Methods to improve GGPP levels in yeast using optimized BTS1p mutants

Abstract

It is provided Bts1p mutants and method of using same to produce geranylgeranyl pyrophosphate (GGPP). Accordingly, it is provided an isolated host cell expressing the Bts1p mutant enzyme as defined herein, and particularly a production host cell a bacterium, a yeast, an algal cell or a plant cell. The Bts1p mutant enzyme provided can be used in any host to increase the level of GGPP.

Description

METHODS TO IMPROVE GGPP LEVELS IN YEAST USING OPTIMIZED BTS1P MUTANTSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is claiming priority from U.S. Provisional Applications Nos. 63 / 642259 filed May 3, 2024 and 63 / 643652 filed May 7, 2024, the content of which are hereby incorporated by reference in their entirety.TECHNICAL FIELD

[0002] It is provided Btsl p mutants and method of using same to produce geranylgeranyl pyrophosphate (GGPP).BACKGROUND

[0003] T erpenoids are the largest class of plant chemical compounds, consisting of over twenty thousand unique molecules. The diterpene class is particularly interesting due to several compounds with reported / potential pharmaceutical applications. Diterpenoids have been associated with potent anticancer, cardiovascular, and anti-inflammatory activities.

[0004] Taxol is the most widely used chemotherapeutic drug. It requires three fully grown Taxus brevifolia trees to obtain 1 g of taxol. Tripterygium wilfordii produces the diterpenoid triptolide. The diterpenoid represents approximately 0.0001% of the plants dry weight, rendering its extraction from plants non-viable.

[0005] Due to the low availability of the compounds in their natural sources and the difficulty of extraction, the production of diterpenoids in microbial cell platforms has been investigated. Saccharomyces cerevisiae has emerged as the premier organism for the production of terpenoids due to its ability to support the activity of essential enzymes in the decoration steps of their pathways. Even if the production of diterpenoids in yeast has been successful, this remains limited compared to other related classes, such as sesquiterpenes.

[0006] A major metabolic bottleneck in diterpene production in yeast is the availability of the geranylgeranyl diphosphate (GGPP) precursor, inherently produced in very low amounts for protein prenylation.

[0007] GGPP is naturally produced in the yeast, although it remains only a side product of the mevalonate pathway used by the RabGGtase complex to modify proteins, essential for cell viability post-translationally.

[0008] It is thus highly desired to be provided with means to improve the production of geranylgeranyl diphosphate (GGPP) in yeast.SUMMARY

[0009] It is provided a Btslp mutant enzyme comprising an amino acid sequence as depicted in SEQ ID NO: 1 and at least one mutation at amino acid K36, R48, V64, S70, S71 , and L257.

[0010] In an embodiment, the Btsl p mutant enzyme comprises at least two mutations, at least three mutations, at least four mutations, at least five mutations, or at least six mutations at amino acid K36, R48, V64, S70, S71 , and L257.

[0011] In an embodiment, the Btsl p mutant enzyme comprises at least one mutation selected from R48G, S71A, S71G, and L257F.

[0012] In an embodiment, the Bts1 p mutant enzyme comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15.

[0013] In a further embodiment, the Btsl p mutant enzyme comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15.

[0014] In an embodiment, the Btsl p mutant enzyme comprises comprising an amino acid sequence that is at least 87% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15. In a further embodiment, the Bts1 p mutant enzyme comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15. In another embodiment, the Btsl p mutant enzyme comprises an amino acid sequence as set forth in to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15.

[0015] In another embodiment, the Btslp mutant enzyme is encoded by a nucleotide sequence having at least 75% identify with SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

[0016] In an embodiment, the Btsl p mutant enzyme is encoded by a nucleotide sequence having at least 85% identify with SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

[0017] In another embodiment, the Btslp mutant enzyme is encoded by a nucleotide sequence having at least 87% identify with SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

[0018] In an embodiment, the Btsl p mutant enzyme is encoded by a nucleotide sequence having at least 95% identify with SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

[0019] In another embodiment, the Btslp mutant enzyme is encoded by a nucleotide sequence comprising a sequence as set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

[0020] It is also provided an isolated nucleic acid molecule comprising a sequence encoding a Btsl p mutant enzyme comprising SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15; a sequence as set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or a sequence which has at least 75% sequence identity with the sequence as defined in (a) or (b).

[0021] In an embodiment, the sequence has at least 85%, 87% or 95% sequence identity with the sequence as defined herein.

[0022] It is provided a vector comprising the nucleotide sequence as defined herein.

[0023] In a further embodiment, the Btsl p mutant enzyme or isolated nucleic acid molecule is isolated from BY4741 yeast strain, Saccharomyces cerevisiae, Saccharomyces boulardi, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces euyanus, Saccharomyces pastorianus, Saccharomyces kudriavzevii, Saccharomyces uvarum, or Saccharomyces arboricola

[0024] It is further provided a host cell comprising the vector or expressing the Btsl p mutant enzyme as provided herewith.

[0025] In an embodiment, the host cell is a bacterium, a yeast, an algal cell or a plant cell.

[0026] It is provided a method of producing geranylgeranyl pyrophosphate (GGPP) in an organism, comprising growing the host cell as provided herewith under conditions wherein GGPP is produced.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Reference will now be made to the accompanying drawings.

[0028] Fig. 1 illustrates HPLC analysis of p-carotene production analysis of BY4741 expressing pYES-BTS1 compared to p-carotene commercial standard.

[0029] Fig. 2 illustrates HPLC analysis of p-carotene production analysis of BY4741 expressing pYES-BTS1 (K36E), and pYES-BTS1.

[0030] Fig. 3 illustrates HPLC analysis of p-carotene production analysis of BY4741 expressing pYES-BTS1 (R48G), and pYES-BTS1.

[0031] Fig. 4 illustrates HPLC analysis of p-carotene production analysis of BY4741 expressing pYES-BTS1 (S70C), and pYES-BTS1.

[0032] Fig. 5 illustrates HPLC analysis of -carotene production analysis of BY4741 expressing pYES-BTS1 (S71A), and pYES-BTS1.

[0033] Fig. 6 illustrates HPLC analysis of p-carotene production analysis of BY4741 expressing pYES-BTS1 (S71G), and pYES-BTS1.

[0034] Fig. 7 illustrates HPLC analysis of p-carotene production analysis of BY4741 expressing pYES-BTS1 (L257F) and pYES-BTS1.

[0035] Fig. 8 illustrates p-carotene production results of BY4741 yeast strain transformed with single mutant pYES-BTS1 constructs, p-carotene production deduced from peak area integration of total carotenoid extract analyzed by HPLC.

[0036] Fig. 9 illustrates p-carotene production results of BY4741 yeast strain transformed with best performing pYES-BTS1 mutant constructs, p-carotene production deduced from peak area integration of total carotenoid extract analyzed by HPLC.

[0037] Fig. 10 illustrates Michaelis-Menten plots for purified Btsl p variants.DETAILED DESCRIPTION

[0038] In accordance with the present description, it is provided Btsl p mutants and method of using same to produce geranylgeranyl pyrophosphate (GGPP).

