of the amino acid (or a nucleotide) occupying a position; and an insertion means adding an amino acid (or a nucleotide) adjacent to and immediately after the amino acid (or a nucleotide) occupying a position. Changes in polynucleotides include, but are not limited to, substitutions, insertions, and/or deletions between one or more nucleotides, represented by their nitrogenous bases such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (II). Changes in amino acids may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the polypeptide/protein; small deletions, typically 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a polyhistidine tag, antigenic epitope, or binding domain. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine, and histidine), amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, thiophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In the context of the present invention, fragments of the nucleotide sequences or fragments of the proteins encoded by said nucleotide sequences are also contemplated, provided that said fragments can perform the function of the complete protein. Thus, the term "functionally "equivalent" refers to a protein or polypeptide (or polynucleotide) with one or more amino acids (or nucleotides) missing from the termini of its sequence, such as at the amino and/or carboxyl terminus of the protein or polypeptide or at the 3' or 5' termini of the nucleotide sequence; where the fragment has the activity of the complete protein (i.e., is a functionally equivalent fragment). Assays to determine whether a fragment of a protein/polynucleotide has the same activity/function as the complete protein/polynucleotide are known to the person skilled in the art.

As understood by one skilled in the art, the codons of the nucleotide sequences of the invention (nucleotide sequences of the invention (i), (i) and (!!!)) may be optimized for expression in Saccharomyces cerevisiae, i.e., the nucleotide sequence has been altered with respect to the original nucleotide sequence such that one or more codons of the nucleotide sequence have been changed to a different codon that encodes the same amino acid, but is used more frequently by the host cell (in the present invention, a Saccharomyces cerevisiae) than the original codon. The degeneracy of the genetic code causes all amino acids except methionine and tryptophan to be encoded by more than one codon. For example, arginine, leucine and serine are encoded by 6 different codons, but many organisms use certain codons more frequently than others. Particularly since the sequences (i) and (i) of the invention are heterologous to the host cell, it is possible to optimize the nucleotide sequence for its expression in Saccharomyces cerevisiae.

In another more preferred embodiment of the cell of the invention: the nucleotide sequence (i) of the invention that codes for the enzyme L-tryptophan decarboxylase comprises a nucleotide sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the nucleotide sequence SEQ ID NO: 4; the nucleotide sequence (ii) of the invention that codes for the enzyme tryptamine 5-hydroxylase comprises the nucleotide sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the nucleotide sequence SEQ ID NO: 5; and the nucleotide sequence (iii) of the invention encoding the ARO4 enzyme comprises a nucleotide sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the nucleotide sequence SEQ ID NO: 6; preferably where the % identity value of the nucleotide sequences (i), (ii) and (iii) (with their respective SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6), is the same % value.

In another more preferred embodiment of the cell of the invention: the nucleotide sequence (i) of the invention that codes for the enzyme L-tryptophan decarboxylase comprises the nucleotide sequence SEQ ID NO: 4; the nucleotide sequence (ii) of the invention that codes for the enzyme tryptamine 5-hydroxylase comprises the nucleotide sequence SEQ ID NO: 5; and the nucleotide sequence (iii) of the invention that codes for the enzyme ARO4 comprises the nucleotide sequence SEQ ID NO: 6;

In a more preferred embodiment, the nucleotide sequences (i), (ii) and (iii) of the invention consist of the nucleotide sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively.

The nucleotide sequences (i), (ii) and (iii) of the cell of the invention may form part of one or more genetic constructs that are introduced into Saccharomyces cerevisiae to obtain the cell of the invention. Therefore, the present invention also relates to a gene construct comprising at least one sequence from among the nucleotide sequences (i), (ii) and (iii) of the cell of the invention. The gene construct may further comprise the elements necessary for the expression of the nucleotide sequences (i), (iii) and (iii) of the cell of the invention. Such elements include, without limitation, promoters, transcription terminators, selection markers, origins of replication, expression enhancers (enhancers) and ribosome binding sites (RBS). Furthermore, as understood by one skilled in the art, the nucleotide sequences (i), (ii) and (iii) of the cell of the invention may be operably linked to a promoter capable of acting in the host Saccharomyces cerevisiae.

The terms "promoter" or "promoter region" each refer to a nucleotide sequence within a gene that is located 5' to a coding sequence and functions to direct transcription of the coding sequence.

