Patent Application
20230313249
Artemisinin was identified as the principal compound in Artemisia annua extract with anti-malarial activity. Its structure was determined to be a sesquiterpene lactone with an endoperoxide bridge. In addition to their anti-malarial properties, artemisinin and its derivatives can be cytotoxic for cancer cells. Artemisinin levels in A. annua are usually in the range of 0.01 to 1% of total dry weight, which together with the fact that complete chemical synthesis of artemisinin is complex and inefficient at least partially accounts for the drug's high price. While methods for chemical synthesis of artemisinin are known, they are not an economical alternative for isolation of artemisinin from the plant. Given the commercial value of artemisinin, cost effective, scalable, and/or sustainable processes for its production are needed.
In various aspects, the present disclosure provides methods and compositions for producing artemisinin, as well as dihydroartemisinic acid (DHAA) and artemisinic acid (AA) (immediate precursors for artemisinin). In various aspects, the present disclosure provides enzymes, polynucleotides encoding said enzymes, and recombinant microbial host cells (or microbial host strains) for the production of artemisinin, DHAA, or AA. The present disclosure further provides methods of making products containing artemisinin.
In some embodiments, the present disclosure provides a microbial host cell expressing an enzyme pathway catalyzing the conversion of farnesyl diphosphate (FPP) to artemisinin, DHAA, or AA, the enzymatic pathway comprising an amorphadiene synthase enzyme, an amorphadiene oxidase enzyme, and a double bond reductase. In some embodiments, the enzymatic pathway further comprises a peroxidase or an alpha-ketoglutarate-dependent dioxygenase enzyme capable of catalyzing conversion of artemisinic acid or dihydroartemisinic acid to artemisinin. In these embodiments, the microbial cells can synthesize artemisinin, DHAA, or AA product from any suitable carbon source. In some embodiments, the enzymes described herein enable high yield production of artemisinin, DHAA, or AA.
In some embodiments, the microbial host cell is prokaryotic or eukaryotic, and may be a bacterium or yeast. In some embodiments, the microbial host cell further expresses or overexpresses one or more enzymes in the methylerythritol phosphate (MEP) and/or the mevalonic acid (MVA) pathway to catalyze the conversion of glucose or other carbon sources to isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP). In some embodiments, the microbial host cell further expresses an enzyme catalyzing the conversion of IPP and/or DMAPP to farnesyl diphosphate (FPP), allowing for artemisinin, DHAA or AA to be produced from sugar or other carbon sources (carbon substrates such as C1, C2, C3, C4, C5, and/or C6 carbon substrates). In some embodiments, the host cell is a bacterium engineered to increase carbon flux through the MEP pathway.
In some embodiments, the microbial host cell expresses an amorphadiene oxidase, which may be a P450 enzyme providing for oxygenation of amorphadiene substrate. In some embodiments, the amorphadiene oxidase converts amorphadiene to artemisinic acid through three oxygenation events with artemisinic alcohol and artemisinic aldehyde as intermediates. In some embodiments, the cell further expresses at least one cytochrome P450 reductase to support P450 enzyme activity.
In some embodiments, the microbial host cell further expresses one or more alcohol dehydrogenases that convert artemisinic alcohol to artemisinic aldehyde. In some embodiments, the microbial host cell further expresses one or more aldehyde dehydrogenases that convert artemisinic aldehyde to artemisinic acid. In some embodiments, AA is recovered from the culture.
In some embodiments, the microbial host cell further expresses one or more double bond reductases converting artemisinic acid to dihydroartemisinic acid (DHAA). In some embodiments, DHAA is recovered from the culture, which can be converted to artemisinin. In still other embodiments, artemisinin is recovered from the culture.
In further embodiments, the microbial cell comprises or further comprises one or more peroxidase or alpha ketoglutarate-dependent dioxygenases (e.g., from A. annua or a derivative enzyme thereof) capable of converting artemisinic acid or dihydroartemisinic acid to artemisinin. For example, in some embodiments, the host cell expresses the entire enzymatic pathway for biosynthesis of artemisinin from C1-C6 carbon substrates, such as glucose or glycerol. In still other embodiments, the host cell expresses the one or more peroxidase and alpha ketoglutarate-dependent dioxygenase enzyme to allow for whole cell or cell lysate bioconversion of fed substrate (e.g., AA or DHAA). In still other embodiments, the one or more peroxidase and alpha ketoglutarate-dependent dioxygenase enzymes described herein are used in purified recombinant form for conversion of AA or DHAA to artemisinin in an in vitro reaction system.