[0039] The production of high-value compounds, such as the medically important diterpenoids and carotenoids, in microorganisms, such as the yeast Saccharomyces cerevisiae, offers a sustainable production method. Diterpenoids have been associated with potent anticancer, cardiovascular, and anti-inflammatory activities. Carotenoids have beneficial roles in human and animal health when ingested in food or as supplements, such as provitamin A function, eye protection, antioxidative effects and protective properties from chronic degenerative diseases. Even if the production of diterpenoids and carotenoids in yeast has been successful, this remains limited compared to other related classes. The main reason for the limited production is the low availability of the classes' first dedicated precursor, geranylgeranyl pyrophosphate (GGPP). The increased production of GGPP would lead to increased production of the downstream compounds. Directed evolution was performed on yeast's native GGPP synthase to increase its enzymatic efficiency. Mutantswere identified and applied to the production of diterpenoids, leading to an improved yield over the native enzyme. These Btsl p mutants could be applied to all GGPP-derived compound production in S. cerevisiae or other organisms.

[0040] Protein prenylation utilizes farnesyl and geranylgeranyl, different types of isoprenoids groups, to modify proteins. These lipophilic moieties attach to carboxyl-terminal cysteine residues to promote the association of soluble proteins to membranes. Most prenylated proteins are geranylgeranylated. BTS1 gene encodes the yeast geranylgeranyl diphosphate synthase.

[0041] The catalytic efficiency of Btsl p was improved through a developed in vivo continuous evolution platform to increase the conversion of farnesyl pyrophosphate (FPP) to GGPP. Highly efficient variants were engineered by introducing genetic variability and a user-designed two-dimensional enzyme-specific evolutionary pressure. The previously reported error-prone of the OrthoRep system was used to introduce the genetic variability component. A BTS1 -specific evolutionary pressure was introduced to the system to select the mutants with improved enzymatic efficiency. A mutant constituent of the RabGGTase complex was used to introduce the enzymatic inefficiency to the complex, selecting for improved GGPP production. Additionally, a competing terpenoid, p-carotene, production pathway was introduced to the system forcing the strain to produce even higher amounts of the precursor to alleviate the RabGGTase enzymatic inefficiency. The product not only puts pressure on the GGPP pool but offered a colorimetric selection method to identify improved Btslp mutants.

[0042] The best-performing strains were identified, and the generated Btsl p mutants were extracted and sequenced. 11 mutants at 8 distinct residues were identified; K36, R48, V64, S70, S71 , and L257. The generated mutations to Btsl p as encompassed herein are not naturally occurring. The use of these mutations for GGPP-derived compounds was shown to increase the production in yeast.

[0043] In an embodiment, it is encompassed a Btsl p mutant enzyme comprising an amino acid sequence as depicted in SEQ ID NO: 1 and at least one mutation at amino acid K36, R48, V64, S70, S71 , and L257. It is further encompassed a Btsl p mutant enzyme comprising an amino acid sequence as depicted in SEQ ID NO: 1 and at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, or eight mutations at amino acid K36, R48, V64, S70, S71 , and

[0044] The identified R48G, S71A, S71G, and L257F mutations were shown to improve the strain's ability to survive under the established selection pressures. The generated mutants were applied to the production of the terpenoid p-carotene. These mutations were then replicated on the episomal pYES2 yeast vector under the expression of the inducible galactose promoter. The constructs were integrated into the BY4741 yeast strain with constructs expressing yeast codon optimized carotenoid related genes phytoene synthase (CrtB), phytoene desaturase (Crtl) and lycopene cyclase (CrtY). The performance of the variant yeast strains was verified by HPLC analysis for the p-carotene production, following galactose induction and extraction by cell distruption. The production of the mutants was compared to strains transformed with the wildtype pYes-BTS1. The Btsl p mutant yeast strains were shown to have an improved yield of p-carotene over the wildtype strain, indicative of improved enzyme performance. The identified Btsl p mutants could thus be used to increase the production of other GGPP-derived compounds in S. cerevisiae or other organisms.

[0045] As encompassed herein, it is provided a Btsl p mutant enzyme comprising at least one mutation selected from R48G, K36E, V64G, S70C, S71A, S71G, and L257F. Also encompassed is a Btsl p mutant enzyme comprising at least two mutations, at least three mutations, at least four mutations, at least five mutations, or at least six mutations selected from R48G, K36E, V64G, S70C, S71A, S71G, R, and L257F.