Promoter selection can allow for the expression of a desired gene product under a variety of conditions. Promoters can be selected for optimal function, such that the vector construct is inserted. Promoters can also be selected based on their regulatory characteristics. Examples of such characteristics include enhanced transcriptional activity and inducibility. Thus, the promoter can be a constitutive promoter or an inducible promoter. In the present invention, the term "inducible promoter" refers to a promoter that activates transcription in the presence of an external stimulus such as, but not limited to, temperature, pH, a hormone, a metabolite (e.g., lactose, mannitol, an amino acid), light (e.g., a specific wavelength), a heavy metal, or an antibiotic. In the present invention, the term "constitutive promoter" refers to a promoter that activates transcription permanently, regardless of environmental conditions. Examples of constitutive promoters for expression in Saccharomyces cerevisisae include, without limitation, promoters such as PGK1 p, TEF1 p or GPDp.

In the present invention, the term “operably linked” refers to the relationship between two nucleic acid regions in such a way that allows them to function as intended. For example, a control sequence “operably linked to a coding sequence” can be ligated in such a way that expression of the coding sequence occurs under conditions compatible with the control sequences. Thus, in some embodiments, the phrase “operably linked” refers to a promoter connected to a coding sequence such that transcription of that coding sequence is controlled and regulated by that promoter. Techniques for operably linking a promoter to a coding sequence are known in the art; the orientation and coding sequence of interest depends on, among other things, the specific nature of the coding sequence, among other things, the specific nature of the promoter. Thus, in another preferred embodiment of the cell of the invention, nucleotide sequences of the invention (i), (i) and/or (iii) are operably linked to a promoter, preferably a constitutive promoter. More preferably, the nucleotide sequence (i) of the invention is operably linked to the constitutive promoter TEF1p, the nucleotide sequence (i) of the invention is operably linked to the PGK1p promoter and the nucleotide sequence (iii) is operably linked to the constitutive promoter GPDp. The TEF1p, PGK1p and GPDp promoters allow the overexpression of the nucleotide sequences whose expression they control.

In another preferred embodiment of the cell of the invention, the enzyme encoded by the nucleotide sequence (i) is overexpressed. More specifically, the enzyme encoded by the nucleotide sequence (i) is overexpressed relative to a non-recombinant or "wild-type" Saccharomyces cerevisiae cell.

In another preferred embodiment of the cell of the invention, the enzyme encoded by the nucleotide sequence (i) is overexpressed. More specifically, the enzyme encoded by the nucleotide sequence (i) is overexpressed relative to a non-recombinant or "wild type" Saccharomyces cerevisiae cell.

In another preferred embodiment of the cell of the invention, the enzyme encoded by the nucleotide sequence (iii) is overexpressed. More specifically, the enzyme encoded by the nucleotide sequence (iii) is overexpressed relative to a non-recombinant or "wild-type" Saccharomyces cerevisiae cell.

In another more preferred embodiment of the cell of the invention, the enzyme encoded by the nucleotide sequence (i), the enzyme encoded by the nucleotide sequence (iii) and the enzyme encoded by the nucleotide sequence (iii) are overexpressed.

Selection markers are widely known in the state of the art. These selection markers, or selection genes, can be auxotrophy markers, antibiotic resistance genes, reporter genes, such as the gene encoding beta-galactosidase of the lactose operon, etc. Genes that confer resistance to antibiotics include, but are not limited to, ampicillin, tetracycline, kanamycin, hygromycin, gentamicin, etc. The markers allow the selection of cells. successfully transformed that grow in a medium containing the corresponding antibiotic because they carry the appropriate resistance gene

The introduction of the nucleotide sequences (i), (i) and (i) into the cell of the invention or into the gene construct(s) comprising said nucleotide sequences can be carried out by using a vector, for example, an expression vector.

In the present description, the term "vector" refers to a nucleic acid molecule that can be used to transport or transfer a nucleotide sequence into a cell. A vector can contain different functional elements, including, but not limited to, transcription control elements, such as promoters or operators, transcription factor binding regions or enhancers, and control elements for initiating and terminating transcription. Vectors include, but are not limited to: plasmids, cosmids, viruses, phages, recombinant expression cassettes, and transposons. Some vectors are capable of replicating or dividing autonomously after being introduced into the host cell, such as bacterial vectors with a bacterial origin of replication. Other vectors can integrate into the host cell's genome and thus replicate along with the cell's genome. Examples of vectors that can be used for the genetic modification of Saccharomyces cerevisiae cells include, but are not limited to, plasmids such as p426GPD, pCfB2803, pCfB2988, or pCfB2628.

In a preferred embodiment of the cell of the invention, the nucleotide sequences (i) and (ii) are integrated into the genome of said cell by means of the multiple integration plasmid pCfB2803, and the nucleotide sequence (iii) is integrated by means of the integration plasmid pCfB2988.

