Biosynthesis of isoprenoids and their precursors

Abstract

The present application describes proteins and host cells involved in methods for producing isoprenoid precursors and / or isoprenoids.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 170,347, filed April 2, 2021, entitled “BIOSYNTHESIS OF ISOPRENOIDS AND PRECURSORS THEREOF,” the entire disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to a sequence listing submitted as a text file via EFS-Web This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII file, created on April 1, 2022, is named G091970078WO00-SEQ-FL.TXT and is 392,553 bytes in size.

[0003] FIELD OF THEINVENTION The present disclosure relates to the production of isoprenoid precursors and isoprenoids in recombinant cells. [Background technology]

[0004] Isoprenoids are a diverse class of organic compounds derived from five carbon building blocks, encompassing at least 50,000 compounds. Due to their structural diversity, isoprenoids have numerous applications as flavorings, fragrances, antioxidants, and pharmaceutical compounds. The biosynthetic pathway of mevalonate has been characterized and is used by eukaryotes, archaea, and some bacteria to produce isoprenoids, but the wide range of isoprenoid isomers often precludes extraction in high yields from naturally occurring sources. Furthermore, the structural complexity of isoprenoids often limits de novo chemical synthesis. Summary of the Invention [Means for solving the problem]

[0005] Aspects of the present disclosure relate to a host cell for producing an isoprenoid precursor or an isoprenoid. In some embodiments, the host cell comprises a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a wild-type lanosterol synthase, and the host cell can produce more isoprenoid or isoprenoid precursor compared to a control host cell that does not contain the heterologous polynucleotide.

[0006] In some embodiments, the wild-type lanosterol synthase comprises SEQ ID NO:1 or SEQ ID NO:313.

[0007] In some embodiments, the lanosterol synthase is selected from the group consisting of 14, 33, 47, 50, 66, 80, 83, 85, 92, 94, 107, 122, 132, 145, 158, 170, 172, 184, 193, 197, 198, 212, 213, 227, 228, 231, 235, 248, 249, 260, 282, 286, 287, 289, 295, 296, 309, 314, 316, 329, 344, 360, 370, 371, 372, 398, 407, 41 and / or 742.

[0008] In some embodiments, the lanosterol synthase contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions and / or deletions relative to SEQ ID NO:1.

[0009] In some embodiments, the lanosterol synthase is: an amino acid Y at a residue corresponding to position 14 of SEQ ID NO:1; an amino acid Q at a residue corresponding to position 33 of SEQ ID NO:1; an amino acid E at a residue corresponding to position 47 of SEQ ID NO:1; an amino acid G at a residue corresponding to position 50 of SEQ ID NO:1; an amino acid R at a residue corresponding to position 66 of SEQ ID NO:1; an amino acid G at a residue corresponding to position 80 of SEQ ID NO:1; an amino acid L at a residue corresponding to position 83 of SEQ ID NO:1; an amino acid N at a residue corresponding to position 85 of SEQ ID NO:1; an amino acid I at a residue corresponding to position 92 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 94 of SEQ ID NO:1; an amino acid D at a residue corresponding to position 107 of SEQ ID NO:1; an amino acid C at a residue corresponding to position 122 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 132 of SEQ ID NO:1; an amino acid C at a residue corresponding to position 145 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 158 of SEQ ID NO:1; an amino acid A at a residue corresponding to position 170 of SEQ ID NO:1; an amino acid N at a residue corresponding to position 172 of SEQ ID NO:1; an amino acid L at a residue corresponding to position 83 of SEQ ID NO:1; an amino acid N at a residue corresponding to position 85 of SEQ ID NO:1; an amino acid I at a residue corresponding to position 92 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 94 of SEQ ID NO:1; an amino acid D at a residue corresponding to position 107 of SEQ ID NO:1; an amino acid C at a residue corresponding to position 122 of SEQ ID NO:1; an amino acid S at Amino acid W; amino acid C or H at the residue corresponding to position 193 of SEQ ID NO:1; amino acid V at the residue corresponding to position 197 of SEQ ID NO:1; amino acid I at the residue corresponding to position 198 of SEQ ID NO:1; amino acid I at the residue corresponding to position 212 of SEQ ID NO:1; amino acid L at the residue corresponding to position 213 of SEQ ID NO:1; amino acid L at the residue corresponding to position 227 of SEQ ID NO:1; amino acid T at the residue corresponding to position 228 of SEQ ID NO:1; amino acid V at the residue corresponding to position 231 of SEQ ID NO:1; amino acid M at the residue corresponding to position 235 of SEQ ID NO:1; amino acid F at the residue corresponding to position 248 of SEQ ID NO:1; amino acid L at the residue corresponding to position 249 of SEQ ID NO:1; amino acid R at the residue corresponding to position 260 of SEQ ID NO:1; amino acid I at the residue corresponding to position 282 of SEQ ID NO:1; amino acid F at the residue corresponding to position 286 of SEQ ID NO:1; amino acid G at the residue corresponding to position 287 of SEQ ID NO:1; amino acid G at the residue corresponding to position 289 of SEQ ID NO:1; amino acid I at the residue corresponding to position 295 of SEQ ID NO:1; amino acid T at the residue corresponding to position 296 of SEQ ID NO:1;The amino acid F at the residue corresponding to position 309 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 314 of SEQ ID NO:1; the amino acid R at the residue corresponding to position 316 of SEQ ID NO:1; the amino acid N at the residue corresponding to position 329 of SEQ ID NO:1; the amino acid A at the residue corresponding to position 344 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 360 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 370 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 371 of SEQ ID NO:1; the amino acid P at the residue corresponding to position 372 of SEQ ID NO:1; the amino acid I at the residue corresponding to position 398 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 407 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 414 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 417 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 423 of SEQ ID NO:1; the amino acid I or S at the residue corresponding to position 432 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 437 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 442 of SEQ ID NO:1; the amino acid M at the residue corresponding to position 444 of SEQ ID NO:1 or S; the amino acid G at the residue corresponding to position 452 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 474 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 479 of SEQ ID NO:1; the amino acid Q at the residue corresponding to position 491 of SEQ ID NO:1; the amino acid N at the residue corresponding to position 498 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 515 of SEQ ID NO:1; the amino acid T at the residue corresponding to position 526 of SEQ ID NO:1; the amino acid T at the residue corresponding to position 529 of SEQ ID NO:1; the amino acid F at the residue corresponding to position 536 of SEQ ID NO:1; the amino acid Y at the residue corresponding to position 544 of SEQ ID NO:1; the amino acid E at the residue corresponding to position 552 of SEQ ID NO:1; the amino acid A at the residue corresponding to position 559 of SEQ ID NO:1; the amino acid M at the residue corresponding to position 560 of SEQ ID NO:1; the amino acid C or N at the residue corresponding to position 564 of SEQ ID NO:1; the amino acid P at the residue corresponding to position 578 of SEQ ID NO:1; the amino acid F at the residue corresponding to position 586 of SEQ ID NO:1; the amino acid T at the residue corresponding to position 608 of SEQ ID NO:1; the amino acid I at the residue corresponding to position 610 of SEQ ID NO:1;the amino acid V at the residue corresponding to position 617 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 619 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 620 of SEQ ID NO:1; the amino acid E or R at the residue corresponding to position 631 of SEQ ID NO:1; the amino acid D at the residue corresponding to position 638 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 650 of SEQ ID NO:1; the amino acid A at the residue corresponding to position 655 of SEQ ID NO:1; the amino acid H at the residue corresponding to position 660 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 679 of SEQ ID NO:1; the amino acid E at the residue corresponding to position 686 of SEQ ID NO:1; the amino acid D at the residue corresponding to position 702 of SEQ ID NO:1; the amino acid Q at the residue corresponding to position 710 of SEQ ID NO:1; the amino acid L or V at the residue corresponding to position 726 of SEQ ID NO:1; the amino acid F at the residue corresponding to position 736 of SEQ ID NO:1; the amino acid M at the residue corresponding to position 738 of SEQ ID NO:1; and / or a truncation resulting in a deletion of the residue corresponding to position 742 of SEQ ID NO:1;

[0010] In some embodiments, the lanosterol synthase contains the amino acid substitutions E617V, G107D, and / or K631E relative to SEQ ID NO:1.

[0011] In some embodiments, for SEQ ID NO:1, the lanosterol synthase has the following sequences: R33Q, R193C, D289G, N295I, S296T, N620S, and Y736F; R184W, L235M, L260R, and E710Q; K47E, L92I, T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, E617V, and F726L;N14Y, N132S, Y145C, R193H, I286F, L316R, F432I, E442V, T444S, I479S, K631R, and T655A;F432S, D452G, and I536F;E287G, K329N, E617V, and F726V; E231V, A407V, Q423L, A529T, and Y564C;V248F, D371V, and G702D;L197V, K282I, N314S, P370L, A608T, G638D, and F650L;L491Q, Y586F, and R660H;G122C, H249L, and K738M; P227L, E474V, V559A, and Y564N; truncations resulting in deletion of K85N, G158S, S515L, P526T, Q619L, and residues corresponding to Q742 of SEQ ID NO:1; G107D and K631E; T212I, W213L, N544Y and V552E; I172N, C414S, L560M and G679S; R193C, D289G, N295I, S296T, N620S and Y736F; K85N and G158S; L197V , K282I, N314S and P370L; I172N, C414S and L560M; D371V, M610I, and G702D; D371V, K498N, M610I, and G702D; D80G, P83L, T170A, T198I, and A228T; T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, and E617V; or L309F, V344A, T398I, and K686E.

[0012] In some embodiments, the lanosterol synthase has the following amino acid substitutions relative to SEQ ID NO:1: R193C, D289G, N295I, S296T, N620S, and Y736F; F432S, D452G, and I536F; K85N, and G158S; L197V, K282I, N314S, and P370L; I172N, C including 414S, L560M, and G679S; I172N, C414S, and L560M; D371V, M610I, and G702D; D371V, K498N, M610I, and G702D; D80G, P83L, T170A, T198I, and A228T; D50G, K66R, N94S, G417S, E617V, and F726L; T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, and E617V; and L309F, V344A, T398I, and K686E.

[0013] In some embodiments, the lanosterol synthase contains the following amino acid substitutions relative to SEQ ID NO:1: D50G, K66R, N94S, G417S, E617V, and F726L; K85N and G158S; K47E, L92I, T360S, S372P, T444M, and R578P; F432S, D452G, and I536F; T360S, S372P, T444M, and R578P; L491Q, Y586F, and R660H; K85N, G158S, S515L, P526T, Q619L, and a truncation resulting in a deletion of a residue corresponding to position 742 of SEQ ID NO:1; or I172N, C414S, L560M, and G679S.

[0014] In some embodiments, the lanosterol synthase comprises amino acid substitutions or deletions relative to SEQ ID NO:1 at one or more residues corresponding to positions 14, 33, 47, 50, 66, 85, 92, 94, 122, 132, 145, 158, 193, 231, 248, 249, 286, 287, 289, 295, 296, 316, 329, 360, 371, 372, 407, 417, 423, 432, 442, 444, 479, 515, 526, 529, 564, 578, 617, 619, 620, 631, 655, 702, 726, 736, 738, and / or 742 of SEQ ID NO:1.

[0015] In some embodiments, the lanosterol synthase is selected from the group consisting of: R33Q, R193C, D289G, N295I, S296T, N620S, and Y736F; K47E, L92I, T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, E617V, and F726L; N14Y, N132S, Y145C, R193H, I286F, L316R, F432I, E442V, T444S, I479S, K631R, and T655A; E287G, K329N, E617V, and F726V; V, A407V, Q423L, A529T, and Y564C; V248F, D371V, and G702D; G122C, H249L, and K738M; or K85N, G158S, S515L, P526T, and Q619L, as well as truncations resulting in the deletion of residues corresponding to Q742 of SEQ ID NO:1.

[0016] In some embodiments, the lanosterol synthase comprises a sequence that is at least 90% identical to SEQ ID NO: 3, 83-87, 89-92, 94-95, 99, 118-120, 316-319, 321-326, 329, or 331.

[0017] In some embodiments, the lanosterol synthase comprises SEQ ID NO: 3, 83-87, 89-92, 94-95, 99, 118-120, 316-319, 321-326, 329, or 331.

[0018] In some embodiments, the heterologous polynucleotide comprises a sequence that is at least 90% identical to SEQ ID NO: 4, 62-66, 68-71, 73-74, 78, 103-109, 111-117, 328, or 330.

[0019] In some embodiments, the heterologous polynucleotide comprises the sequence of SEQ ID NO: 4, 62-66, 68-71, 73-74, 78, 103-109, 111-117, 328, or 330.

[0020] A further aspect of the present disclosure relates to a host cell comprising a heterologous polynucleotide encoding a lanosterol synthase, wherein the lanosterol synthase comprises a sequence at least 90% identical to SEQ ID NO: 3, 83-87, 89-92, 94-95, 99, 100-102, 118-120, 316-319, 321-326, 329, or 331.

[0021] In some embodiments, the lanosterol synthase comprises SEQ ID NO: 3, 83-87, 89-92, 94-95, 99, 100-102, 118-120, 316-319, 321-326, 329, or 331.

[0022] A further aspect of the disclosure relates to a host cell comprising a heterologous polynucleotide encoding a lanosterol synthase, wherein the lanosterol synthase is selected from the group consisting of: R33Q, R193C, D289G, N295I, S296T, N620S, and Y736F; K47E, L92I, T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, E617V, and F726L; N14Y, N132S, Y145C, R193H, I2 86F, L316R, F432I, E442V, T444S, I479S, K631R, and T655A; E287G, K329N, E617V, and F726V; E231V, A407V, Q423L, A529T, and Y564C; V248F, D371V, and G702D; G122C, H249L, and K738M; or K85N, G158S, S515L, P526T, and Q619L, and truncations resulting in a deletion of residues corresponding to Q742 of SEQ ID NO:1.

[0023] A further aspect of the present disclosure relates to a host cell comprising a heterologous polynucleotide encoding a lanosterol synthase, wherein the heterologous polynucleotide comprises a sequence that is at least 90% identical to SEQ ID NO: 4, 62-66, 68-71, 73-74, 78, 80-82, 103-109, 111-117, 328, or 330.

[0024] In some embodiments, the heterologous polynucleotide comprises SEQ ID NO: 4, 62-66, 68-71, 73-74, 78, 80-82, 103-109, 111-117, 328, or 330.

[0025] In some embodiments, the host cell comprises a heterologous polynucleotide encoding a lanosterol synthase, where the lanosterol synthase comprises amino acid substitutions or deletions relative to SEQ ID NO:313 at one or more residues corresponding to positions 64, 120, 121, 136, 226, 268, 275, 281, 300, 322, 333, 438, 502, 604, 619, 628, 656, 693, 726, 727, 728, 729, 730, and / or 731.

[0026] In some embodiments, the lanosterol synthase is: an amino acid G at a residue corresponding to position 64 of SEQ ID NO:313; an amino acid V at a residue corresponding to position 120 of SEQ ID NO:313; an amino acid S at a residue corresponding to position 121 of SEQ ID NO:313; an amino acid V at a residue corresponding to position 136 of SEQ ID NO:313; an amino acid I at a residue corresponding to position 226 of SEQ ID NO:313; an amino acid S at a residue corresponding to position 268 of SEQ ID NO:313; an amino acid I at a residue corresponding to position 275 of SEQ ID NO:313; an amino acid A at a residue corresponding to position 281 of SEQ ID NO:313; an amino acid G at a residue corresponding to position 300 of SEQ ID NO:313; 313; the amino acid G at the residue corresponding to position 322 of SEQ ID NO:313; the amino acid A at the residue corresponding to position 333 of SEQ ID NO:313; the amino acid E at the residue corresponding to position 438 of SEQ ID NO:313; the amino acid L at the residue corresponding to position 502 of SEQ ID NO:313; the amino acid N at the residue corresponding to position 604 of SEQ ID NO:313; the amino acid S at the residue corresponding to position 619 of SEQ ID NO:313; the amino acid E at the residue corresponding to position 628 of SEQ ID NO:313; the amino acid T at the residue corresponding to position 656 of SEQ ID NO:313; the amino acid G at the residue corresponding to position 693 of SEQ ID NO:313; and / or a deletion of residues corresponding to positions 726 to 731 of SEQ ID NO:313.

[0027] In some embodiments, the lanosterol synthase includes, relative to SEQ ID NO:313: P121S, A136V, S300G, V322G, K438E, F502L, K628E, and a deletion of residues corresponding to positions 726-731 of SEQ ID NO:313; K268S, T281A, F502L, T604N, A656T, and E693G; or C619S, F275I, I120V, M226I, R64G, and T333A.

[0028] In some embodiments, the lanosterol synthase comprises a sequence that is at least 90% identical to any one of SEQ ID NOs:100-102.

[0029] In some embodiments, the lanosterol synthase comprises a sequence selected from SEQ ID NOs:100-102.

[0030] In some embodiments, the heterologous polynucleotide encoding lanosterol synthase comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs:80-82.

[0031] In some embodiments, the heterologous polynucleotide encoding lanosterol synthase comprises a sequence selected from SEQ ID NOs:80-82.

[0032] In some embodiments, the host cell is capable of producing mevalonate.

[0033] In some embodiments, the host cell is capable of producing at least 0.2 g / L of mevalonate.

[0034] In some embodiments, the host cell is capable of producing at least 0.7 g / L of mevalonate.

[0035] In some embodiments, the host cell is capable of producing at least 9 mg / L of isoprenoid.

[0036] In some embodiments, the host cells are capable of producing at least 1.1-fold more isoprenoids than a control host cell comprising SEQ ID NO:1 and / or a control host cell comprising SEQ ID NO:313.

[0037] In some embodiments, the host cells are capable of producing at least 3-fold more isoprenoids than a control host cell comprising SEQ ID NO:1 and / or a control host cell comprising SEQ ID NO:313.

[0038] In some embodiments, the host cell is capable of producing up to 200 mg / L of lanosterol.

[0039] In some embodiments, the host cell is capable of producing at least 5 mg / L of oxidosqualene.

[0040] In some embodiments, the host cell is capable of producing more mevalonate than a control host cell that does not include a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a wild-type lanosterol synthase.

[0041] In some embodiments, the host cell is capable of producing more 2-3-oxidosqualene compared to a host cell that does not include a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a wild-type lanosterol synthase.

[0042] In some embodiments, the host cell further comprises (a) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a wild-type squalene epoxidase; or (b) a heterologous polynucleotide that reduces squalene epoxidase activity, wherein the host cell is capable of producing more isoprenoid or isoprenoid precursor compared to a control host cell that does not contain the heterologous polynucleotides of (a) and / or (b).

[0043] In some embodiments, the wild-type squalene epoxidase comprises SEQ ID NO: 9 or 312.

[0044] A further aspect of the present disclosure relates to a host cell for producing an isoprenoid precursor or an isoprenoid, wherein the host cell comprises: (a) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a wild-type squalene epoxidase; or (b) a heterologous polynucleotide that reduces squalene epoxidase activity, and wherein the host cell is capable of producing more isoprenoid or isoprenoid precursor compared to a control host cell that does not contain the heterologous polynucleotides of (a) and / or (b).

[0045] In some embodiments, the wild-type squalene epoxidase comprises SEQ ID NO: 9 or 312.

[0046] In some embodiments, the heterologous polynucleotide encodes a squalene epoxidase that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and / or deletions relative to SEQ ID NO: 9 or 312.

[0047] In some embodiments, the host cell is capable of producing mevalonate.

[0048] In some embodiments, the host cell is capable of producing at least 0.2 g / L of mevalonate.

[0049] In some embodiments, the host cell is capable of producing at least 0.7 g / L of mevalonate.

[0050] In some embodiments, the host cell is capable of producing more mevalonate than a control host cell that does not contain: (a) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a wild-type squalene epoxidase; and / or (b) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0051] In some embodiments, the host cell is capable of producing more 2-3-oxidosqualene compared to a host cell that does not contain: (a) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a wild-type squalene epoxidase; and / or (b) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0052] In some embodiments, the host cell further comprises (a) a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a lanosterol synthase; or (b) a heterologous polynucleotide that reduces lanosterol synthase activity, wherein the host cell is able to produce more isoprenoid or isoprenoid precursor compared to a control host cell that does not contain the heterologous polynucleotides of (a) and / or (b).

[0053] In some embodiments, the wild-type lanosterol synthase comprises SEQ ID NO:1 or 313.

[0054] Further aspects of the present disclosure relate to host cells comprising: (a) one or more enzymes of the yeast mevalonate pathway; and (b) a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to wild-type lanosterol synthase; and / or (c) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to wild-type squalene epoxidase; or (d) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0055] In some embodiments, one or more enzymes of the yeast mevalonate pathway are selected from enzymes having one of the following enzyme classification numbers: EC 2.3.1.9, EC 2.3.3.10, EC 1.1.1.88, EC 1.1.1.34, EC 2.7.1.36, EC 2.7.4.2, EC 4.1.1.33, and / or EC 5.3.3.2. Further aspects of the present disclosure provide host cells comprising: (a) one or more enzymes of the archaeal I mevalonate pathway; and (b) a heterologous polynucleotide encoding a lanosterol synthase with reduced activity compared to wild-type lanosterol synthase; or (c) a heterologous polynucleotide that reduces lanosterol synthase activity; and / or (d) a heterologous polynucleotide encoding a squalene epoxidase with reduced activity compared to wild-type squalene epoxidase; or (e) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0056] In some embodiments, one or more enzymes of the archaeal I mevalonate pathway are selected from enzymes having one of the following enzyme classification numbers: EC 4.1.1.99, EC 2.7.4.26, EC 2.3.1.9, EC 2.3.3.10, EC 1.1.1.88, EC 1.1.1.34, EC 2.7.1.36, and / or EC 5.3.3.2.

[0057] Further aspects of the present disclosure provide host cells comprising: (a) one or more enzymes of the archaeal II mevalonate pathway; and (b) a heterologous polynucleotide encoding a lanosterol synthase with reduced activity compared to wild-type lanosterol synthase; or (c) a heterologous polynucleotide that reduces lanosterol synthase activity; and / or (d) a heterologous polynucleotide encoding a squalene epoxidase with reduced activity compared to wild-type squalene epoxidase; or (e) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0058] In some embodiments, one or more enzymes of the Archaeal II mevalonate pathway are selected from enzymes having one of the following enzyme classification numbers: EC 2.7.1.185, EC 2.7.1.186, EC 2.7.4.26, EC 4.1.1.99, EC 2.3.1.9, EC 2.3.3.10, EC 1.1.1.88, EC 1.1.1.34, EC 2.7.1.36, and / or EC 5.3.3.2.

[0059] Further aspects of the present disclosure provide host cells comprising: (a) one or more enzymes of the MEP pathway; and (b) a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to wild-type lanosterol synthase; or (c) a heterologous polynucleotide that reduces lanosterol synthase activity; and / or (d) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to wild-type squalene epoxidase; or (e) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0060] In some embodiments, one or more enzymes of the MEP pathway are selected from enzymes having one of the following enzyme classification numbers: EC 2.2.1.7, EC 1.1.1.267, EC 2.7.7.60, EC 2.7.1.148, EC 4.6.1.12, EC 1.17.7.1, and / or EC 1.17.1.2.

[0061] In some embodiments, the host cell is a yeast cell, a plant cell, or a bacterial cell.

[0062] In some embodiments, the host cell is a yeast cell.

[0063] In some embodiments, the yeast cell is a Saccharomyces cerevisiae cell.

[0064] In some embodiments, the yeast cell is a Yarrowia lipolytica cell.

[0065] In some embodiments, the host cell is a bacterial cell.

[0066] In some embodiments, the bacterial cell is an E. coli cell.

[0067] A further aspect of the present disclosure provides a method of producing mevalonate comprising culturing any of the host cells associated with the present disclosure.

[0068] A further aspect of the present disclosure provides a method for producing an isoprenoid precursor or an isoprenoid comprising culturing any of the host cells associated with the present disclosure.

[0069] A further aspect of the present disclosure relates to a method for producing 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP) comprising culturing any of the host cells associated with the present disclosure.

[0070] Further aspects of the present disclosure relate to methods of producing an isoprenoid precursor or isoprenoid comprising culturing a host comprising: (a) a heterologous polynucleotide encoding a lanosterol synthase having reduced activity compared to wild-type lanosterol synthase; and / or (b) a heterologous polynucleotide encoding a squalene epoxidase having reduced activity compared to wild-type squalene epoxidase; or (c) a heterologous polynucleotide that reduces squalene epoxidase activity, wherein the host cell is capable of producing more isoprenoid or isoprenoid precursor compared to a control host cell that does not contain one or more of (a)-(c).

[0071] In some embodiments, the wild-type lanosterol synthase comprises SEQ ID NO:1 or 313.

[0072] In some embodiments, the heterologous polynucleotide of (a) is selected from the group consisting of 14, 33, 47, 50, 66, 80, 83, 85, 92, 94, 107, 122, 132, 145, 158, 170, 172, 184, 193, 197, 198, 212, 213, 227, 228, 231, 235, 248, 249, 260, 282, 286, 287, 289, 295, 296, 309, 314, 316, 329, 344, 360, 370, 371, 372, 398, 407, 414, 417, 1 at one or more residues corresponding to positions 423, 432, 437, 442, 444, 452, 474, 479, 491, 498, 515, 526, 529, 536, 544, 552, 559, 560, 564, 578, 586, 608, 610, 617, 619, 620, 631, 638, 650, 655, 660, 679, 686, 702, 710, 726, 736, 738, and / or 742.

[0073] In some embodiments, the heterologous polynucleotide of (a) encodes a lanosterol synthase that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions and / or deletions relative to SEQ ID NO:1.