[0046] As encompassed herewith, it is provided the following Btsl p mutants:-BTS1 K36E a. a. sequence (SEQ ID NO: 3): MEAKIDELINNDPVWSSQNESLISKPYNHILLKPGENFRLNLIVQINRVMNLPKDQLAIVSQI VELLHNSSLLIDDIEDNAPLRRGQTTSHLIFGVPSTINTANYMYFRAMQLVSQLTTKEPLYH NLITIFNEELINLHRGQGLDIYWRDFLPEIIPTQEMYLNMVMNKTGGLFRLTLRLMEALSPS SHHGHSLVPFINLLGIIYQIRDDYLNLKDFQMSSEKGFAEDITEGKLSFPIVHALNFTKTKGQ TEQHNEILRILLLRTSDKDIKLKLIQILEFDTNSLAYTKNFINQLVNMIKNDNENKYLPDLASH SDTATNLHDELLYIIDHLSEL-BTS1 K36E nt. sequence (SEQ ID NO: 4):ATGGAGGCCAAGATAGATGAGCTGATCAATAATGATCCTGTTTGGTCCAGCCAAAATG AAAGCTTGATTTCAAAACCTTATAATCACATCCTTTTGAAACCTGGCgAGAACTTTAGA CTAAATTTAATAGTTCAAATTAACAGAGTTATGAATTTGCCCAAAGACCAGCTGGCCAT AGTTTCGCAAATTGTTGAGCTCTTGCATAATTCCAGCCTTTTAATCGACGATATAGAAG ATAATGCTCCCTTGAGAAGGGGACAGACCACTTCTCACTTAATCTTCGGTGTACCCTCCACTATAAACACCGCAAATTATATGTATTTCAGAGCCATGCAACTTGTATCGCAGCTAA CCACAAAAGAGCCTTTGTATCATAATTTGATTACGATTTTCAACGAAGAATTGATCAAT CTACATAGGGGACAAGGCTTGGATATATACTGGAGAGACTTTCTGCCTGAAATCATAC CTACTCAGGAGATGTATTTGAATATGGTTATGAATAAAACAGGCGGCCTTTTCAGATTA ACGTTG AG ACTCATGG AAG CG CTGTCTCCTTCCTC ACACC ACG G CCATTCG TTG GTT CCTTTCATAAATCTTCTGGGTATTATTTATCAGATTAGAGATGATTACTTGAATTTGAAA GATTTCCAAATGTCCAGCGAAAAAGGCTTTGCTGAGGACATTACAGAGGGGAAGTTAT CTTTTCCCATCGTCCACGCCCTTAACTTCACTAAAACGAAAGGTCAAACTGAGCAACA CAATGAAATTCTAAGAATTCTCCTGTTGAGGACAAGTGATAAAGATATAAAACTAAAGC TGATTCAAATACTGGAATTCGACACCAATTCATTGGCCTACACCAAAAATTTTATTAAT CAATTAGTGAATATGATAAAAAATGATAATGAAAATAAGTATTTACCTGATTTGGCTTC GCATTCCGACACCGCCACCAATTTACATGACGAATTGTTATATATAATAGACCACTTATCCGAATTGTGA-BTS1 R48G a. a. sequence (SEQ ID NO: 5):MEAKIDELINNDPVWSSQNESLISKPYNHILLKPGKNFRLNLIVQINGVMNLPKDQLAIVSQI VELLHNSSLLIDDIEDNAPLRRGQTTSHLIFGVPSTINTANYMYFRAMQLVSQLTTKEPLYH NLITIFNEELINLHRGQGLDIYWRDFLPEIIPTQEMYLNMVMNKTGGLFRLTLRLMEALSPS SHHGHSLVPFINLLGIIYQIRDDYLNLKDFQMSSEKGFAEDITEGKLSFPIVHALNFTKTKGQ TEQHNEILRILLLRTSDKDIKLKLIQILEFDTNSLAYTKNFINQLVNMIKNDNENKYLPDLASH SDTATNLHDELLYIIDHLSEL-BTS1 R48G nt. sequence (SEQ ID NO: 6):ATGGAGGCCAAGATAGATGAGCTGATCAATAATGATCCTGTTTGGTCCAGCCAAAATG AAAGCTTGATTTCAAAACCTTATAATCACATCCTTTTGAAACCTGGCAAGAACTTTAGA CTAAATTTAATAGTTC AAATTAACG G AGTTATG AATTTG CCCAAAG ACCAG CTGG COAT AGTTTCGCAAATTGTTGAGCTCTTGCATAATTCCAGCCTTTTAATCGACGATATAGAAG ATAATGCTCCCTTGAGAAGGGGACAGACCACTTCTCACTTAATCTTCGGTGTACCCTC CACTATAAACACCGCAAATTATATGTATTTCAGAGCCATGCAACTTGTATCGCAGCTAA CCACAAAAGAGCCTTTGTATCATAATTTGATTACGATTTTCAACGAAGAATTGATCAAT CTACATAGGGGACAAGGCTTGGATATATACTGGAGAGACTTTCTGCCTGAAATCATAC CTACTCAGGAGATGTATTTGAATATGGTTATGAATAAAACAGGCGGCCTTTTCAGATTA ACGTTG AG ACTCATGG AAG CG CTGTCTCCTTCCTC ACACC ACG G CCATTCG TTG GTTCCTTTCATAAATCTTCTGGGTATTATTTATCAGATTAGAGATGATTACTTGAATTTGAAA GATTTCCAAATGTCCAGCGAAAAAGGCTTTGCTGAGGACATTACAGAGGGGAAGTTAT CTTTTCCCATCGTCCACGCCCTTAACTTCACTAAAACGAAAGGTCAAACTGAGCAACACAATGAAATTCTAAGAATTCTCCTGTTGAGGACAAGTGATAAAGATATAAAACTAAAGC TGATTCAAATACTGGAATTCGACACCAATTCATTGGCCTACACCAAAAATTTTATTAAT CAATTAGTGAATATGATAAAAAATGATAATGAAAATAAGTATTTACCTGATTTGGCTTC GCATTCCGACACCGCCACCAATTTACATGACGAATTGTTATATATAATAGACCACTTAT CCGAATTGTGA-BTS1 V64G a. a. sequence (SEQ ID NO: 7): MEAKIDELINNDPVWSSQNESLISKPYNHILLKPGKNFRLNLIVQINRVMNLPKDQLAIVSQI GELLHNSSLLIDDIEDNAPLRRGQTTSHLIFGVPSTINTANYMYFRAMQLVSQLTTKEPLYH NLITIFNEELINLHRGQGLDIYWRDFLPEIIPTQEMYLNMVMNKTGGLFRLTLRLMEALSPS SHHGHSLVPFINLLGIIYQIRDDYLNLKDFQMSSEKGFAEDITEGKLSFPIVHALNFTKTKGQ TEQHNEILRILLLRTSDKDIKLKLIQILEFDTNSLAYTKNFINQLVNMIKNDNENKYLPDLASH SDTATNLHDELLYIIDHLSEL-BTS1 V64G nt. sequence (SEQ ID NO: 8):ATGGAGGCCAAGATAGATGAGCTGATCAATAATGATCCTGTTTGGTCCAGCCAAAATG AAAGCTTGATTTCAAAACCTTATAATCACATCCTTTTGAAACCTGGCAAGAACTTTAGA CTAAATTTAATAGTTCAAATTAACAGAGTTATGAATTTGCCCAAAGACCAGCTGGCCAT AGTTTCGCAAATTGGTGAGCTCTTGCATAATTCCAGCCTTTTAATCGACGATATAGAA GATAATGCTCCCTTGAGAAGGGGACAGACCACTTCTCACTTAATCTTCGGTGTACCCT CCACTATAAACACCGCAAATTATATGTATTTCAGAGCCATGCAACTTGTATCGCAGCTA ACCACAAAAGAGCCTTTGTATCATAATTTGATTACGATTTTCAACGAAGAATTGATCAA TCTAC ATAG G GG ACAAG G CTTGG ATATATACTG G AG AG ACTTTCTG CCTG AAATCATA CCTACTCAGGAGATGTATTTGAATATGGTTATGAATAAAACAGGCGGCCTTTTCAGAT TAACGTTG AG ACTCATG G AAG CG CTGTCTCCTTCCTC ACACCACG G CO ATTCGTTG G TTCCTTTCATAAATCTTCTGGGTATTATTTATCAGATTAGAGATGATTACTTGAATTTGA AAG ATTTCCAAATGTCCAG CG AAAAAG G CTTTG CTG AG G ACATTACAG AG GG G AAG T TATCTTTTCCCATCGTCCACGCCCTTAACTTCACTAAAACGAAAGGTCAAACTGAGCA ACACAATGAAATTCTAAGAATTCTCCTGTTGAGGACAAGTGATAAAGATATAAAACTAA AGCTGATTCAAATACTGGAATTCGACACCAATTCATTGGCCTACACCAAAAATTTTATT AATCAATTAGTGAATATGATAAAAAATGATAATGAAAATAAGTATTTACCTGATTTGGCT TCGCATTCCGACACCGCCACCAATTTACATGACGAATTGTTATATATAATAGACCACTT ATCCGAATTGTGA-BTS1 S70C a. a. sequence (SEQ ID NO: 9): MEAKIDELINNDPVWSSQNESLISKPYNHILLKPGKNFRLNLIVQINRVMNLPKDQLAIVSQI VELLHNCSLLIDDIEDNAPLRRGQTTSHLIFGVPSTINTANYMYFRAMQLVSQLTTKEPLYHNLITIFNEELINLHRGQGLDIYWRDFLPEIIPTQEMYLNMVMNKTGGLFRLTLRLMEALSPS SHHGHSLVPFINLLGIIYQIRDDYLNLKDFQMSSEKGFAEDITEGKLSFPIVHALNFTKTKGQ TEQHNEILRILLLRTSDKDIKLKLIQILEFDTNSLAYTKNFINQLVNMIKNDNENKYLPDLASH SDTATNLHDELLYIIDHLSEL-BTS1 S70C nt. sequence (SEQ ID NO: 10):ATGGAGGCCAAGATAGATGAGCTGATCAATAATGATCCTGTTTGGTCCAGCCAAAATG AAAGCTTGATTTCAAAACCTTATAATCACATCCTTTTGAAACCTGGCAAGAACTTTAGA CTAAATTTAATAGTTCAAATTAACAGAGTTATGAATTTGCCCAAAGACCAGCTGGCCAT AGTTTCGCAAATTGTTGAGCTCTTGCATAATTGCAGCCTTTTAATCGACGATATAGAAG