The obtaining of said vectors can be carried out by conventional methods known to those skilled in the art, just as for the transformation of Saccharomyces cerevisiae different widely known methods can be used - chemical transformation, liposomes, transfection, electroporation, particle bombardment, gene bullets ("gene gun"), microinjection, etc., described in various manuals widely known to those skilled in the art.

In another aspect, the present invention relates to a composition, hereinafter "composition of the invention", comprising the cell of the invention. As demonstrated in the examples of the present invention, by culturing the cell of the invention it is possible to produce serotonin from glucose.

Therefore, in another aspect, the present invention relates to the use of the cell of the invention or the composition of the invention, for the production of serotonin, hereinafter “use of the invention”.

In a preferred embodiment of the use of the invention, the production of serotonin is from a carbon source and a nitrogen source, where preferably the carbon source is glucose and the nitrogen source is ammonium.

In another aspect, the present invention relates to a method for the production of serotonin, hereinafter “method of the invention”, comprising:

(a) culturing the cell of the invention, or the composition of the invention, in a culture medium comprising at least one carbon source and one nitrogen source.

The cell of the invention has been defined in previous paragraphs of this document, and applies equally to the use of the invention and the method of the invention, as well as all its particular embodiments.

As understood by those skilled in the art, the growth or cultivation of the cell or of a population comprising the Saccharomyces cerevisiae cell of the invention is carried out in a culture medium comprising the components necessary for said microorganism to proliferate. Thus, in this document, the term "culture medium" refers to a substance, compound, mixture, or solution comprising the source of carbon and nitrogen necessary for the survival, growth, and/or metabolism of the microorganism, wherein said culture medium may be solid, semi-solid, semi-liquid, or liquid.

In this document, the term "nitrogen source" refers to molecules, chemical compounds, organic matter, comprising nitrogen (N) atoms and which are metabolized by microorganisms to carry out their growth and development, where said nitrogen source may have inorganic or organic origin. In a preferred embodiment of the method of the invention, the nitrogen source is selected from the list consisting of: ammonium, urea, the twenty-two different proteinogenic amino acids, and any combination thereof. In another particular embodiment of the method of the invention, the nitrogen source is ammonium. Preferably, the ammonium is in salt form. In the present invention, the term "ammonium salt" refers to a compound comprising an ammonium cation and an anion, which may be inorganic, such as the chloride anion.

In a more preferred embodiment of the method of the invention, the nitrogen source is ammonium in the form of ammonium chloride.

In this document, the term "carbon source" refers to molecules, chemical compounds, organic matter, comprising carbon atoms (C) and which are metabolized by microorganisms to carry out their growth and development, where said carbon source may have inorganic or organic origin.

In a preferred embodiment of the method of the invention, the carbon source is selected from the list consisting of: glucose, fructose, mannose, galactose, sucrose, maltose, and raffinose. In another more preferred embodiment of the method of the invention, the carbon source is glucose.

In another preferred embodiment of the method of the invention, the culture medium comprises glucose in a concentration of between 10 and 300 grams of glucose per liter of culture medium (g/L), more preferably between 20 and 250 grams of glucose per liter of culture medium, between 30 and 150 grams of glucose per liter of culture medium, and even more preferably between 50 and 100 grams of glucose per liter of culture medium.

In another preferred embodiment of the method of the invention, the culture medium comprises ammonium salt, preferably ammonium chloride, in a concentration of between 0.1 and 10 grams of ammonium salt per liter of culture medium, more preferably between 0.2 and 5 grams of ammonium salt per liter of culture medium, between 0.2 and 2 grams of ammonium salt per liter of culture medium, and even more preferably between 0.4 and 1.5 grams of ammonium per liter of culture medium. The present inventors have demonstrated that the cell of the invention can also synthesize serotonin from other nitrogen sources such as the amino acid L-tryptophan.

Thus in another preferred embodiment of the method of the invention, the culture medium comprises tryptophan, more preferably the culture medium comprises L-tryptophan and ammonium as culture sources.

In another preferred embodiment of the method of the invention, the culture medium comprises between 0.5 and 5 grams of L-tryptophan per liter of culture medium, more preferably between 1 and 3 grams of L-tryptophan per liter of culture medium.

As understood by those skilled in the art, the cultivation of the cell or a population comprising the cell of the invention must be carried out under suitable conditions so that the yeast can grow and synthesize serotonin. The suitable conditions of pH, temperature, humidity, etc., necessary to cultivate a strain of Saccharomyces cerevisiae are widely known in the state of the art.

In general, the growth of Saccharomyces cerevisiae yeasts requires a temperature above about 10°C and below about 40°C. Thus, in a preferred embodiment of the method of the invention, step (a) is carried out at a temperature between 15°C and 40°C, preferably between 20°C and 35°C, more preferably between 24°C and 32°C. In another even more preferred embodiment, step (a) of the method of the invention is carried out at a temperature of 28°C.