[0074] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity comprises: an amino acid Y at a residue corresponding to position 14 of SEQ ID NO:1; an amino acid Q at a residue corresponding to position 33 of SEQ ID NO:1; an amino acid E at a residue corresponding to position 47 of SEQ ID NO:1; an amino acid G at a residue corresponding to position 50 of SEQ ID NO:1; an amino acid R at a residue corresponding to position 66 of SEQ ID NO:1; an amino acid G at a residue corresponding to position 80 of SEQ ID NO:1; an amino acid L at a residue corresponding to position 83 of SEQ ID NO:1; an amino acid N at a residue corresponding to position 85 of SEQ ID NO:1; an amino acid I at a residue corresponding to position 92 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 94 of SEQ ID NO:1; an amino acid D at a residue corresponding to position 107 of SEQ ID NO:1; an amino acid C at a residue corresponding to position 122 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 132 of SEQ ID NO:1; an amino acid C at a residue corresponding to position 145 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 158 of SEQ ID NO:1; an amino acid A at a residue corresponding to position 170 of SEQ ID NO:1; an amino acid A at a residue corresponding to position 172 of SEQ ID NO:1. amino acid N; amino acid W at the residue corresponding to position 184 of SEQ ID NO:1; amino acid C or H at the residue corresponding to position 193 of SEQ ID NO:1; amino acid V at the residue corresponding to position 197 of SEQ ID NO:1; amino acid I at the residue corresponding to position 198 of SEQ ID NO:1; amino acid I at the residue corresponding to position 212 of SEQ ID NO:1; amino acid L at the residue corresponding to position 213 of SEQ ID NO:1; amino acid L at the residue corresponding to position 227 of SEQ ID NO:1; amino acid T at the residue corresponding to position 228 of SEQ ID NO:1; amino acid V at the residue corresponding to position 231 of SEQ ID NO:1; amino acid M at the residue corresponding to position 235 of SEQ ID NO:1; amino acid F at the residue corresponding to position 248 of SEQ ID NO:1; amino acid L at the residue corresponding to position 249 of SEQ ID NO:1; amino acid R at the residue corresponding to position 260 of SEQ ID NO:1; amino acid I at the residue corresponding to position 282 of SEQ ID NO:1; amino acid F at the residue corresponding to position 286 of SEQ ID NO:1; amino acid G at the residue corresponding to position 287 of SEQ ID NO:1; amino acid G at the residue corresponding to position 289 of SEQ ID NO:1; amino acid I at the residue corresponding to position 295 of SEQ ID NO:1;the amino acid T at the residue corresponding to position 296 of SEQ ID NO:1; the amino acid F at the residue corresponding to position 309 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 314 of SEQ ID NO:1; the amino acid R at the residue corresponding to position 316 of SEQ ID NO:1; the amino acid N at the residue corresponding to position 329 of SEQ ID NO:1; the amino acid A at the residue corresponding to position 344 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 360 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 370 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 371 of SEQ ID NO:1; the amino acid P at the residue corresponding to position 372 of SEQ ID NO:1; the amino acid I at the residue corresponding to position 398 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 407 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 414 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 417 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 423 of SEQ ID NO:1; the amino acid I or S at the residue corresponding to position 432 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 437 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 442 of SEQ ID NO:1; the sequence amino acid M or S at the residue corresponding to position 444 of SEQ ID NO:1; amino acid G at the residue corresponding to position 452 of SEQ ID NO:1; amino acid V at the residue corresponding to position 474 of SEQ ID NO:1; amino acid S at the residue corresponding to position 479 of SEQ ID NO:1; amino acid Q at the residue corresponding to position 491 of SEQ ID NO:1; amino acid N at the residue corresponding to position 498 of SEQ ID NO:1; amino acid L at the residue corresponding to position 515 of SEQ ID NO:1; amino acid T at the residue corresponding to position 526 of SEQ ID NO:1; amino acid T at the residue corresponding to position 529 of SEQ ID NO:1; amino acid F at the residue corresponding to position 536 of SEQ ID NO:1; amino acid Y at the residue corresponding to position 544 of SEQ ID NO:1; amino acid E at the residue corresponding to position 552 of SEQ ID NO:1; amino acid A at the residue corresponding to position 559 of SEQ ID NO:1; amino acid M at the residue corresponding to position 560 of SEQ ID NO:1; amino acid C or N at the residue corresponding to position 564 of SEQ ID NO:1; amino acid P at the residue corresponding to position 578 of SEQ ID NO:1; amino acid F at the residue corresponding to position 586 of SEQ ID NO:1; amino acid T at the residue corresponding to position 608 of SEQ ID NO:1;lanosterol synthase comprising a truncation resulting in the deletion of an amino acid I at the residue corresponding to position 610 of SEQ ID NO:1; an amino acid V at the residue corresponding to position 617 of SEQ ID NO:1; an amino acid L at the residue corresponding to position 619 of SEQ ID NO:1; an amino acid S at the residue corresponding to position 620 of SEQ ID NO:1; an amino acid E or R at the residue corresponding to position 631 of SEQ ID NO:1; an amino acid D at the residue corresponding to position 638 of SEQ ID NO:1; an amino acid L at the residue corresponding to position 650 of SEQ ID NO:1; an amino acid A at the residue corresponding to position 655 of SEQ ID NO:1; an amino acid H at the residue corresponding to position 660 of SEQ ID NO:1; an amino acid S at the residue corresponding to position 679 of SEQ ID NO:1; an amino acid E at the residue corresponding to position 686 of SEQ ID NO:1; an amino acid D at the residue corresponding to position 702 of SEQ ID NO:1; an amino acid Q at the residue corresponding to position 710 of SEQ ID NO:1; an amino acid L or V at the residue corresponding to position 726 of SEQ ID NO:1; an amino acid F at the residue corresponding to position 736 of SEQ ID NO:1; an amino acid M at the residue corresponding to position 738 of SEQ ID NO:1; and / or a truncation resulting in the deletion of a residue corresponding to position 742 of SEQ ID NO:1;

[0075] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity encodes a lanosterol synthase that includes the following amino acid substitutions relative to SEQ ID NO:1: E617V, G107D, and / or K631E.

[0076] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity is selected from the group consisting of: R33Q, R193C, D289G, N295I, S296T, N620S, and Y736F; R184W, L235M, L260R, and E710Q; K47E, L92I, T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, E617V, and F726L; N14Y, N132S, Y145C, R193 H, I286F, L316R, F432I, E442V, T444S, I479S, K631R, and T655A;F432S, D452G, and I536F;E287G, K329N, E617V, and F726V;E231V, A407V, Q423L, A529T, and Y564C;V248F, D371V, and G702D;L197V, K282I, N314S, P370L, A608T, G638D, and F650L;L491Q, Y586F, and R660H;G122C , H249L, and K738M; P227L, E474V, V559A, and Y564N; K85N, G158S, S515L, P526T, Q619L, and truncations resulting in deletion of residues corresponding to Q742 in SEQ ID NO:1; G107D and K631E; T212I, W213L, N544Y, and V552E; I172N, C414S, L560M, and G679S; R193C, D289G, N295I, S296T, N620S, and Y736F; K85N and G158S; L197V, encoding a lanosterol synthase that includes K282I, N314S, and P370L; I172N, C414S, and L560M; D371V, M610I, and G702D; D371V, K498N, M610I, and G702D; D80G, P83L, T170A, T198I, and A228T; T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, and E617V; or L309F, V344A, T398I, and K686E.

[0077] In some embodiments, the lanosterol synthase has the following amino acid substitutions relative to SEQ ID NO:1: R193C, D289G, N295I, S296T, N620S, and Y736F; F432S, D452G, and I536F; K85N, and G158S; L197V, K282I, N314S, and P370L; I172N, C414S, L560M, and G679S; I172N, C414S, and L560M; D371V , M610I, and G702D; D371V, K498N, M610I, and G702D; D80G, P83L, T170A, T198I, and A228T; D50G, K66R, N94S, G417S, E617V, and F726L; T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, and E617V; and L309F, V344A, T398I, and K686E.

[0078] In some embodiments, the lanosterol synthase contains the following amino acid substitutions relative to SEQ ID NO:1: D50G, K66R, N94S, G417S, E617V, and F726L; K85N and G158S; K47E, L92I, T360S, S372P, T444M, and R578P; F432S, D452G, and I536F; T360S, S372P, T444M, and R578P; L491Q, Y586F, and R660H; K85N, G158S, S515L, P526T, Q619L, and a truncation resulting in a deletion of a residue corresponding to position 742 of SEQ ID NO:1; or I172N, C414S, L560M, and G679S.

[0079] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity encodes a lanosterol synthase that includes amino acid substitutions or deletions relative to SEQ ID NO:1 at one or more residues corresponding to positions 14, 33, 47, 50, 66, 85, 92, 94, 122, 132, 145, 158, 193, 231, 248, 249, 286, 287, 289, 295, 296, 316, 329, 360, 371, 372, 407, 417, 423, 432, 442, 444, 479, 515, 526, 529, 564, 578, 617, 619, 620, 631, 655, 702, 726, 736, 738, and / or 742 of SEQ ID NO:1.

[0080] In some embodiments, the heterologous polynucleotide is selected from the group consisting of: R33Q, R193C, D289G, N295I, S296T, N620S, and Y736F; K47E, L92I, T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, E617V, and F726L; N14Y, N132S, Y145C, R193H, I286F, L316R, F432I, E442V, T444S, I4 79S, K631R, and T655A; E287G, K329N, E617V, and F726V; E231V, A407V, Q423L, A529T, and Y564C; V248F, D371V, and G702D; G122C, H249L, and K738M; or K85N, G158S, S515L, P526T, and Q619L, and encodes a lanosterol synthase that includes a truncation resulting in a deletion of residues corresponding to Q742 of SEQ ID NO:1.

[0081] In some embodiments, the heterologous polynucleotide encodes a lanosterol synthase comprising a sequence that is at least 90% identical to SEQ ID NO: 3, 83-87, 89-92, 94-95, 99, 118-120, 316-319, 321-326, 329, or 331.

[0082] In some embodiments, the lanosterol synthase comprises SEQ ID NO: 3, 83-87, 89-92, 94-95, 99, 118-120, 316-319, 321-326, 329, or 331.

[0083] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase comprises a sequence that is at least 90% identical to SEQ ID NO: 4, 62-66, 68-71, 73-74, 78, 103-109, 111-117, 328, or 330.

[0084] In some embodiments, the heterologous polynucleotide comprises the sequence of SEQ ID NO: 4, 62-66, 68-71, 73-74, 78, 103-109, 111-117, 328, or 330.

[0085] In some embodiments, the lanosterol synthase contains amino acid substitutions or deletions relative to SEQ ID NO:313 at one or more residues corresponding to positions 64, 120, 121, 136, 226, 268, 275, 281, 300, 322, 333, 438, 502, 604, 619, 628, 656, 693, 726, 727, 728, 729, 730, and / or 731.

[0086] In some embodiments, the lanosterol synthase is: an amino acid G at a residue corresponding to position 64 of SEQ ID NO:313; an amino acid V at a residue corresponding to position 120 of SEQ ID NO:313; an amino acid S at a residue corresponding to position 121 of SEQ ID NO:313; an amino acid V at a residue corresponding to position 136 of SEQ ID NO:313; an amino acid I at a residue corresponding to position 226 of SEQ ID NO:313; an amino acid S at a residue corresponding to position 268 of SEQ ID NO:313; an amino acid I at a residue corresponding to position 275 of SEQ ID NO:313; an amino acid A at a residue corresponding to position 281 of SEQ ID NO:313; an amino acid G at a residue corresponding to position 300 of SEQ ID NO:313; 313; the amino acid G at the residue corresponding to position 322 of SEQ ID NO:313; the amino acid A at the residue corresponding to position 333 of SEQ ID NO:313; the amino acid E at the residue corresponding to position 438 of SEQ ID NO:313; the amino acid L at the residue corresponding to position 502 of SEQ ID NO:313; the amino acid N at the residue corresponding to position 604 of SEQ ID NO:313; the amino acid S at the residue corresponding to position 619 of SEQ ID NO:313; the amino acid E at the residue corresponding to position 628 of SEQ ID NO:313; the amino acid T at the residue corresponding to position 656 of SEQ ID NO:313; the amino acid G at the residue corresponding to position 693 of SEQ ID NO:313; and / or a deletion of residues corresponding to positions 726 to 731 of SEQ ID NO:313.

[0087] In some embodiments, the lanosterol synthase includes, relative to SEQ ID NO:313: P121S, A136V, S300G, V322G, K438E, F502L, K628E, and a deletion of residues corresponding to positions 726-731 of SEQ ID NO:313; K268S, T281A, F502L, T604N, A656T, and E693G; or C619S, F275I, I120V, M226I, R64G, and T333A.

[0088] In some embodiments, the lanosterol synthase comprises a sequence that is at least 90% identical to any one of SEQ ID NOs:100-102.

[0089] In some embodiments, the lanosterol synthase comprises a sequence selected from SEQ ID NOs:100-102.

[0090] In some embodiments, the heterologous polynucleotide encoding lanosterol synthase comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs:80-82.

[0091] In some embodiments, the heterologous polynucleotide encoding lanosterol synthase comprises a sequence selected from SEQ ID NOs:80-82.

[0092] In some embodiments, the host cell is capable of producing mevalonate.

[0093] In some embodiments, the host cell is capable of producing at least 0.2 g / L of mevalonate.

[0094] In some embodiments, the host cell is capable of producing at least 0.7 g / L of mevalonate.

[0095] In some embodiments, the host cell is capable of producing at least 9 mg / L of isoprenoid.

[0096] In some embodiments, the host cells are capable of producing at least 1.1-fold more isoprenoids than a control host cell comprising SEQ ID NO:1 and / or a control host cell comprising SEQ ID NO:313.

[0097] In some embodiments, the host cells are capable of producing at least 3-fold more isoprenoids than a control host cell comprising SEQ ID NO:1 and / or a control host cell comprising SEQ ID NO:313.

[0098] In some embodiments, the host cell is capable of producing up to 200 mg / L of lanosterol.

[0099] In some embodiments, the host cell is capable of producing at least 5 mg / L of oxidosqualene.

[0100] In some embodiments, the host cell is capable of producing more mevalonate than a control host cell that does not contain: (a) a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a wild-type lanosterol synthase; and / or (b) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a wild-type squalene epoxidase; or (c) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0101] In some embodiments, the host cell is capable of producing more 2-3-oxidosqualene compared to a host cell that does not contain: (a) a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a wild-type lanosterol synthase; and / or (b) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a wild-type squalene epoxidase; or (c) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0102] In some embodiments, the wild-type squalene epoxidase comprises SEQ ID NO: 9 or 312.

[0103] In some embodiments, the heterologous polynucleotide encodes a squalene epoxidase that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and / or deletions compared to SEQ ID NO: 9 or 312.

[0104] In some embodiments, the host cell is a yeast cell, a plant cell, or a bacterial cell.

[0105] In some embodiments, the host cell is a yeast cell.

[0106] In some embodiments, the yeast cell is a Saccharomyces cerevisiae cell.

[0107] In some embodiments, the yeast cell is a Yarrowia lipolytica cell.

[0108] In some embodiments, the host cell is a bacterial cell.

[0109] In some embodiments, the bacterial cell is an E. coli cell.

[0110] In some embodiments, the isoprenoid precursor is mevalonate, 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP), and / or 2-3-oxidosqualene.

[0111] In some embodiments, the host cell is capable of producing more isoprenoid or isoprenoid precursor compared to a control host cell that does not include: a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a control wild-type lanosterol synthase; and / or a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a control wild-type squalene epoxidase; or a heterologous polynucleotide that reduces squalene epoxidase activity.

[0112] In some embodiments, the host cell is capable of producing more isoprenoid or isoprenoid precursor compared to a control host cell that does not contain: a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a control wild-type lanosterol synthase; a heterologous polynucleotide that reduces lanosterol synthase activity; and / or a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a control wild-type squalene epoxidase; or a heterologous polynucleotide that reduces squalene epoxidase activity.

[0113] In some embodiments, the wild-type lanosterol synthase comprises SEQ ID NO:1 or 313.

[0114] In some embodiments, the wild-type squalene epoxidase comprises SEQ ID NO: 9 or 312.

[0115] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity is selected from the group consisting of 14, 33, 47, 50, 66, 80, 83, 85, 92, 94, 107, 122, 132, 145, 158, 170, 172, 184, 193, 197, 198, 212, 213, 227, 228, 231, 235, 248, 249, 260, 282, 286, 287, 289, 295, 296, 309, 314, 316, 329, 344, 360, 370, 371, 372, 398, 40 1 at one or more residues corresponding to positions 7, 414, 417, 423, 432, 437, 442, 444, 452, 474, 479, 491, 498, 515, 526, 529, 536, 544, 552, 559, 560, 564, 578, 586, 608, 610, 617, 619, 620, 631, 638, 650, 655, 660, 679, 686, 702, 710, 726, 736, 738, and / or 742.

[0116] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity encodes a lanosterol synthase that contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions and / or deletions relative to SEQ ID NO:1.

[0117] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity comprises: an amino acid Y at a residue corresponding to position 14 of SEQ ID NO:1; an amino acid Q at a residue corresponding to position 33 of SEQ ID NO:1; an amino acid E at a residue corresponding to position 47 of SEQ ID NO:1; an amino acid G at a residue corresponding to position 50 of SEQ ID NO:1; an amino acid R at a residue corresponding to position 66 of SEQ ID NO:1; an amino acid G at a residue corresponding to position 80 of SEQ ID NO:1; an amino acid L at a residue corresponding to position 83 of SEQ ID NO:1; an amino acid N at a residue corresponding to position 85 of SEQ ID NO:1; an amino acid I at a residue corresponding to position 92 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 94 of SEQ ID NO:1; an amino acid D at a residue corresponding to position 107 of SEQ ID NO:1; an amino acid C at a residue corresponding to position 122 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 132 of SEQ ID NO:1; an amino acid C at a residue corresponding to position 145 of SEQ ID NO:1; an amino acid S at a residue corresponding to position 158 of SEQ ID NO:1; an amino acid A at a residue corresponding to position 170 of SEQ ID NO:1; an amino acid A at a residue corresponding to position 172 of SEQ ID NO:1. amino acid N; amino acid W at the residue corresponding to position 184 of SEQ ID NO:1; amino acid C or H at the residue corresponding to position 193 of SEQ ID NO:1; amino acid V at the residue corresponding to position 197 of SEQ ID NO:1; amino acid I at the residue corresponding to position 198 of SEQ ID NO:1; amino acid I at the residue corresponding to position 212 of SEQ ID NO:1; amino acid L at the residue corresponding to position 213 of SEQ ID NO:1; amino acid L at the residue corresponding to position 227 of SEQ ID NO:1; amino acid T at the residue corresponding to position 228 of SEQ ID NO:1; amino acid V at the residue corresponding to position 231 of SEQ ID NO:1; amino acid M at the residue corresponding to position 235 of SEQ ID NO:1; amino acid F at the residue corresponding to position 248 of SEQ ID NO:1; amino acid L at the residue corresponding to position 249 of SEQ ID NO:1; amino acid R at the residue corresponding to position 260 of SEQ ID NO:1; amino acid I at the residue corresponding to position 282 of SEQ ID NO:1; amino acid F at the residue corresponding to position 286 of SEQ ID NO:1; amino acid G at the residue corresponding to position 287 of SEQ ID NO:1; amino acid G at the residue corresponding to position 289 of SEQ ID NO:1; amino acid I at the residue corresponding to position 295 of SEQ ID NO:1;the amino acid T at the residue corresponding to position 296 of SEQ ID NO:1; the amino acid F at the residue corresponding to position 309 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 314 of SEQ ID NO:1; the amino acid R at the residue corresponding to position 316 of SEQ ID NO:1; the amino acid N at the residue corresponding to position 329 of SEQ ID NO:1; the amino acid A at the residue corresponding to position 344 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 360 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 370 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 371 of SEQ ID NO:1; the amino acid P at the residue corresponding to position 372 of SEQ ID NO:1; the amino acid I at the residue corresponding to position 398 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 407 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 414 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 417 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 423 of SEQ ID NO:1; the amino acid I or S at the residue corresponding to position 432 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 437 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 442 of SEQ ID NO:1; the sequence amino acid M or S at the residue corresponding to position 444 of SEQ ID NO:1; amino acid G at the residue corresponding to position 452 of SEQ ID NO:1; amino acid V at the residue corresponding to position 474 of SEQ ID NO:1; amino acid S at the residue corresponding to position 479 of SEQ ID NO:1; amino acid Q at the residue corresponding to position 491 of SEQ ID NO:1; amino acid N at the residue corresponding to position 498 of SEQ ID NO:1; amino acid L at the residue corresponding to position 515 of SEQ ID NO:1; amino acid T at the residue corresponding to position 526 of SEQ ID NO:1; amino acid T at the residue corresponding to position 529 of SEQ ID NO:1; amino acid F at the residue corresponding to position 536 of SEQ ID NO:1; amino acid Y at the residue corresponding to position 544 of SEQ ID NO:1; amino acid E at the residue corresponding to position 552 of SEQ ID NO:1; amino acid A at the residue corresponding to position 559 of SEQ ID NO:1; amino acid M at the residue corresponding to position 560 of SEQ ID NO:1; amino acid C or N at the residue corresponding to position 564 of SEQ ID NO:1; amino acid P at the residue corresponding to position 578 of SEQ ID NO:1; amino acid F at the residue corresponding to position 586 of SEQ ID NO:1; amino acid T at the residue corresponding to position 608 of SEQ ID NO:1;lanosterol synthase comprising a truncation resulting in the deletion of an amino acid I at the residue corresponding to position 610 of SEQ ID NO:1; an amino acid V at the residue corresponding to position 617 of SEQ ID NO:1; an amino acid L at the residue corresponding to position 619 of SEQ ID NO:1; an amino acid S at the residue corresponding to position 620 of SEQ ID NO:1; an amino acid E or R at the residue corresponding to position 631 of SEQ ID NO:1; an amino acid D at the residue corresponding to position 638 of SEQ ID NO:1; an amino acid L at the residue corresponding to position 650 of SEQ ID NO:1; an amino acid A at the residue corresponding to position 655 of SEQ ID NO:1; an amino acid H at the residue corresponding to position 660 of SEQ ID NO:1; an amino acid S at the residue corresponding to position 679 of SEQ ID NO:1; an amino acid E at the residue corresponding to position 686 of SEQ ID NO:1; an amino acid D at the residue corresponding to position 702 of SEQ ID NO:1; an amino acid Q at the residue corresponding to position 710 of SEQ ID NO:1; an amino acid L or V at the residue corresponding to position 726 of SEQ ID NO:1; an amino acid F at the residue corresponding to position 736 of SEQ ID NO:1; an amino acid M at the residue corresponding to position 738 of SEQ ID NO:1; and / or a truncation resulting in the deletion of a residue corresponding to position 742 of SEQ ID NO:1;

[0118] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity encodes a lanosterol synthase that includes the following amino acid substitutions relative to SEQ ID NO:1: E617V, G107D, and / or K631E.

[0119] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity has the following sequence(s) relative to SEQ ID NO:1: R33Q, R193C, D289G, N295I, S296T, N620S, and Y736F; R184W, L235M, L260R, and E710Q; K47E, L92I, T360S, S372P, T444M, and R578P; D50G, K66R, N 94S, G417S, E617V, and F726L;N14Y, N132S, Y145C, R193H, I286F, L316R, F432I, E442V, T444S, I479S, K631R, and T655A;F432S, D452G, and I536F;E287G, K329N, E617V, and F726V;E231V, A407V, Q423L, A529T, and Y564C; truncations resulting in deletion of residues corresponding to V248F, D371V, and G702D; L197V, K282I, N314S, P370L, A608T, G638D, and F650L; L491Q, Y586F, and R660H; G122C, H249L, and K738M; P227L, E474V, V559A, and Y564N; K85N, G158S, S515L, P526T, Q619L, and Q742 of SEQ ID NO:1; G107D and K631E; T212I, W213L, N544Y, and V552E; I172N, C414S, L560M, and G679S; R193C, D289G , N295I, S296T, N620S, and Y736F; K85N and G158S; L197V, K282I, N314S, and P370L; I172N, C414S, and L560M; D371V, M610I, and G702D; D371V, K498N, M610I, and G702D; D80G, P83L, T170A, T198I, and A228T; T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, and E617V; or L309F, V344A, T398I, and K686E.

[0120] In some embodiments, the lanosterol synthase has the following amino acid substitutions relative to SEQ ID NO:1: R193C, D289G, N295I, S296T, N620S, and Y736F; F432S, D452G, and I536F; K85N, and G158S; L197V, K282I, N314S, and P370L; I172N, C414S, L560M, and G679S; I172N, C414S, and L560M; D371V , M610I, and G702D; D371V, K498N, M610I, and G702D; D80G, P83L, T170A, T198I, and A228T; D50G, K66R, N94S, G417S, E617V, and F726L; T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, and E617V; and L309F, V344A, T398I, and K686E.

[0121] In some embodiments, the lanosterol synthase contains the following amino acid substitutions relative to SEQ ID NO:1: D50G, K66R, N94S, G417S, E617V, and F726L; K85N and G158S; K47E, L92I, T360S, S372P, T444M, and R578P; F432S, D452G, and I536F; T360S, S372P, T444M, and R578P; L491Q, Y586F, and R660H; K85N, G158S, S515L, P526T, Q619L, and a truncation resulting in a deletion of a residue corresponding to position 742 of SEQ ID NO:1; or I172N, C414S, L560M, and G679S.

[0122] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase with reduced activity encodes a lanosterol synthase that includes amino acid substitutions or deletions relative to SEQ ID NO:1 at one or more residues corresponding to positions 14, 33, 47, 50, 66, 85, 92, 94, 122, 132, 145, 158, 193, 231, 248, 249, 286, 287, 289, 295, 296, 316, 329, 360, 371, 372, 407, 417, 423, 432, 442, 444, 479, 515, 526, 529, 564, 578, 617, 619, 620, 631, 655, 702, 726, 736, 738, and / or 742 of SEQ ID NO:1.

[0123] In some embodiments, the heterologous polynucleotide is selected from the group consisting of: R33Q, R193C, D289G, N295I, S296T, N620S, and Y736F; K47E, L92I, T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, E617V, and F726L; N14Y, N132S, Y145C, R193H, I286F, L316R, F432I, E442V, T444S, I4 79S, K631R, and T655A; E287G, K329N, E617V, and F726V; E231V, A407V, Q423L, A529T, and Y564C; V248F, D371V, and G702D; G122C, H249L, and K738M; or K85N, G158S, S515L, P526T, and Q619L, and encodes a lanosterol synthase that includes a truncation resulting in a deletion of residues corresponding to Q742 of SEQ ID NO:1.

[0124] In some embodiments, the heterologous polynucleotide encodes a lanosterol synthase comprising a sequence that is at least 90% identical to SEQ ID NO: 33, 83-87, 89-92, 94-95, 99, 118-120, 316-319, 321-326, 329, or 331.

[0125] In some embodiments, the lanosterol synthase comprises SEQ ID NO: 33, 83-87, 89-92, 94-95, 99, 118-120, 316-319, 321-326, 329, or 331.

[0126] In some embodiments, the heterologous polynucleotide encoding a lanosterol synthase comprises a sequence that is at least 90% identical to SEQ ID NO: 4, 62-66, 68-71, 73-74, 78, 103-109, 111-117, 328, or 330.

[0127] In some embodiments, the heterologous polynucleotide comprises the sequence of SEQ ID NO: 4, 62-66, 68-71, 73-74, 78, 103-109, 111-117, 328, or 330.

[0128] In some embodiments, the host cell comprises a heterologous polynucleotide encoding a lanosterol synthase, where the lanosterol synthase comprises amino acid substitutions or deletions relative to SEQ ID NO:313 at one or more residues corresponding to positions 64, 120, 121, 136, 226, 268, 275, 281, 300, 322, 333, 438, 502, 604, 619, 628, 656, 693, 726, 727, 728, 729, 730, and / or 731.

[0129] In some embodiments, the lanosterol synthase is: an amino acid G at a residue corresponding to position 64 of SEQ ID NO:313; an amino acid V at a residue corresponding to position 120 of SEQ ID NO:313; an amino acid S at a residue corresponding to position 121 of SEQ ID NO:313; an amino acid V at a residue corresponding to position 136 of SEQ ID NO:313; an amino acid I at a residue corresponding to position 226 of SEQ ID NO:313; an amino acid S at a residue corresponding to position 268 of SEQ ID NO:313; an amino acid I at a residue corresponding to position 275 of SEQ ID NO:313; an amino acid A at a residue corresponding to position 281 of SEQ ID NO:313; an amino acid G at a residue corresponding to position 300 of SEQ ID NO:313; 313; the amino acid G at the residue corresponding to position 322 of SEQ ID NO:313; the amino acid A at the residue corresponding to position 333 of SEQ ID NO:313; the amino acid E at the residue corresponding to position 438 of SEQ ID NO:313; the amino acid L at the residue corresponding to position 502 of SEQ ID NO:313; the amino acid N at the residue corresponding to position 604 of SEQ ID NO:313; the amino acid S at the residue corresponding to position 619 of SEQ ID NO:313; the amino acid E at the residue corresponding to position 628 of SEQ ID NO:313; the amino acid T at the residue corresponding to position 656 of SEQ ID NO:313; the amino acid G at the residue corresponding to position 693 of SEQ ID NO:313; and / or a deletion of residues corresponding to positions 726 to 731 of SEQ ID NO:313.