ATAATGCTCCCTTGAGAAGGGGACAGACCACTTCTCACTTAATCTTCGGTGTACCCTC CACTATAAACACCGCAAATTATATGTATTTCAGAGCCATGCAACTTGTATCGCAGCTAA CCACAAAAGAGCCTTTGTATCATAATTTGATTACGATTTTCAACGAAGAATTGATCAAT CTACATAGGGGACAAGGCTTGGATATATACTGGAGAGACTTTCTGCCTGAAATCATAC CTACTCAGGAGATGTATTTGAATATGGTTATGAATAAAACAGGCGGCCTTTTCAGATTA ACGTTG AG ACTCATGG AAG CG CTGTCTCCTTCCTC ACACC ACG G CCATTCG TTG GTTCCTTTCATAAATCTTCTGGGTATTATTTATCAGATTAGAGATGATTACTTGAATTTGAAA GATTTCCAAATGTCCAGCGAAAAAGGCTTTGCTGAGGACATTACAGAGGGGAAGTTAT CTTTTCCCATCGTCCACGCCCTTAACTTCACTAAAACGAAAGGTCAAACTGAGCAACA CAATGAAATTCTAAGAATTCTCCTGTTGAGGACAAGTGATAAAGATATAAAACTAAAGC TGATTCAAATACTGGAATTCGACACCAATTCATTGGCCTACACCAAAAATTTTATTAAT CAATTAGTGAATATGATAAAAAATGATAATGAAAATAAGTATTTACCTGATTTGGCTTC GCATTCCGACACCGCCACCAATTTACATGACGAATTGTTATATATAATAGACCACTTAT CCGAATTGTGA-BTS1 S71 A a.a. sequence (SEQ ID NO: 1 1):MEAKIDELINNDPVWSSQNESLISKPYNHILLKPGKNFRLNLIVQINRVMNLPKDQLAIVSQI VELLHNSALLIDDIEDNAPLRRGQTTSHLIFGVPSTINTANYMYFRAMQLVSQLTTKEPLYH NLITIFNEELINLHRGQGLDIYWRDFLPEIIPTQEMYLNMVMNKTGGLFRLTLRLMEALSPS SHHGHSLVPFINLLGIIYQIRDDYLNLKDFQMSSEKGFAEDITEGKLSFPIVHALNFTKTKGQ TEQHNEILRILLLRTSDKDIKLKLIQILEFDTNSLAYTKNFINQLVNMIKNDNENKYLPDLASH SDTATNLHDELLYIIDHLSELBTS1 S71A nt. sequence (SEQ ID NO: 12):ATGGAGGCCAAGATAGATGAGCTGATCAATAATGATCCTGTTTGGTCCAGCCAAAATG AAAGCTTGATTTCAAAACCTTATAATCACATCCTTTTGAAACCTGGCAAGAACTTTAGACTAAATTTAATAGTTCAAATTAACAGAGTTATGAATTTGCCCAAAGACCAGCTGGCCAT AGTTTCGCAAATTGTTGAGCTCTTGCATAATTGCAGCCTTTTAATCGACGATATAGAAG ATAATGCTCCCTTGAGAAGGGGACAGACCACTTCTCACTTAATCTTCGGTGTACCCTC CACTATAAACACCGCAAATTATATGTATTTCAGAGCCATGCAACTTGTATCGCAGCTAA CCACAAAAGAGCCTTTGTATCATAATTTGATTACGATTTTCAACGAAGAATTGATCAAT CTACATAGGGGACAAGGCTTGGATATATACTGGAGAGACTTTCTGCCTGAAATCATAC CTACTCAGGAGATGTATTTGAATATGGTTATGAATAAAACAGGCGGCCTTTTCAGATTA ACGTTG AG ACTCATGG AAG CG CTGTCTCCTTCCTC ACACC ACG G CCATTCG TTG GTT CCTTTCATAAATCTTCTGGGTATTATTTATCAGATTAGAGATGATTACTTGAATTTGAAA GATTTCCAAATGTCCAGCGAAAAAGGCTTTGCTGAGGACATTACAGAGGGGAAGTTAT CTTTTCCCATCGTCCACGCCCTTAACTTCACTAAAACGAAAGGTCAAACTGAGCAACA CAATGAAATTCTAAGAATTCTCCTGTTGAGGACAAGTGATAAAGATATAAAACTAAAGC TGATTCAAATACTGGAATTCGACACCAATTCATTGGCCTACACCAAAAATTTTATTAAT CAATTAGTGAATATGATAAAAAATGATAATGAAAATAAGTATTTACCTGATTTGGCTTC GCATTCCGACACCGCCACCAATTTACATGACGAATTGTTATATATAATAGACCACTTATCCGAATTGTGA-BTS1 S71 G a. a. sequence (SEQ ID NO: 13):MEAKIDELINNDPVWSSQNESLISKPYNHILLKPGKNFRLNLIVQINRVMNLPKDQLAIVSQI VELLHNSGLLIDDIEDNAPLRRGQTTSHLIFGVPSTINTANYMYFRAMQLVSQLTTKEPLYH NLITIFNEELINLHRGQGLDIYWRDFLPEIIPTQEMYLNMVMNKTGGLFRLTLRLMEALSPS SHHGHSLVPFINLLGIIYQIRDDYLNLKDFQMSSEKGFAEDITEGKLSFPIVHALNFTKTKGQ TEQHNEILRILLLRTSDKDIKLKLIQILEFDTNSLAYTKNFINQLVNMIKNDNENKYLPDLASH SDTATNLHDELLYIIDHLSEL-BTS1 S71 G a.a. sequence (SEQ ID NO: 14):ATGGAGGCCAAGATAGATGAGCTGATCAATAATGATCCTGTTTGGTCCAGCCAAAATG AAAGCTTGATTTCAAAACCTTATAATCACATCCTTTTGAAACCTGGCAAGAACTTTAGA CTAAATTTAATAGTTCAAATTAACAGAGTTATGAATTTGCCCAAAGACCAGCTGGCCAT AGTTTCGCAAATTGTTGAGCTCTTGCATAATTCCGGCCTTTTAATCGACGATATAGAAG ATAATGCTCCCTTGAGAAGGGGACAGACCACTTCTCACTTAATCTTCGGTGTACCCTC CACTATAAACACCGCAAATTATATGTATTTCAGAGCCATGCAACTTGTATCGCAGCTAA CCACAAAAGAGCCTTTGTATCATAATTTGATTACGATTTTCAACGAAGAATTGATCAAT CTACATAGGGGACAAGGCTTGGATATATACTGGAGAGACTTTCTGCCTGAAATCATAC CTACTCAGGAGATGTATTTGAATATGGTTATGAATAAAACAGGCGGCCTTTTCAGATTA ACGTTG AG ACTCATGG AAG CG CTGTCTCCTTCCTC ACACC ACG G CCATTCG TTG GTTCCTTTCATAAATCTTCTGGGTATTATTTATCAGATTAGAGATGATTACTTGAATTTGAAA GATTTCCAAATGTCCAGCGAAAAAGGCTTTGCTGAGGACATTACAGAGGGGAAGTTAT CTTTTCCCATCGTCCACGCCCTTAACTTCACTAAAACGAAAGGTCAAACTGAGCAACACAATGAAATTCTAAGAATTCTCCTGTTGAGGACAAGTGATAAAGATATAAAACTAAAGC TGATTCAAATACTGGAATTCGACACCAATTCATTGGCCTACACCAAAAATTTTATTAATCAATTAGTGAATATGATAAAAAATGATAATGAAAATAAGTATTTACCTGATTTGGCTTC GCATTCCGACACCGCCACCAATTTACATGACGAATTGTTATATATAATAGACCACTTAT CCGAATTGTGA-BTS1 L257 a. a. sequence (SEQ ID NO: 15):MEAKIDELINNDPVWSSQNESLISKPYNHILLKPGKNFRLNLIVQINRVMNLPKDQLAIVSQI VELLHNSSLLIDDIEDNAPLRRGQTTSHLIFGVPSTINTANYMYFRAMQLVSQLTTKEPLYH NLITIFNEELINLHRGQGLDIYWRDFLPEIIPTQEMYLNMVMNKTGGLFRLTLRLMEALSPSSHHGHSLVPFINLLGIIYQIRDDYLNLKDFQMSSEKGFAEDITEGKLSFPIVHALNFTKTKGQ TEQHNEIFRILLLRTSDKDIKLKLIQILEFDTNSLAYTKNFINQLVNMIKNDNENKYLPDLASH SDTATNLHDELLYIIDHLSEL-BTS1 L257 nt. sequence (SEQ ID NO: 16):ATGGAGGCCAAGATAGATGAGCTGATCAATAATGATCCTGTTTGGTCCAGCCAAAATG AAAGCTTGATTTCAAAACCTTATAATCACATCCTTTTGAAACCTGGCAAGAACTTTAGA CTAAATTTAATAGTTCAAATTAACAGAGTTATGAATTTGCCCAAAGACCAGCTGGCCAT AGTTTCGCAAATTGTTGAGCTCTTGCATAATTCCAGCCTTTTAATCGACGATATAGAAG ATAATGCTCCCTTGAGAAGGGGACAGACCACTTCTCACTTAATCTTCGGTGTACCCTCCACTATAAACACCGCAAATTATATGTATTTCAGAGCCATGCAACTTGTATCGCAGCTAA CCACAAAAGAGCCTTTGTATCATAATTTGATTACGATTTTCAACGAAGAATTGATCAAT CTACATAGGGGACAAGGCTTGGATATATACTGGAGAGACTTTCTGCCTGAAATCATACCTACTCAGGAGATGTATTTGAATATGGTTATGAATAAAACAGGCGGCCTTTTCAGATTA ACGTTG AG ACTCATGG AAG CG CTGTCTCCTTCCTC ACACC ACG G CCATTCG TTG GTT CCTTTCATAAATCTTCTGGGTATTATTTATCAGATTAGAGATGATTACTTGAATTTGAAA GATTTCCAAATGTCCAGCGAAAAAGGCTTTGCTGAGGACATTACAGAGGGGAAGTTAT CTTTTCCCATCGTCCACGCCCTTAACTTCACTAAAACGAAAGGTCAAACTGAGCAACA CAATG AAATTTTTAG AATTCTCCTG TTG AG G ACAAG TG ATAAAG ATATAAAACTAAAG C TGATTCAAATACTGGAATTCGACACCAATTCATTGGCCTACACCAAAAATTTTATTAAT CAATTAGTGAATATGATAAAAAATGATAATGAAAATAAGTATTTACCTGATTTGGCTTCGCATTCCGACACCGCCACCAATTTACATGACGAATTGTTATATATAATAGACCACTTAT CCGAATTGTGA