In another preferred embodiment of the method of the invention, step a) is carried out for a period of time between 24 and 120 hours, more preferably for a period between 48 and 96 hours, and even more preferably for 72 hours.

Preferably, step a) of the method of the invention is carried out under stirring conditions.

Once step a) of the method of the invention has been completed, the serotonin produced can be isolated by methods known to the person skilled in the art, which include examples such as, but not limited to, centrifugation, chromatography, decantation, filtration, lyophilization or by means of organic solvents. Thus, in another preferred embodiment, the method of the invention further comprises the following step: b) purifying the serotonin after step a).

DESCRIPTION OF THE FIGURES

Figure 1. Comparison of serotonin concentration in mg/L between control strains BY4743, the SER strain, and the SER+ARO4K229L strain in a tryptophan-enriched medium.

Figure 2. Comparison of serotonin concentration in mg/L between the control strains BY4743, the SER strain and the SER+ARO4K229L strain in a medium with glucose and ammonium as nitrogen sources.

Figure 3. Evolution of the different variables over time. The variables measured are CO2, O2, Ethanol, Biomass, and Serotonin.

EXAMPLES

The invention will then be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

Example 1. Serotonin production

The inventors have optimized the production of serotonin from glucose with the recombinant Saccharomyces cerevisiae strain, the S. cerevisiae TDC + T5H + ARO4K229L strain, by combining the multiple integration into the genome of the Clostridium sporogenes L-tryptophan decarboxylase (CsTDC) genes, which generates the decarboxylation of L-tryptophan to tryptamine, and the Oryza sativa T5H hydroxylase (OsT5H), which produces the hydroxylation of tryptamine to serotonin, and the overexpression of S. cerevisiae ARO4 with the point mutation K229L (ARO4 K229L), also by integration into the genome.

Materials and methods

2. 7. Construction of serotonin-producing plasmids and strains The sequences of the Clostridium sporogenes TDC and Oryza sativa T5H genes were chemically synthesized by Twist Bioscience. These sequences were optimized for S. cerevisiae codon usage. To clone them into plasmids, they were amplified from chemically synthesized DNA fragments with the primers TDC BamHI F, TDC Xho\ R, T5H BamHI F, and T5H Xho\ R (Table 1). These primers include tails containing the recognition sequence for the restriction enzymes BamHI and X/?ol, which were used to clone both genes into plasmid p426GPD (Mumberg et al., 1995), giving rise to plasmids p426GPD-TDC and p426GPD-T5H (Table 2).

Tabla 2. Plásmidos utilizados para la obtención de cepas de levadura modificadas 5 genéticamente para la producción de serotonina. Table 1. Primers used to obtain genetically modified yeast strains for serotonin production.

Table 2. Plasmids used to obtain genetically modified yeast strains for serotonin production.

For the modification of an amino acid change in the ARO4 gene, the ARO4 gene was amplified by PCR from the genomic DNA of the S. cerevisiae Sc288C strain, using the primers ARO4 F BamHI and ARO4 R Xho\. These primers include tails containing the recognition sequence of the restriction enzymes BamHI and Xho\, which were used for the cloning of ARO4 in the plasmid p426GPD (Mumberg et al., 1995), originating the plasmid p426GPD-aro4. From the plasmid p426GPD-aro4, PCR-directed mutagenesis was performed to generate a point mutation in the ARO4 sequence using the primers ARO4 F BamHI / ARO4 R Xhol / ARO4 K229L F / ARO4 K229L R. Specifically, the sequence AAG (coding for the amino acid lysine, K) was replaced by TTG (coding for the amino acid leucine, L) at nucleotide position 685 of the nucleotide sequence encoding ARO4 (SEQ ID NO: 8), obtaining the nucleotide sequence encoding ARO4 K229L (SEQ ID NO: 6).