[0130] In some embodiments, the lanosterol synthase includes, relative to SEQ ID NO:313: P121S, A136V, S300G, V322G, K438E, F502L, K628E, and a deletion of residues corresponding to positions 726-731 of SEQ ID NO:313; K268S, T281A, F502L, T604N, A656T, and E693G; or C619S, F275I, I120V, M226I, R64G, and T333A.

[0131] In some embodiments, the lanosterol synthase comprises a sequence that is at least 90% identical to any one of SEQ ID NOs:100-102.

[0132] In some embodiments, the lanosterol synthase comprises a sequence selected from SEQ ID NOs:100-102.

[0133] In some embodiments, the heterologous polynucleotide encoding lanosterol synthase comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs:80-82.

[0134] In some embodiments, the heterologous polynucleotide encoding lanosterol synthase comprises a sequence selected from SEQ ID NOs:80-82.

[0135] In some embodiments, the host cell is capable of producing mevalonate.

[0136] In some embodiments, the host cell is capable of producing at least 0.2 g / L of mevalonate.

[0137] In some embodiments, the host cell is capable of producing at least 0.7 g / L of mevalonate.

[0138] In some embodiments, the host cell is capable of producing more mevalonate than a control host cell that does not contain (a) a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a wild-type lanosterol synthase; and / or (b) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a wild-type squalene epoxidase; or (c) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0139] In some embodiments, the host cell is capable of producing more mevalonate than a control host cell that does not contain: (a) a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to wild-type lanosterol synthase; or (b) a heterologous polynucleotide that reduces lanosterol synthase activity; and / or (c) a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to wild-type squalene epoxidase; or (d) a heterologous polynucleotide that reduces squalene epoxidase activity.

[0140] In some embodiments, the host cell is capable of producing more 2-3-oxidosqualene compared to a host cell that does not comprise a heterologous polynucleotide encoding a lanosterol synthase that has reduced activity compared to a wild-type lanosterol synthase; and / or a heterologous polynucleotide encoding a squalene epoxidase that has reduced activity compared to a wild-type squalene epoxidase; or a heterologous polynucleotide that reduces squalene epoxidase activity.

[0141] In some embodiments, the host cell is capable of producing more 2-3-oxidosqualene compared to a host cell that does not contain a heterologous polynucleotide encoding a lanosterol synthase with reduced activity compared to a wild-type lanosterol synthase; or a heterologous polynucleotide that reduces lanosterol synthase activity; and / or a heterologous polynucleotide encoding a squalene epoxidase with reduced activity compared to a wild-type squalene epoxidase; or a heterologous polynucleotide that reduces squalene epoxidase activity.

[0142] In some embodiments, the heterologous polynucleotide encoding a squalene epoxidase with reduced activity encodes a squalene epoxidase that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions and / or deletions relative to SEQ ID NO: 9 or 312.

[0143] In some embodiments, the host cell is a yeast cell, a plant cell, or a bacterial cell.

[0144] In some embodiments, the host cell is a yeast cell.

[0145] In some embodiments, the yeast cell is a Saccharomyces cerevisiae cell.

[0146] In some embodiments, the yeast cell is a Yarrowia lipolytica cell.

[0147] In some embodiments, the host cell is a bacterial cell.

[0148] In some embodiments, the bacterial cell is an E. coli cell.

[0149] Each limitation of the present invention may encompass various embodiments of the present invention. It is therefore anticipated that each limitation of the present invention involving any one element or combination of elements may be included in each aspect of the present invention. The present invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present invention is capable of other embodiments and of being practiced or carried out in various ways.

[0150] The accompanying drawings are not intended to be to scale. The drawings are merely illustrative and are not necessary to practice the present disclosure. For clarity, not every component is shown in every drawing. [Brief description of the drawings]

[0151] [Figure 1A]Figure 1A-1D provide four biosynthetic pathways for forming the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMPP) from acetyl-CoA, including the mevalonate (MEV) pathway from Saccharomyces cerevisiae (Figure 1A), Archaeon I (Figure 1B) and Archaeon II (Figure 1C), as well as the non-mevalonate or methylerythritol phosphate (MEP) pathway found in eubacteria, algae and plant plastids (Figure 1D). Structures of intermediates and pathway enzymes are shown. [Figure 1B] Figure 1A-1D provide four biosynthetic pathways for forming the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMPP) from acetyl-CoA, including the mevalonate (MEV) pathway from Saccharomyces cerevisiae (Figure 1A), Archaeon I (Figure 1B) and Archaeon II (Figure 1C), as well as the non-mevalonate or methylerythritol phosphate (MEP) pathway found in eubacteria, algae and plant plastids (Figure 1D). Structures of intermediates and pathway enzymes are shown. [Figure 1C] Figure 1A-1D provide four biosynthetic pathways for forming the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMPP) from acetyl-CoA, including the mevalonate (MEV) pathway from Saccharomyces cerevisiae (Figure 1A), Archaeon I (Figure 1B) and Archaeon II (Figure 1C), as well as the non-mevalonate or methylerythritol phosphate (MEP) pathway found in eubacteria, algae and plant plastids (Figure 1D). Structures of intermediates and pathway enzymes are shown. [Figure 1D]Figure 1A-1D provide four biosynthetic pathways for forming the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMPP) from acetyl-CoA, including the mevalonate (MEV) pathway from Saccharomyces cerevisiae (Figure 1A), Archaeon I (Figure 1B) and Archaeon II (Figure 1C), as well as the non-mevalonate or methylerythritol phosphate (MEP) pathway found in eubacteria, algae and plant plastids (Figure 1D). Structures of intermediates and pathway enzymes are shown. [Diagram 2] 2 shows the sterol biosynthetic pathway in which IPP and DMPP are converted to lanosterol by various enzymatic steps. ERG7 is shown as a non-limiting example of a lanosterol synthase. [Diagram 3] FIG. 3 is a graph depicting mevalonate production by Yarrowia strains containing lanosterol synthase. [Figure 4] 4 is a graph showing cucurbitadienol production by strains containing lanosterol synthase (erg7 allele). Strain 870688 containing SEQ ID NO:1 was used as a control. [Diagram 5] 5 is a graph showing cucurbitadienol, ergosterol, lanosterol and mevalonate production by strains containing lanosterol synthase (erg7 allele). Strain 887779 containing SEQ ID NO:1 was used as a control. [Figure 6] Figure 6 is a graph depicting oxidosqualene production in lanosterol synthase temperature sensitive mutant (erg7 mutant) strains at 30° C. and 35° C. Three lanosterol synthase mutant strains 756247, 756248 and 756249, containing SEQ ID NOs: 100-102, respectively, were tested and the parental BY4742 Saccharomyces cerevisiae strain was included as a negative control. [Figure 7]7 is a graph depicting the production of ergosterol, ethanol, and mevalonate and the consumption of glucose in lanosterol synthase temperature sensitive mutant (erg7 mutant) strains at 30° C. Three lanosterol synthase mutant strains 756247, 756248, and 756249 containing SEQ ID NOs: 100-102 were tested, respectively, and the parental BY4742 Saccharomyces cerevisiae strain was included as a negative control. [Figure 8] 8 is a graph depicting the production of ergosterol, ethanol, and mevalonate and the consumption of glucose in lanosterol synthase temperature sensitive mutant (erg7 mutant) strains at 35° C. Three lanosterol synthase mutant strains 756247, 756248, and 756249 containing SEQ ID NOs: 100-102 were tested, respectively, and the parental BY4742 Saccharomyces cerevisiae strain was included as a negative control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0152] The structural diversity of isoprenoids makes these compounds suitable for many applications, including use as flavorings, pharmaceutical drug production, and aroma compounds.However, purification and de novo chemical synthesis of isoprenoids from natural sources often have high production costs and low yields.In addition, although the mevalonate pathway is used by eukaryotes and other natural sources to produce isoprenoid building blocks, bottlenecks and the production of off-target compounds limit the flux through the mevalonate pathway.

[0153] The present disclosure is premised on the unexpected discovery that a somewhat low-character lanosterol synthase and / or squalene epoxidase (SQE) can be utilized to increase production of isoprenoids and isoprenoid precursors. In some embodiments, the lanosterol synthase variant is encoded by a variant of the ERG7 coding sequence. In some embodiments, the squalene epoxidase is encoded by the ERG1 gene. Thus, in some embodiments, host cells are provided herein that have been engineered to efficiently produce isoprenoids and their precursors. Methods include heterologous expression of lanosterol synthase and / or squalene epoxidase. Examples 1 and 3-4 describe the identification and functional characterization of lanosterol synthase that can be used to increase isoprenoids and isoprenoid production. The proteins and host cells described in the present disclosure can be used to make isoprenoids and their precursors.

[0154] Synthesis of isoprenoids and their precursors Isoprenoids and their precursors can be synthesized from acetyl-CoA via a mevalonate intermediate (the mevalonate ("MEV" or "MVA") pathway) or from pyruvate and glyceraldehyde-3-phosphate via a 1-deoxyxylulose-5-phosphate (DXP) intermediate (the non-mevalonate or methylerythritol phosphate (MEP) pathway).

[0155] Synthesis of isoprenoids in many eukaryotes, such as yeast, archaea, and some bacteria, begins with the MEV pathway, which converts acetyl-CoA to isopentenyl pyrophosphate (IPP). In the mevalonate pathway, two acetyl-COA molecules condense to form acetoacetyl-CoA, which in turn condenses to form 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA). HMG-CoA is then reduced to form mevalonate.

[0156] From mevalonate, the isoprenoid precursor IPP can be formed in three ways.

[0157] As shown in Figure 1A, mevalonate can be phosphorylated to form mevalonate-5-phosphate, which can be phosphorylated to form mevalonate pyrophosphate. Mevalonate pyrophosphate can be decarboxylated to form IPP. IPP can then be isomerized to form dimethylallyl pyrophosphate (DMAPP). Exemplary enzymes useful for forming IPP from acetyl-CoA as shown in Figure 1A (yeast MEV pathway) are within the classes summarized in the table below.

[0158] [Table 1]

[0159] Alternatively, mevalonate can be phosphorylated to form mevalonate-5-phosphate, as shown in FIG. 1B, which depicts the mevalonate pathway from Archaeal I bacteria. Mevalonate-5-phosphate can be decarboxylated to form isopentenyl phosphate, which can be further phosphorylated to form isopentenyl pyrophosphate (IPP). Exemplary enzymes that can be used to form IPP from acetyl-COA using the Archaeal I mevalonate (MEV-A1) pathway are within the classes summarized in the table below.

[0160] [Table 2]

[0161] Additionally, as shown in FIG. 1C, which depicts the mevalonate pathway from Archaeal II bacteria (MEV-AII), mevalonate can be converted to IPP in four steps. Mevalonate can be phosphorylated to form mevalonate-3-phosphate, which can be phosphorylated to form mevalonate-3,5-bisphosphate. Mevalonate-3,5-bisphosphate can be decarboxylated to form isopentenyl phosphate, which can be phosphorylated to IPP. Exemplary enzymes that can be used to form IPP from acetyl-COA using the Archaeal II mevalonate pathway are within the classes summarized in the table below.

[0162] [Table 3]

[0163] IPP and DMPP can also be formed in the non-mevalonate or methylerythritol phosphate (MEP) pathway as illustrated in Figure 1D. In the MEP pathway (derived from eubacteria, algae and higher plants), pyruvate and glyceraldehyde-3-phosphate can condense to form 1-deoxyxylulose-5-phosphate (DXP). This is followed by NADPH-dependent reduction and isomerization of DXP to 2C-methyl-D-erythritol 4-phosphate (MEP), which is catalyzed by DXP reductoisomerase (DXR). MEP then reacts with CTP and is converted to 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME) by the enzymatic action of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS). CDP-ME undergoes phosphorylation by ATP-dependent 4-diphosphocytidyl-2-C-methyl-D-erythritol (erythrito) kinase (CME) to produce 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (CDP-MEP), which is then cyclized by 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) with concomitant elimination of CMP to form 2C-methyl-D-erythritol 2,4-cyclodiphosphate (2-C-methyl-D-erythritol-2,4-cyclopyrophosphate, MEC or MEcPP). MEC then undergoes reductive ring-opening catalyzed by NADPH-dependent 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS) to produce 4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMB-PP). Finally, HMB-PP is reduced by 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR) to produce a mixture of IPP and DMAPP (12, 18). Exemplary enzymes that can be used for the formation of IPP from pyruvate and glyceraldehyde-3-phosphate using the non-mevalonate pathway are within the classes summarized in the table below.

[0164] [Table 4]

[0165] For example, the isoprenoid precursors IPP and / or DMAPP produced from the MEV or MEP pathway can be used to produce a variety of isoprenoids, as shown in FIG. 2, which illustrates that prenyltransferase catalyzes the elongation of isoprenoid chains to produce prenyl diphosphates of various lengths. For example, geranyl pyrophosphate synthase catalyzes the formation of GPP, farnesyl pyrophosphate synthase catalyzes the production of FPP, and geranylgeranyl pyrophosphate synthase catalyzes the formation of GGPP. As used herein, the term "prenyl diphosphate" encompasses monoprenyl diphosphates with only one prenyl group and polyprenyl diphosphates that contain at least two prenyl groups. IPP and DMAPP are non-limiting examples of monoprenyl diphosphates. Geranylgeranyl diphosphate (GGPP) is a non-limiting example of polyprenyl diphosphate. Exemplary prenyltransferases useful for producing isoprenoids are within the classes summarized in the table below.

[0166] [Table 5]

[0167] Prenyl diphosphate serves as a substrate for a number of isoprenoid synthesis pathways. As a non-limiting example, FIG. 2 shows how IPP and DMAPP are incorporated into a sterol biosynthetic pathway from Saccharomyces cerevisiae. Squalene synthase catalyzes the formation of squalene from FPP. Squalene synthase may be encoded by the ERG9 gene. A non-limiting example of a squalene synthase is provided by UniProtKB Accession No. P29704. Squalene epoxidase (SQE) then oxidizes squalene to form 2-3-oxidosqualene, which serves as a substrate for lanosterol synthase to produce lanosterol. Lanosterol can then be converted to the sterol ergosterol through a series of steps known in the art. See, for example, Klug and Daum, FEMS Yeast Res. 2014 May;14(3):369-88. Additionally, prenyl diphosphate substrates can also be used as substrates by terpene synthases to produce isoprenoids.

[0168] Isoprenoids and Isoprenoid Precursors Non-limiting examples of isoprenoids and isoprenoid precursors that can be produced as described herein include the following:

[0169] Isoprenoid precursors include, but are not limited to, acetyl-CoA, acetoacetyl-CoA, HMG-CoA, mevalonate, mevalonate-5-phosphate, mevalonate pyrophosphate, isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl diphosphate (FPP), squalene and 2-3-oxidosqualene. In some embodiments, the isoprenoid precursor is a compound shown in Figures 1A-1D and / or 2.

[0170] As used herein, unless otherwise specified, an isoprenoid is an organic compound containing an isoprene (i.e., C5H8) unit and its derivatives. The terms "isoprenoid", "terpene" and "terpenoid" are used interchangeably in this application. For example, an isoprenoid is an isoprenoid having the molecular formula (C5H8): nwhere n represents the number of isoprene subunits. Isoprenoids may contain more than five carbon atoms. Non-limiting examples include isoprenoids having 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 300, 305, 310, 320, 330, 340, 350, 360, 370, 385, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700 5, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 52 0, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 77 5, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1,000 or more than 1,000 carbons. In some embodiments, the isoprenoid is an irregular isoprenoid. Isoprenoids also include oxygenated compounds.Isoprenoids are structurally diverse compounds and can be, for example, cyclic (e.g., monocyclic, polycyclic, homocyclic and heterocyclic compounds) or acyclic (e.g., linear and branched compounds). In some embodiments, isoprenoids can have a flavor and / or odor. As used herein, aroma compounds refer to compounds that have an odor.

[0171] Non-limiting examples of isoprenoids include monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes and tetraterpenes. Monoterpenes contain 10 carbons. Non-limiting examples of monoterpenes include, but are not limited to, myrcene, methanol, carvone, hinokitiol, linalool, limonene, sabinene, thujene, carene, borneol, eucalyptol and camphene. Sesquiterpenes contain 15 carbons. As used herein, sesquiterpenes include sesquiterpene hydrocarbons and sesquiterpene alcohols (sesquiterpenols). Non-limiting examples of sesquiterpenes include delta-cadinene, epi-cubenol, tau-cadinol, alpha-cadinol, gamma-selinene, 10-epi-gamma-eudesmol, gamma-eudesmol, alpha / beta-eudesmol, juniper camphor, 7-epi-alpha-eudesmol, cryptomeridiol isomer 1, cryptomeridiol isomer 2, cryptomeridiol isomer 3, humulene, alpha-guayene, delta-guayene, zingiberene, beta-bisabolene, beta- Examples of terpenes include, but are not limited to, farnesene, beta-sesquiphellandrene, cubenol, alpha-bisabolol, alpha-curcumene, trans-nerolidol, gamma, bisabolene, beta-caryophyllene, trans-sesquisabinene hydrate, delta-elemene, cis-eudesma-6-en-11-ol, daucene, isodaucene, trans-bergamotene, alpha-zingiberene, sesquisabinene hydrate, and 8-isopropenyl-1,5-dimethyl-1,5-cyclodecadiene. Diterpenes contain 20 carbons. Non-limiting examples of diterpenes include, but are not limited to, cembrene and sclareol. Sesterterpenes contain 25 carbons. Non-limiting examples of sesterterpenes include geranylfarnesol. Triterpenes contain 30 carbons. Non-limiting examples of triterpenes include squalene, polypodatetraene, melavaricane, lanostane, cucurbitacin, hopane, oleanane, and ursolic acid. Tetraterpenes contain 40 carbons.Non-limiting examples of tetraterpenes include carotenoids, such as xanthophylls and carotenes.See, for example, WO2019 / 161141.In some embodiments, the isoprenoid is a cannabinoid.See, for example, WO2020 / 176547.

[0172] To identify an isoprenoid precursor or isoprenoid of interest, any method known in the art can be used, including mass spectrometry (eg, gas chromatography-mass spectrometry).

[0173] In some embodiments, the isoprenoid is mogrol (11,24,25-trihydroxycucurbitadienol), a mogrol precursor, or a mogroside.

[0174] In some embodiments, decreasing ERG7 expression, levels or activity reduces the amount of 2-3-oxido-squalene that is converted to lanosterol and increases the amount of 2-3-oxido-squalene available to be converted to the mogrol precursor, mogrol and / or mogroside via one or more enzymatic steps.

[0175] In some embodiments, mogrol precursors include, but are not limited to, 2,3,22,23-dioxidosqualene, cucurbitadienol, 24,25-expoxy cucurbitadienol, 11-hydroxycucurbitadienol, 11-hydroxy-24,25-epoxy cucurbitadienol, 11-hydroxy-cucurbitadienol, 11-oxo-cucurbitadienol, and 24,25-dihydroxycucurbitadienol.

[0176] In some embodiments, precursors to mogrosides include mogrol precursors, mogrol and other mogrosides.

[0177] In some embodiments, the mogrosides include mogroside I-A1 (MIA1), mogroside IE (MIE or M1E), mogroside II-A1 (MIIA1 or M2A1), mogroside II-A2 (MIIA2 or M2A2), mogroside III-A1 (MIIIA1 or M3A1), mogroside II-E (MIIE or M2E), mogroside III (MIII or M3), chamenoside I, mogroside IV ... Mogrosides include, but are not limited to, mogroside IV (MIV or M4), mogroside IVa (MIVA or M4A), isomogroside IV, mogroside III-E (MIIIE or M3E), mogroside V (MV or M5), mogroside VIA (MVIA), mogroside VIB (MVIB), isomogroside V, mogroside VIa1 (MVIa1 or MVIa1) and mogroside VI (MVI or M6). In some embodiments, the mogroside is chamenoside I, which may also be referred to as chamenoside or sham. In some embodiments, the mogroside is MIIIE.

[0178] Enzymes for increasing production of isoprenoids or isoprenoid precursors In various aspects, the present disclosure relates to a method for increasing production of isoprenoids or isoprenoid precursors in a host cell, the host cell expressing (1) reduced levels of one or more enzymes in the MEV, MEV-A1, MEV-AII or MEP pathway, (2) reduced levels of one or more enzymes involved in the conversion of IPP or DMAPP to sterols, such as lanosterol or ergosterol, (3) attenuated forms of one or more of the enzymes, or (4) any combination thereof. For example, a host cell with increased production of isoprenoids may contain mutants of lanosterol synthase and / or squalene epoxidase (SQE) with reduced (e.g., reduced, but not eliminated) activity. In some embodiments, the lanosterol synthase mutant is encoded by a mutant of the ERG7 coding sequence. In some embodiments, the squalene epoxidase is encoded by the ERG1 gene.

[0179] In some embodiments, the decrease in lanosterol synthase or squalene epoxidase activity is associated with a surprising increase in the abundance of mevalonate (which is neither a substrate nor a product of lanosterol synthase or squalene epoxidase), and the increase in mevalonate can promote increased synthesis of compounds derived directly or indirectly (e.g., via one or more enzymatic steps) from mevalonate, including various isoprenoids and isoprenoid precursors. In some embodiments, the lanosterol synthase variant is encoded by a variant of the ERG7 coding sequence. In some embodiments, the squalene epoxidase is encoded by the ERG1 gene.

[0180] In some embodiments, a decrease in lanosterol synthase or squalene epoxidase activity can also decrease the amount of 2-3-oxido-squalene converted to lanosterol and increase the amount of 2-3-oxido-squalene available to be diverted into alternative pathways, such as to the mogrol precursor, mogrol, and / or mogroside. In some embodiments, the lanosterol synthase variant is encoded by a variant of the ERG7 coding sequence. In some embodiments, the squalene epoxidase is encoded by the ERG1 gene.

[0181] 1. Lanosterol synthase Isoprenoids and isoprenoid production can be enhanced by upregulating or downregulating the expression of one or more genes or the activity of their gene products or encoded enzymes, such as lanosterol synthase.

[0182] As used in the present disclosure, lanosterol synthase is an enzyme that can catalyze the cyclization of 2-3-oxidosqualene to produce lanosterol. In some embodiments, the lanosterol synthase disclosed herein is a hypomorphic form of lanosterol synthase (e.g., a mutant with reduced but not eliminated lanosterol synthase activity). Without wishing to be bound by theory, complete inactivation of lanosterol synthase is lethal in yeast, since lanosterol synthase may be required to produce hydrophobic components of the cell membrane that are important for maintaining cell integrity. In some embodiments, the lanosterol synthase disclosed herein is useful for isoprenoid precursor and / or isoprenoid production, since the reduction of lanosterol synthase activity increases the flux through the terpene synthesis pathway. In some embodiments, the lanosterol synthase disclosed herein increases the flux through the terpene synthesis pathway and / or reduces the competition for oxidosqualene. In some embodiments, the terpene synthesis pathway comprises one or more enzymes shown in Figures 1A-1D, Figure 2, Tables 1-5 and / or disclosed herein. Structurally, the lanosterol synthase may comprise the catalytic motif DCTAE (SEQ ID NO:5). See, for example, Corey et al. PNAS 1994 Mar 15;91(6):2211-5 and Shi et al. 1994 Jul 19;91(15):7370-4. In some embodiments, the lanosterol synthase corresponds to the enzyme classification number EC5.4.99.7.

[0183] As a non-limiting example, lanosterol synthase has the amino acid sequence: (SEQ ID NO:1) may include.

[0184] SEQ ID NO:1 may be encoded by the ERG7 gene. In some embodiments, SEQ ID NO:1 has the nucleotide sequence: is coded by

[0185] In some embodiments, the lanosterol synthase comprises the amino acid sequence set forth in UniProtKB Accession No. P38604 (SEQ ID NO: 313, Tables 15-16).

[0186] In some embodiments, the lanosterol synthase comprising SEQ ID NO:313 is encoded by the polynucleotide: is coded by

[0187] In some embodiments, the lanosterol synthase has the amino acid sequence: (SEQ ID NO:3) Includes.

[0188] In some embodiments, the lanosterol synthase comprising SEQ ID NO:3 has the nucleotide sequence: is coded by

[0189] In some embodiments, the lanosterol synthase of the present disclosure is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least a little, or at least a little of any one of SEQ ID NOs: 1-4, 8, 61-66, 68-71, 73-74, 78, 80-87, 89-92, 94-95, 99-109, 111-120, 304, 313, 316-319, 321-326, and 328-331. The present invention also includes a sequence (e.g., a nucleic acid or amino acid sequence) that is at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical (including all values ​​in between) to any lanosterol synthase in Tables 15-16 or any lanosterol synthase sequence disclosed in this application or known in the art.

[0190] In some embodiments, the lanosterol synthase has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 9, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100 amino acid changes.

[0191] In some embodiments, the lanosterol synthase is at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, up to 49, up to 50, up to 51, up to 52, up to 53, up to 54, up to 55, up to 56, up to 57, up to 58, up to 59, up to 60, up to 61, up to 62, up to 63, up to 64, up to 65, up to 66, up to 67, up to 68, up to 69, up to 70, up to 71, up to 72, up to 73, up to 74, up to 75, up to 76, up to 77, up to 78, up to 79, up to 80, up to 81, up to 82, up to 83, up to 84, up to 85, up to 86, up to 87, up to 88, up to 89, up to 90, up to 91, up to 92, up to 93, up to 94, up to 95, up to 96, up to 97, up to 98, up to 99, or up to 100 amino acid changes.

[0192] In some embodiments, the lanosterol synthase comprises 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 5-60, 5-70, 5-80, 5-90, 5-100, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, or 10-100 amino acid changes (including all values ​​in between) relative to SEQ ID NO:1 or 313.

[0193] In some embodiments, the lanosterol synthase is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 6, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 11 3, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 , 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 2 07, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 23 8, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269,270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577 , 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635 , 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 69 3, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, or 742.

[0194] In some embodiments, the lanosterol synthase is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 5, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 , 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 1 75, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 20 6, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237 , 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 5 74, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629 , 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, or 731.

[0195] In some embodiments, the amino acid change is a substitution, insertion, or deletion.

[0196] In some embodiments, the amino acid changes result in truncation of the lanosterol synthase relative to the control. In some embodiments, the control is a wild-type lanosterol synthase. In some embodiments, the control is a different lanosterol synthase. As non-limiting examples, the lanosterol synthase can include one or more changes as shown in Tables 7, 9, 10A-10B, 11-14, or relative to SEQ ID NO: 1 or 313.