Other aspects and embodiments of the invention will be apparent from the following detailed disclosure.
Artemisinin (
A biosynthetic pathway for artemisinin is shown in
In various aspects, the present disclosure provides enzymes (including variants thereof) related to biosynthesis of artemisinin (or its intermediate precursors DHAA or AA), microbial host cells (or microbial host strains) comprising such enzymes, methods for producing dihydroartemisinic acid (DHAA), AA or artemisinin, and methods of making pharmaceutical products containing these compounds. In some aspects, the present invention provides polynucleotides encoding for the enzymes disclosed herein.
In embodiments, the present disclosure provides a microbial host cell expressing a biosynthetic pathway catalyzing the conversion of farnesyl diphosphate (FPP) to AA, DHAA, or artemisinin. In various embodiments, the biosynthetic pathway comprises an amorphadiene synthase (ADS), an amorphadiene oxidase (AO), and a double bond reductase enzyme. In some embodiments, the microbial host cell may further comprise one or more alcohol dehydrogenase enzymes (ADH) and aldehyde dehydrogenase (ALDH) enzymes. In some embodiments, the microbial host cell may further comprise one or more peroxidases capable of converting artemisinic acid (AA) or dihydroartemisinic acid (DHAA) to artemisinin. In some embodiments, the microbial host cell may further comprise one or more alpha-ketoglutarate-dependent dioxygenases capable of converting artemisinic acid (AA) or dihydroartemisinic acid (DHAA) to artemisinin. In some embodiments, one or more enzymes are engineered for productivity, stability, and or expression in the microbial cell resulting in a high production of AA, DHAA, or artemisinin. In these embodiments, the microbial cells can synthesize AA, DHAA or artemisinin product from any suitable carbon source.
One aspect of the present invention is related to a microbial host cell for producing AA, DHAA, or artemisinin, the microbial cell expressing a biosynthetic pathway comprising: a heterologous enzyme having an amorphadiene synthase activity (ADS), a heterologous enzyme having an amorphadiene oxidase activity (AO), and a heterologous enzyme having a double bond reductase activity (DBR). DHAA is the immediate precursor of artemisinin, and its transformation to artemisinin has been shown to occur spontaneously through photo-oxidation, without enzyme intervention (Sy et al., The mechanism of the spontaneous autoxidation of dihydroartemisinic acid, Tetrahedron, vol. 58, 2002, 897-908 and Wallart et al., Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin, J. Nat. Prod., vol. 62, 1999, 430-433, which are hereby incorporated by reference in their entirety).
Amorphadiene synthase refers to a terpene synthase that catalyzes formation of amorphadiene from farnesyl diphosphate (FPP), as shown in
In some embodiments, the ADS comprises the amino acid sequence of SEQ ID NO: 1, 2, or a variant thereof. In some embodiments, the ADS enzyme comprises an amino acid sequence that has 50% or more sequence identity with SEQ ID NO: 1 or 2. In some embodiments, the ADS enzyme comprises an amino acid sequence that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the ADS enzyme includes from 1 to about 20, or from 1 to about 10, or from 1 to about 5 amino acid modifications independently selected from substitutions, deletions, and insertions to the amino acid sequence of SEQ ID NO: 1 or 2.
In some embodiments, the ADS enzyme comprises a substitution to one or more of the substrate binding site or active site, as compared to the wild type enzyme. In some embodiments, the amino acid modifications can be selected to improve one or more of the following properties in the microbial cell: enzyme productivity, selectivity for the desired substrate and/or product, stability, temperature tolerance, and expression.