Nucleotide sequence encoding the ARO4 enzyme of Saccharomyces cerevisisae, SEQ ID NO: 8:

ATGAGTGAATCTCCAATGTTCGCTGCCAACGGCATGCCAAAGGTAAATCAAGGTG CTGAAGAAGATGTCAGAATTTTAGGTTACGACCCATTAGCTTCTCCAGCTCTCCTT CAAGTGCAAATCCCAGCCACACCAACTTCTTTGGAAACTGCCAAGAGAGGTAGAA GAGAAGCTATAGATATTATTACCGGTAAAGACGACAGAGTTCTTGTCATTGTCGGT CCTTGTTCCATCCATGATCTAGAAGCCGCTCAAGAATACGCTTTGAGATTAAAGAA ATTGTCAGATGAATTAAAAGGTGATTTATCCATCATTATGAGAGCATACTTGGAGA AGCCAAGAACAACCGTCGGCTGGAAAGGTCTAATTAATGACCCTGATGTTAACAA CACTTTCAACATCAACAAGGGTTTGCAATCCGCTAGACAATTGTTTGTCAACTTGA CAAATATCGGTTTGCCAATTGGTTCTGAAATGCTTGATACCATTTCTCCTCAATACT TGGCTGATTTGGTCTCCTTCGGTGCCATTGGTGCCAGAACCACCGAATCTCAACT GCACAGAGAATTGGCCTCCGGTTGTCTTTCCCAGTTGGTTTCAAGAACGGTACC GATGGTACCTTAAATGTTGCTGTGGATGCTTGTCAAGCCGCTGCTCATTCTCACCA TTTCATGGGTGTTACTAAGCATGGTGTTGCTGCTATCACCACTACTAAGGGTAACG AACACTGCTTCGTTATTCTAAGAGGTGGTAAAAAGGGTACCAACTACGACGCTAA GTCCGTTGCAGAAGCTAAGGCTCAATTGCCTGCCGGTTCCAACGGTCTAATGATT GACTACTCTCACGGTAACTCCAATAAGGATTTCAGAAACCAACCAAAGGTCAATGA CGTTGTTTGTGAGCAAATCGCTAACGGTGAAAACGCCATTACCGGTGTCATGATT GAATCAAACATCAACGAAGGTAACCAAGGCATCCCAGCCGAAGGTAAAGCCGGCT TGAAATATGGTGTTTCCATCACTGATGCTTGTATAGGTTGGGAAACTACTGAAGAC GTCTTGAGGAAATTGGCTGCTGCTGTCAGACAAAGAAGAGAAGTTAACAAGAAAT AG

To construct the integration plasmids pCfB2803 TDC+T5H and pCfB2988 ARO4*, they were assembled using the cloning technique known as USER (uracil-specific cleavage reaction). To do this, the following biobricks were amplified by PCR: TDC, T5H, TEF1-PGK1 dual promoter, and GPDp-ARO4*. To amplify these biobricks, primers containing uracils and Phusion U Hot Start DNA polymerase (Thermo Scientific) were used. The primers used for the amplification of the “biobricks” were: OsT5H-GV2R, CsTDC-GV1 R, CsTDC-GP1 F, OsT5H-GV2R, PG1 R (TEF1 p), PG2R (PGK1 p), PV2F (GPDp), and GV2R (ARO4).

To assemble the serotonin-producing genes in plasmid pCfB2803, three sequences or “biobricks” were amplified, one containing TDC, another containing T5H and a third containing two strong constitutive promoters PGK1p and TEF1p, amplified from the previously constructed plasmids p426GPD-TDC, p426GPD-T5H and pCfB2628 (Germann et al., 2016), respectively. For this purpose, the primers OsT5H-GV2, CsTDC-GV1 R, CsTDC-GP1 F, OsT5H-GV2R, PG1 R (TEF1 p), PG2R (PGK1p) were used.

To assemble the modified ARO4 in pCfB2988, after directed mutagenesis, the plasmid p426GPD-ARO4* was constructed, from which both the promoter and the mutated ARO4 sequence were amplified with the primers PV2F (GPDp) / GV2R (ARO4), thus obtaining the “biobrick” GPDp-ARO4*.

In parallel, the vectors pCfB2988 and pCfB2803 used (Maury et al., 2016) were prepared by sequential treatment with the enzymes AsiSI (SfaAl) (Thermo Fisher Scientific) and Bsml (New England Biolabs). After purification, the prepared vectors and the amplified sequences were mixed and treated with the enzyme USER™ (New England Biolabs) and after the reaction, the mixture was used directly for bacterial transformation, obtaining pCfB2803 TDC+T5H and pCfB2988 ARO4*. The correct construction of the plasmids pCfB2803 TDC+T5H and pCfB2988 ARO4* was confirmed by Sanger sequencing using primers ADH1_test_fw and CYC1_test_rv.

The pCfB2803 T5H + TDC plasmid is a vector that allows multiple TDC and T5H integrations at sites that share homology with Ty4 elements, and the pCfB2988 ARO4* plasmid at sites that share homology with Ty1 elements, both of which are widely distributed throughout the yeast genome (Maury et al., 2016). In order to integrate the TDC+T5H gene cassette, as well as the gene cassette with the mutated ARO4 gene, the BY4743 strain, a laboratory S. cerevisiae strain that has auxotrophy for the URA3, LEU2, and HIS3 genes, was used. The cassette that complemented URA auxotrophy was pCfB2988, while the cast that complemented LEU auxotrophy was pCfB2803.