[0047] In an embodiment, the Bts1 p mutant enzyme encompassed herein comprises an amino acid sequence that is at least 75% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15. Also encompassed is a Btsl p mutant enzyme comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13 or, 15.

[0048] Also encompassed is an isolated nucleic acid molecule comprising a sequence encoding a Btsl p mutant enzyme comprising SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15, ; a sequence as set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, or 1 ; or a sequence which has at least 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with a sequence encoding a Bts1 p mutant enzyme comprising SEQ ID NO: 3, 5, 7, 9, 11 , 13, or with a sequence as set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, or 1.

[0049] Further encompassed is a vector comprising the nucleotide sequence as defined herein.

[0050] As provided, the enzymatic fitness of the enzyme was assessed through the yeast-based production of the terpenoid p-carotene. Carotenoids are downstream products of GGPP. The increased production of the carotenoids would thus be indicative of increased production of GGPP from Btsl p. To establish the productions platforms, the R48G, K36E, V64G, S70C, S71A, S71G, and L257F Btslp mutants were replicated on the episomal pYES2 yeast vector, under the expression of the inducible galactose promoter. The generated constructs were integrated into the BY4741 yeast strain, expressing the phytoene synthase (CrtB), phytoene desaturase (Crtl) and lycopene cyclase (CrtY). The expression of the mutant Btsl p was shown to improve p-carotene production over the wild-type Btsl p. The mutants and the combinations of the mutations could be applied to GGPP-derived compound production in S. cerevisiae or other organisms.

[0051] As seen in Fig. 8, the R48G, S70C, and S17G mutations were shown to be the best performers showing a 10.5, 4,5, and 3-5-fold production improvement over the wildtype enzyme, respectively. Tp-carotene production was also tested of BY4741 yeast strain transformed with combination mutant pYES-BTS1 constructs. Of the assayed constructs the R48G-V64G BTS1 construct was shown to be the best performing leading to a 23-fold improvement in production (Fig. 9).

[0052] Accordingly, it is provided an isolated host cell comprising the vector as defined herein or expressing the Btsl p mutant enzyme as defined herein. Particularly, it is encompassed a yeast production host cell comprising the vector as defined herein or expressing the Btslp mutant enzyme as defined herein. The “host cell” as encompassed herein means cells that express the Btslp mutant enzyme as defined herein, such as e.g. and not limited to, bacteria, yeast or plant cells to produce GGPP-derived compounds.

[0053] There are naturally occurring GGPP synthases, specifically in closely related yeast species that have a high sequence identity with BTS1 , as provided in Table 1 .Table 1 : GGPP synthases - closely related yeast species with high sequence identity with BTS1.GGPPs from other yeast species % identity with ScBTSISaccharomyces boulardii ( KOH47307.1 ) 99.7%Saccharomyces paradoxus (XP_033769695.1) 96.1%Saccharomyces mikatae (CAI4036808.1) 91.9%Saccharomyces eubayanus ( XP_018218962.1) 87.8%Saccharomyces pastorianus (QID88364.1 ) 87.5%Saccharomyces kudriavzevii ( EJT44504.1 ) 87.8%Saccharomyces uvarum ( CAI4054475.1 ) 87.2%Saccharomyces arboricola ( EJS41366. 1) 87.8%

[0054] Accordingly, closely related yeast species that have a high sequence identity with BTS1 (i.e. greater than 85% identity) are Saccharomyces cerevisiae, Saccharomyces boulardi, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces euyanus, Saccharomyces pastorianus, Saccharomyces kudriavzevii, Saccharomyces uvarum, or Saccharomyces arboricola.