In some embodiments, the ADS enzyme comprises one or more amino acid substitutions at positions shown in
In some embodiments, the ADS enzyme comprises an amino acid substitution at one or more positions (e.g., from 1 to 20, or from 1 to 10, or from 1 to 5) corresponding to the following positions of SEQ ID NO: 1: 396, 104, 162, 474, 118, 363, 322, 173, 112, 431, 151, 291, 134, 341, 230, 245, 44, 385, 100, 469, 500, 292, 471, 207, 463, 189, 340, 510, 260, 247, 211, 430, 277, 318, 275, 170, 124, 125, 145, 169, 445, 155, 152, 507, 520, 393, 447, 455, 498, 409, 204, and 261. In some embodiments, the ADS enzyme comprises one or more substitutions (e.g., from 1 to 20, or from 1 to 10, or from 1 to 5) selected from the following substitutions numbered according to SEQ ID NO: 1: V396A, S104A, D162E, Y474E, T118S, G363A, S322D, I173S, K112Q, L431I, S151H, A291V, Q134E, M341L, E230D, V245I, K44E, H385Y, G100L, N469G, I500V, V292I, N471S, A207R, S463C, I189V, F340L, Y510N, C260F, K247R, P211S, P430K, Y277F, V318I, S275V, R170H, N124K, Y125F, E145S, S169T, L445I, P155H, M152L, A507R, A520K, D393M, T447S, K455G, K498T, C409S, I204L, and L261A. In some embodiments, the ADS enzyme comprises at least two, at least three, at least four, or at least five amino acid substitutions selected from V396A, S104A, D162E, Y474E, T118S, G363A, S322D, I173S, K112Q, L431I, S151H, A291V, Q134E, M341L, E230D, V245I, K44E, H385Y, G100L, N469G, I500V, V292I, N471S, A207R, S463C, I189V, F340L, Y510N, C260F, K247R, P211S, P430K, Y277F, V318I, S275V, R170H, N124K, Y125F, E145S, S169T, L445I, P155H, M152L, A507R, A520K, D393M, T447S, K455G. K498T, C409S, I204L, and L261A numbered according to SEQ ID NO: 1. In some embodiments, the ADS enzyme comprises the substitutions T118S, D162E, I173S, S322D, G363A, V396A, and Y474E numbered according to SEQ ID NO: 1.
In some embodiments, the ADS enzyme exhibits increased production of amorphadiene in a microbial cell producing farnesyl diphosphate as compared to ADS comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the ADS enzyme is isolated or partially purified, or is heterologously expressed in a host cell. Some embodiments of the present invention are related to recombinant nucleic acid molecules comprising a nucleotide sequence encoding the ADS enzyme described herein. The recombinant nucleic acid could be a part of extrachromosomal elements such as plasmids, or bacterial artificial chromosomes, or may be chromosomally integrated.
In some embodiments, the present invention is related to amorphadiene oxidase (AO) or a variant thereof. In other embodiments, the present invention is related to microbial cells that express AO or its variants as described herein. Artemisia annua Amorphadiene oxidase, also known as CYP71AV1, is a cytochrome P450 (CYP450) that can provide for three-step oxidation of amorphadiene, along with its cognate reductase, to artemisinic acid.
CYP450 enzymes are involved in the formation (synthesis) and breakdown (metabolism) of various molecules and chemicals within cells. CYP450 enzymes have been identified in all kingdoms of life (i.e., animals, plants, fungi, protists, bacteria, archaea, and even in viruses). Illustrative structure and function of CYP450 enzymes are described in Uracher et al., TRENDS in Biotechnology, 24(7): 324-330 (2006). In some embodiments, the P450 enzymes are engineered to have a deletion of all or part of the wild type N-terminal transmembrane region, and the addition of a transmembrane domain derived from a bacterial inner membrane cytoplasmic C-terminus protein. In various embodiments, the transmembrane domain is a single-pass transmembrane domain. See U.S. Patent Publication No. 2018/0251738, which is hereby incorporated by reference in its entirety. In various embodiments, the transmembrane domain (or “N-terminal anchor”) is derived from an E. coli gene (or ortholog thereof) selected from waaA, ypfN, yhcB, yhbM, yhhm, zipA, ycgG, djlA, sohB, lpxK, F11O, motA, htpx, pgaC, ygdD, hemr, and ycls. These genes were identified as inner membrane cytoplasmic C-terminus proteins through bioinformatics prediction as well as experimental validation. The AO enzyme may employ an N-terminal anchor sequence that is a derivative of the E. coli wild-type transmembrane domain that has one or more (e.g., one, two, or three) mutations (e.g., amino acid substitutions) with respect to the wild-type sequence. Engineered P450 enzymes in accordance with these embodiments are described in U.S. Patent Publication No. 2018/0251738, which is hereby incorporated by reference in its entirety.