For the integration of the previously mentioned modifications in the strain, after verifying the correct construction of the vectors pCfB2803 TDC+T5H and pCfB2988 ARO4* by Sanger sequencing, both vectors were amplified for the extraction of the gene cassettes, by digesting both plasmids with the restriction enzyme Not\ and column purification, prior to the transformation of the yeast.

For the transformation of S. cerevisiae, the lithium acetate transformation protocol described by Gietz et al., (2004) was used, in which cells are incubated in the presence of lithium acetate, polyethylene glycol, carrier DNA and the plasmid of interest and subjected to a heat shock of 28°C for 30 minutes, followed by a heat shock of 42°C for 30 minutes. Subsequently, the cells are seeded in the different selection media and incubated at 28°C for 2 or 3 days. The correct integration of the sequences was confirmed by colony PCR using the primers CsTDC R Xhol / OsT5H R Xhol for the integration of TDC Y T5H, and the primers GPD_test_Fw / ARO4 R Xhol for the integration of pGPD_ARO4 K229L.

The strain resulting from the integration of the gene cassette obtained from the Not\ digestion of pCfB2803 TDC+T5H is identified as “SER strain”. The strain resulting from the integration of the gene cassette obtained from the Not\ digestion of pCfB2988 ARO4K229L into the SER strain is identified as “SER + ARO4K229L strain”.

2.2. Growth conditions and sample preparation To determine serotonin production, the modified SER and SER + ARO4K229L strains were inoculated into 5 mL of YNB medium overnight at 28°C with shaking. The following morning, the culture was inoculated into fresh medium at a cell count of 2x10 ⁶ cells/ml and grown in two media: one with all the nitrogen source coming from ammonium, and another where there was a 1:1 ratio between ammonium and L-tryptophan (Table 3). Strains were inoculated into Erlenmeyer flasks with a medium volume / total volume ratio of 1/5. This culture was incubated with constant shaking at 28°C, and after 72 h of growth, a sample was taken for analysis by HPLC-FLD. Samples were diluted to 50% with HPLC-grade absolute methanol and filtered with 0.22 pm nylon filters prior to injection into the chromatograph.

Table 3. Composition of the minimal medium (YNB) used for serotonin production.

2.3. Detection and quantification of serotonin by liquid chromatography

The serotonin produced was detected by high performance liquid chromatography (HPLC) on a Waters ACQ Arc Sys Core chromatograph using an Accucore™ C18 reversed phase column (Thermo Scientific) with dimensions of 4.6 x 150 mm and 2.6 pm particle size. The mobile phases used were A (acetonitrile) and B (0.01% trifluoroacetic acid in water) with a constant flow rate of 0.8 mL-mirr ¹ , the injection volume was 10 pL and the gradient program was set as follows: initial flow rate of 5:95% (A:B) 0-7 min, 90:10 % (A:B) 7-11 min, 5:95 % (A:B), 11-17 min. The detection of The chromatography of the analytes took place in a Waters 2475 fluorescence detector (FLR) where the chromatogram corresponding to excitation A = 295 nm and emission A = 330 nm was extracted. The retention time for serotonin was 5.5 min.

Results

The S. cerevisiae strain with the TDC and T5H genes produced approximately 567 mg L' ¹ from a medium enriched with L-tryptophan. In contrast, in a medium containing only glucose and ammonium, the SER strain was able to produce 22 mg L' ¹ of serotonin after 72 h of growth in said medium. Modifying this strain with the overexpression of the AR04 gene modified with the K229L substitution produced an increase of more than 5 times the production of serotonin compared to the strain with only TDC + T5H, exceeding 120 mg/L (Table 4).

Table 4. Concentrations in mg L' ¹ of serotonin produced by the modified strains after 72 h in minimal medium with 80 g L' ¹ of glucose and different nitrogen sources.

Example 2 - Scale-up in 1L bioreactor.

The inventors have carried out a fermentation in 1 L bioreactors as proof that the SER + ARO4K229L strain is capable of producing serotonin at an industrial level, being a first stage of scaling up to industrial volumes.