[0197] In some embodiments, the lanosterol synthase is selected from the group consisting of 14, 33, 47, 50, 66, 80, 83, 85, 92, 94, 107, 122, 132, 145, 158, 170, 172, 184, 193, 197, 198, 212, 213, 227, 228, 231, 235, 248, 249, 260, 282, 286, 287, 289, 295, 296, 309, 314, 316, 329, 344, 360, 370, 371, 372, 398, 407, 41 and / or 742. In some embodiments, the lanosterol synthase is: the amino acid Y at the residue corresponding to position 14 of SEQ ID NO:1; the amino acid Q at the residue corresponding to position 33 of SEQ ID NO:1; the amino acid E at the residue corresponding to position 47 of SEQ ID NO:1; the amino acid G at the residue corresponding to position 50 of SEQ ID NO:1; the amino acid R at the residue corresponding to position 66 of SEQ ID NO:1; the amino acid G at the residue corresponding to position 80 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 83 of SEQ ID NO:1; the amino acid N at the residue corresponding to position 85 of SEQ ID NO:1; the amino acid I at the residue corresponding to position 92 of SEQ ID NO:1; the amino acid I at the residue corresponding to position 94 of SEQ ID NO:1 the amino acid S; the amino acid D at the residue corresponding to position 107 of SEQ ID NO:1; the amino acid C at the residue corresponding to position 122 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 132 of SEQ ID NO:1; the amino acid C at the residue corresponding to position 145 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 158 of SEQ ID NO:1; the amino acid A at the residue corresponding to position 170 of SEQ ID NO:1; the amino acid N at the residue corresponding to position 172 of SEQ ID NO:1; the amino acid W at the residue corresponding to position 184 of SEQ ID NO:1; the amino acid C or H at the residue corresponding to position 193 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 197 of SEQ ID NO:1;Amino acid I at the residue corresponding to position 198 of SEQ ID NO:1; Amino acid I at the residue corresponding to position 212 of SEQ ID NO:1; Amino acid L at the residue corresponding to position 213 of SEQ ID NO:1; Amino acid L at the residue corresponding to position 227 of SEQ ID NO:1; Amino acid T at the residue corresponding to position 228 of SEQ ID NO:1; Amino acid V at the residue corresponding to position 231 of SEQ ID NO:1; Amino acid M at the residue corresponding to position 235 of SEQ ID NO:1; Amino acid F at the residue corresponding to position 248 of SEQ ID NO:1; Amino acid L at the residue corresponding to position 249 of SEQ ID NO:1; Amino acid R at the residue corresponding to position 260 of SEQ ID NO:1; Amino acid I at the residue corresponding to position 282 of SEQ ID NO:1; Amino acid F at the residue corresponding to position 286 of SEQ ID NO:1; Amino acid G at the residue corresponding to position 287 of SEQ ID NO:1; Amino acid G at the residue corresponding to position 289 of SEQ ID NO:1; Amino acid I at the residue corresponding to position 295 of SEQ ID NO:1; Amino acid T at the residue corresponding to position 296 of SEQ ID NO:1; Amino acid F at the residue corresponding to position 309 of SEQ ID NO:1; Amino acid S at the residue corresponding to position 314 of SEQ ID NO:1; the amino acid R at the residue corresponding to position 316; the amino acid N at the residue corresponding to position 329 of SEQ ID NO:1; the amino acid A at the residue corresponding to position 344 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 360 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 370 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 371 of SEQ ID NO:1: the amino acid P at the residue corresponding to position 372 of SEQ ID NO:1; the amino acid I at the residue corresponding to position 398 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 407 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 414 of SEQ ID NO:1; the amino acid S at the residue corresponding to position 417 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 423 of SEQ ID NO:1: the amino acid I or S at the residue corresponding to position 432 of SEQ ID NO:1; the amino acid L at the residue corresponding to position 437 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 442 of SEQ ID NO:1; the amino acid M or S at the residue corresponding to position 444 of SEQ ID NO:1; the amino acid G at the residue corresponding to position 452 of SEQ ID NO:1; the amino acid V at the residue corresponding to position 474 of SEQ ID NO:1;S amino acid at the residue corresponding to position 479 of SEQ ID NO:1; Q amino acid at the residue corresponding to position 491 of SEQ ID NO:1; N amino acid at the residue corresponding to position 498 of SEQ ID NO:1; L amino acid at the residue corresponding to position 515 of SEQ ID NO:1; T amino acid at the residue corresponding to position 526 of SEQ ID NO:1; T amino acid at the residue corresponding to position 529 of SEQ ID NO:1; F amino acid at the residue corresponding to position 536 of SEQ ID NO:1; Y amino acid at the residue corresponding to position 544 of SEQ ID NO:1; E amino acid at the residue corresponding to position 552 of SEQ ID NO:1; A amino acid at the residue corresponding to position 559 of SEQ ID NO:1; M amino acid at the residue corresponding to position 560 of SEQ ID NO:1; C or N amino acid at the residue corresponding to position 564 of SEQ ID NO:1; P amino acid at the residue corresponding to position 578 of SEQ ID NO:1; F amino acid at the residue corresponding to position 586 of SEQ ID NO:1; T amino acid at the residue corresponding to position 608 of SEQ ID NO:1; I amino acid at the residue corresponding to position 610 of SEQ ID NO:1; 1; the amino acid V at a residue corresponding to position 7; the amino acid L at a residue corresponding to position 619 of SEQ ID NO:1; the amino acid S at a residue corresponding to position 620 of SEQ ID NO:1; the amino acid E or R at a residue corresponding to position 631 of SEQ ID NO:1; the amino acid D at a residue corresponding to position 638 of SEQ ID NO:1; the amino acid L at a residue corresponding to position 650 of SEQ ID NO:1; the amino acid A at a residue corresponding to position 655 of SEQ ID NO:1; the amino acid H at a residue corresponding to position 660 of SEQ ID NO:1; the amino acid S at a residue corresponding to position 679 of SEQ ID NO:1; the amino acid E at a residue corresponding to position 686 of SEQ ID NO:1; the amino acid D at a residue corresponding to position 702 of SEQ ID NO:1; the amino acid Q at a residue corresponding to position 710 of SEQ ID NO:1; the amino acid L or V at a residue corresponding to position 726 of SEQ ID NO:1; the amino acid F at a residue corresponding to position 736 of SEQ ID NO:1; the amino acid M at a residue corresponding to position 738 of SEQ ID NO:1; and / or a truncation resulting in a deletion of a residue corresponding to position 742 of SEQ ID NO:1. In some embodiments, the lanosterol synthase contains the amino acid substitutions E617V, G107D, and / or K631E relative to SEQ ID NO:1.

[0198] In some embodiments, with respect to SEQ ID NO:1, the lanosterol synthase is: R33Q, R193C, D289G, N295I, S296T, N620S, and Y736F; R184W, L235M, L260R, and E710Q; K47E, L92I, T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, E617V, and F726L; N14Y, N132S, Y145C, R193H, I286F, L316R, F 432I, E442V, T444S, I479S, K631R, and T655A;F432S, D452G, and I536F;E287G, K329N, E617V, and F726V;E231V, A407V, Q423L, A529T, and Y564C;V248F, D371V, and G702D;L197V, K282I, N314S, P370L, A608T, G638D, and F650L;L491Q, Y586F, and R660H;G122C, H249L , and K738M; P227L, E474V, V559A, and Y564N; K85N, G158S, S515L, P526T, Q619L, and truncations resulting in deletion of residues corresponding to Q742 in SEQ ID NO:1; G107D and K631E; T212I, W213L, N544Y, and V552E; I172N, C414S, L560M, and G679S; R193C, D289G, N295I, S296T, N620S, and Y736F; K85N, and G158S ; L197V, K282I, N314S, and P370L; I172N, C414S, and L560M; D371V, M610I, and G702D; D371V, K498N, M610I, and G702D; D80G, P83L, T170A, T198I, and A228T; T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, and E617V; or L309F, V344A, T398I, and K686E.

[0199] In some embodiments, the lanosterol synthase has the following amino acid substitutions relative to SEQ ID NO:1: R193C, D289G, N295I, S296T, N620S, and Y736F; F432S, D452G, and I536F; K85N, and G158S; L197V, K282I, N314S, and P370L; I172N, C414S, L560M, and G679S; I172N, C414S, and L560M; D371V , M610I, and G702D; D371V, K498N, M610I, and G702D; D80G, P83L, T170A, T198I, and A228T; D50G, K66R, N94S, G417S, E617V, and F726L; T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, and E617V; and L309F, V344A, T398I, and K686E.

[0200] In some embodiments, the lanosterol synthase comprises the following amino acid substitutions relative to SEQ ID NO:1: D50G, K66R, N94S, G417S, E617V, and F726L; K85N and G158S; K47E, L92I, T360S, S372P, T444M, and R578P; F432S, D452G, and I536F; T360S, S372P, T444M, and R578P; L491Q, Y586F, and R660H; K85N, G158S, S515L, P526T, Q619L, and a truncation resulting in a residue corresponding to position 742 of SEQ ID NO:1; or I172N, C414S, L560M, and G679S.

[0201] In some embodiments, lanosterol contains amino acid substitutions or deletions relative to SEQ ID NO:1 at one or more residues corresponding to positions 14, 33, 47, 50, 66, 85, 92, 94, 122, 132, 145, 158, 193, 231, 248, 249, 286, 287, 289, 295, 296, 316, 329, 360, 371, 372, 407, 417, 423, 432, 442, 444, 479, 515, 526, 529, 564, 578, 617, 619, 620, 631, 655, 702, 726, 736, 738, and / or 742 of SEQ ID NO:1. In some embodiments, relative to SEQ ID NO:1, the lanosterol synthase is: R33Q, R193C, D289G, N295I, S296T, N620S, and Y736F; K47E, L92I, T360S, S372P, T444M, and R578P; D50G, K66R, N94S, G417S, E617V, and F726L; N14Y, N132S, Y145C, R193H, I286F, L316R, F432I, E442V , T444S, I479S, K631R, and T655A; E287G, K329N, E617V, and F726V; E231V, A407V, Q423L, A529T, and Y564C; V248F, D371V, and G702D; G122C, H249L, and K738M; or K85N, G158S, S515L, P526T, and Q619L, and truncations resulting in a deletion of residues corresponding to Q742 of SEQ ID NO:1.

[0202] In some embodiments, the host cell comprises a heterologous polynucleotide encoding a lanosterol synthase, where the lanosterol synthase comprises amino acid substitutions or deletions relative to SEQ ID NO:313 at one or more residues corresponding to positions 64, 120, 121, 136, 226, 268, 275, 281, 300, 322, 333, 438, 502, 604, 619, 628, 656, 693, 726, 727, 728, 729, 730, and / or 731.

[0203] In some embodiments, the lanosterol synthase is: an amino acid G at a residue corresponding to position 64 of SEQ ID NO:313; an amino acid V at a residue corresponding to position 120 of SEQ ID NO:313; an amino acid S at a residue corresponding to position 121 of SEQ ID NO:313; an amino acid V at a residue corresponding to position 136 of SEQ ID NO:313; an amino acid I at a residue corresponding to position 226 of SEQ ID NO:313; an amino acid S at a residue corresponding to position 268 of SEQ ID NO:313; an amino acid I at a residue corresponding to position 275 of SEQ ID NO:313; an amino acid A at a residue corresponding to position 281 of SEQ ID NO:313; an amino acid G at a residue corresponding to position 300 of SEQ ID NO:313; 313; the amino acid G at the residue corresponding to position 322 of SEQ ID NO:313; the amino acid A at the residue corresponding to position 333 of SEQ ID NO:313; the amino acid E at the residue corresponding to position 438 of SEQ ID NO:313; the amino acid L at the residue corresponding to position 502 of SEQ ID NO:313; the amino acid N at the residue corresponding to position 604 of SEQ ID NO:313; the amino acid S at the residue corresponding to position 619 of SEQ ID NO:313; the amino acid E at the residue corresponding to position 628 of SEQ ID NO:313; the amino acid T at the residue corresponding to position 656 of SEQ ID NO:313; the amino acid G at the residue corresponding to position 693 of SEQ ID NO:313; and / or a deletion of residues corresponding to positions 726 to 731 of SEQ ID NO:313.

[0204] In some embodiments, the lanosterol synthase includes, relative to SEQ ID NO:313: P121S, A136V, S300G, V322G, K438E, F502L, K628E, and a deletion of residues corresponding to positions 726-731 of SEQ ID NO:313; K268S, T281A, F502L, T604N, A656T, and E693G; or C619S, F275I, I120V, M226I, R64G, and T333A.

[0205] It should be understood that the activity, such as the specific activity, of lanosterol synthase can be measured by any means known to those skilled in the art. In some embodiments, the production of one or more isoprenoid precursors and / or isoprenoids can be used to measure lanosterol activity. As a non-limiting example, the production of mevalonate can be used as a readout of lanosterol synthase activity. For example, a lanosterol synthase with reduced activity (e.g., reduced but not eliminated activity) can increase mevalonate production in a host cell relative to a control. In some embodiments, the control is a host cell with a different lanosterol synthase. In some embodiments, the control is a host cell with a wild-type lanosterol synthase.

[0206] The activity of lanosterol synthase can be altered using any suitable method known in the art. In some embodiments, one or more amino acid changes reduce the activity of lanosterol synthase compared to a control lanosterol synthase. In some embodiments, the control lanosterol synthase is a wild-type lanosterol synthase. In some embodiments, the expression of lanosterol synthase is altered to affect lanosterol synthase activity. In some embodiments, the host cell comprises a heterologous polynucleotide that can reduce lanosterol synthase activity. In some embodiments, reducing lanosterol synthase expression in the host cell reduces lanosterol synthase activity. In some embodiments, the activity of lanosterol synthase is reduced using a weak promoter driving the expression of lanosterol synthase, one or more codons that are not optimized for a particular host cell, the use of antisense nucleic acid, genetic modification to change the expression of a gene and / or introduce one or more changes, changing the promoter driving the expression of lanosterol synthase, and / or changing the coding sequence of lanosterol synthase.

[0207] In some embodiments, lanosterol synthase enhances production of isoprenoid precursors and / or isoprenoids by a host cell by at least 0.01%, at least 0.05%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 ... The increase may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, or at least 1000% (including all values ​​in between). In some embodiments, lanosterol synthase enhances the production of isoprenoid precursors and / or isoprenoids by the host cell by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 10 ... In some embodiments, the lanosterol synthase can increase the production of isoprenoid precursors and / or isoprenoids by the host cell by up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 450%, up to 500%, up to 550%, up to 600%, up to 650%, up to 700%, up to 750%, up to 800%, up to 850%, up to 900%, up to 950%, or up to 1000% (including all values ​​in between).As compared to production of isoprenoid precursors and / or isoprenoids by a host cell that does not contain lanosterol synthase, the production may be increased by 0.01%-1%, 1%-10%, 10%-20%, 10%-50%, 50%-100%, 100%-200%, 200%-300%, 300%-400%, 400%-500%, 500%-600%, 600%-700%, 700%-800%, 800%-900%, 900%-1000%, 1%-50%, 1%-100%, 1%-500%, or 1%-1000% (including all values ​​in between). In some embodiments, the lanosterol synthase enhances the production of isoprenoid precursors and / or isoprenoids by the host cell by at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.1 fold, at least 3.2 fold, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 3.6 fold, at least 3.7 fold, at least 3.8 fold, at least 3.9 fold, at least 3.8 fold, at least 3.9 fold, at least 3.1 fold, at least 3.2 fold, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 3.6 ...1 fold, at least 3.2 fold, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least fold, at least 3.7 fold, at least 3.8 fold, at least 3.9 fold, at least 4 fold, at least 4.1 fold, at least 4.2 fold, at least 4.3 fold, at least 4.4 fold, at least 4.5 fold, at least 4.6 fold, at least 4.7 fold, at least 4.8 fold, at least 4.9 fold, at least 5 fold, at least 5.1 fold, at least 5.2 fold, at least 5.3 fold, at least 5.4 fold, at least 5.5 fold, at least 5.6 fold, at least 5.7 fold, at least 5.8 fold, at least 5.9 fold, at least 6 fold, at least 6.1 fold, at least 6.2 fold, at least 6.3 fold, at least 6.4 fold, at least 6.5 fold, at least 6.6 fold, at least 6.7 fold, at least 6.8 fold, at least 6.9 fold, at least 7 fold, at least 7.1 fold, at least 7.2 fold, at least 7.3 fold, at least 7.4 fold,at least 7.5 times, at least 7.6 times, at least 7.7 times, at least 7.8 times, at least 7.9 times, at least 8 times, at least 8.1 times, at least 8.2 times, at least 8.3 times, at least 8.4 times, at least 8.5 times, at least 8.6 times, at least 8.7 times, at least 8.8 times, at least 8.9 times, at least 9 times, at least 9.1 times, at least 9.2 times, at least 9.3 times, at least 9.4 times, at least 9.5 times, at least 9.6 times, at least 9.7 times, at least 9.8 times, at least 9.9 times, at least at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 58-fold, at least 59-fold, at least 60-fold, at least 61-fold, at least 62-fold, at least 63-fold, at least 64-fold, at least At least 65 times, at least 66 times, at least 67 times, at least 68 times, at least 69 times, at least 70 times, at least 71 times, at least 72 times, at least 73 times, at least 74 times, at least 75 times, at least 76 times, at least 77 times, at least 78 times, at least 79 times, at least 80 times, at least 81 times, at least 82 times, at least 83 times, at least 84 times, at least 85 times, at least 86 times, at least 87 times, at least 88 times, at least 89 times, at least 90 times, at least 91 times, at least 92 times,It can be increased by at least 93-fold, at least 94-fold, at least 95-fold, at least 96-fold, at least 97-fold, at least 98-fold, at least 99-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, or at least 1000-fold (including all values ​​in between). In some embodiments, the isoprenoid precursor is mevalonate. In some embodiments, the isoprenoid precursor is IPP, GPP, FPP. In some embodiments, the isoprenoid precursor is mevalonate or 2-3-oxidosqualene.

[0208] In some embodiments, the host cell comprising a lanosterol synthase has a lanosterol synthesis enzyme activity of at least 0.01 mg / L, at least 0.05 mg / L, at least 1 mg / L, at least 5 mg / L, at least 10 mg / L, at least 15 mg / L, at least 20 mg / L, at least 25 mg / L, at least 30 mg / L, at least 35 mg / L, at least 40 mg / L, at least 45 mg / L, at least 50 mg / L, at least 55 mg / L, at least 60 mg / L, at least 65 mg / L, at least 70 mg / L, at least 75 mg / L, at least 80 mg / L, at least 85 mg / L, at least 90 mg / L, at least 95 mg / L, at least 100 mg / L, at least 10 ... L, at least 80mg / L, at least 85mg / L, at least 90mg / L, at least 95mg / L, at least 100mg / L, at least 150mg / L, at least 200mg / L, at least 250mg / L, at least 300mg / L, at least 350mg / L, at least 400mg / L, at least 450mg / L, at least 500mg / L, at least 550mg / L, at least 600mg / L, at least 650mg / L, at least 700mg / L, at least 750mg / L, at least 800mg / L, at least 850mg / L, at least 900mg / L, at least 950mg / L, at least 1g / L, at least 1.1g / L, at least 1.2g / L, at least 1.3g / L, at least 1.4g / L, at least 1.5g / L, at least 1.6g / L, at least 1.7g / L, at least 1.8g / L, at least 1.9g / L, at least 2g / L, at least 2.1g / L, at least 2.2g / L, at least 2.3g / L, at least 2.4g / L, at least 2.5g / L, at least 2.6g / L, at least 2.7g / L, at least Also 2.8g / L, at least 2.9g / L, at least 3g / L, at least 3.1g / L, at least 3.2g / L, at least 3.3g / L, at least 3.4g / L, at least 3.5g / L, at least 3.6g / L, at least 3.7g / L, at least 3.8g / L, at least 3.9g / L, at least 4g / L, at least 4.1g / L, at least 4.2g / L, at least 4.3g / L, at least 4.4g / L, at least 4.5g / L, at least 4.6g / L, at least 4.7g / L, at least 4.8g / L, at least 4.9g / L, at least 5g / L, at least 5.1g / L, at least 5.2g / L, at least 5.3g / L, at least 5.4g / L, at least 5.5g / L, at least 5.6g / L, at least 5.7g / L, at least 5.8g / L, at least 5.9g / L, at least 6g / L, at least 6.1g / L, at least 6.2g / L, at least 6.3g / L, at least 6.4g / L, at least 6.5g / L, at least 6.6g / L, at least 6.7g / L, at least 6.8g / L, at least 6.9g / L, at least 7g / L, at least 7.1g / L, at least 7.2g / L, at least 7.3g / L, at least 7.4 g / L, at least 7.5g / L, at least 7.6g / L, at least 7.7g / L, at least 7.8g / L, at least 7.9g / L, at least 8g / L, at least 8.1g / L, at least 8.2g / L, at least 8.3g / L, at least 8.4g / L, at least 8.5g / L, at least 8.6g / L, at least 8.7g / L, at least 8.8g / L, at least 8.9g / L, at least 9g / L, at least 9.1g / L, at least 9.2g / L, at least 9.3g / L, at least 9.4g / L, at least 9.5g / L, at least 9.6g / L, at least 9.7g / L, at least 9.8g / L, at least 9.In one embodiment, the present invention can produce at least 9 g / L, at least 10 g / L, at least 20 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, at least 70 g / L, at least 80 g / L, at least 90 g / L, at least 100 g / L, at least 200 g / L, at least 300 g / L, at least 400 g / L, at least 500 g / L, at least 600 g / L, at least 700 g / L, at least 800 g / L, at least 900 g / L, or at least 1000 g / L (including all values ​​in between). In some embodiments, the host cell comprising a lanosterol synthase has an activity of up to 5 mg / L, up to 10 mg / L, up to 15 mg / L, up to 20 mg / L, up to 25 mg / L, up to 30 mg / L, up to 35 mg / L, up to 40 mg / L, up to 45 mg / L, up to 50 mg / L, up to 55 mg / L, up to 60 mg / L, up to 65 mg / L, up to 70 mg / L, up to 75 mg / L, up to 80 mg / L, up to 85 mg / L, up to 90 mg / L, up to 95 mg / L, up to 100 mg / L, up to 10 ... 5mg / L, max 90mg / L, max 95mg / L, max 100mg / L, max 150mg / L, max 200mg / L, max 250mg / L, max 300mg / L, max 350mg / L, max 400m g / L, up to 450mg / L, up to 500mg / L, up to 550mg / L, up to 600mg / L, up to 650mg / L, up to 700mg / L, up to 750mg / L, up to 800mg / L, up to 850mg / L, maximum 900mg / L, maximum 950mg / L, maximum 1g / L, maximum 1.1g / L, maximum 1.2g / L, maximum 1.3g / L, maximum 1.4g / L, maximum 1.5g / L, maximum 1.6g / L, maximum 1.7g / L, maximum 1.8g / L, maximum 1.9g / L, maximum 2g / L, maximum 2.1g / L, maximum 2.2g / L, maximum 2.3g / L, maximum 2.4g / L, maximum 2.5g / L, maximum 2.6g / L, maximum 2.7g / L , maximum 2.8g / L, maximum 2.9g / L, maximum 3g / L, maximum 3.1g / L, maximum 3.2g / L, maximum 3.3g / L, maximum 3.4g / L, maximum 3.5g / L, maximum 3.6g / L, maximum 3.7g / L, maximum Large 3.8g / L, Max 3.9g / L, Max 4g / L, Max 4.1g / L, Max 4.2g / L, Max 4.3g / L, Max 4.4g / L, Max 4.5g / L, Max 4.6g / L, Max 4.7g / L, Max 4.8g / L, maximum 4.9g / L, maximum 5g / L, maximum 5.1g / L, maximum 5.2g / L, maximum 5.3g / L, maximum 5.4g / L, maximum 5.5g / L, maximum 5.6g / L, maximum 5.7g / L L, maximum 5.8g / L, maximum 5.9g / L, maximum 6g / L, maximum 6.1g / L, maximum 6.2g / L, maximum 6.3g / L, maximum 6.4g / L, maximum 6.5g / L, maximum 6.6g / L, maximum Large 6.7g / L, Max 6.8g / L, Max 6.9g / L, Max 7g / L, Max 7.1g / L, Max 7.2g / L, Max 7.3g / L, Max 7.4g / L, Max 7.5g / L, Max 7. 6g / L, maximum 7.7g / L, maximum 7.8g / L, maximum 7.9g / L, maximum 8g / L, maximum 8.1g / L, maximum 8.2g / L, maximum 8.3g / L, maximum 8.4g / L, maximum 8.5g / L L, maximum 8.6g / L, maximum 8.7g / L, maximum 8.8g / L, maximum 8.9g / L, maximum 9g / L, maximum 9.1g / L, maximum 9.2g / L, maximum 9.3g / L, maximum 9.4g / L, maximum Large 9.5g / L, Max 9.6g / L, Max 9.7g / L, Max 9.8g / L, Max 9.9g / L, Max 10g / L, Max 20g / L, Max 30g / L, Max 40g / L, Max 50g / L L, up to 60 g / L, up to 70 g / L, up to 80 g / L, up to 90 g / L, up to 100 g / L, up to 200 g / L, up to 300 g / L, up to 400 g / L, up to 500 g / L, up to 600 g / L, up to 700 g / L, up to 800 g / L, up to 900 g / L, or up to 1000 g / L of isoprenoid precursors and / or isoprenoids. In some embodiments, the host cell comprising lanosterol synthase can produce 0.01mg / L~1mg / L, 1mg / L~10mg / L, 10mg / L~20mg / L, 10mg / L~50mg / L, 50mg / L~100mg / L , 100mg / L~200mg / L, 200mg / L~300mg / L, 300mg / L~400mg / L, 400mg / L~500mg / L, 500 mg / L~600mg / L, 600mg / L~700mg / L, 700mg / L~800mg / L, 800mg / L~900mg / L, 900mg / L ~1000mg / L, 1mg / L~50mg / L, 1mg / L~100mg / L, 1mg / L~500mg / L, 1mg / L~1000mg / L, 1g In one embodiment, the present invention can produce isoprenoid precursors and / or isoprenoids at a concentration of 10 g / L, 10 g / L to 20 g / L, 10 g / L to 50 g / L, 50 g / L to 100 g / L, 100 g / L to 200 g / L, 200 g / L to 300 g / L, 300 g / L to 400 g / L, 400 g / L to 500 g / L, 500 g / L to 600 g / L, 600 g / L to 700 g / L, 700 g / L to 800 g / L, 800 g / L to 900 g / L, 900 g / L to 1000 g / L, 1 g / L to 50 g / L, 1 g / L to 100 g / L, 1 g / L to 500 g / L, or 1 g / L to 1000 g / L (including all values ​​in between). In some embodiments, the isoprenoid precursor is mevalonate. In some embodiments, the isoprenoid precursor is IPP, GPP, FPP. In some embodiments, the isoprenoid precursor is mevalonate or 2-3-oxidosqualene.

[0209] In some embodiments, lanosterol is used as a readout of lanosterol synthase activity.For example, lanosterol synthase with reduced activity can produce less lanosterol from 2-3-oxidosqualene compared to a control.In some embodiments, the control is a different lanosterol synthase.In some embodiments, the control is wild-type lanosterol synthase.Lanosterol synthase activity can be determined using cell lysate, purified enzyme, or in host cells.

[0210] In some embodiments, lanosterol synthase enhances the production of lanosterol by a host cell by at least 0.01%, at least 0.05%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 100% relative to the production of lanosterol by a host cell that does not contain lanosterol synthase. The amount of the ion exchange reaction may be decreased by at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, or at least 1000% (including all values ​​in between). In some embodiments, lanosterol synthase enhances the production of lanosterol by a host cell by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80% compared to the production of lanosterol by a host cell that does not contain lanosterol synthase. , up to 85%, up to 90%, up to 95%, up to 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 450%, up to 500%, up to 550%, up to 600%, up to 650%, up to 700%, up to 750%, up to 800%, up to 850%, up to 900%, up to 950%, or up to 1000% (including all values ​​in between).In some embodiments, lanosterol synthase can reduce lanosterol production by a host cell by 0.01% to 1%, 1% to 10%, 10% to 20%, 10% to 50%, 50% to 100%, 100% to 200%, 200% to 300%, 300% to 400%, 400% to 500%, 500% to 600%, 600% to 700%, 700% to 800%, 800% to 900%, 900% to 1000%, 1% to 50%, 1% to 100%, 1% to 500%, or 1% to 1000% (including all values ​​in between) compared to lanosterol production by a host cell that does not contain lanosterol synthase.