In some embodiments, the AO enzyme comprises an amino acid sequence of SEQ ID NO: 3 or a variant thereof. In embodiments, the AO enzyme comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof. In some embodiments, the AO enzyme comprises an amino acid sequence that has 50% or more sequence identity with SEQ ID NO: 3 or 4. In some embodiments, the AO enzyme comprises an amino acid sequence that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least or about 99% sequence identity with the amino acid sequence of SEQ ID NO: 3 or 4. In some embodiments, the AO enzyme comprises an amino acid sequence that includes from 1 to about 20, or from 1 to about 10, or from 1 to about 5 amino acid modifications independently selected from substitutions, deletions, and insertions to an amino acid sequence of SEQ ID NO: 3 or 4.
In some embodiments, the AO enzyme comprises a substitution to one or more of the substrate binding site or active site. In some embodiments, modifications to enzymes can be informed by available structures including construction of a homology model.
In some embodiments, the amino acid modifications can be selected to improve one or more properties upon expression in the microbial cell, such as those selected from: enzyme productivity, selectivity for the desired substrate and/or product, stability, temperature tolerance, and expression. In some embodiments, selection and modification of enzymes is informed by assaying activity on AD substrate or by expression in microbial cells producing AD.
In some embodiments, the AO enzyme comprises one or more amino acid substitutions at positions shown in
In some embodiments, the AO enzyme comprises an amino acid substitution at one or more positions (e.g., from 1 to 20, or from 1 to 10, or from 1 to 5) corresponding to the following positions of SEQ ID NO: 3: 239, 257, 408, 410, 421, 320, 130, 489, 198, 119, 102, 186, 252, 294, 314, 457, 474, 319, 322, 390, 125, 155, 251, 445, 424, 250, 387, 462, 153, 151, 243, 308, 495, 73, 103, 59, 123, 124, 146, 256, 261, 369, 469, and 64. In some embodiments, the AO enzyme comprises one or more (e.g., from 1 to 20, or from 1 to 10, or from 1 to 5) substitutions selected from the following substitutions numbered according to SEQ ID NO:3: A239R, A257D, A408P, A410E, A421I, C320N, E130D, E489D, G198K, H119G, I102L, I186T, I252L, I294V, I314M, I457L, I474L, K319R, K322R, K390R, L125F, L155I, L25II, L445F, M424K, N250R, N387A, N462D, Q153R, S151Q, S243K, S308T, S495T, S73P, T103A, T59L, V123I, V124A, V146T, V256I, V261E, V369L, V469M, and V64L. In some embodiments, the AO enzyme comprises at least two, at least three, at least four, or at least five amino acid substitutions selected from A239R, A257D, A408P, A410E, A421I, C320N, E130D, E489D, G198K, H119G, I102L, I186T, I252L, I294V, I314M, I457L, I474L, K319R, K322R, K390R, L125F, L155I, L25II, L445F, M424K, N250R, N387A, N462D, Q153R, S151Q, S243K, S308T, S495T, S73P, T103A, T59L, V123I, V124A, V146T, V256I, V261E, V369L, V469M, and V64L numbered according to SEQ ID NO: 3. In some embodiments, the AO enzyme comprises V64L, S73P, L155I, C320N, K322R, and V369L substitutions numbered according to SEQ ID NO: 3.
In some embodiments, the AO enzyme exhibits increased production of artemisinic alcohol (A-OH) as compared to AO comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the AO enzyme exhibits increased production of artemisinic aldehyde (A-CHO) as compared to AO comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the AO enzyme exhibits increased production of artemisinic acid (AA) as compared to AO comprising the amino acid sequence of SEQ ID NO: 3.
In some embodiments, the AO or variant thereof, as described herein, is isolated or partially purified, or is heterologously expressed in a host cell. Some embodiments of the present invention are related to recombinant nucleic acid molecules comprising a nucleotide sequence encoding the AO or variant thereof described herein. The recombinant nucleic acid could be a part of extrachromosomal elements such as plasmids, or bacterial artificial chromosomes, or may be chromosomally integrated.
In some embodiments, the AO or its variant comprises a leader sequence that supports expression and activity in E. coli, a linker sequence, or a CPR or derivative thereof sufficient to regenerate the AO variant. In embodiments, the AO or its variant requires the presence of an electron transfer protein capable of transferring electrons to the CYP450 protein. In some embodiments, this electron transfer protein is a cytochrome P450 reductase (CPR), which can be expressed by the microbial host cell. Various reductases that may be used are described in U.S. Patent Publication No. 2018/0135081, which is hereby incorporated by reference in its entirety.