Materials and methods

2. 1. Bioreactor culture conditions

The modified strain SER + ARO4K229L was inoculated into 20 mL of minimal medium overnight at 28°C with shaking. The following morning, the culture was inoculated into 1 L of fresh medium inside the bioreactors at a cell count of 2x10 ⁶ cells/ml. final. The medium used was as defined in Table 4. The agitator was set at 300 rpm. The pH was set at 5 and was controlled by external calibrated pumps to introduce HCl or NaOH depending on the pH during the fermentation. An air flow of 0.75 L/min was introduced continuously throughout the fermentation, as well as maintaining the temperature at 28 ° C. The gas outlet was connected to a mass spectrometer, so real-time data were collected on the percentage of CO2, O2 and Ethanol. Samples were taken at various time points to measure OD600, biomass dry weight and serotonin concentration by HPLC-FLD. Serotonin samples were diluted to 50% with HPLC grade absolute methanol and filtered with 0.22 pm nylon filters prior to injection into the chromatograph.

Table 5. Composition of the minimal medium used for serotonin production.

2.3. Detection and quantification of serotonin by liquid chromatography

The serotonin produced was detected by high performance liquid chromatography (HPLC) on a Waters ACQ Arc Sys Core chromatograph using a reversed phase Accucore™ C18 column (Thermo Scientific) with dimensions of 4.6 x 150 mm and 2.6 pm particle size. The mobile phases used were A (acetonitrile) and B (0.01% thiofluoroacetic acid in water) with a constant flow rate of 0.8 mL-mirr ¹ , the injection volume was 10 pL and the gradient program was set as follows: initial flow rate of 5:95% (A:B) 0-7 min, 90:10 % (A:B) 7-11 min, 5:95 % (A:B), 11-17 min. Analyte detection took place in a Waters 2475 fluorescence detector (FLR) where the chromatogram corresponding to excitation A = 295 nm and emission A = 330 nm was extracted. The retention time for serotonin was 5.5 min.

Results

Fermentation was carried out for 72 h. Figure 3 shows the values obtained for each variable over time. CO2 production increases exponentially starting at 8 h, peaking at 32 h, followed by a decline that stabilizes after 48 h. This point indicates the end of the exponential phase, where the biomass stabilizes and the cells enter the stationary phase. Regarding serotonin production, we see that peak levels are reached around 36 h, a few hours after the end of the exponential phase, reaching average levels of 240 mg/L of serotonin. Calculating the yields, we see that the batch fermentation achieved a substrate/product yield of 3 mg of serotonin per gram of glucose. Regarding productivity, values of 6.66 mg/L/h of serotonin were obtained in the fermentation, calculated at 36 h, which is when a stable serotonin concentration is observed.