[0211] In some embodiments, the lanosterol synthase activity in the host cell is determined by the level of ergosterol produced by the cell. Ergosterol is a fungal cell membrane sterol produced from lanosterol. See, e.g., Klug and Daum, FEMS Yeast Res. 2014 May;14(3):369-88. In some embodiments, the host cell containing lanosterol synthase has a lanosterol synthase activity of up to 5 mg / L, up to 10 mg / L, up to 15 mg / L, up to 20 mg / L, up to 25 mg / L, up to 30 mg / L, up to 35 mg / L, up to 40 mg / L, up to 45 mg / L, up to 50 mg / L, up to 55 mg / L, up to 60 mg / L, up to 65 mg / L, up to 70 mg / L, up to 75 mg / L, up to 80 mg / L, up to 85 mg / L, up to 90 mg / L, up to 95 mg / L, up to 100 mg / L, up to 150 mg / L, up to 20 mg / L, up to 25 mg / L, up to 30 mg / L, up to 35 mg / L, up to 40 mg / L, up to 45 mg / L, up to 50 mg / L, up to 55 mg / L, up to 60 mg / L, up to 65 mg / L, up to 70 mg / L, up to 75 mg / L, up to 80 mg / L, up to 85 mg / L, up to 90 mg / L, up to 95 mg / L, up to 10 ... Max 100mg / L, Max 150mg / L, Max 200mg / L, Max 250mg / L, Max 300mg / L, Max 350mg / L, Max 400mg / L, Max 450mg / L, Max 500mg / L, Max 550mg / L, Max 600mg / L, maximum 650mg / L, maximum 700mg / L, maximum 750mg / L, maximum 800mg / L, maximum 850mg / L, maximum 900mg / L, maximum 950mg / L, maximum 1g / L, maximum 1.1g / L, maximum 1.2g / L, Max 1.3g / L, Max 1.4g / L, Max 1.5g / L, Max 1.6g / L, Max 1.7g / L, Max 1.8g / L, Max 1.9g / L, Max 2g / L, Max 2.1g / L, Max 2.2g / L, Max 2.3g / L, Max 2.4 g / L, maximum 2.5g / L, maximum 2.6g / L, maximum 2.7g / L, maximum 2.8g / L, maximum 2.9g / L, maximum 3g / L, maximum 3.1g / L, maximum 3.2g / L, maximum 3.3g / L, maximum 3.4g / L, maximum 3.5g / L, maximum 3.6g / L, maximum 3.7g / L, maximum 3.8g / L, maximum 3.9g / L, maximum 4g / L, maximum 4.1g / L, maximum 4.2g / L, maximum 4.3g / L, maximum 4.4g / L, maximum 4.5g / L, maximum 4.6g / L, maximum 4.7g / L L, Max 4.8g / L, Max 4.9g / L, Max 5g / L, Max 5.1g / L, Max 5.2g / L, Max 5.3g / L, Max 5.4g / L, Max 5.5g / L, Max 5.6g / L, Max 5.7g / L, Max 5.8g / L, Max 5.It can produce ergosterol at a maximum of 9 g / L, 6 g / L, 6.1 g / L, 6.2 g / L, 6.3 g / L, 6.4 g / L, 6.5 g / L, 6.6 g / L, 6.7 g / L, 6.8 g / L, 6.9 g / L, 7 g / L, 7.1 g / L, 7.2 g / L, 7.3 g / L, 7.4 g / L, 7.5 g / L, 7.6 g / L, 7.7 g / L, 7.8 g / L, 7.9 g / L, 8 g / L, 8.1 g / L, 8.2 g / L, 8.3 g / L, 8.4 g / L, 8.5 g / L, 8.6 g / L, 8.7 g / L, 8.8 g / L, 8.9 g / L, 9 g / L, 9.1 g / L, 9.2 g / L, 9.3 g / L, 9.4 g / L, 9.5 g / L, 9.6 g / L, 9.7 g / L, 9.8 g / L, 9.9 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L, 700 g / L, 800 g / L, 900 g / L, or 1000 g / L. In some embodiments, lanosterol synthase is 0.01mg / L~1mg / L, 1mg / L~10mg / L, 10mg / L~20mg / L, 10mg / L~50mg / L, 50mg / L~100mg / L, 100mg / L~200mg / L, 200mg / L~300mg / L, 300mg / L~400mg / L, 400mg / L~500mg / L , 500mg / L~600mg / L, 600mg / L~700mg / L, 700mg / L~800mg / L, 800mg / L~900mg / L, 900mg / L~1000mg / L, 1mg / L~50mg / L, 1mg / L~100mg / L, 1mg / L~500mg / L, 1mg / L~100 The enzyme can produce 0 mg / L, 1 g / L to 10 g / L, 10 g / L to 20 g / L, 10 g / L to 50 g / L, 50 g / L to 100 g / L, 100 g / L to 200 g / L, 200 g / L to 300 g / L, 300 g / L to 400 g / L, 400 g / L to 500 g / L, 500 g / L to 600 g / L, 600 g / L to 700 g / L, 700 g / L to 800 g / L, 800 g / L to 900 g / L, 900 g / L to 1000 g / L, 1 g / L to 50 g / L, 1 g / L to 100 g / L, 1 g / L to 500 g / L, or 1 g / L to 1000 g / L (including all values ​​in between). .

[0212] In some embodiments, the lanosterol synthase is at most 5 mg / L, at most 10 mg / L, at most 15 mg / L, at most 20 mg / L, at most 25 mg / L, at most 30 mg / L, at most 35 mg / L, at most 40 mg / L, at most 45 mg / L, at most 50 mg / L, at most 55 mg / L, at most 60 mg / L, at most 65 mg / L, at most 70 mg / L, at most 75 mg / L, at most 80 mg / L, at most 85 mg / L, at most 90 mg / L, at most 95 mg / L, at most 100 mg / L, at most 150 mg / L, at most 200 mg / L, at most 250 mg / L, at most 300 mg / L, at most 350 mg / L, at most 400 mg / L, at most 450 mg / L, at most 500 mg / L, at most 550 mg / L, at most 600 mg / L, at most 650 mg / L, at most 700 mg / L, at most 750 mg / L, at most 800 mg / L, at most 850 mg / L, at most 900 mg / L, at most 950 mg / L, at most 1 g / L, at most 1.1 g / L, at most 1.2 g / L, at most 1.3 g / L, at most 1.4 g / L, at most 1.5 g / L, at most 1.6 g / L, at most 1.7 g / L, at most 1.8 g / L, at most 1.9 g / L, at most 2 g / L, at most 2.1 g / L, at most 2.2 g / L, at most 2.3 g / L, at most 2.4 g / L, at most 2.5 g / L, at most 2.6 g / L, at most 2.7 g / L, at most 2.8 g / L, at most 2.9 g / L, at most 3 g / L, at most 3.1 g / L, at most 3.2 g / L, at most 3.3 g / L, at most 3.4 g / L, at most 3.5 g / L, at most 3.6 g / L, at most 3.7 g / L, at most 3.8 g / L, at most 3.9 g / L, at most 4 g / L, at most 4.1 g / L, at most 4.2 g / L, at most 4.3 g / L, at most 4.4 g / L, at most 4.5 g / L, at most 4.6 g / L, at most 4.7 g / L, at most 4.8 g / L, at most 4.9 g / L, at most 5 g / L, at most 5.1 g / L, at most 5.2 g / L, at most 5.3 g / L, at most 5.4 g / L, at most 5.5 g / L, at most 5.6 g / L, at most 5.7 g / L, at most 5.8 g / L, at most 5.9 g / L, at most 6 g / L, at most 6.1 g / L, at most 6.2 g / L, at most 6.3 g / L, at most 6.4 g / L, at most 6.5 g / L, at most 6.6 g / L, at most 6.7 g / L, at most 6.8 g / L, at most 6.9 g / L, at most 7 g / L, at most 7.1 g / L, at most 7.2 g / L, at most 7.3 g / L, at most 7.4 g / L, at most 7.5 g / L, at most 7.6 g / L, at most 7.7 g / L, at most 7.8 g / L, at most 7.9g / L, maximum 8g / L, maximum 8.1g / L, maximum 8.2g / L, maximum 8.3g / L, maximum 8.4g / L, maximum 8.5g / L, maximum 8.6g / L, maximum 8.7g / L, maximum 8.8g / L, maximum 8.9g / L L, Max 9g / L, Max 9.1g / L, Max 9.2g / L, Max 9.3g / L, Max 9.4g / L, Max 9.5g / L, Max 9.6g / L, Max 9.7g / L, Max 9.8g / L, Max 9.9g / L, Max It is possible to produce 10g / L, up to 20g / L, up to 30g / L, up to 40g / L, up to 50g / L, up to 60g / L, up to 70g / L, up to 80g / L, up to 90g / L, up to 100g / L, up to 200g / L, up to 300g / L, up to 400g / L, up to 500g / L, up to 600g / L, up to 700g / L, up to 800g / L, up to 900g / L, or up to 1000g / L of ergosterol.

[0213] In some embodiments, lanosterol synthase is 0.01 mg / L to 1 mg / L, 1 mg / L to 10 mg / L, 10 mg / L to 20 mg / L, 10 mg / L to 50 mg / L, 50 mg / L to 100 mg / L, 100 mg / L to 200 mg / L, 200 mg / L to 300 mg / L, 300 mg / L to 400 mg / L, 400 mg / L to 500 mg / L, 500 mg / L to 600 mg / L, 600 mg / L to 700 mg / L, 700 mg / L to 800 mg / L, 800 mg / L to 900 mg / L, 900 mg / L to 1000 mg / L, 1 mg / L to 50 mg / L, 1 mg / L to 100 mg / L, 1 mg / L to 500 mg / L, mg / L, 1mg / L~1000mg / L, 1g / L~10g / L, 10g / L~20g / L, 10g / L~50g / L, 50g / L~100g / L, 1 00g / L~200g / L, 200g / L~300g / L, 300g / L~400g / L, 400g / L~500g / L, 500g / L~600g / L, 6 In one embodiment, the yeast can produce ergosterol from 00g / L to 700g / L, 700g / L to 800g / L, 800g / L to 900g / L, 900g / L to 1000g / L, 1g / L to 50g / L, 1g / L to 100g / L, 1g / L to 500g / L, or 1g / L to 1000g / L (including all values ​​in between).

[0214] 2. Squalene epoxidase enzyme (SQE) The production of isoprenoids and isoprenoids can be enhanced by upregulating or downregulating the expression of one or more genes, or the activity of the gene product or the encoded enzyme, including, for example, squalene epoxidase. In some embodiments, squalene epoxidase corresponds to the enzyme classification number EC 1.14.14.17.

[0215] Aspects of the present disclosure provide a squalene epoxidase (SQE) capable of oxidizing squalene (e.g., squalene or 2-3-oxidosqualene) to produce squalene epoxide (e.g., 2-3-oxidosqualene or 2-3,22-23-diepoxysqualene). SQE may also be referred to as squalene monooxygenase. In some embodiments, the squalene epoxidase is encoded by ERG1.

[0216] In some embodiments, the SQE comprises the sequence set forth in GenBank Accession No. AOW05469.1: MVTQQSAAETSATQTNEYDVVIVGAGIAGPALAVALGNQGRKVLVVERDLSEPDRIVGELLQPGGVAALKTLGLGSCIEDIDAIPCQGYNVIYSGEECVLKYPKVPRDIQQDYNELYRSGKSAD ISNEAPRGVSFHHGRFVMNLRRAARDTPNVTLLEATVTEVVKNPYTGHIIGVKTFSKTGGAKIYKHFFAPLTVVCDGTFSKFRKDFSTNKTSVRSHFAGLILKDAVLPSPQHGHVILSPNSCPV LVYQVGARETRILCDIQGPVPSNATGALKEHMEKNVMPHLPKSIQPSFQAALKEQTIRVMPNSFLSASKNDHHGLILLGDALNMRHPLTGGGMTVALNDALLLSRLLTGVNLEDTYAVSSVMSS QFHWQRKHLDSIVNILSMALYSLFAADSDYLRILQLGCFNYFKLGGICVDHPVMLLAGVLPRPMYLFTHFFVVAIYGGICNMQANGIAKLPASLLQFVASLVTACIVIFPYIWSELT (SEQ ID NO: 9)

[0217] In some embodiments, SEQ ID NO:9 is the nucleotide sequence: is coded by

[0218] In some embodiments, the SQE comprises the amino acid sequence set forth in GenBank Accession No. CAA97201.1 (SEQ ID NO:312).

[0219] In some embodiments, the nucleotide sequence encoding SEQ ID NO:312 is set forth in SEQ ID NO:303.

[0220] The SQEs of the disclosure have a repeat sequence that is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 109, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 109, at least 109, at least 200, at least 201, at least 201, at least 202, at least 203, at least 204, at least 205, at least 206, at least 207, at least 208, at least 209, at least 209, at least 210, at %, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% (including all values ​​in between) identical.

[0221] In some embodiments, the SQEs of the present disclosure can promote the formation of epoxides in squalene compounds (e.g., epoxidation of squalene or 2,3-oxidosqualene). In some embodiments, the SQEs of the present disclosure catalyze the formation of mogrol precursors (e.g., 2-3-oxidosqualene or 2-3,22-23-diepoxysqualene).

[0222] The activity, such as the specific activity, of the recombinant SQE may be measured as the concentration of isoprenoid precursor (e.g., 2-3-oxidosqualene or 2-3,22-23-diepoxysqualene) produced per unit of enzyme per unit time. In some embodiments, the SQE of the present disclosure has an activity, such as a specific activity, of at least 0.0000001 μmol / min / mg (e.g., at least 0.000001 μmol / min / mg, at least 0.0001 μmol / min / mg, at least 0.001 μmol / min / mg, at least 0.01 μmol / min / mg, at least 0.1 μmol / min / mg, at least 1 μmol / min / mg, at least 10 μmol / min / mg, or at least 100 μmol / min / mg (including all values ​​therebetween).

[0223] In some embodiments, the activity, such as the specific activity, of SQE is at least 1.1 times greater [e.g., at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or at least 100 times (including all values ​​in between)] greater than the activity of a control SQE.

[0224] The activity of squalene epoxidase can be altered using any suitable method or technique known in the art. In some embodiments, one or more amino acid changes alter the activity of the squalene epoxidase compared to a control squalene epoxidase. In some embodiments, the control squalene epoxidase is a wild-type squalene epoxidase. In some embodiments, the expression of the squalene epoxidase is altered to affect squalene epoxidase activity. In some embodiments, the host cell comprises a heterologous polynucleotide capable of decreasing squalene epoxidase activity. In some embodiments, decreasing squalene epoxidase expression in the host cell decreases squalene epoxidase activity. In some embodiments, the host cell comprises a heterologous polynucleotide capable of increasing squalene epoxidase activity. In some embodiments, increasing squalene epoxidase expression in the host cell increases squalene epoxidase activity.

[0225] In some embodiments, the activity of squalene epoxidase is reduced using a weak promoter driving expression of the squalene epoxidase, one or more codons that are not optimized for a particular host cell, the use of antisense nucleic acids, genetic modifications that alter gene expression and / or introduce one or more modifications, altering the promoter driving expression of the squalene epoxidase, and / or altering the coding sequence of the squalene epoxidase.

[0226] In some embodiments, the activity of squalene epoxidase is increased using a strong promoter driving expression of the squalene epoxidase, one or more codons optimized for a particular host cell, a nucleic acid encoding the squalene epoxidase, genetic modifications to alter expression of the gene and / or introduce one or more modifications, alterations to the promoter driving expression of the squalene epoxidase, and / or alterations to the coding sequence of the squalene epoxidase.

[0227] In some embodiments, the squalene epoxidase enhances the production of isoprenoid precursors and / or isoprenoids by the host cell by at least 0.01%, at least 0.05%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least The production of isoprenoid precursors and / or isoprenoids may be increased by 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, or at least 1000% (including all values ​​in between) compared to production of isoprenoid precursors and / or isoprenoids by a host cell that does not contain squalene epoxidase. In some embodiments, the squalene epoxidase enhances the production of isoprenoid precursors and / or isoprenoids by the host cell by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 150%, up to 200%, , up to 250%, up to 300%, up to 350%, up to 400%, up to 450%, up to 500%, up to 550%, up to 600%, up to 650%, up to 700%, up to 750%, up to 800%, up to 850%, up to 900%, up to 950%, or up to 1000% (including all values ​​in between) compared to production of isoprenoid precursors and / or isoprenoids by a host cell that does not contain squalene epoxidase.In some embodiments, squalene epoxidase can increase production of isoprenoid precursors and / or isoprenoids by a host cell by 0.01% to 1%, 1% to 10%, 10% to 20%, 10% to 50%, 50% to 100%, 100% to 200%, 200% to 300%, 300% to 400%, 400% to 500%, 500% to 600%, 600% to 700%, 700% to 800%, 800% to 900%, 900% to 1000%, 1% to 50%, 1% to 100%, 1% to 500%, or 1% to 1000% (including all values ​​therebetween) compared to production of isoprenoid precursors and / or isoprenoids by a host cell that does not contain squalene epoxidase.

[0228] In some embodiments, the host cell comprising squalene epoxidase has an activity of at least 0.01 mg / L, at least 0.05 mg / L, at least 1 mg / L, at least 5 mg / L, at least 10 mg / L, at least 15 mg / L, at least 20 mg / L, at least 25 mg / L, at least 30 mg / L, at least 35 mg / L, at least 40 mg / L, at least 45 mg / L, at least 50 mg / L, at least 55 mg / L, at least 60 mg / L, at least 65 mg / L, at least 70 mg / L, at least 75 mg / L, at least 80 mg / L, at least 8 ... 5mg / L, at least 80mg / L, at least 85mg / L, at least 90mg / L, at least 95mg / L, at least 100mg / L, at least 150mg / L, at least 200mg / L, at least 250mg / L, at least 300mg / L, at least 350mg / L, at least 400mg / L, at least 450mg / L, at least 500mg / L, at least 550mg / L, at least 600mg / L, at least 650mg / L, at least 700mg / L, at least 750mg / L, at least 800mg / L , at least 850mg / L, at least 900mg / L, at least 950mg / L, at least 1g / L, at least 1.1g / L, at least 1.2g / L, at least 1.3g / L, at least 1.4g / L, at least 1.5g / L, at least 1.6g / L, at least 1.7g / L, at least 1.8g / L, at least 1.9g / L, at least 2g / L, at least 2.1g / L, at least 2.2g / L, at least 2.3g / L, at least 2.4g / L, at least 2.5g / L, at least 2.6g / L, at least 2. 7g / L, at least 2.8g / L, at least 2.9g / L, at least 3g / L, at least 3.1g / L, at least 3.2g / L, at least 3.3g / L, at least 3.4g / L, at least 3.5g / L, at least 3.6g / L, at least 3.7g / L, at least 3.8g / L, at least 3.9g / L, at least 4g / L, at least 4.1g / L, at least 4.2g / L, at least 4.3g / L, at least 4.4g / L, at least 4.5g / L, at least 4.6g / L, at least 4.7g / L, at least 4.8g / L, at least 4.9g / L, at least 5g / L, at least 5.1g / L, at least 5.2g / L, at least 5.3g / L, at least 5.4g / L, at least 5.5g / L, at least 5.6g / L, at least 5.7g / L, at least 5.8g / L, at least 5.9g / L, at least 6g / L, at least 6.1g / L, at least 6.2g / L, at least 6.3g / L, at least 6.4g / L, at least 6.5g / L, at least 6.6g / L, at least 6.7g / L, at least 6.8g / L, at least 6.9g / L, at least 7g / L, at least 7.1g / L, at least 7.2g / L, at least 7.3g / L, at least at least 7.4g / L, at least 7.5g / L, at least 7.6g / L, at least 7.7g / L, at least 7.8g / L, at least 7.9g / L, at least 8g / L, at least 8.1g / L, at least 8.2g / L, at least 8.3g / L, at least 8.4g / L, at least 8.5g / L, at least 8.6g / L, at least 8.7g / L, at least 8.8g / L, at least 8.9g / L, at least 9g / L, at least 9.1g / L, at least 9.2g / L, at least 9.3g / L, at least 9.4g / L, at least 9.5g / L, at least 9.6g / L, at least 9.7g / L, at least 9.8g / L, at least 9.In one embodiment, the present invention can produce at least 9 g / L, at least 10 g / L, at least 20 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, at least 70 g / L, at least 80 g / L, at least 90 g / L, at least 100 g / L, at least 200 g / L, at least 300 g / L, at least 400 g / L, at least 500 g / L, at least 600 g / L, at least 700 g / L, at least 800 g / L, at least 900 g / L, or at least 1000 g / L (including all values ​​in between). In some embodiments, the host cell comprising squalene epoxidase has an activity of up to 5 mg / L, up to 10 mg / L, up to 15 mg / L, up to 20 mg / L, up to 25 mg / L, up to 30 mg / L, up to 35 mg / L, up to 40 mg / L, up to 45 mg / L, up to 50 mg / L, up to 55 mg / L, up to 60 mg / L, up to 65 mg / L, up to 70 mg / L, up to 75 mg / L, up to 80 mg / L, up to 85mg / L, max 90mg / L, max 95mg / L, max 100mg / L, max 150mg / L, max 200mg / L, max 250mg / L, max 300mg / L, max 350mg / L, max 400 mg / L, up to 450mg / L, up to 500mg / L, up to 550mg / L, up to 600mg / L, up to 650mg / L, up to 700mg / L, up to 750mg / L, up to 800mg / L, up to 850m g / L, max. 900mg / L, max. 950mg / L, max. 1g / L, max. 1.1g / L, max. 1.2g / L, max. 1.3g / L, max. 1.4g / L, max. 1.5g / L, max. 1.6g / L, max. 1.7 g / L, maximum 1.8g / L, maximum 1.9g / L, maximum 2g / L, maximum 2.1g / L, maximum 2.2g / L, maximum 2.3g / L, maximum 2.4g / L, maximum 2.5g / L, maximum 2.6g / L, maximum 2.7g / L , maximum 2.8g / L, maximum 2.9g / L, maximum 3g / L, maximum 3.1g / L, maximum 3.2g / L, maximum 3.3g / L, maximum 3.4g / L, maximum 3.5g / L, maximum 3.6g / L, maximum 3.7g / L, maximum Large 3.8g / L, Max 3.9g / L, Max 4g / L, Max 4.1g / L, Max 4.2g / L, Max 4.3g / L, Max 4.4g / L, Max 4.5g / L, Max 4.6g / L, Max 4.7g / L, Max 4.8g / L, maximum 4.9g / L, maximum 5g / L, maximum 5.1g / L, maximum 5.2g / L, maximum 5.3g / L, maximum 5.4g / L, maximum 5.5g / L, maximum 5.6g / L, maximum 5.7g / L L, maximum 5.8g / L, maximum 5.9g / L, maximum 6g / L, maximum 6.1g / L, maximum 6.2g / L, maximum 6.3g / L, maximum 6.4g / L, maximum 6.5g / L, maximum 6.6g / L, maximum Large 6.7g / L, Max 6.8g / L, Max 6.9g / L, Max 7g / L, Max 7.1g / L, Max 7.2g / L, Max 7.3g / L, Max 7.4g / L, Max 7.5g / L, Max 7. 6g / L, maximum 7.7g / L, maximum 7.8g / L, maximum 7.9g / L, maximum 8g / L, maximum 8.1g / L, maximum 8.2g / L, maximum 8.3g / L, maximum 8.4g / L, maximum 8.5g / L L, maximum 8.6g / L, maximum 8.7g / L, maximum 8.8g / L, maximum 8.9g / L, maximum 9g / L, maximum 9.1g / L, maximum 9.2g / L, maximum 9.3g / L, maximum 9.4g / L, maximum Large 9.5g / L, Max 9.6g / L, Max 9.7g / L, Max 9.8g / L, Max 9.9g / L, Max 10g / L, Max 20g / L, Max 30g / L, Max 40g / L, Max 50g / L L, up to 60 g / L, up to 70 g / L, up to 80 g / L, up to 90 g / L, up to 100 g / L, up to 200 g / L, up to 300 g / L, up to 400 g / L, up to 500 g / L, up to 600 g / L, up to 700 g / L, up to 800 g / L, up to 900 g / L, or up to 1000 g / L of isoprenoid precursors and / or isoprenoids. In some embodiments, the host cell comprising squalene epoxidase can produce 0.01mg / L~1mg / L, 1mg / L~10mg / L, 10mg / L~20mg / L, 10mg / L~50mg / L, 50mg / L~100mg / L , 100mg / L~200mg / L, 200mg / L~300mg / L, 300mg / L~400mg / L, 400mg / L~500mg / L, 500 mg / L~600mg / L, 600mg / L~700mg / L, 700mg / L~800mg / L, 800mg / L~900mg / L, 900mg / L ~1000mg / L, 1mg / L~50mg / L, 1mg / L~100mg / L, 1mg / L~500mg / L, 1mg / L~1000mg / L, 1g The present invention can produce isoprenoid precursors and / or isoprenoids at a concentration of 1000 g / L, ... In some embodiments, the isoprenoid precursor is mevalonate. In some embodiments, the isoprenoid precursor is IPP, GPP, FPP. In some embodiments, the isoprenoid precursor is mevalonate or 2-3-oxidosqualene.

[0229] In some embodiments, squalene epoxidase enhances the production of lanosterol or isoprenoid precursors by a host cell by at least 0.01%, at least 0.05%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least The production of lanosterol or isoprenoid precursors may be reduced by 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, or at least 1000% (including all values ​​in between) compared to production of lanosterol or isoprenoid precursors by a host cell that does not contain squalene epoxidase. In some embodiments, the squalene epoxidase enhances the production of lanosterol or an isoprenoid precursor by a host cell by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 150%, up to 200%. , up to 250%, up to 300%, up to 350%, up to 400%, up to 450%, up to 500%, up to 550%, up to 600%, up to 650%, up to 700%, up to 750%, up to 800%, up to 850%, up to 900%, up to 950%, or up to 1000% (including all values ​​in between) reduced compared to production of lanosterol or isoprenoid precursors by a host cell that does not contain squalene epoxidase.In some embodiments, squalene epoxidase can reduce lanosterol or isoprenoid precursor production by a host cell by 0.01% to 1%, 1% to 10%, 10% to 20%, 10% to 50%, 50% to 100%, 100% to 200%, 200% to 300%, 300% to 400%, 400% to 500%, 500% to 600%, 600% to 700%, 700% to 800%, 800% to 900%, 900% to 1000%, 1% to 50%, 1% to 100%, 1% to 500%, or 1% to 1000% (including all values ​​therebetween) compared to lanosterol or isoprenoid precursor production by a host cell that does not contain squalene epoxidase.

[0230] In some embodiments, increasing the activity of squalene epoxidase promotes the production of 2-3-oxidosqualene, lanosterol, 2-3;22,23-diepoxysqualene and / or isoprenoids derived from these compounds.In some embodiments, decreasing the activity of squalene epoxidase promotes the production of isoprenoids derived from farnesyl diphosphate, except for 2-3-oxidosqualene and isoprenoids derived therefrom, promotes the production of intermediate molecules of the mevalonate pathway, such as mevalonate, promotes the production of intermediate molecules of the MEP pathway, such as 2C-methyl-D-erythritol-2,4-cyclodiphosphate, and / or reduces the production of 2-3-oxidosqualene, lanosterol, 2-3;22,23-diepoxysqualene or isoprenoids derived from these compounds.