Exemplary cytochrome P450 reductase enzymes (CPR), which may be used in the present invention, include that shown herein as SEQ ID NO: 5, or a variant thereof. Variants generally include enzymes comprising an amino acid sequence that has 50% or more sequence identity with of SEQ ID NO: 5. In some embodiments, the P450 reductase enzyme comprises an amino acid sequence that has at least about 60% sequence identity, or at least about 70% sequence identity, or at least about 80% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence identity, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the P450 reductase enzyme comprises an amino acid sequence that includes from 1 to about 20, or from 1 to about 10, or from 1 to about 5 amino acid modifications independently selected from substitutions, deletions, and insertions to the amino acid sequence of SEQ ID NO: 5.
In some embodiments, the alcohol intermediate (i.e., artemisinic alcohol) is converted to artemisinic aldehyde by an alcohol dehydrogenase (ADH). In embodiments, the microbial host cell expresses one or more ADHs. In some embodiments, modifications to enzymes can be informed by construction of a homology model. Structural coordinates from known structures of alcohol dehydrogenases can be used for constructing homology models of ADH enzymes, which are useful for guiding the engineering of ADH enzymes with improved specificity and productivity.
In some embodiments, the ADH enzyme comprises an amino acid sequence selected from SEQ ID NOs: 6, 7, or a variant thereof. Variants generally include enzymes comprising an amino acid sequence that has 50% or more sequence identity with any one of SEQ ID NOs: 6 and 7. In some embodiments, the ADH enzyme comprises an amino acid sequence that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, the ADH enzyme comprises an amino acid sequence that includes from 1 to about 20, or from 1 to about 10, or from 1 to about 5 amino acid modifications independently selected from substitutions, deletions, and insertions to an amino acid sequence of SEQ ID NO: 6 or 7.
In some embodiments, the ADH enzyme comprises a substitution to one or more of the substrate binding site or active site. In some embodiments, the amino acid modifications can be selected to improve one or more of the following properties in microbial cells: enzyme productivity, selectivity for the desired substrate and/or product, stability, temperature tolerance, and expression.
In some embodiments, the ADH enzyme comprises one or more amino acid substitutions at positions shown in
In some embodiments, the ADH enzyme comprises an amino acid substitution at one or more positions (e.g., from 1 to 20, or from 1 to 10, or from 1 to 5) corresponding to the following positions of SEQ ID NO: 6: 82, 302, 155, 360, 299, 258, 304, 19, 107, 193, 263, 168, 78, 20, 253, 75, 191, 302, 80, 153, 203, 169, 229, 221, 329, 150, 6, 305, 60, 25, 310, 92, 233, 257, and 170. In embodiments, the ADH enzyme comprises one or more substitutions (e.g., from 1 to 20, from 1 to 10, or from 1 to 5) selected from the following substitutions numbered according to SEQ ID NO: 6: A82V, E302G, A155V, Q360N, A299V, V258I, S304E, S19A, T107S, S193E, H263D, S168P, 178V, S20G, S253P, 175V, Q191K, E302G, K80E, A153T, I203L, Y169L, Q229P, K221R, Q329R, T150S, P6G, V305I, I60E, L25I, L310F, M92I, A233E, L257V, and P170D. In some embodiments, the ADH enzyme comprises at least two, at least three, at least four, or at least five amino acid substitutions selected from A82V, E302G, A155V, Q360N, A299V, V258I, S304E, S19A, T107S, S193E, H263D, S168P, 178V, S20G, S253P, 175V, Q191K, E302G, K80E, A153T, I203L, Y169L, Q229P, K221R, Q329R, T150S, P6G, V305I, I60E, L25I, L310F, M92I, A233E, L257V, and P170D numbered according to SEQ ID NO: 6. In embodiments, the ADH comprises the substitution A82V numbered according to SEQ ID NO: 6.
In some embodiments, the ADH enzyme exhibits increased production of artemisinic aldehyde (A-CHO) in microbial cells producing artemisinic alcohol as compared to ADH comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the ADH enzyme described herein is isolated or partially purified, or is heterologously expressed in a host cell. Some embodiments of the present invention are related to recombinant nucleic acid molecules comprising a nucleotide sequence encoding the ADH enzymes described herein. The recombinant nucleic acid could be a part of extrachromosomal elements such as plasmids, or bacterial artificial chromosomes, or may be chromosomally integrated.