Claims

1. A recombinant Saccharomyces cerevisiae cell comprising: (i) a nucleotide sequence encoding an L-tryptophan decarboxylase enzyme or a functionally equivalent fragment thereof, wherein the L-tryptophan decarboxylase enzyme comprises an amino acid sequence with at least 80% identity to the amino acid sequence SEQ ID NO: 1; (i) a nucleotide sequence encoding a tryptamine 5-hydroxylase enzyme or a functionally equivalent fragment thereof, wherein the tryptamine 5-hydroxylase enzyme comprises an amino acid sequence with at least 80% identity to the amino acid sequence SEQ ID NO: 2; and (iii) a nucleotide sequence encoding an ARO4 enzyme or a functionally equivalent fragment thereof, wherein the ARO4 enzyme comprises an amino acid sequence with at least 80% identity with the amino acid sequence SEQ ID NO: 3, and wherein said amino acid sequence comprises the substitution of K (lysine) for L (leucine) at position 229 (ARO4 K229L). 2. Recombinant Saccharomyces cerevisiae cell according to claim 1, wherein said cell comprises: the nucleotide sequence (i) encoding an L-tryptophan decarboxylase enzyme or a functionally equivalent fragment thereof, wherein the L-tryptophan decarboxylase enzyme comprises an amino acid sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence SEQ ID NO: 1; the nucleotide sequence (ii) encoding a tryptamine 5-hydroxylase enzyme or a functionally equivalent fragment thereof, wherein the tryptamine 5-hydroxylase enzyme comprises an amino acid sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence SEQ ID NO: 2; and the nucleotide sequence (iii) encoding an ARO4 enzyme or a functionally equivalent fragment thereof, wherein the ARO4 enzyme comprises an amino acid sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the amino acid sequence SEQ ID NO: 3, and wherein said amino acid sequence comprises the substitution of K (lysine) for L (leucine) at position 229 (ARO4 K229L). 3. Recombinant Saccharomyces cerevisiae cell according to claim 1 or 2, wherein said cell comprises: the nucleotide sequence (i) encoding the enzyme L-tryptophan decarboxylase comprises a nucleotide sequence with at least 80% identity with the nucleotide sequence SEQ ID NO: 4; the nucleotide sequence (ii) encoding the enzyme tryptamine 5-hydroxylase comprises a nucleotide sequence with at least 80% identity with the nucleotide sequence SEQ ID NO: 5; and the nucleotide sequence (iii) encoding the enzyme ARO4 K229L comprises a nucleotide sequence with at least 80% identity with the nucleotide sequence SEQ ID NO: 6. 4. Recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 3, wherein said cell comprises: the nucleotide sequence (i) encoding the enzyme L-tryptophan decarboxylase comprises a nucleotide sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the nucleotide sequence SEQ ID NO: 4; the nucleotide sequence (ii) encoding the enzyme tryptamine 5-hydroxylase comprises a nucleotide sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the nucleotide sequence SEQ ID NO: 5; and the nucleotide sequence (iii) encoding the ARO4 K229L enzyme comprises a nucleotide sequence with at least 85, 90, 95, 96, 97, 98 or 99% identity with the nucleotide sequence SEQ ID NO: 6. 5. Recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 4, wherein said cell comprises: the nucleotide sequence (i) encoding an L-tryptophan decarboxylase enzyme or a functionally equivalent fragment thereof, wherein the L-tryptophan decarboxylase enzyme comprises the amino acid sequence SEQ ID NO: 1; the nucleotide sequence (ii) encoding a tryptamine 5-hydroxylase enzyme or a functionally equivalent fragment thereof, wherein the tryptamine 5-hydroxylase enzyme comprises the amino acid sequence SEQ ID NO: 2; and the nucleotide sequence (iii) encoding an ARO4 enzyme wherein the ARO4 enzyme consists of the amino acid sequence SEQ ID NO: 3, and wherein said sequence comprises the substitution of K (lysine) for L (leucine) at position 229 (ARO4 K229L). 6. Recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 5, wherein said cell comprises: the nucleotide sequence (i) encoding an L-tryptophan decarboxylase enzyme or a functionally equivalent fragment thereof, wherein the L-tryptophan decarboxylase enzyme consists of the amino acid sequence SEQ ID NO: 1; the nucleotide sequence (ii) encoding a tryptamine 5-hydroxylase enzyme or a functionally equivalent fragment thereof, wherein the tryptamine 5-hydroxylase enzyme consists of the amino acid sequence SEQ ID NO: 2; and the nucleotide sequence (iii) encoding an ARO4 enzyme or a functionally equivalent fragment thereof, wherein the ARO4 enzyme consists of the amino acid sequence SEQ ID NO: 3, and wherein said amino acid sequence comprises the substitution of K (lysine) for L (leucine) at position 229 (ARO4K229L). 7. Recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 6, wherein the nucleotide sequences (i), (ii) and (iii) comprise the nucleotide sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively. 8. Recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 7, wherein the nucleotide sequences (i), (ii) and (iii) consist of the nucleotide sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively. 9. Recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 8, wherein the enzyme encoded by sequence (iii) is overexpressed. 10. Recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 9, wherein the enzyme encoded by sequence (i) is overexpressed. 11. Recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 10, wherein the enzyme encoded by the sequence (i) is overexpressed. 12. A composition comprising the recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 11. 13. Use of the recombinant Saccharomyces cerevisiae strain according to any one of claims 1 to 11, or the composition according to claim 12, for the production of serotonin. 14. A method for the production of serotonin comprising: (a) culturing the recombinant Saccharomyces cerevisiae cell according to any one of claims 1 to 11, or the composition according to claim 12, in a culture medium comprising at least one carbon source and one nitrogen source. 15. Method for the production of serotonin according to claim 14, wherein the carbon source is glucose. 16. Method for the production of serotonin according to claim 14 or 15, wherein the carbon source is glucose and/or the nitrogen source is ammonium. 17. Method for the production of serotonin according to any one of claims 14 to 16, wherein the ammonium is in salt form, preferably as ammonium chloride. 18. Method for the production of serotonin according to any one of claims 14 to 17, wherein the culture medium comprises glucose in a concentration of between 10 and 300 grams of glucose per liter of culture medium. 19. Method for the production of serotonin according to any one of claims 14 to 18, wherein the culture medium comprises the ammonium salt, preferably ammonium chloride, in a concentration of between 0.1 and 10 grams of ammonium salt per liter of culture medium. 20. Method for the production of serotonin according to any one of claims 14 to 19, wherein step (a) is carried out at a temperature between 24°C and 32°C. 21. Method for the production of serotonin according to any one of claims 14 to 20, wherein step a) is carried out for a period of between 24 hours and 120 hours. 22. Method for the production of serotonin according to any one of claims 14 to 21, wherein said method further comprises the following step: (b) purify serotonin after step (a).