[0231] 3. Mevalonate (MEV) pathway enzymes Isoprenoids and isoprenoid production can be enhanced by upregulating or downregulating the expression of one or more genes or the activity of their gene products or encoded enzymes, including, for example, one or more enzymes in the MEV pathway, such as:

[0232] FIG. 1A provides non-limiting examples of enzymes involved in the mevalonate (MEV) pathway. First, acetoacetyl-CoA thiolase condenses two acetyl-CoA molecules to form acetoacetyl-CoA. Acetoacetyl-CoA thiolase can be encoded by the ERG10 gene. UniProtKB accession numbers P41338 and P10551 provide non-limiting examples of acetoacetyl-CoA thiolase. Increasing the expression of the ERG10 gene or increasing the activity of the ERG10 enzyme can be used to increase the production of isoprenoids or isoprenoid precursors.

[0233] Acetoacetyl-CoA synthase synthesizes acetoacetyl-CoA by catalyzing the condensation of acetyl-CoA and malonyl-CoA to form acetoacetyl-CoA and CoA. Increasing the expression of the acetoacetyl-CoA synthase gene or increasing the enzyme activity of acetoacetyl-CoA synthase can be used to increase the production of isoprenoids or isoprenoid precursors.

[0234] HMG-CoA synthase condenses acetoacetyl-CoA to form 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA). HMG-CoA synthase can be encoded by the ERG13 gene. UniProtKB accession numbers P54839 and A0A1D8PTW6 provide non-limiting examples of HMG-CoA synthases. Increasing the expression of the ERG13 gene or increasing the activity of the ERG13 enzyme can be used to increase the production of isoprenoids or isoprenoid precursors.

[0235] HMG-CoA reductase then reduces HMG-CoA to produce mevalonate. HMG-CoA reductase can be encoded by the HMG1 gene. UniProtKB Accession No. P12683 provides a non-limiting example of an HMG-CoA reductase encoded by HMG1. HMG-CoA reductase can be encoded by the HMG2 gene. UniProtKB Accession No. P12684 provides a non-limiting example of an HMG-CoA reductase encoded by HMG2. Increasing the expression of the HMG1 and / or HMG2 genes or increasing the activity of the HMG1 and / or HMG2 enzymes can be used to increase the production of isoprenoids or isoprenoid precursors.

[0236] Mevalonate-5-kinase phosphorylates mevalonate to form mevalonate-5-phosphate. Mevalonate-5-kinase can be encoded by the ERG12 gene. UniProtKB Accession Nos. P07277 and A0A1D8PEL1 provide non-limiting examples of mevalonate-5-kinase. Increasing the expression of the ERG12 gene or increasing the activity of the ERG12 enzyme can be used to increase the production of isoprenoids or isoprenoid precursors.

[0237] Mevalonate-5-phosphate is phosphorylated by phosphomevalonate kinase to form mevalonate pyrophosphate. Phosphomevalonate kinase can be encoded by ERG8 gene. UniProtKB Accession No. P24521 provides a non-limiting example of phosphomevalonate kinase. Increasing the expression of ERG8 gene or increasing the activity of ERG8 enzyme can be used to increase the production of isoprenoids or isoprenoid precursors.

[0238] Mevalonate pyrophosphate decarboxylase converts mevalonate pyrophosphate to IPP. Mevalonate pyrophosphate decarboxylase can be encoded by ERG19 gene. UniProtKB Accession No. P32377 provides a non-limiting example of mevalonate pyrophosphate decarboxylase. Increasing the expression of ERG19 gene or increasing the activity of ERG19 enzyme can be used to increase the production of isoprenoids or isoprenoid precursors.

[0239] Isopentenyl pyrophosphate isomerase catalyzes the conversion of IPP to dimethylallyl pyrophosphate (DMAPP). Since DMAPP is an electrophile and more reactive than IPP, IPP isomerization to DMAPP promotes isoprenoid biosynthesis. Isopentenyl pyrophosphate isomerase can be encoded by the IDI1 gene. UniProtKB Accession No. P15496 provides a non-limiting example of isopentenyl pyrophosphate isomerase.

[0240] In some embodiments, increasing the activity of one or more of the mevalonate (MEV) pathway genes promotes production of isoprenoids.

[0241] Archaeal mevalonate 1 (MEV-A1) pathway enzymes Isoprenoids and isoprenoid production can be enhanced by upregulating or downregulating the expression of one or more genes or the activity of their gene products or encoded enzymes, including, for example, one or more enzymes in the MEV-A1 pathway, such as:

[0242] FIG. 1B provides non-limiting examples of enzymes involved in the archaeal mevalonate 1 (MEV-A1) pathway. First, acetoacetyl-CoA thiolase condenses two acetyl-CoA molecules to form acetoacetyl-CoA. Acetoacetyl-CoA thiolase can be encoded by the ERG10 gene. UniProtKB accession numbers P41338 and P10551 provide non-limiting examples of acetoacetyl-CoA thiolases.

[0243] Acetoacetyl-CoA synthase also synthesizes acetoacetyl-CoA by catalyzing the condensation of acetyl-CoA and malonyl-CoA to form acetoacetyl-CoA and CoA.

[0244] HMG-CoA synthase then condenses acetoacetyl-CoA to form 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA). HMG-CoA synthase can be encoded by the ERG13 gene. UniProtKB accession numbers P54839 and A0A1D8PTW6 provide non-limiting examples of HMG-CoA synthases.

[0245] HMG-CoA reductase then reduces HMG-CoA to produce mevalonate. HMG-CoA reductase may be encoded by the HMG1 gene. UniProtKB Accession No. P12683 provides a non-limiting example of an HMG-CoA reductase encoded by HMG1. HMG-CoA reductase may be encoded by the HMG2 gene. UniProtKB Accession No. P12684 provides a non-limiting example of an HMG-CoA reductase encoded by HMG2.

[0246] Mevalonate-5-kinase then phosphorylates mevalonate to form mevalonate-5-phosphate. Mevalonate-5-kinase can be encoded by the ERG12 gene. UniProtKB Accession Nos. P07277 and A0A1D8PEL1 provide non-limiting examples of mevalonate-5-kinase.

[0247] Mevalonate-5-phosphate is decarboxylated by mevalonate-5-phosphate decarboxylase to form isopentenyl pyrophosphate. Mevalonate-5-phosphate decarboxylase can be encoded by PMD gene. UniProtKB Accession Nos. D4GXZ3 and Q18K00 provide non-limiting examples of mevalonate-5-phosphate decarboxylases.

[0248] Isopentenyl phosphate kinase converts isopentenyl pyrophosphate to IPP. Isopentenyl phosphate kinase can be encoded by the IPK gene. UniProtKB Accession Nos. Q60352 and Q56187 provide non-limiting examples of isopentenyl phosphate kinase.

[0249] Isopentenyl pyrophosphate isomerase catalyzes the conversion of IPP to DMAPP. Since DMAPP is an electrophile and more reactive than IPP, the isomerization of IPP to DMAPP promotes isoprenoid biosynthesis. Isopentenyl pyrophosphate isomerase can be encoded by the IDI1 gene. UniProtKB Accession No. P15496 provides a non-limiting example of isopentenyl pyrophosphate isomerase.

[0250] In some embodiments, increasing the activity of one or more of the archaeal mevalonate I (MEV-A1) pathway genes promotes production of isoprenoids.

[0251] Archaeal mevalonate 2 (MEV-A2) pathway enzymes Isoprenoids and isoprenoid production can be enhanced by upregulating or downregulating the expression of one or more genes or the activity of their gene products or encoded enzymes, including, for example, one or more enzymes in the MEV-A2 pathway, such as:

[0252] FIG. 1C provides non-limiting examples of enzymes involved in the archaeal mevalonate 2 (MEV-A1) pathway. First, acetoacetyl-CoA thiolase condenses two acetyl-CoA molecules to form acetoacetyl-CoA. Acetoacetyl-CoA thiolase can be encoded by the ERG10 gene. UniProtKB accession numbers P41338 and P10551 provide non-limiting examples of acetoacetyl-CoA thiolases.

[0253] Acetoacetyl-CoA synthase also synthesizes acetoacetyl-CoA by catalyzing the condensation of acetyl-CoA and malonyl-CoA to form acetoacetyl-CoA and CoA.

[0254] HMG-CoA synthase then condenses acetoacetyl-CoA to form 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA). HMG-CoA synthase can be encoded by the ERG13 gene. UniProtKB accession numbers P54839 and A0A1D8PTW6 provide non-limiting examples of HMG-CoA synthases.

[0255] HMG-CoA reductase then reduces HMG-CoA to produce mevalonate. HMG-CoA reductase may be encoded by the HMG1 gene. UniProtKB Accession No. P12683 provides a non-limiting example of an HMG-CoA reductase encoded by HMG1. HMG-CoA reductase may be encoded by the HMG2 gene. UniProtKB Accession No. P12684 provides a non-limiting example of an HMG-CoA reductase encoded by HMG2.

[0256] Mevalonate-3-kinase then phosphorylates mevalonate to form mevalonate-3-phosphate. Mevalonate-3-kinase can be encoded by the M3K gene. UniProtKB Accession Nos. Q9HIN1 and Q6KZB1 provide non-limiting examples of mevalonate-3-kinases.

[0257] Mevalonate-3-phosphate is phosphorylated by mevalonate-3-phosphate-5-kinase to form mevalonate-3,5-bisphosphate. Mevalonate-3-phosphate-5-kinase can be encoded by the M3K gene. UniProtKB Accession Nos. Q9HIN1 and Q6KZB1 provide non-limiting examples of mevalonate-3-kinase.

[0258] Mevalonate-3,5-phosphate is then decarboxylated by mevalonate-5-phosphate decarboxylase to form isopentenyl pyrophosphate. Mevalonate-5-phosphate decarboxylase can be encoded by PMD gene. UniProtKB Accession Nos. D4GXZ3 and Q18K00 provide non-limiting examples of mevalonate-5-phosphate decarboxylases.

[0259] Isopentenyl phosphate kinase converts isopentenyl pyrophosphate to IPP. Isopentenyl phosphate kinase can be encoded by the IPK gene. UniProtKB Accession Nos. Q60352 and Q56187 provide non-limiting examples of isopentenyl phosphate kinase.

[0260] Isopentenyl pyrophosphate isomerase catalyzes the conversion of IPP to DMAPP. Since DMAPP is an electrophile and more reactive than IPP, the isomerization of IPP to DMAPP promotes isoprenoid biosynthesis. Isopentenyl pyrophosphate isomerase can be encoded by the IDI1 gene. UniProtKB Accession No. P15496 provides a non-limiting example of isopentenyl pyrophosphate isomerase.

[0261] In some embodiments, increasing the activity of one or more of the archaeal mevalonate 2 (MEV-A2) pathway genes promotes production of isoprenoids.

[0262] Methylerythritol phosphate (MEP) pathway enzymes Isoprenoids and isoprenoid production can be enhanced by upregulating or downregulating the expression of one or more genes or the activity of their gene products or encoded enzymes, including, for example, one or more enzymes in the MEP pathway, such as:

[0263] Figure 1D provides a non-limiting example of the enzyme involved in the methylerythritol phosphate (MEP) pathway. First, 1-deoxy-D-xylulose-5-phosphate synthase condenses pyruvate and glyceraldehyde 3-phosphate to form 1-deoxy-D-xylulose-5-phosphate (DXP). 1-deoxy-D-xylulose-5-phosphate synthase can be encoded by the DXS gene. UniProtKB accession numbers P77488 and A0A3D8XGB8 provide a non-limiting example of 1-deoxy-D-xylulose-5-phosphate synthase.

[0264] 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) then reduces DXP to form 2C-methyl-D-erythritol 4-phosphate (MEP). 1-deoxy-D-xylulose-5-phosphate reductoisomerase can be encoded by the IspC gene or the DXR gene. UniProtKB accession numbers P45568 and O96693 provide non-limiting examples of 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

[0265] 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (CMS) then converts DXP to 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME). 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase can be encoded by the YgpP gene or the IspD gene. UniProtKB accession numbers Q46893 and A0A5E7ZFQ6 provide non-limiting examples of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase.

[0266] CDP-ME then undergoes phosphorylation by ATP-dependent 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK) to produce 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-MEP). 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase can be encoded by the YchB gene or the IspE gene. UniProtKB accession numbers P62615 and A0A535X269 provide non-limiting examples of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase.

[0267] CDP-MEP is cyclized by 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) to form 2C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC or MEcPP). 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase can be encoded by the IspF gene. UniProtKB accession numbers P62617 and Q8RQP5 provide non-limiting examples of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase.

[0268] 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS) converts MEC to 4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMB-PP or HMBPP). 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate synthase can be encoded by the GcpE gene or the IspG gene. UniProtKB accession numbers P62620 and Q8DK70 provide non-limiting examples of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase.

[0269] 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR) converts mevalonate HMB-PP to a mixture of IPP and DMAPP. 4-Hydroxy-3-methylbut-2-en-1-yl diphosphate reductase can be encoded by the LytB gene or the IspH gene. UniProtKB accession numbers W1F471 and A0A113QNS4 provide non-limiting examples of 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase.

[0270] Isopentenyl pyrophosphate isomerase catalyzes the conversion of IPP to DMAPP. Since DMAPP is an electrophile and more reactive than IPP, the isomerization of IPP to DMAPP promotes isoprenoid biosynthesis. Isopentenyl pyrophosphate isomerase can be encoded by the IDI1 gene. UniProtKB Accession No. P15496 provides a non-limiting example of isopentenyl pyrophosphate isomerase.

[0271] Increasing the activity of one or more of the methylerythritol phosphate (MEP) pathway genes promotes production of isoprenoids.

[0272] Prenyltransferase As used herein, "prenyltransferase" refers to a protein that promotes the transfer of a prenyl group to a substrate. In some embodiments, prenyltransferase promotes the condensation of IPP with an allyl substrate to generate prenyl diphosphates of different lengths. Geranyl pyrophosphate synthase catalyzes the formation of GPP. Geranyl pyrophosphate synthase can be encoded by the ERG20 gene.

[0273] Farnesyl diphosphate synthase catalyzes the conversion of GPP to FPP. Farnesyl diphosphate synthase can be encoded by the ERG20 gene. UniProtKB Accession Nos. P08524 and A0A1D8PH78 provide non-limiting examples of farnesyl diphosphate synthase.

[0274] Geranylgeranyl pyrophosphate synthase catalyzes the formation of GGPP. Geranylgeranyl pyrophosphate synthase can be encoded by the GGPPS gene. UniProtKB Accession No. Q64KQ5 provides a non-limiting example of geranylgeranyl pyrophosphate synthase.

[0275] In some embodiments, increasing the activity of one or more of the prenyltransferases enhances production of an isoprenoid.

[0276] Squalene synthase As used herein, "squalene synthase" refers to a protein that catalyzes the production of squalene from farnesyl diphosphate. Squalene synthase may be encoded by the ERG9 gene. UniProtKB Accession Nos. P36596, P29704 and Q9HGZ6 provide non-limiting examples of squalene synthase.

[0277] In some embodiments, increasing the activity of squalene synthase promotes the production of squalene, 2-3-oxidosqualene, lanosterol, 2-3;22,23-diepoxysqualene and / or isoprenoids derived therefrom. In some embodiments, decreasing the activity of squalene synthase reduces the production of isoprenoids derived from farnesyl diphosphate, except for squalene and isoprenoids derived therefrom, promotes the production of intermediate molecules in the mevalonate pathway, such as mevalonate, promotes the production of intermediate molecules in the MEP pathway, such as 2C-methyl-D-erythritol 2,4-cyclodiphosphate, and / or reduces the production of squalene, 2-3-oxidosqualene, lanosterol, 2-3;22,23-diepoxysqualene or isoprenoids derived therefrom.

[0278] Terpene synthase As used herein, "terpene synthase" refers to a protein that can produce isoprenoids that can use prenyl diphosphate as substrate.At least two types of terpene synthases have been characterized: classical terpene synthases and isoprenyl diphosphate synthase-type terpene synthases.Classical terpene synthases are found in prokaryotes (e.g., bacteria) and eukaryotes (e.g., plants, fungi and amoebae), while isoprenyl diphosphate synthase-type terpene synthases are found in insects (see, for example, Chen et al., Terpene synthase genes in eukaryotes beyond plants and fungi: Occurrence in social amoebae. Proc Natl Acad Sci US A. 2016;113(43):12132-12137, the entirety of which is incorporated herein by reference). In classical terpene synthases, several highly conserved structural motifs have been reported, including the aspartic acid-rich "DDxx(x)D / E" motif and the "NDxxSxxxD / E" (SEQ ID NO: 55) motif, both of which are involved in the coordination of substrate binding (see, e.g., Starks et al., Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase. Science. 1997 Sep 19;277(5333):1815-20 and Christianson et al., Unearthing the roots of the terpenome. Curr Opin Chem Biol. 2008 Apr;12(2):141-50, each of which is incorporated herein by reference in its entirety for this purpose). See also, e.g., WO2019 / 161141 and WO2020 / 176547.

[0279] In some embodiments, increasing the activity of an isoprenoid-specific terpene synthase enhances the production of the isoprenoid.

[0280] Acetoacetyl-CoA synthase An embodiment of the present invention provides an acetoacetyl-CoA synthase that catalyzes the condensation of acetyl-CoA and malonyl-CoA to form acetoacetyl-CoA and CoA, but does not accept malonyl-[acyl-carrier-protein] as a substrate. Acetoacetyl-CoA synthase can also convert malonyl-CoA to acetyl-CoA by decarboxylation of malonyl-CoA. An embodiment of the present invention provides an acetoacetyl-CoA synthase that increases the level of acetoacetyl-CoA.

[0281] In some embodiments, the acetoacetyl-CoA synthase is encoded by the NphT7 gene. NphT7 catalyzes an alternative pathway to acetoacetyl-CoA and is present in the MEV pathway but not in the MEP pathway. See, e.g., FIG. 1A. In some embodiments, the acetoacetyl-CoA synthase has the amino acid sequence: MTDVRFRIIGTGAYVPERIVSNDEVGAPAGVDDDWITRKTGIRQRRWAADDQATSDLATAAGRAALKAAGITPEQLTVIAVATSTPDRPQPPTAAYVQHHLGATGTAAFDVNAVCSGTVFALSSVAGTLVYRGGYALVIGADLYSRILNPADRKTVVLFGDGAGAMVL GPTSTGTGPIVRRVALHTFGGLTDLIRVPAGGSRQPLDTGLDAGLQYFAMDGREVRRFVTEHLPQLIKGFLHEAGVDAADISHFVPHQANGVMLDEVFGELHLPRATMHRTVETYGNTGAASIPITMDAAVRAGSFRPGELVLLAGFGGGMAASFALIEW (SEQ ID NO: 6) Includes.

[0282] In some embodiments, the acetoacetyl-CoA synthase is encoded by a polynucleotide having the following sequence: ATGACCGACGTCCGATTCCGAATTATCGGTACTGGTGCCTACGTTCCCGAACGAATCGTTTCCAACGATGAAGTCGGTGCTCCTGCCGGTGTTGACGACGACTGGATCACCCGAAAGACCGGTATTCGACAGCGACGATGGGCTGCCGATGACCAGGCCACCTCTGATCTGGCCACTGCTGCCGGTCGAGCTGCCCTGAAGGCCGCTGGTATCACTCCCGAGCAGCTGACCGTTATTGCTGTTGCCACCTCCACTCCCGATCGACCCCAGCCTCCCACTGCTGCCTATGTTCAGCACCACCTCGGAGCCACCGGTACTGCTGCCTTCGACGTCAACGCTGTCTGCTCCGGTACCGTTTTCGCCCTGTCCTCTGTTGCTGGCACCCTCGTTTACCGAGGTGGTTACGCTCTGGTCATTGGCGCTGACCTGTACTCTCGAATCCTCAACCCTGCCGACCGAAAGACCGTCGTTCTGTTCGGTGATGGTGCCGGTGCCATGGTTCTCGGTCCTACCTCCACCGGTACTGGTCCCATTGTTCGACGAGTTGCCCTGCACACCTTCGGTGGTCTGACCGACCTGATTCGAGTCCCCGCTGGTGGTTCTCGACAGCCCCTGGACACTGATGGCCTCGATGCTGGACTGCAGTACTTCGCTATGGACGGTCGTGAGGTCCGACGATTCGTCACTGAGCACCTCCCCCAGCTGATCAAGGGTTTCCTGCACGAGGCCGGTGTCGACGCTGCCGACATCTCTCACTTCGTCCCTCATCAGGCCAACGGTGTCATGCTCGACGAGGTCTTCGGCGAGCTGCATCTGCCTCGAGCTACCATGCACCGAACTGTCGAGACTTACGGCAACACCGGAGCTGCCTCCATTCCCATCACCATGGACGCTGCCGTTCGAGCCGGTTCCTTCCGACCTGGTGAGCTGGTCCTGCTGGCCGGTTTCGGTGGCGGTATGGCCGCTTCCTTCGCCCTGATCGAGTGGTAG(SEQ ID NO: 7)

[0283] The acetoacetyl-CoA synthase of the disclosure may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 122%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at The acetoacetyl-CoA synthase sequence may comprise at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% (including all values ​​in between) identical to the acetoacetyl-CoA synthase sequence set forth as SEQ ID NO:6 or 7, or to any acetoacetyl-CoA synthase disclosed in this application or known in the art. The present disclosure also relates to host cells comprising such acetoacetyl-CoA synthases, polynucleotides encoding such acetoacetyl-CoA synthases, and / or methods of using such host cells.

[0284] In some embodiments, an acetoacetyl-CoA synthase of the disclosure can promote the formation of acetoacetyl-CoA.

[0285] The activity, such as the specific activity, of the recombinant acetoacetyl-CoA synthase may be measured as the concentration of acetoacetyl-CoA produced per unit of enzyme per unit time. In some embodiments, the acetoacetyl-CoA synthase of the present disclosure has an activity, such as a specific activity, of at least 0.0000001 μmol / min / mg (e.g., at least 0.000001 μmol / min / mg, at least 0.0001 μmol / min / mg, at least 0.001 μmol / min / mg, at least 0.01 μmol / min / mg, at least 0.1 μmol / min / mg, at least 1 μmol / min / mg, at least 10 μmol / min / mg, or at least 100 μmol / min / mg (including all values ​​therebetween).

[0286] In some embodiments, the activity, such as the specific activity, of the acetoacetyl-CoA synthase is at least 1.1 times greater [e.g., at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 1.9 times, at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, or at least 100 times (including all values ​​in between)] greater than the activity of a control acetoacetyl-CoA synthase.

[0287] In various embodiments, the disclosure relates to: an acetoacetyl-CoA synthase as set forth in SEQ ID NO:6; a polynucleotide encoding the acetoacetyl-CoA synthase as set forth in SEQ ID NO:7; a host cell comprising the acetoacetyl-CoA synthase as set forth in SEQ ID NO:6; or a host cell comprising a polynucleotide encoding the acetoacetyl-CoA synthase as set forth in SEQ ID NO:7. In some embodiments, the disclosure relates to: a method of making an isoprenoid or an isoprenoid precursor, comprising producing an isoprenoid or an isoprenoid precursor in a host cell comprising the acetoacetyl-CoA synthase as set forth in SEQ ID NO:6, and / or a polynucleotide encoding the acetoacetyl-CoA synthase as set forth in SEQ ID NO:7.

[0288] In various embodiments, any of the host cells described herein may further comprise an acetoacetyl-CoA synthase as described herein; any of the methods described herein may be practiced using any of the host cells described herein that further comprise an acetoacetyl-CoA synthase as described herein.

[0289] Mutants Aspects of the present disclosure relate to polynucleotides encoding any of the described recombinant polypeptides, such as lanosterol synthase, squalene epoxidase, MEV pathway enzymes, MEP pathway enzymes, squalene synthase, prenyltransferase, terpene synthase, and any of the proteins related to the present disclosure. Variants of the polynucleotide or amino acid sequences described in this application are also encompassed by the present disclosure. A variant may share at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (including all values ​​in between) sequence identity with a reference sequence.

[0290] Unless otherwise specified, the term "sequence identity" refers to the relationship between two polypeptide or polynucleotide sequences as determined by sequence comparison (alignment), as known in the art. In some embodiments, sequence identity is determined over the entire length of the sequences, while in other embodiments, sequence identity is determined over a region of the sequences.

[0291] Identity may also refer to the degree of sequence relatedness between two sequences, as determined by the number of matches between strings of two or more residues (e.g., nucleic acid or amino acid residues). Identity measures the percentage of identical matches between the smaller of two or more sequences with gap alignment (if any) accommodated by a particular mathematical model, algorithm, or computer program.

[0292] The identity of related polypeptide or nucleic acid sequences can be easily calculated by any method known to those skilled in the art. The "percent identity" of two sequences (e.g., nucleic acid or amino acid sequences) can be determined, for example, using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68,1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77,1993. Such an algorithm is incorporated into the NBLAST® and XBLAST® programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10,1990. BLAST® protein searches can be performed, for example, with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the present invention. When gaps exist between the two sequences, Gapped BLAST® can be utilized, for example, as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST® and Gapped BLAST® programs, the default parameters of the respective programs (e.g., XBLAST® and NBLAST®) can be used, or the parameters can be appropriately adjusted, as understood by those of skill in the art.

[0293] Other local alignment techniques that may be used are, for example, based on the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol.147:195-197). A general global alignment technique that may be used is, for example, the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol.48:443-453), which is based on dynamic programming.

[0294] More recently, the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed, which is said to generate global alignments of nucleic acid and amino acid sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. In some embodiments, the identity of two polypeptides is determined by aligning two amino acid sequences, calculating the number of identical amino acids, and dividing by the length of one of the amino acid sequences. In some embodiments, the identity of two nucleic acids is determined by aligning two nucleotide sequences, calculating the number of identical nucleotides, and dividing by the length of one of the nucleic acids.

[0295] For multiple sequence alignment, computer programs including Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct 11;7:539) may be used.

[0296] In preferred embodiments, sequences, including nucleic acid or amino acid sequences, are found to have a particular percent identity to a reference sequence, such as a sequence disclosed and / or claimed in this application, when sequence identity is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77,1993 (e.g., the BLAST®, NBLAST®, XBLAST® or Gapped BLAST® programs, using the default parameters of each program).

[0297] In some embodiments, sequences, including nucleic acid or amino acid sequences, are found to have a particular percent identity to a reference sequence, such as the sequences disclosed and / or claimed in this application, when determining sequence identity using the Smith-Waterman algorithm (Smith, TF & Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol.147:195-197) or the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol.48:443-453).

[0298] In some embodiments, sequences, including nucleic acid sequences or amino acid sequences, are found to have a particular percent identity to a reference sequence, such as a sequence disclosed and / or claimed in this application, when sequence identity is determined using the Fast Optimal Global Sequence Alignment Algorithm (FOGSAA).

[0299] In some embodiments, sequences, including nucleic acid or amino acid sequences, are found to have a particular percent identity to a reference sequence, such as a sequence disclosed and / or claimed in this application, when sequence identity is determined using Clustal Omega (Sievers et al., Mol Syst Biol. 2011 Oct 11:7:539).

[0300] As used in this application, a residue (e.g., a nucleic acid or amino acid residue) in sequence "X" is said to correspond to a position or residue (e.g., a nucleic acid or amino acid residue) "Z" in a different sequence "Y" if the residue in sequence "X" is at the corresponding position of "Z" in sequence "Y" when sequences X and Y are aligned using amino acid sequence alignment tools known in the art.