In an artemisinin biosynthetic pathway, aldehyde dehydrogenase (ALDH) can catalyze conversion of artemisinic aldehyde to artemisinic acid (
By way of example, in some embodiments, the ALDH enzyme comprises an amino acid sequence of SEQ ID NO: 8 or a variant thereof. ALDH variants generally include enzymes comprising an amino acid sequence that has 50% or more sequence identity with SEQ ID NO: 8. In some embodiments, the ALDH enzyme comprises an amino acid sequence that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 8. In some embodiments, the ALDH enzyme comprises an amino acid sequence that includes from 1 to about 20, or from 1 to about 10, or from 1 to about 5 amino acid modifications independently selected from substitutions, deletions, and insertions to an amino acid sequence of SEQ ID NO: 8.
In some embodiments, the ALDH enzyme comprises a substitution to one or more of the substrate binding site or active site. In some embodiments, the amino acid modifications can be selected to improve one or more of the following properties in microbial cells: enzyme productivity, selectivity for the desired substrate and/or product, stability, temperature tolerance, and expression. In some embodiments, selection and modification of enzymes is informed by assaying activity on artemisinic alcohol or assaying activity in microbial cells producing artemisinic alcohol. In embodiments, the ALDH has increased production of artemisinic acid (AA) as compared to ALDH comprising the amino acid sequence of SEQ ID NO: 8 (AaALDH).
In some embodiments, the ALDH enzyme described herein, is isolated or partially purified, or is heterologously expressed in a host cell. Some embodiments of the present invention are related to recombinant nucleic acid molecules comprising a nucleotide sequence encoding the ALDH enzyme. The recombinant nucleic acid could be a part of extrachromosomal elements such as plasmids, or bacterial artificial chromosomes, or may be chromosomally integrated.
In some embodiments, the microbial host cell expresses one or more double bond reductases (DBRs). As used herein, a double-bond reductase is an enzyme that enhances production of DHAA in the microbial cells, by action on artemisinic acid and/or artemisinic aldehyde. In some embodiments, modifications to enzymes can be informed by construction of a homology model.
In some embodiments, the DBR comprises an amino acid sequence of SEQ ID NOs: 9, 10, or a variant thereof. In some embodiments, the variants of DBR include enzymes comprising an amino acid sequence that has 50% or more sequence identity with SEQ ID NO: 9 or 10. In some embodiments, the DBR enzyme comprises an amino acid sequence that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 9 or 10. In some embodiments, the DBR enzyme comprises an amino acid sequence that includes from 1 to about 20, or from 1 to about 10, or from 1 to about 5 amino acid modifications independently selected from substitutions, deletions, and insertions to an amino acid sequence of SEQ ID NO: 9 or 10.
In some embodiments, the DBR enzyme comprises a substitution to one or more of the substrate binding site or active site. In some embodiments, the amino acid modifications can be selected to improve one or more of the following properties in microbial cells: enzyme productivity, selectivity for the desired substrate and/or product, stability, temperature tolerance, and expression. In some embodiments, the DBR enzyme comprises an amino acid sequence that has at least about 80% or at least about 90%, or at least about 95% sequence identity to SEQ ID NO: 9, with an amino acid substitution at position 241 with respect to SEQ ID NO: 9. In some embodiments, the DBR enzyme comprises a substitution of asparagine at position 241 with respect to SEQ ID NO: 9.
In some embodiments, the DBR variant exhibits increased production of DHAA as compared to DBR comprising the amino acid sequence of SEQ ID NO: 9, when coexpressed with ADS, AO, CPR, ADH, and ALDH (as described). In some embodiments, the DBR variant exhibits increased production of DHAA as compared to DBR comprising the amino acid sequence of SEQ ID NO: 10, when coexpressed with ADS, AO, CPR, ADH, and ALDH.
In some embodiments, the DBR or variant thereof, as described herein, is isolated or partially purified, or is heterologously expressed in a host cell. Some embodiments of the present invention are related to recombinant nucleic acid molecules comprising a nucleotide sequence encoding the DBR or variant thereof as described herein. The recombinant nucleic acid could be a part of extrachromosomal elements such as plasmids, or bacterial artificial chromosomes, or may be chromosomally integrated.