[0055] As Btsl p encodes for a GGPP synthase, which is a conserved enzyme from archaea to humans, therefore the Btslp mutant enzyme as defined herein can be used in any host to increase the level of GGPP. It is thus encompassed a method of producing geranylgeranyl pyrophosphate (GGPP) in an organism, comprising growing the isolated host cell or the yeast production host cell as defined herein under conditions wherein GPP is produced.

[0056] As provided herewith, microbial production platforms offer a sustainable production method for highly valuable and difficult to source compounds. The compounds yield is often restricted due to inherent limitations in the specific microbes used.Sesquiterpenoids are produced at much higher yield than diterpenoids in yeast due to relative higher availability of their precursor, farnesyl pyrophosphate (FPP) and geranyl geranyl pyrophosphate (GGPP) respectively. The low production titer of diterpenoids and carotenoids is a matter of high importance due to the high therapeutic and nutraceutical potential of the compound classes. To address this limitation a directed evolution strategy was employed to improve the GGPP production of yeast’s native GGPP synthase - Btsl p. Exploiting GGPP natural role in yeast, Btsl p was evolved under introduced temperature sensitivity, rendering excess GGPP required for cell viability. An additional competing carotenoid production pathway was introduced to further increase the evolutionary pressure as well ass offering a colorimetric selection system for identification of high performing variants. The evolved Btslp variants displayed mutations at 6 distinct residues. The highest performing mutants lead to an over 23-fold improvement in diterpenoid production attributed to increased substrate affinity, as confirmed by in vitro kinetic analysis. Notably, the most efficient variants harboured combined mutations at residues in close proximity to the active site and at the enzyme surface, indicating long-range modulation of the enzyme binding pocket.EXAMPLE I Establishing BTS1 -specific continuous directed evolution system and mutants identification

[0057] The BTS1 -specific continuous directed evolution system was based on the OrthoRep system previously established by Ravikumar et al. (2018, Cell, 175: 1946-1957).- BTS1 wt a. a. sequence (SEQ ID NO: 1):MEAKIDELINNDPVWSSQNESLISKPYNHILLKPGKNFRLNLIVQINRVMNLPKDQLAIVSQI VELLHNSSLLIDDIEDNAPLRRGQTTSHLIFGVPSTINTANYMYFRAMQLVSQLTTKEPLYH NLITIFNEELINLHRGQGLDIYWRDFLPEIIPTQEMYLNMVMNKTGGLFRLTLRLMEALSPS SHHGHSLVPFINLLGIIYQIRDDYLNLKDFQMSSEKGFAEDITEGKLSFPIVHALNFTKTKGQ TEQHNEILRILLLRTSDKDIKLKLIQILEFDTNSLAYTKNFINQLVNMIKNDNENKYLPDLASH SDTATNLHDELLYIIDHLSEL-BTS1 wt nt. sequence (SEQ ID NO: 2):ATGGAGGCCAAGATAGATGAGCTGATCAATAATGATCCTGTTTGGTCCAGCCAAAATG AAAGCTTGATTTCAAAACCTTATAATCACATCCTTTTGAAACCTGGCAAGAACTTTAGA CTAAATTTAATAGTTCAAATTAACAGAGTTATGAATTTGCCCAAAGACCAGCTGGCCAT AGTTTCGCAAATTGTTGAGCTCTTGCATAATTCCAGCCTTTTAATCGACGATATAGAAGATAATGCTCCCTTGAGAAGGGGACAGACCACTTCTCACTTAATCTTCGGTGTACCCTC CACTATAAACACCGCAAATTATATGTATTTCAGAGCCATGCAACTTGTATCGCAGCTAA CCACAAAAGAGCCTTTGTATCATAATTTGATTACGATTTTCAACGAAGAATTGATCAAT CTACATAGGGGACAAGGCTTGGATATATACTGGAGAGACTTTCTGCCTGAAATCATAC CTACTCAGGAGATGTATTTGAATATGGTTATGAATAAAACAGGCGGCCTTTTCAGATTA ACGTTG AG ACTCATGG AAG CG CTGTCTCCTTCCTC ACACC ACG G CCATTCG TTG GTT CCTTTCATAAATCTTCTGGGTATTATTTATCAGATTAGAGATGATTACTTGAATTTGAAA GATTTCCAAATGTCCAGCGAAAAAGGCTTTGCTGAGGACATTACAGAGGGGAAGTTAT CTTTTCCCATCGTCCACGCCCTTAACTTCACTAAAACGAAAGGTCAAACTGAGCAACA CAATGAAATTCTAAGAATTCTCCTGTTGAGGACAAGTGATAAAGATATAAAACTAAAGC TGATTCAAATACTGGAATTCGACACCAATTCATTGGCCTACACCAAAAATTTTATTAAT CAATTAGTGAATATGATAAAAAATGATAATGAAAATAAGTATTTACCTGATTTGGCTTC GCATTCCGACACCGCCACCAATTTACATGACGAATTGTTATATATAATAGACCACTTAT CCGAATTGTGA

[0058] The system introduces the genetic variability component to the user-defined gene, one of the two necessary components for any directed evolution system. The evolutionary pressure, selecting for the user-defined gene of interest, must be adapted to the system to select for the desired improved fitness. To establish the BTS1 mutation in the OrthoRep strain, Ga-Y319, BTS1 , amplified from yeast genomic DNA, was inserted into the linear pGLK1. The insertion is mediated by the shuttle vector pFDP-10B2. BTS1 was inserted into pFDP-10B2 by Gibson assembly, replacing the reporter mKATE2 gene. The FDP-10B2 vector backbone and the BTS1 gene fragment were amplified by PCR, using New England Biolabs (NEB) Q5® High-Fidelity DNA Polymerase (Cat no.M0491) using primers listed in Table 2. The construct assembly was completed with the NEBuilder® HiFi DNA Assembly Master Mix (Cat no. E2621) and transformed into NEB 5-alpha Competent E. col cells (Cat no. C2987). Following plasmid extraction, the construct assembly was verified by NEB EcoRI-HF® (Cat no. R3101) digestion. The assembled pFDP-10B2-BTS1 construct was linearized by Scal-HF® (Cat no. R3122) digestion, gel purified. The linearization of the construct allows for the homologous recombination of the construct with pGLK1 in vivo. The linearized pFDP-10B2-BTS1 and the error-prone polymerase plasmid pAR-Ec633 (Purchased from https: / / www.addgene.org / 130873 / ) were transformed in the Ga-Y319 yeast strain, provided by Prof. Chang Liu (UC Irvine) using the LiAc / SS carrier DNA / PEG method.Table 2: Primer usedConstructs Sequence (5’-3’)Primers for pFDP-BTS1 Gibson assemblyBTS1 Forward Primer tgtaaaatggaggccaagatagatgagct (SEQ ID NO: 17)BTS1 Reverse Primer ccccagttttcacaattcggataagtggtctattatatataacaattcg (SEQ ID NO: 18)FDP-10B2 Forward gaattgtgaaaactggggcacagaggat (SEQ ID NO: 19)PrimerFDP-10B2 Reverse cttggcctccattttacatgtctatgagcttatcacatgtttctaca (SEQ ID NO: 20)PrimerPrimers for Bet2 mutant PCR amplification.BET2 Forward Primer gaattcaaaatgtcaggatctcttacg (SEQ ID NO: 21 )BET2 Reverse Primer ctcgagctatttgtatggatacttttt (SEQ ID NO: 22) Primers for pUG27 PCR amplification forBET2 KO.BET2 KO forward aaatctaccggaagattttcaagtgcaatatatttactcatattcccagctgaagcttcgtac (SEQ IDNO: 23)BET2 KO Reverse ttaaaacaatactagaccagggtcatttatcgccatttcttgattccttactagtggatctgatatcac(SEQ ID NO: 24)Primers for pGLK1-BST1 amplificationOrtho_seq_new_for tccaacagtaccaccgaagtcg (SEQ ID NO: 25)Ortho_seq_new_rev gtcctcacggactcatcagacc (SEQ ID NO: 26)Primers for pYES2-BST 1 construct assemblyPyesVec.FOR cgaattgtgatgactcgagcatgcatctagaggg (SEQ ID NO: 27)BTS1(String).REV ggctcgagtcatcacaattcggataagtggtctattatatataacaattcg (SEQ ID NO: 28)Bts1(string).FOR aatattaagcttggatccaaaatggaggccaaga (SEQ ID NO: 29) pYESVec.REV ccattttggatccaagcttaatattccctatagtgagtcgtattacag (SEQ ID NO: 30)Primers for BST1 site-directed mutagenesisBTS1 R48G ctggtctttgggcaaattcataactccgttaatttg (SEQ ID NO: 31)BTS1 K36E tttagtctaaagttctcgccaggtttc (SEQ ID NO: 32)BTS1 V64G tgcaagagctcaccaatttgc (SEQ ID NO: 33)BTS1 S70C gtcgattaaaaggctgcaattatgcaagag (SEQ ID NO: 34)BTS1 S71A tatcttctatatcgtcgattaaaagggcggaattatgcaagag (SEQ ID NO: 35)BTS1 S71G cttctatatcgtcgattaaaaggccggaattatg (SEQ ID NO: 36)BTS1 L257F gtcctcaacaggaaaattcttag (SEQ ID NO: 37)