[0301] A variant sequence may be a homologous sequence. As used herein, a homologous sequence refers to a sequence that has a particular percent identity (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 1 %, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity (including all values ​​in between), including but not limited to paralogous sequences, orthologous sequences, or sequences resulting from convergent evolution (e.g., nucleic acid sequences or amino acid sequences). Paralogous sequences result from the duplication of genes within the genome of a species, while orthologous sequences diverge after speciation. Two different species may have evolved independently, but as a result of convergent evolution, each may contain sequences that share a certain percent identity with sequences from the other species.

[0302] In some embodiments, a polypeptide variant (e.g., a variant of a lanosterol synthase, squalene epoxidase, MEV pathway enzyme, MEP pathway enzyme, squalene synthase, prenyltransferase, terpene synthase, or any protein related to this disclosure) comprises a domain that shares secondary structure (e.g., alpha helices, beta sheets) with a reference polypeptide (e.g., a reference lanosterol synthase, MEV pathway enzyme, MEP pathway enzyme, squalene epoxidase, squalene synthase, prenyltransferase, terpene synthase, or any protein related to this disclosure). In some embodiments, a polypeptide variant (e.g., a variant of a lanosterol synthase, squalene epoxidase, MEV pathway enzyme, MEP pathway enzyme, squalene synthase, prenyltransferase, terpene synthase, or any protein related to this disclosure) shares a tertiary structure with a reference polypeptide (e.g., a reference lanosterol synthase, squalene epoxidase, MEV pathway enzyme, MEP pathway enzyme, squalene synthase, prenyltransferase, terpene synthase, or any protein related to this disclosure). As a non-limiting example, a variant polypeptide may have low primary sequence identity (e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% sequence identity) compared to a reference polypeptide, but may share one or more secondary structures (e.g., including but not limited to, loops, alpha helices, beta sheets) or have the same tertiary structure as the reference polypeptide. For example, a loop may be located between a beta sheet and an alpha helix, between two alpha helices, or between two beta sheets. Homology modeling can be used to compare two or more tertiary structures.

[0303] Mutation of nucleotide sequence can be performed by various methods known to those skilled in the art.For example, mutation can be generated by PCR-induced mutation, site-directed mutagenesis by the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. USA 82: 488-492, 1985), chemical synthesis of the gene encoding the polypeptide, gene editing tools, or insertion such as insertion of tag (e.g., HIS tag or GFP tag).Mutation can include, for example, substitution, deletion, and translocation, which are generated by any method known in the art.Methods for making mutations can be found in references such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York, 2010.

[0304] In some embodiments, methods for generating variants include circular shuffling (Yu and Lutz, Trends Biotechnol. 2011 Jan;29(1):18-25). In circular shuffling, the linear primary sequence of a polypeptide can be circularized (e.g., by joining the N-terminus and C-terminus of the sequence) and the polypeptide can be truncated ("broken") at different positions. Thus, the linear primary sequence of the new polypeptide can have low sequence identity [e.g., less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% (including all values ​​in between)] as determined by linear sequence alignment methods (e.g., Clustal Omega or BLAST). However, topology analysis of the two proteins can reveal that the tertiary structures of the two polypeptides are similar or dissimilar. Without being bound by any particular theory, variant polypeptides produced by circular shuffling of a reference polypeptide and having a similar tertiary structure to the reference polypeptide may share similar functional properties (e.g., enzyme activity, enzyme kinetics, substrate specificity, or product specificity). In some instances, circular shuffling may alter the secondary, tertiary, or quaternary structure to produce proteins with different functional properties (e.g., increased or decreased enzyme activity, different substrate specificity, or different product specificity). See, e.g., Yu and Lutz, Trends Biotechnol. 2011 Jan;29(1):18-25.

[0305] It should be understood that in the protein that has undergone circular permutation, the linear amino acid sequence of the protein is different from that of the reference protein that has not undergone circular permutation.However, those skilled in the art can determine which residues in the protein that has undergone circular permutation correspond to those in the reference protein that has not undergone circular permutation, for example, by aligning sequences and detecting conserved motifs, and / or by comparing the structure or predicted structure of the protein, for example, by homology modeling.

[0306] In some embodiments, the algorithm for determining the percent identity between the sequence of interest and the reference sequence described in this application considers the presence of circular permutation between the sequences. The presence of circular permutation can be detected using any method known in the art, including, for example, RASPODOM (Weiner et al., Bioinformatics. 2005 Apr 1;21(7):932-7). In some embodiments, the presence of circular permutation is corrected (e.g., at least one domain in the sequence is rearranged) before calculating the percent identity between the sequence of interest and the sequence described in this application. It should be understood that the claims of this application encompass sequences whose percent identity to the reference sequence is calculated after considering potential circular permutations of the sequence.

[0307] Functional variants of recombinant lanosterol synthase, MEV pathway enzymes, non-mevalonate pathway enzymes, squalene synthase, squalene epoxidase, prenyltransferase, terpene synthase, and any other proteins disclosed in this application are also encompassed by this disclosure. For example, functional variants may bind to one or more of the same substrates (e.g., mogrol, mogroside, or precursors thereof) or produce one or more of the same products (e.g., mogrol, mogroside, or precursors thereof). Functional variants can be identified using any method known in the art. For example, the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68,1990, mentioned above, can be used to identify homologous proteins with known functions.

[0308] Putative functional variants can also be identified by searching for polypeptides with functionally annotated domains. Databases including Pfam (Sonnhammer et al., Proteins. 1997 Jul;28(3):405-20) can be used to identify polypeptides with specific domains. For example, in some cases, among oxidosqualene cyclases, additional CDS enzymes can be identified by searching for polypeptides with a leucine residue corresponding to position 123 of SEQ ID NO:256. This leucine residue is involved in determining the product specificity of CDS enzymes; mutation of this residue can result in, for example, cycloartenol or parkeol as product (Takase et al. Org Biomol Chem. 2015 Jul 13(26):7331-6).

[0309] Homology modeling can also be used to identify amino acid residues that are amenable to mutation without affecting function.Non-limiting examples of such methods include using position-specific scoring matrices (PSSMs) and energy minimization protocols.See, for example, Stormo et al., Nucleic Acids Res. 1982 May 11;10(9):2997-3011.

[0310] The PSSM can be combined with the calculation of a Rosetta energy function that determines the difference between the wild type and a single point mutant. Without being bound to a particular theory, potentially stabilizing mutations are desirable for protein engineering (e.g., producing functional homologs). In some embodiments, potentially stabilizing mutations have a ΔΔG of less than -0.1 (e.g., less than -0.2, less than -0.3, less than -0.35, less than -0.4, less than -0.45, less than -0.5, less than -0.55, less than -0.6, less than -0.65, less than -0.7, less than -0.75, less than -0.8, less than -0.85, less than -0.9, less than -0.95, or less than -1.0) Rosetta Energy Units (Reu). calc See, e.g., Goldenzweig et al., Mol Cell. 2016 Jul 21;63(2):337-346.doi:10.1016 / j.molcel.2016.06.012.

[0311] In some embodiments, the coding sequence for lanosterol synthase, MEV or MEP pathway enzymes, squalene synthase, squalene epoxidase, prenyltransferase, terpene synthase, or any protein related to the disclosure corresponds to a reference coding sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more than 100 positions. In some embodiments, the coding sequence for lanosterol synthase, MEV pathway enzyme, MEP pathway enzyme, squalene synthase, squalene epoxidase, prenyltransferase, terpene synthase, or any protein related to the disclosure is at least one amino acid sequence greater than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 1 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more codons contain mutations. As will be appreciated by those of skill in the art, a mutation in a codon may or may not change the amino acid encoded by the codon, due to the degeneracy of the genetic code.In some embodiments, the one or more mutations in the coding sequence do not change the amino acid sequence of the coding sequence relative to the amino acid sequence of a reference polypeptide.

[0312] In some embodiments, one or more mutations in a recombinant lanosterol synthase, MEV pathway enzyme, MEP pathway enzyme, squalene synthase, squalene epoxidase, prenyltransferase, terpene synthase, or other recombinant protein sequence related to the present disclosure alter the amino acid sequence of the polypeptide relative to the amino acid sequence of a reference polypeptide. In some embodiments, the one or more mutations alter the amino acid sequence of the recombinant polypeptide relative to the amino acid sequence of the reference polypeptide, altering (enhancing or decreasing) the activity of the polypeptide relative to the reference polypeptide.

[0313] The activity of the enzymes of the present disclosure may be altered using any suitable method or technique known in the art. In some embodiments, one or more amino acid changes alter the activity of the enzyme compared to a control enzyme. In some embodiments, the control enzyme is a wild-type enzyme. In some embodiments, the expression of the enzyme is altered to affect the enzyme activity. In some embodiments, the host cell comprises a heterologous polynucleotide capable of enzyme activity. In some embodiments, reducing the enzyme expression in the host cell reduces the enzyme activity. In some embodiments, the host cell comprises a heterologous polynucleotide capable of increasing the enzyme activity. In some embodiments, increasing the enzyme expression in the host cell increases the enzyme activity.

[0314] In some embodiments, the activity of the enzyme is reduced using a weak promoter driving expression of the enzyme, one or more codons that are not optimized for a particular host cell, the use of antisense nucleic acids, genetic modifications that alter the expression of a gene and / or introduce one or more modifications, altering the promoter driving expression of the enzyme, and / or altering the coding sequence of the enzyme.

[0315] Reduced enzyme activity can mean reduced enzyme expression, reduced enzyme stability, reduced enzyme specific activity, and / or reduced enzyme function due to interference with other protein, nucleic acid or small molecule inhibitors known in the art.

[0316] In some embodiments, the activity of the enzyme is increased using a strong promoter driving expression of the enzyme, one or more codons optimized for a particular host cell, a nucleic acid encoding the enzyme, genetic modifications that alter expression of the gene and / or introduce one or more modifications, changes to the promoter driving expression of the enzyme, and / or changes to the coding sequence of the enzyme.

[0317] The activity, including specific activity, of any of the recombinant polypeptides described in this application can be measured using methods known in the art. As a non-limiting example, the activity of a recombinant polypeptide can be determined by measuring its substrate specificity, the product(s) produced, the concentration of the product(s) produced, or any combination thereof. As used in this application, the "specific activity" of a recombinant polypeptide refers to the amount (e.g., concentration) of a particular product produced for a given amount (e.g., concentration) of the recombinant polypeptide per unit time.

[0318] Those skilled in the art will also understand that mutations in recombinant polypeptide coding sequences may result in conservative amino acid substitutions to provide functionally equivalent variants of the aforementioned polypeptides, e.g., variants that retain the activity of the polypeptide. As used in this application, "conservative amino acid substitutions" or "conservatively substituted" refers to amino acid substitutions that do not alter the relative charge, or size characteristics or functional activity of the protein in which the amino acid substitution is made.

[0319] In some instances, an amino acid is characterized by its R group (see, e.g., Table 6). For example, an amino acid may contain a non-polar aliphatic R group, a positively charged R group, a negatively charged R group, a non-polar aromatic R group, or a polar uncharged R group. Non-limiting examples of amino acids containing a non-polar aliphatic R group include alanine, glycine, valine, leucine, methionine, and isoleucine. Non-limiting examples of amino acids containing a positively charged R group include lysine, arginine, and histidine. Non-limiting examples of amino acids containing a negatively charged R group include aspartic acid and glutamic acid. Non-limiting examples of amino acids containing a non-polar aromatic R group include phenylalanine, tyrosine, and tryptophan. Non-limiting examples of amino acids containing a polar uncharged R group include serine, threonine, cysteine, proline, asparagine, and glutamine.

[0320] Non-limiting examples of functionally equivalent variants of polypeptides include conservative amino acid substitutions in the amino acid sequences of proteins disclosed in the present application.Conservative amino acid substitutions include substitutions between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Further non-limiting examples of conservative amino acid substitutions are shown in Table 6.

[0321] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 residues may be altered in preparing a variant polypeptide, in some embodiments, amino acids are replaced by conservative amino acid substitutions.

[0322] [Table 6]

[0323] Amino acid substitutions in the amino acid sequence of a polypeptide to produce a recombinant polypeptide variant having desired properties and / or activity can be made by altering the coding sequence of the polypeptide. Similarly, conservative amino acid substitutions in the amino acid sequence of a polypeptide to produce a functionally equivalent variant of the polypeptide are typically made by altering the coding sequence of the recombinant polypeptide (e.g., lanosterol synthase, MEV pathway enzyme, MEP pathway enzyme, squalene synthase, squalene epoxidase, prenyltransferase, terpene synthase, or any protein related to the present disclosure).

[0324] Expression of Nucleic Acids in Host Cells Aspects of the present disclosure relate to recombinant expression of one or more genes encoding one or more enzymes in the MEV or MEP pathway for the synthesis of isoprenoids or isoprenoid precursors, functional variants and mutants thereof, and related uses. For example, the methods described in the present application can be used to produce isoprenoid precursors and / or isoprenoids.

[0325] The term "heterologous" with respect to polynucleotides, such as polynucleotides containing genes, is used interchangeably with the terms "exogenous" and "recombinant" and refers to the following: polynucleotides artificially provided to a biological system; polynucleotides modified in a biological system; or polynucleotides whose expression or regulation has been manipulated in a biological system. A heterologous polynucleotide introduced or expressed in a host cell may be a polynucleotide derived from an organism or species different from the host cell, or may be a synthetic polynucleotide, or may be a polynucleotide endogenously expressed in the same organism or species as the host cell. For example, a polynucleotide that is endogenously expressed in a host cell may be considered heterologous if it is non-naturally located in the host cell; recombinantly, stably or transiently expressed in the host cell; modified in the host cell; selectively edited in the host cell; expressed in a copy number different from the copy number naturally occurring in the host cell; or non-naturally expressed in the host cell, such as by manipulating the regulatory region that controls the expression of the polynucleotide. In some embodiments, a heterologous polynucleotide is a polynucleotide that is endogenously expressed in a host cell, but whose expression is driven by a promoter that does not naturally control the expression of the polynucleotide. In other embodiments, a heterologous polynucleotide is a polynucleotide that is endogenously expressed in a host cell, but whose expression is driven by a promoter that naturally controls the expression of the polynucleotide, but the promoter or another control region is modified. In some embodiments, the promoter is recombinantly activated or repressed. For example, gene editing-based techniques can be used to regulate the expression of a polynucleotide, including an endogenous polynucleotide, from a promoter, including an endogenous promoter. See, for example, Chavez et al., Nat Methods. 2016 Jul; 13(7): 563-567. A heterologous polynucleotide may include a wild-type sequence or a mutant sequence compared to a reference polynucleotide sequence.

[0326] The nucleic acid encoding any of the recombinant polypeptides described in this application, such as lanosterol synthase, MEV or MEP pathway enzymes, squalene synthase, squalene epoxidase, prenyltransferase, terpene synthase, or any protein related to this disclosure, can be incorporated into any suitable vector by any method known in the art.For example, the vector can be an expression vector, including but not limited to a viral vector (e.g., lentivirus, retrovirus, adenovirus, or adeno-associated virus vector), any vector suitable for transient expression, any vector suitable for constitutive expression, or any vector suitable for inducible expression (e.g., galactose-inducible or doxycycline-inducible vector).

[0327] In some embodiments, the vector replicates autonomously within the cell. The vector may contain one or more endonuclease restriction sites that are cleaved by a restriction endonuclease to insert and ligate a nucleic acid containing a gene described in this application to produce a recombinant vector capable of replicating within the cell. The vector is typically composed of DNA, but RNA vectors are also available. Cloning vectors include, but are not limited to, plasmids, fosmids, phagemids, viral genomes, and artificial chromosomes. As used in this application, the term "expression vector" or "expression construct" refers to a recombinantly or synthetically generated nucleic acid construct that has a set of specific nucleic acid elements that allow transcription of a particular nucleic acid in a host cell, such as a yeast cell. In some embodiments, the nucleic acid sequence of a gene described in this application is inserted into the cloning vector so as to be operably linked to a regulatory sequence and, in some embodiments, expressed as an RNA transcript. In some embodiments, the vector contains one or more markers, such as a selectable marker as described in this application, to identify cells that have been transformed or transfected with the recombinant vector. In some embodiments, the nucleic acid sequence of a gene described in this application is codon-optimized. Codon optimization may increase production of a gene product by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (including all values ​​in between) relative to a non-codon optimized reference sequence.

[0328] A coding sequence and a regulatory sequence are said to be "operably associated" or "operably linked" when they are covalently linked and expression or transcription of the coding sequence is under the influence or control of the regulatory sequence. A coding sequence and a regulatory sequence are said to be operably associated or linked if the coding sequence is transcribed upon induction of a promoter in the 5' regulatory sequence when the coding sequence is to be translated into a functional protein, and if the nature of the linkage between the coding sequence and the regulatory sequence does not result in (1) the introduction of a frameshift mutation, (2) interference with the ability of the promoter region to direct transcription of the coding sequence, or (3) interference with the ability of the corresponding RNA transcript to be translated into a protein.

[0329] In some embodiments, the nucleic acid encoding any of the proteins described in this application is under the control of a regulatory sequence (e.g., an enhancer sequence). In some embodiments, the nucleic acid is expressed under the control of a promoter. The promoter can be a native promoter, e.g., a promoter of the gene in its endogenous context that provides normal control of expression of the gene. Alternatively, the promoter can be a promoter different from the native promoter of the gene, e.g., a promoter different from the promoter of the gene in its endogenous context.

[0330] In some embodiments, the promoter is a eukaryotic promoter. Non-limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1 GAL1, GAL10, GAL7, GAL3, GAL2, MET3, MET25, HXT3, HXT7, ACT1, ADH1, ADH2, CUP1-1, ENO2, and SOD1, which will be known to those skilled in the art (see, for example, Addgene's website: blog.addgene.org / plasmids-101-the-promoter-region). In some embodiments, the promoter is a prokaryotic promoter (e.g., a bacteriophage or bacterial promoter). Non-limiting examples of bacteriophage promoters include Pls1con, T3, T7, SP6, and PL. Non-limiting examples of bacterial promoters include Pbad, PmgrB, Ptrc2, Plac / ara, Ptac, and Pm.

[0331] In some embodiments, the promoter is an inducible promoter. As used in this application, an "inducible promoter" is a promoter that is controlled by the presence or absence of a molecule. Non-limiting examples of inducible promoters include chemically controlled promoters and physically controlled promoters. For chemically controlled promoters, transcriptional activity can be controlled by one or more compounds, such as alcohol, tetracycline, galactose, steroids, metals, or other compounds. For physically controlled promoters, transcriptional activity can be controlled by phenomena such as light or temperature. Non-limiting examples of tetracycline-controlled promoters include anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, such as tetracycline repressor protein (tetR), tetracycline operator sequence (tetO) and tetracycline transactivator fusion protein (tTA). Non-limiting examples of steroid-regulated promoters include promoters based on rat glucocorticoid receptor, human estrogen receptor, gaecdysone receptor, and promoters from the steroid / retinoid / thyroid receptor superfamily. Non-limiting examples of metal-regulated promoters include promoters from metallothionein (proteins that bind and sequester metal ions) genes. Non-limiting examples of pathogenesis-regulated promoters include promoters induced by salicylic acid, ethylene, or benzothiadiazole (BTH). Non-limiting examples of temperature / heat-inducible promoters include heat shock promoters. Non-limiting examples of light-regulated promoters include light-responsive promoters from plant cells. In certain embodiments, the inducible promoter is a galactose-inducible promoter. In some embodiments, the inducible promoter is induced by one or more physiological conditions (e.g., pH, temperature, radiation, osmolality, saline gradient, cell surface binding, or concentration of one or more exogenous or endogenous inducers).Non-limiting examples of exogenous inducers or inducers include amino acids and amino acid analogs, sugars and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal-containing compounds, salts, ions, enzyme substrate analogs, hormones, or any combination thereof.

[0332] In some embodiments, the promoter is a constitutive promoter.As used in this application, "constitutive promoter" refers to an unregulated promoter that allows continuous transcription of gene.Non-limiting examples of constitutive promoters include TDH3, PGK1, PKC1, PDC1, TEF1, TEF2, RPL18B, SSA1, TDH2, PYK1, TPI1, HXT3, HXT7, ACT1, ADH1, ADH2, ENO2 and SOD1.

[0333] Other inducible or constitutive promoters known to those of skill in the art are also contemplated.

[0334] The regulatory sequences required for gene expression vary depending on the species or cell type, but generally include 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, as necessary, such as TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed regulatory sequences include promoter regions containing promoter sequences for transcriptional control of operably linked genes. Regulatory sequences may also include enhancer sequences or upstream activator sequences. Vectors may include 5' leader sequences or signal sequences. Regulatory sequences may also include terminator sequences. In some embodiments, terminator sequences mark the end of a gene in DNA during transcription. The selection and design of one or more suitable vectors suitable for directing expression of one or more genes described in this application in a host cell is within the ability and discretion of one skilled in the art.

[0335] Expression vectors containing the necessary elements for expression are commercially available and known to those of skill in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012).

[0336] In some embodiments, introduction of a polynucleotide, such as a polynucleotide encoding a recombinant polypeptide, into a host cell results in genomic integration of the polynucleotide. In some embodiments, the host cell is capable of genomic integration of at least 1 copy, at least 2 copies, at least 3 copies, at least 4 copies, at least 5 copies, at least 6 copies, at least 7 copies, at least 8 copies, at least 9 copies, at least 10 copies, at least 11 copies, at least 12 copies, at least 13 copies, at least 14 copies, at least 15 copies, at least 16 copies, at least 17 copies, at least 18 copies, at least 19 copies, at least 20 copies, at least 21 copies, at least 22 copies, at least 23 copies, at least 24 copies, at least 25 copies, at least 26 copies, at least 27 copies, at least 28 copies, at least 29 copies, at least 30 copies, at least 31 copies, at least 32 copies, at least 33 copies, at least 34 copies, at least 35 copies, at least 36 copies, at least 37 copies, at least 38 copies, at least 39 copies, at least 40 copies, at least 41 copies, at least 42 copies, at least 43 copies, at least 44 copies, at least 45 copies, at least 46 copies, at least 47 copies, at least 48 copies, at least 49 copies, at least 50 copies, at least 51 copies, at least 52 copies, at least 53 copies, at least 54 copies, at least 55 copies, at least 56 copies, at least 57 copies, at least 58 copies, at least 59 copies, at least 60 copies, at least 61 copies, at least 62 copies, at least 63 copies, at least 64 copies, at least 65 copies, at least 66 copies, at least 67 copies, at In one embodiment, the recombinant polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more copies (including all values ​​in between) of a polynucleotide sequence, such as a polynucleotide sequence encoding any of the recombinant polypeptides described herein, in its genome.

[0337] host cell Any protein of the present disclosure can be expressed in a host cell. As used in this application, the term "host cell" refers to a cell that can be used to express polynucleotides, such as polynucleotides encoding proteins used in the production of isoprenoids and their precursors.

[0338] Any suitable host cell, including eukaryotic or prokaryotic cells, can be used to produce any of the recombinant polypeptides including lanosterol synthase, MEV or MEP pathway enzymes, squalene synthase, squalene epoxidase, prenyltransferase, terpene synthase, and other proteins disclosed in this application. Suitable host cells include, but are not limited to, fungal cells (e.g., yeast cells), bacterial cells (e.g., E. coli cells), algae cells, plant cells, insect cells, and animal cells, including mammalian cells.

[0339] Suitable yeast host cells include, but are not limited to, Candida, Hansenula, Saccharomyces (e.g., S. cerepsiae), Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia (e.g., Y. lipolytica). In some embodiments, the yeast cell is selected from the group consisting of Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Saccharomyces pombe, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, and the like. opuntiae, Pichia pastoris, Pichia pseudopastoris, Pichia membranifaciens, Komagataella pseudopastoris, Komagataella pastoris, Komagataella kurtzmanii, Komagataella mondaviorum, Pichia thermotolerans, Pichia salictaria, Pichia quercumquercuum, Pichia pijperi, Pichia stipites, Pichia methanolica, Pichia angusta, Komagataella phaffii, Komagataella pastoris, Kluyveromyces lactis, Candida albicans, Candida boidinii or Yarrowia lipolytica.

[0340] In some embodiments, the yeast strain is an industrial polyploid yeast strain. Other non-limiting examples of fungal cells include cells obtained from the genera Aspergillus, Penicillium, Fusarium, Rhizopus, Acremonium, Neurospora, Sordaria, Magnaporthe, Allomyces, Ustilago, Botrytis, and Trichoderma.

[0341] In certain embodiments, the host cells are algal cells such as Chlamydomonas (eg, C. Reinhardtii) and Phormidium (P. sp. ATCC29409).

[0342] In other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include gram-positive, gram-negative, and gram-variable bacterial cells. Host cells may be selected from the following species: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacter, and the like. Brevibacterium, Butyrivibrio, Buchnera, Campestris, Campylobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus acillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Mycobacterium, Neisseria, Pantoea,Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Saccharopolyspora, Staphylococcus The bacteria may be, but are not limited to, Bacillus subtilis ...

[0343] In some embodiments, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes, A. rubi), Arthrobacter species (e.g., A. aurescens, A. citreus, A. globformis, A. hydrocarboglutamicus, A. mysores), or a host cell selected from the group consisting of Agrob ... ns, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparaffinus, A. sulfureus, A. ureafaciens)], or Bacillus species (e.g., B. thuringiensis, B. anthracis, B. megateriu, megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii ), Bacillus subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausiai, B. stearothermophilus, and B. amyloliquefaciens. In certain embodiments, the host cell is an industrial Bacillus strain, including, but not limited to, B. subtilis, B. pumilus, B. licheniformis, B. megaterium, B. clausiai, B. stearothermophilus, and B. amyloliquefaciens.In some embodiments, the host cell is an industrial Clostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, C. beijerinckii). In some embodiments, the host cell is an industrial Corynebacterium species (e.g., C. glutamicum, C. acetoacidophilum). In some embodiments, the host cell is an industrial Escherichia species (e.g., E. coli). In some embodiments, the host cell is an industrial Erwinia species (e.g., E. uredovora, E. carotovora, E. (ananas), E. herbicola, E. punctata, E. terreus). In some embodiments, the host cell is an industrial Pantoea species (e.g., P. citrea, P. agglomerans). In some embodiments, the host cell is an industrial Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii). In some embodiments, the host cell is an industrial Streptococcus species (e.g., S. equisimiles, S. pyogenes, S. uberis). In some embodiments, the host cell is an industrial Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungidicus, S. griseus, S. lividans).In some embodiments, the host cell is an industrial Zymomonas species (eg, Z. mobilis, Z. lipolytica).

[0344] The present disclosure is also suitable for use with a variety of animal cell types, including mammalian cells, such as human (including 293, HeLa, WI38, PER.C6 and Bowes melanoma cells), mouse (including 3T3, NS0, NS1, Sp2 / 0), hamster (CHO, BHK), monkey (COS, FRhL, Vero), and hybridoma cell lines.

[0345] The present disclosure is also suitable for use in a variety of plant cell types.

[0346] The term "cell" as used in this application may refer to a single cell or a population of cells, such as a population of cells belonging to the same cell line or lineage. Use of the singular term "cell" should not be construed as specifically referring to a single cell rather than a population of cells.