[0059] Following the insertion of the BTS1 and the error-prone polymerase in the Ga- Y319 strain, the evolutionary pressure was added to the system. The first level of evolutionary pressure was added by the insertion of the BET2 mutant to the strain. A BET2 mutant strain was obtained from the Dr. Boone (Li et al., 2011 , Nat Biotechnol, 29: 361-367) and Dr. Ferro-Novick Labs (Jiang et al., 1995, Journal of Biological Chemistry, 270: 21793- 21799). Genomic DNA extraction was performed on the strain following the Winston prep protocol. The resulting extracted DNA was used as a PCR template to amplify the mutant BET2. The PCR gene fragment was then inserted into the pCR™-TOPO™ Vector using the TOPO™ TA Cloning™ Kit from ThermoFischer Scientific (Cat no. K450002). The circularized construct was transformed in NEB 5-alpha Competent E. coli cells (Cat no. C2987). The plasmid was then extracted and sent for Sanger sequencing by Genome Quebec. The mutation responsible for temperature sensitivity was identified as BET2(F126S). BET2(F126S) was then integrated into the low-copy number yeast vector pDAY-L(CENZARS) developed in Ignea Lab. The pDAY-U BET2(F126S) was transformed in the strain using the LiAc / SS carrier DNA / PEG method. Following successful insertion, the wild-type chromosomal BET2 was deleted. The deletion cassette targeting the BET2 locus by homologous recombination was amplified by PCR from the pUG27 vector. The BET2-Ko deletion cassette and the pBF-3060 vector were transformed into the strain using the LiAc / SS carrier DNA / PEG protocol. The deletion cassette was then excised from the chromosome by Cre-recombinase, harbored on the pBF-3060 vector.

[0060] A second-fold evolutionary pressure was introduced to the system through a competing terpenoid production pathway. B-carotene production was chosen as the target terpenoid due to its applicability for colorimetric selection. The p-carotene biosynthetic pathway was introduced to the strain through the CrtYBI tri-domain fusion construct. The construct was sourced synthetically in the pMK vector from ThermoFischer Scientific. The gene fragment was then inserted in the pESC-W vector using restriction digestion and ligation protocol using the BamHI-HF® (cat no. R2136), Sall-HF® (cat no. R3138) and T4 DNA ligase (M0202) enzymes from NEB. The assembled construct was inserted into theyeast strain using the LiAc / SS carrier DNA / PEG method. Following insertion, the BTS1- specific continuous directed evolution system was fully established.

[0061] Following the establishment of the directed evolution system, individual colonies were selected and grown in 96 well plates at 30 and 37 °C. The colonies were passaged at 1 / 1600 dilutions to encourage error-prone pGLK1-BTS1 replication. The colonies were passaged ten times. The colonies were plated on six replica plates, with and without |3- carotene induction, and grown at three temperatures 24, 30, and 37 °C. Biomass accumulation and colorimetric selection of the colonies across the replica plates were used to identify the best-performing colonies, indicative of the colonies with the improved Btslp variants. The pGLK1-BTS1 constructs from the identified colonies were extracted using a modified zymolase (Zymo Research Cat no. E1005) / proteinase K (ThermoFischer Scientific cat no. EO0491) total DNA extraction method (Rvikumar et al., 2018, Cell, 175: 1946-1957). The resulting total DNA extract was used as a PCR template for BTS1 amplification by NEB Q5® High-Fidelity DNA Polymerase (Cat no.M0491). The resulting PCR fragments were analyzed by Sanger sequencing by Genome Quebec, identifying the sequence mutations.

[0062] BTS1 amplified by the genome was inserted in pYES2 construct by Gibson assembly using the primers form Table 1. The construct pYES2-BTS1 was also sourced synthetically from ThermoFischer Scientific. The identified mutations were replicated by site- directed mutagenesis on the pYES2-BTS1 construct. A two-step PCR strategy, using the Phusion® High-Fidelity DNA Polymerase (Cat no. M0530) from NEB, was performed to complete the mutations, a BTS1 fragment containing the target mutations were first amplified. The mutated gene fragment was then used as a mega primer to amplify the entire pYES2-BTS1 construct incorporating the target mutations. The resulting PCRs were digested by Dpnl (NEB Cat no. R0176), to digest the wild-type pYES2-BTS1 , before transforming in NEB 5-alpha Competent E. coli cells (Cat no. C2987). Following plasmid extraction, the mutated pYES2-BTS1 were transformed with pESC-W / CrtYBI in BY4741 yeast strain using the LiAc / SS carrier DNA / PEG method. Carotenoid production was induced by galactose and grown in Erlenmeyer flasks at 24 °C 75 rpm for 48 hours. To extract P-carotene the cells first collected by centrifugation and subsequently mechanically lysed with glass beads and cell disruptor. The carotenoids were extracted in a hexane acetone mixture (2:1). The carotenoid containing organic layer was then extracted, following centrifugation to separate it from cell debris. The extract was subsequently injected in a HPLC. The HPLC system used is a Thermo Ultimate 3000 HPLC equipped with a UV / visible light detector and (with the option of DAD and fraction collector) a C-18 column (5 pm, 100 A, 250 x 4.6 mm, Waters) with a C18 guard column. The pigments were eluted at 20 °C ata flow rate of 1 mL min-1with the following steps: 0 to 20 min, linear gradient of buffer A (methanol: water = 90:10) from 100 to 0; 20 to 22 min, 100% buffer B (ethyl acetate); 22 to 23 min, linear gradient of buffer B from 100 to 0; and 23 to 28 min, 100% buffer A. The injection volume was set to 20 pL.EXAMPLE IIKinetics parameters of Btsl p variants

[0063] To test the enzymatic activity of engineered Btsl p mutants, variants were selected that would present the best efficiency using FPP as substrate in yeast in vivo systems. As a model reaction to assess enzymatic activity, the phosphate transfer from IPP (Isopentenyl-pyrophosphate) to FPP (Farnesyl pyrophosphate) was chosen, which was reported as an efficient reaction for Btsl p and Btslp mutants with substitutions at one of the three amino acids residues Lys36, Arg48and Ser71. Apparent kinetic constants of Btsl p mutants were determined and compared with the Btsl p wt (Table 3). In particular, the R48G mutations implied a 2-fold decreased the apparent Michaelis constant K indicating a higher FPP affinity. Similarly, the R48G-S71A mutation leads to a 7.8-fold increase in feat.

[0064] The mutant’s kinetics parameters were also compared with GPP and DMAPP as substrates, previous intermediate of the mevalonate pathway. The single mutants R48G was found to have the best fold improvement for GPP.

[0065] The best kinetic (featapp / / <Mapp) results for DMAPP were obtained in the presence of the single mutations K36E and R48G. The absence in activity in the mutant K36E implies that the amino acid mutation Lys36leads to a destabilization at the active site of Btsl p. However, the mutant Arg48implied a 2-fold decreased the apparent Michaelis constant KM indicating a higher DMAPP affinity.Table 3: Enzyme kinetics of Btsl p mutants.

[0066] The enzyme purification was conducted at 4 °C. Cell paste was suspended in 10 ml of lysis buffer (25 mM TrisHCI pH 7.5, 150 mM NaCI, 1 mM PMSF, 10 mM imidazole, 20 mM B-mercaptoethanol, 10% glycerol, lysozyme 1 mg / ml_). The cells were disrupted by sonication, and the lysate was then centrifuged at 3 900 X rpm at 4°C for 30 min. The cell- free extract was loaded onto a Ni2+-NTA column, which had been previously equilibrated with equilibration buffer (25 mM TrisHCI pH 7.5, 150 mM NaCI, 10 mM imidazole, B- mercaptoethanol, 10% glycerol). The column was washed with 10 mM imidazole followed by 20 mM imidazole-containing buffer. His-tagged Bts1 p were eluted with 250 mM imidazole.

[0067] For enzyme activity measurement, WT and each Btsl p mutant (2,3 pM) was used. The reaction was started in 200 pL of solution containing 10 pM MOPS buffer (pH 7,0), various concentration of FPP, GPP, DMAPP, or IPP as specified below, 5 mM MgCI2, 1 mM DTT, 0.75 mM of carbonate / bicarbonate buffer. For FPP Kmdeterminations 10 pM of IPP was utilized and FPP concentration from 0.1 to 16 pM. For GPP and DMAPP Kmdeterminations 20 pM of IPP was utilized and the concentrations of GPP and DMAPP from 0.25 to 50 pM. The enzyme reaction cas initiated by adding 0.01 mg / mL Btsl p at 30°C during 1 h, then the hydrolyze of the acid-labile diphosphates The reaction was terminated by adding 10 mM EDTA and the product was extracted with 1 -butanol. Reactions were carried out for 30 min at 30 °C; and then, they were terminated by the addition of 0.2 mL 2N HCI in 83% ethanol and overlaid with 0.1 mL decane to trap the volatile products. After 30 min at 30 °C to hydrolyze the acid-labile diphosphates, reactions were neutralized by adding 0.35 mL of 10% NaOH. The decane phase (1 pL) was analyzed by GC-MS. The experiments were carried out in triplicate.

[0068] While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations and including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:1 . A Bts1 p mutant enzyme comprising an amino acid sequence as depicted in SEQ ID NO: 1 and at least one mutation at amino acid K36, R48, Q56, L57, V64, S70, S71 , and L257.

2. The Btsl p mutant enzyme of claim 1 , comprising at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, or eight mutations at amino acid K36, R48, Q56, L57, V64, S70, S71 , and L257.

3. The Btsl p mutant enzyme of claim 1 or 2, comprising at least one mutation selected from R48G, K36E, K36Q, V64G, S70C, S71A, S71G, and L257F.

4. The Btsl p mutant enzyme of any one of claims 1-3, comprising an amino acid sequence that is at least 75% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15.

5. The Btsl p mutant enzyme of any one of claims 1-4, comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15.

6. The Btsl p mutant enzyme of any one of claims 1-5, comprising an amino acid sequence that is at least 87% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15.

7. The Btsl p mutant enzyme of any one of claims 1-6, comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15.

8. The Btsl p mutant enzyme of any one of claims 1-7, comprising an amino acid sequence as set forth in to SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15.

9. The Btsl p mutant enzyme of any one of claims 1-8, encoded by a nucleotide sequence having at least 75% identify with SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

10. The Btsl p mutant enzyme of any one of claims 1-9, encoded by a nucleotide sequence having at least 85% identify with SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

11. The Btsl p mutant enzyme of any one of claims 1-10, encoded by a nucleotide sequence having at least 87% identify with SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24.

12. The Btsl p mutant enzyme of any one of claims 1-11 , encoded by a nucleotide sequence having at least 95% identify with SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

13. The Btsl p mutant enzyme of any one of claims 1-12, encoded by a nucleotide sequence comprising a sequence as set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

14. The Btsl p mutant enzyme of any one of claims 1-13, wherein said Btsl p mutant enzyme is isolated from BY4741 yeast strain, Saccharomyces cerevisiae, Saccharomyces boulardi, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces euyanus, Saccharomyces pastorianus, Saccharomyces kudriavzevii, Saccharomyces uvarum, or Saccharomyces arboricola.

15. An isolated nucleic acid molecule comprising:(a) a sequence encoding a Btsl p mutant enzyme comprising SEQ ID NO: 3, 5, 7, 9, 11 , 13, or 15;(b) a sequence as set forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or(c) a sequence which has at least 75% sequence identity with the sequence as defined in (a) or (b).

16. The isolated nucleic acid molecule of claim 15, wherein the sequence has at least 85%, 87% or 95% sequence identity with the sequence as defined in (a) or (b).

17. The isolated nucleic acid molecule of claim 15 or 16, wherein said isolated nucleic acid molecule is isolated from Saccharomyces cerevisiae, Saccharomyces boulardi, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces euyanus, Saccharomyces pastorianus, Saccharomyces kudriavzevii, Saccharomyces uvarum, or Saccharomyces arboricola.

18. A vector comprising the nucleotide sequence of any one of claims 15-17.

19. A host cell comprising the vector of claim 18 or expressing the Bts1 p mutant enzyme of any one of claims 1-14.

20. The host cell of claim 19, wherein said host cell is a bacterium, a yeast, an algal cell or a plant cell.

21. A method of producing geranylgeranyl pyrophosphate (GGPP) in an organism, comprising growing the host cell of claim 19 or 20 under conditions wherein GPP is produced.

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