[0347] The host cell may contain genetic modifications relative to its wild-type counterpart. As a non-limiting example, the host cell (e.g., S. cerevisiae or Y. lipolytica) may be modified to reduce or inactivate one or more of the following genes: hydroxymethylglutaryl-CoA (HMG-CoA) reductase (HMG1), acetyl-CoA C-acetyltransferase (acetoacetyl-CoA thiolase) (ERG10), 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (ERG13), farnesyl-diphosphate farnesyltransferase (squalene synthase) (ERG9), may be modified to overexpress squalene epoxidase, or may be modified to downregulate lanosterol synthase. In some embodiments, the host cell (e.g., S. cerevisiae) may be modified to reduce or inactivate one or more of the following genes: hydroxymethylglutaryl-CoA (HMG-CoA) reductase (HMG1), acetyl-CoA C-acetyltransferase (acetoacetyl-CoA thiolase), 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, farnesyl-diphosphate farnesyltransferase (squalene synthase), squalene epoxidase, or lanosterol synthase. In some embodiments, the host cell may be modified to reduce or inactivate the activity of lanosterol synthase or squalene epoxidase. In some embodiments, squalene epoxidase is encoded by the ERG1 gene. In some embodiments, lanosterol synthase is encoded by the ERG7 gene. In some embodiments, the host cell is modified to reduce or eliminate expression of one or more transporter genes, such as PDR1 or PDR3, and / or the glucanase gene EXG1.

[0348] In some embodiments, the host cell is modified to reduce or inactivate at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 genes.

[0349] In some embodiments, the host cell is modified to reduce or inactivate 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 genes.

[0350] The reduction of gene expression and / or inactivation of gene can be achieved by any suitable method, including but not limited to deleting gene, introducing point mutation into gene, truncating gene, introducing insertion into gene, introducing tag or fusion into gene, or selectively editing gene. For example, polymerase chain reaction (PCR)-based method can be used (see, for example, Gardner et al., Methods Mol Biol. 2014;1205:45-78), or well-known gene editing technology can be used. As a non-limiting example, gene can be deleted by gene replacement (e.g., replacement with marker containing selection marker). Gene can also be truncated by using transposon system (see, for example, Poussu et al., Nucleic Acids Res. 2005;33(12):e104).

[0351] A vector encoding any of the recombinant polypeptides described in this application can be introduced into a suitable host cell using any method known in the art. A non-limiting example of a yeast transformation protocol is described in Gietz et al., Yeast transformation can be conducted by LiAc / SS Carrier DNA / PEG method. Methods Mol Biol. 2006;313:107-20, which is incorporated by reference in its entirety. The host cell can be cultured under any suitable conditions as understood by those skilled in the art. For example, any medium, temperature, culture condition known in the art can be used. In the case of a host cell carrying an inducible vector, the cell can be cultured with an appropriate inducer to promote expression.

[0352] Any of the cells disclosed in the present application can be cultured in any type of medium (rich or minimal) and in any composition before, during, and after contact with and / or incorporation of nucleic acid. The conditions of the culture or culture process can be optimized through routine experimentation, as would be understood by one of ordinary skill in the art. In some embodiments, the selected medium is supplemented with various supplementary components. In some embodiments, the concentrations and amounts of the supplementary components are optimized. In some embodiments, other aspects of the medium and growth conditions (e.g., pH, temperature, etc.) are optimized through routine experimentation. In some embodiments, the frequency with which the medium is supplemented with one or more supplementary components and the time for which the cells are cultured are optimized.

[0353] The cultivation of the cells described in this application can be carried out in any cultivation vessel known and used in the art. In some embodiments, an aerated reaction vessel (e.g., a stirred tank reactor) is used to cultivate the cells. In some embodiments, a bioreactor or fermenter is used to cultivate the cells. Thus, in some embodiments, the cells are used in fermentation. As used in this application, the terms "bioreactor" and "fermenter" are used interchangeably and refer to an enclosure, or partial enclosure, in which biological, biochemical, and / or chemical reactions occur involving living organisms, parts of living organisms, or purified proteins. A "large-scale bioreactor" or "industrial-scale bioreactor" is a bioreactor used to make products on a commercial or semi-commercial scale. Large-scale bioreactors typically have volumes ranging from liters, hundreds of liters, thousands of liters, or more.

[0354] Non-limiting examples of bioreactors include: stirred tank fermenters, bioreactors agitated by rotary mixers, chemostats, bioreactors agitated by shaking devices, airlift fermenters, packed bed reactors, fixed bed reactors, fluidized bed bioreactors, bioreactors using wave induced agitation, centrifuge bioreactors, roller bottles, and hollow fiber bioreactors, roller apparatus (e.g., benchtop, cart mounted, and / or automated), vertical stack plates, spinner flasks, stirred or rocking flasks, shaken multi-well plates, MD bottles, T-flasks, Roux bottles, multi-surface tissue culture growth apparatus, modified fermenters, and coated beads (e.g., beads coated with serum proteins, nitrocellulose, or carboxymethylcellulose to prevent cell attachment).

[0355] In some embodiments, the bioreactor comprises a cell culture system in which the cells (e.g., yeast cells) are in contact with moving liquid and / or bubbles. In some embodiments, the cells or cell cultures are grown in suspension. In other embodiments, the cells or cell cultures are attached to a solid support. Non-limiting examples of support systems include microcarriers (e.g., polymer spheres, microbeads, and microdisks that may be porous or nonporous), crosslinked beads (e.g., dextran) charged with specific chemical groups (e.g., tertiary amine groups), 2D microcarriers containing cells entrapped in nonporous polymer fibers, 3D supports (e.g., support fibers, hollow fibers, multi-cartridge reactors, and semipermeable membranes that may include porous fibers), microcarriers with reduced ion exchange capacity, encapsulated cells, capillaries, and aggregates. In some embodiments, the supports are made of materials such as dextran, gelatin, glass, or cellulose.

[0356] In some embodiments, the industrial scale process is operated in a continuous, semi-continuous, or discontinuous mode. Non-limiting examples of operation modes are batch, fed batch, expanded batch, repeated batch, draw / fill, rotating wall, rotating flask, and / or perfusion operation modes. In some embodiments, the bioreactor allows for continuous or semi-continuous replenishment of a substrate stock, e.g., a carbohydrate source, and / or continuous or semi-continuous separation of product from the bioreactor.

[0357] In some embodiments, the bioreactor or fermenter includes sensors and / or control systems for measuring and / or adjusting reaction parameters. Non-limiting examples of reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type, or cell state, etc.), chemical parameters (e.g., pH, redox potential, concentration of reaction substrates and / or products, dissolved gas concentrations such as oxygen concentration and CO2 concentration, nutrient concentration, metabolite concentration, oligopeptide concentration, amino acid concentration, vitamin concentration, hormone concentration, additive concentration, serum concentration, ionic strength, ion concentration, relative humidity, molar concentration, osmolality, concentration of other chemicals such as buffers, adjuvants, or reaction by-products), physical / mechanical parameters (e.g., density, conductivity, agitation, pressure, and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, and thermodynamic parameters such as temperature, light intensity / quality). Sensors for measuring the parameters described in this application are well known to those skilled in the relevant mechanical and electronic arts. Control systems for adjusting parameters within a bioreactor based on input from the sensors described in this application are well known to those skilled in the art of bioreactor engineering.

[0358] In some embodiments, the method involves batch fermentation (e.g., shake flask fermentation). General considerations for batch fermentation (e.g., shake flask fermentation) include oxygen and glucose levels. For example, batch fermentation (e.g., shake flask fermentation) may be oxygen and glucose limited, so in some embodiments, the ability of the strain in a well-designed fed-batch fermentation is underestimated. Also, the end product (e.g., isoprenoid precursor or isoprenoid) may show some differences from the substrate (e.g., isoprenoid precursor or isoprenoid) in terms of solubility, toxicity, cellular accumulation and secretion, and in some embodiments, may have different fermentation kinetics.

[0359] The methods described in this application encompass the production of precursors and isoprenoids using recombinant cells, cell lysates, or isolated recombinant polypeptides (e.g., lanosterol synthase, squalene epoxidase, MEV pathway enzymes, MEP pathway enzymes, squalene synthase, prenyltransferase, terpene synthase, and any of the proteins related to this disclosure).

[0360] The isoprenoid precursors and isoprenoids produced by any of the recombinant cells disclosed in this application can be identified and extracted using any method known in the art. Mass spectrometry (e.g., LC-MS, GC-MS) is a non-limiting example of a method for identification and can be used to aid in the extraction of the compound of interest.

[0361] In some embodiments, a host cell comprising one or more proteins described herein (e.g., lanosterol synthase, MEV pathway enzymes, MEP pathway enzymes, squalene epoxidase, squalene synthase, prenyltransferase, terpene synthase, and / or any protein associated with the present disclosure) has a cytotoxicity of at least 0.005 mg / L, at least 0.01 mg / L, at least 0.02 mg / L, at least 0.03 mg / L, at least 0.04 mg / L, at least 0.05 mg / L, at least 0.06 mg / L, at least 0.08 mg / L, at least 0.09 mg / L, at least 0.10 mg / L, at least 0.11 mg / L, at least 0.12 mg / L, at least 0.13 mg / L, at least 0.14 mg / L, at least 0.15 mg / L, at least 0.16 mg / L, at least 0.17 mg / L, at least 0.18 mg / L, at least 0.19 mg / L, at least 0.20 mg / L, at least 0.21 mg / L, at least 0.22 mg / L, at least 0.23 mg / L, at least 0.24 mg / L, at least 0.25 mg / L, at least 0.26 mg / L, at least 0.27 mg / L, at least 0.28 mg / L, at least 0.29 mg / L, at least 0.30 mg / L, at least 0.31 mg / L, at least 0.32 mg / L, at least 0.33 mg / L, at least 0.34 mg / L, at least 0.35 mg / L, at least 0.36 mg / L, at least 0.37 mg / L, at least 0.38 mg / L, at at least 0.07mg / L, at least 0.08mg / L, at least 0.09mg / L, at least 0.1mg / L, at least 0.2mg / L, at least 0.3mg / L, at least 0.4mg / L, at least 0.5mg / L, at least 0.6mg / L, at least 0.7mg / L, at least 0.8mg / L, at least 0.9mg / L, at least 1mg / L, at least 2mg / L, at least 3mg / L, at least 4mg / L, at least 5mg / L, at least 6mg / L, at least 7mg / L, at least 8mg / L, at least at least 9 mg / L, at least 10 mg / L, at least 11 mg / L, at least 12 mg / L, at least 13 mg / L, at least 14 mg / L, at least 15 mg / L, at least 16 mg / L, at least 17 mg / L, at least 18 mg / L, at least 19 mg / L, at least 20 mg / L, at least 21 mg / L, at least 22 mg / L, at least 23 mg / L, at least 24 mg / L, at least 25 mg / L, at least 26 mg / L, at least 27 mg / L, at least 28 mg / L, at least 29 mg / L, at least at least 30mg / L, at least 31mg / L, at least 32mg / L, at least 33mg / L, at least 34mg / L, at least 35mg / L, at least 36mg / L, at least 37mg / L, at least 38mg / L, at least 39mg / L, at least 40mg / L, at least 41mg / L, at least 42mg / L, at least 43mg / L, at least 44mg / L, at least 45mg / L, at least 46mg / L, at least 47mg / L, at least 48mg / L, at least 49mg / L, at least 50mg / L,at least 51mg / L, at least 52mg / L, at least 53mg / L, at least 54mg / L, at least 55mg / L, at least 56mg / L, at least 57mg / L, at least 58mg / L, at least 59mg / L, at least 60mg / L, at least 61mg / L, at least 62mg / L, at least 63mg / L, at least 64mg / L, at least 65mg / L, at least 66mg / L, at least 67mg / L, at least 68mg / L, at least 69mg / L, at least 70mg / L, at least 75mg / L mg / L, at least 80 mg / L, at least 85 mg / L, at least 90 mg / L, at least 95 mg / L, at least 100 mg / L, at least 125 mg / L, at least 150 mg / L, at least 175 mg / L, at least 200 mg / L, at least 225 mg / L, at least 250 mg / L, at least 275 mg / L, at least 300 mg / L, at least 325 mg / L, at least 350 mg / L, at least 375 mg / L, at least 400 mg / L, at least 425 mg / L, at least 450 mg / L, at least 475mg / L, at least 500mg / L, at least 1,000mg / L, at least 2,000mg / L, at least 3,000mg / L, at least 4,000mg / L, at least 5,000mg / L, at least 6,000mg / L, at least 7,000mg / L, at least 8,000mg / L, at least 9,000mg / L, at least 10,000mg / L, at least 11g / L, at least 12g / L, at least 13g / L, at least 14g / L, at least 15g / L, at least 16g / L, at least 17g / L, at least 18g / L, at least 19g / L, at least 20g / L, at least 21g / L, at least 22g / L, at least 23g / L, at least 24g / L, at least 25g / L, at least 26g / L, at least 27g / L, at least 28g / L, at least 29g / L, at least 30g / L, at least 31g / L, at least 32g / L, at least 33g / L, at least 34g / L, at least 35g / L, at least 36g / L, at least 37g / L, at least 38g / L, at least 39g / L,at least 40g / L, at least 41g / L, at least 42g / L, at least 43g / L, at least 44g / L, at least 45g / L, at least 46g / L, at least 47g / L, at least 48g / L, at least 49g / L, at least 50g / L, at least 51g / L, at least 52g / L, at least 53g / L, at least 54g / L, at least 55g / L, at least 56g / L, at least 57g / L , at least 58g / L, at least 59g / L, at least 60g / L, at least 61g / L, at least 62g / L, at least 63g / L, at least 64g / L, at least 65g / L, at least 66g / L, at least 67g / L, at least 68g / L, at least 69g / L, at least 70g / L, at least 75g / L, at least 80g / L, at least 85g / L, at least 90g / L, at least 95g / L L, at least 100g / L, at least 125g / L, at least 150g / L, at least 175g / L, at least 200g / L, at least 225g / L, at least 250g / L, at least 275g / L, at least 300g / L, at least 325g / L, at least 350g / L, at least 375g / L, at least 400g / L, at least 425g / L, at least 450g / L, at least 475g / L, at least At least 500 g / L, at least 1,000 g / L, at least 2,000 g / L, at least 3,000 g / L, at least 4,000 g / L, at least 5,000 g / L, at least 6,000 g / L, at least 7,000 g / L, at least 8,000 g / L, at least 9,000 g / L, or at least 10,000 g / L of one or more isoprenoids and / or isoprenoid precursors. In some embodiments, the isoprenoid precursor is mevalonate.

[0362] In some embodiments, the host cell comprises one or more enzymes of a yeast mevalonate pathway and a heterologous polynucleotide encoding a lanosterol synthase with reduced activity compared to a control lanosterol synthase; or a heterologous polynucleotide that reduces lanosterol synthase activity; and / or a heterologous polynucleotide encoding a squalene epoxidase with reduced activity compared to a control squalene epoxidase; or a heterologous polynucleotide that reduces squalene epoxidase activity. In some embodiments, the one or more enzymes of the yeast mevalonate pathway are selected from the enzymes listed in Table 1.

[0363] In some embodiments, the host cell comprises one or more enzymes in the archaeal mevalonate I pathway and a heterologous polynucleotide encoding a lanosterol synthase with reduced activity compared to a control lanosterol synthase; or a heterologous polynucleotide that reduces lanosterol synthase activity; and / or a heterologous polynucleotide encoding a squalene epoxidase with reduced activity compared to a control squalene epoxidase; or a heterologous polynucleotide that reduces squalene epoxidase activity. In some embodiments, the one or more enzymes of the archaeal mevalonate I pathway are selected from the enzymes listed in Table 2.

[0364] In some embodiments, the host cell comprises one or more enzymes in an archaeal type II mevalonate pathway and a heterologous polynucleotide encoding a lanosterol synthase with reduced activity compared to a control lanosterol synthase; or a heterologous polynucleotide that reduces lanosterol synthase activity; and / or a heterologous polynucleotide encoding a squalene epoxidase with reduced activity compared to a control squalene epoxidase; or a heterologous polynucleotide that reduces squalene epoxidase activity. In some embodiments, the one or more enzymes in the archaeal type II mevalonate pathway are selected from the enzymes listed in Table 3.

[0365] In some embodiments, the host cell comprises one or more enzymes in the MEP pathway and a heterologous polynucleotide encoding a lanosterol synthase with reduced activity compared to a control lanosterol synthase; or a heterologous polynucleotide that reduces lanosterol synthase activity; and / or a heterologous polynucleotide encoding a squalene epoxidase with reduced activity compared to a control squalene epoxidase; or a heterologous polynucleotide that reduces squalene epoxidase activity. In some embodiments, the one or more enzymes of the MEP pathway are selected from the enzymes listed in Table 4.

[0366] The phraseology and terminology used in this application are for purposes of description and should not be regarded as limiting. The use of terms such as "including," "comprising," "having," "containing," "involving," and / or variations thereof in this application is meant to encompass the items listed thereafter and equivalents thereof, as well as additional items.

[0367] The present invention is further illustrated by the following examples, which should not be construed as further limiting in any way. The entire contents of all documents cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference. EXAMPLES

[0368] [Example 1] Identification of lanosterol synthase with reduced activity This example describes the identification of a lanosterol synthase with reduced activity.

[0369] Mutagenic PCR was performed with the ERG7 template, and the PCR mixture was digested with BsaI and ligated into pERG7.NatR digested with HindIII and NcoI to generate libraries of mutants ranging from low (2-4 mutations per gene), medium (6-9 mutations per gene), to high (12-20 mutations per gene).Cleavage of these plasmids with PacI and SspI and introduction into a Yarrowia strain (genotype pTEF-HMGt erg7Δ13[GPR1-1 ERG7 HygR]) yielded plates of nourseothricin-resistant (NatR) transformants (grown at 22°C or 30°C) that were replica plated onto YNBAc (YNB + 30 mM glacial acetic acid) at the appropriate temperature. 372 acetate-resistant (AcR) clones were identified, picked on YPD medium, grown at the appropriate temperature, then plated on YPD4 medium, grown at 30°C for 3 days, and the supernatants were assayed for mevalonate by LC-RIA. AcR cells can grow on medium containing acetate. Concurrently, clones originally grown at 22°C were tested for temperature-sensitive growth at 32°C, while those grown at 30°C were tested for cold sensitivity at 18°C.

[0370] As shown in Table 7 and Figure 3, nine temperature-sensitive (Ts) and three partially cold-sensitive (Cs) clones were identified that had increased mevalonate titers relative to the parent. These strains were 1A3, 2F9, 2F6, 2C5, 2B3, 2A5, 2F1, 3B9 and 3D11. Of the strains tested, 2F6, which has the lanosterol synthase shown in SEQ ID NO:3, showed the highest mevalonate titers. 4A6 and 4F11 have the same mutation. Strains not designated as Ts or Cs are neither temperature-sensitive nor cold-sensitive.

[0371] [Table 7]

[0372] Many of the mevalonate-efflux ERG7 alleles significantly perturbed the steady-state levels of other metabolites; 2F6 specifically decreased squalene and increased oxidosqualene, dioxidosqualene, and ergosterol. [Example 2]

[0373] Characterization of acetoacetyl-CoA synthetase that enhances squalene production in Yarrowia host cells This example describes the characterization of the effect of acetoacetyl-CoA synthase on squalene production in a host cell. An acetoacetyl-CoA synthase containing SEQ ID NO:6 and encoded by SEQ ID NO:7 was constructed. Various constructs were constructed, each expressing acetoacetyl-CoA synthase under the control of a different promoter. The constructs were then randomly inserted into a Yarrowia host cell strain that produced approximately 17.2 mg / L of squalene. As shown in Table 8, acetoacetyl-CoA synthase (represented by SEQ ID NOs:6 and 7) increased the squalene titer to approximately 23.8-33 mg / L.

[0374] [Table 8]

[0375] Several of the nphT7 cassettes also induced extremely high mevalonate secretion, up to 5 g / L, corresponding to a significant fraction of the theoretical yield. [Example 3]

[0376] Cucurbitadienol production in ERG7 mutant host cells This example describes the characterization of cucurbitadienol synthase (CDS) in various Yarrowia host cells, including mutants of SEQ ID NO:1.

[0377] The pERG7-NatR plasmid was used to generate acetate-resistant (AcR) cells as in Example 1, resulting in clones with high mevalonate titers. AcR cells can grow in medium containing acetate. Constructs encoding specific CDSs were randomly inserted into these cells. All strains except strains 887779 and 870688 express AquAgaCDS16 (SEQ ID NOs: 226 and 327). Strains 887779 and 870688 express SgCDS1 (SEQ ID NOs: 256 and 332). Strains 950910 and 950917 also express NphT7 (SEQ ID NO: 6). The resulting nourseothricin-resistant (NatR) isolates were picked and grown in 0.5 mL YPD medium in 96-deep-well plates at 30° C. for 2 days, subcultured into 0.5 mL YPD10 medium at 30° C. for 4 days, and the cultures were then assayed by GC-MS for cucurbitadienol. Nourseothricin resistance allows for the selection of cells containing heterologous nucleic acid encoding the CDS. Strain 870688 containing SEQ ID NO:1 was used as a control.

[0378] As shown in Table 9 and FIG. 4, the cucurbitadienol titers of Yarrowia strains containing the mutant lanosterol synthase are significantly greater than the strain containing SEQ ID NO:1.

[0379] Selection of strains was then performed in ambr 250 bioreactors and assayed for cucurbitadienol, ergosterol and lanosterol by GC-MS and mevalonate by HPLC. Strain 887779 containing SEQ ID NO:1 was used as a control. As shown in FIG. 5 and Tables 10A-10B, Yarrowia strains with mutant lanosterol synthase alleles accumulate less lanosterol and more mevalonate and cucurbitadienol relative to strains containing wild-type lanosterol synthase containing SEQ ID NO:1.

[0380] [Table 9]

[0381] [Table 10]

[0382] [Table 11] [Example 4]

[0383] Production of oxidosqualene in Saccharomyces cerevisiae host cells carrying the mutant of SEQ ID NO:313 This example describes the identification of a lanosterol synthase with reduced activity using SEQ ID NO:313 as a mutational template.

[0384] Three different temperature sensitive lanosterol synthase mutants were tested and host cells containing each of these lanosterol synthase mutants were analyzed for glucose consumption and for the production of oxidosqualene, mevalonate, ergosterol and ethanol. The parent strain with the native lanosterol synthase (SEQ ID NO: 313) was used as a negative control.

[0385] Strain 756247 expressed a lanosterol synthase comprising the protein sequence of SEQ ID NO: 100. The nucleotide sequence encoding SEQ ID NO: 100 contains the following mutations relative to SEQ ID NO: 8 (mutations in SEQ ID NO: 100 relative to SEQ ID NO: 313 are shown in brackets): C361T (P121S), C407T (A136V), G474A (silent), A898G (S300G), A909G (silent), T965G (V322G), A1312G (K438E), T1506A (F502L), T1732C (silent), A1882G (K628E) and T2178G (Y726*-truncating mutation). Silent mutations do not result in a change in the amino acid sequence.

[0386] Strain 756248 expressed a lanosterol synthase comprising the protein sequence of SEQ ID NO: 101. The nucleotide sequence encoding SEQ ID NO: 101 contains the following mutations relative to SEQ ID NO: 8 (mutations in SEQ ID NO: 101 relative to SEQ ID NO: 313 are shown in brackets): C333T (silent), A803G / A804T (K268S), A841G (T281A), T1504C (F502L), C1811A (T604N), G1966A (A656T) and A2078G (E693G).

[0387] Strain 756249 expressed a lanosterol synthase comprising the protein sequence of SEQ ID NO: 102. The nucleotide sequence encoding SEQ ID NO: 102 contains the following mutations relative to SEQ ID NO: 8 (mutations in SEQ ID NO: 102 relative to SEQ ID NO: 313 are shown in brackets): A190G (R64G), A358G (I120V), G678T (M226I), T823A (F275I), A997G (T333A) and T1855A (C619S).

[0388] To measure 2-3-oxidosqualene production, strains were first grown overnight at 30° C., diluted to a starting OD of 0.2, and grown for an additional 16 h in triplicate in 96-well deep-well plates at either 30° C. or 35° C. The cell culture volume was 500 μL, and the medium used for this experiment was YPD (10 g / L yeast extract, 20 g / L pectone, and 20 g / L dextrose).

[0389] 400 μL of ethyl acetate containing 200 μL of culture and internal standards (100 μm tridecane and 100 mg / L pregnenolone) were transferred to a 96-well deep-well plate containing 100 μL of silica / zirconia beads (0.5 mm diameter, catalog number 11079105z Biospec) per well. The plate containing the samples was heat sealed and stirred at 1750 rpm for 5 minutes using a Genogrinder. The plate was then centrifuged at 4000 rpm for 10 minutes at 4°C to separate the aqueous and organic layers. The plate was then stored at -30°C for 2 hours to freeze the aqueous layer, and 100 μL from the top layer was transferred to a glass vial and analyzed by GC-FID. A gas chromatograph (Thermo Scientific Trace 1310) equipped with a TG-5MS column (15 m x 0.25 mm x 0.25 μm) was used at a flow rate of 1.5 mL / min. Elutants were determined by comparing peak retention times with those of known standards and amounts quantified by comparing analyte peak areas to those of standards of known concentration.

[0390] As shown in FIG. 7 and Table 11, at 30° C., Saccharomyces cerevisiae host cells containing any one of SEQ ID NOs: 100-102 produced less ergosterol than the parent strain (negative control) and the lanosterol synthase containing any one of SEQ ID NOs: 100-102 was less active compared to the wild-type lanosterol synthase containing SEQ ID NO: 313 at this temperature, indicating impaired lanosterol synthase activity. At 30° C., 5-10 mg / L oxidosqualene was detected in all three lanosterol synthase mutant strains, while the control strain did not produce detectable levels of oxidosqualene (FIG. 6 and Table 11). Thus, host cells with reduced lanosterol synthase activity showed increased oxidosqualene production.

[0391] At 35°C, the lanosterol synthase mutant strains were unable to grow or could only grow minimally compared to the control strain, as indicated by the residual glucose counts (Figure 8 and Table 12). The starting glucose concentration for all strains was 20 g / L. Without wishing to be bound by any particular theory, it is possible that since the lanosterol synthase mutants are temperature sensitive, the cells cannot survive at high temperatures in the absence of a functional lanosterol synthase containing SEQ ID NO:313. Only strain 756249 accumulated some oxidosqualene at 35°C. The control strain with the native lanosterol synthase gene encoding SEQ ID NO:313 was able to consume all the glucose at 30°C and 35°C, but did not produce detectable levels of oxidosqualene. Thus, the results suggest that a complete knockout of lanosterol synthase activity is detrimental to these cells.

[0392] [Table 12]

[0393] [Table 13]

[0394] Table 13. Non-limiting examples of amino acid changes relative to SEQ ID NO:1* [Table 14] [Table 15] *Indicates disconnection

[0395] Table 14. Non-limiting examples of amino acid changes relative to SEQ ID NO: 313* [Table 16] *Indicates a truncation resulting in the deletion of residues 726-731 in SEQ ID NO:313

[0396] Table 15. Non-limiting examples of lanosterol synthase sequences [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30] [Table 31] [Table 32] [Table 33]

Table 34

Table 35

Table 36

Table 37

Table 38

Table 39

Table 40

Table 41

Table 42

Table 43

Table 44

Table 45

Table 46

Table 47

Table 48

Table 49

Table 50

Table 51

Table 52

Table 53

Table 54

Table 55

Table 56

Table 57

Table 58

Table 59

Table 60

Table 61

Table 62

Table 63

Table 64

Table 65

Table 66

Table 67

Table 68

[0397] Table 16. Additional enzyme sequences relevant to the present disclosure [Table 78] [Table 79] [Table 80] [Table 81] [Table 82] [Table 83] [Table 84] [Table 85] [Table 86] [Table 87]

[0398] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

[0399] All references disclosed in this application, including patent documents, are incorporated by reference in their entirety, specifically with respect to the disclosures referenced in this application.

Claims

[Claim 1] 23. The invention substantially as herein described and illustrated with reference to the examples and drawings.