In some embodiments, the microbial host cell may further comprise one or more peroxidases capable of converting artemisinic acid (AA) or dihydroartemisinic acid (DHAA) to artemisinin. In some embodiments, the peroxidase comprises any one of the amino acid sequences of SEQ ID NOs: 11-200, or a variant thereof. In some embodiments, the peroxidase comprises an amino acid sequence that has 50% or more sequence identity with any one of SEQ ID NOs: 11-200. In some embodiments, the peroxidase comprises an amino acid sequence that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 11-200. In some embodiments, the peroxidase comprises an amino acid sequence that includes from 1 to about 20, or from 1 to about 10, or from 1 to about 5 amino acid modifications independently selected from substitutions, deletions, and insertions to an amino acid sequence of any one of SEQ ID NOs: 11-200.
In some embodiments, the microbial host cell may further comprise one or more alpha-ketoglutarate-dependent dioxygenases capable of converting artemisinic acid (AA) or dihydroartemisinic acid (DHAA) to artemisinin. In some embodiments, the alpha-ketoglutarate-dependent dioxygenase comprises any one of the amino acid sequences of SEQ ID NOs: 201-488, or a variant thereof. In some embodiments, the alpha-ketoglutarate-dependent dioxygenase comprises an amino acid sequence that has 50% or more sequence identity with any one of SEQ ID NOs: 201-488. In some embodiments, the alpha-ketoglutarate-dependent dioxygenase comprises an amino acid sequence that has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 201-488. In some embodiments, the alpha-ketoglutarate-dependent dioxygenase comprises an amino acid sequence that includes from 1 to about 20, or from 1 to about 10, or from 1 to about 5 amino acid modifications independently selected from substitutions, deletions, and insertions to an amino acid sequence of any one of SEQ ID NOs: 201-488.
In various embodiments, any one of the enzyme variants described herein including, but not limited to, ADS (variants of SEQ ID NO: 1), AO (variants of SEQ ID NO: 3), ADH (variants of SEQ ID NO: 6), ALDH (variants of SEQ ID NO: 8), DBR (variants of SEQ ID NO: 9 or 10), peroxidase (variants of SEQ ID NOs: 11-200), and alpha-ketoglutarate-dependent dioxygenase (variants of SEQ ID NOs: 201-488) result in higher product yields and higher overall productivity of their corresponding product in microbial cells. For example, in various embodiments, the microbial cell expresses an enzyme variant that results in at least 1.5-fold, or at least 2-fold, or at least 4-fold, or at least 10-fold higher titers for the corresponding product in microbial cells, as compared to its wild type counterpart.
The amino acid substitutions, for any one of the enzyme variants described herein including, but not limited to, ADS, AO, CPR, ADH, ALDH, DBR, peroxidase, and alpha-ketoglutarate-dependent dioxygenase may be (independently) conservative or non-conservative substitutions. “Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups:
As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt α-helices. Some preferred conservative substitutions within the above six groups are exchanges within the following sub-groups: (i) Ala, Val, Leu and Ile; (ii) Ser and Thr; (ii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe.
As used herein, “non-conservative substitutions” or “non-conservative amino acid exchanges” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.
The similarity of nucleotide and amino acid sequences can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, such as with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80). The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410. BLAST protein searches may be performed with the BLASTP program, score=50, word length=3. To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields.
In various embodiments, the one or more heterologous enzymes selected from ADS, AO, CPR, ADH, ALDH, DBR, peroxidase, and alpha-ketoglutarate-dependent dioxygenase are expressed together in one or more operons, or are expressed individually in a host cell. The enzymes may be expressed from extrachromosomal elements such as plasmids, or bacterial artificial chromosomes, or may be chromosomally integrated.
In some embodiments, the microbial host cell is also engineered to express or overexpress one or more enzymes in the methylerythritol phosphate (MEP) and/or the mevalonic acid (MVA) pathway to catalyze isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) from glucose or other carbon source.
In some embodiments, the microbial host cell is engineered to express or overexpress one or more enzymes of the MEP pathway. In some embodiments, the MEP pathway is increased and balanced with downstream pathways by providing duplicate copies of certain rate-limiting enzymes. The MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, also called the MEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/l-deoxy-D-xylulose 5-phosphate) pathway or the non-mevalonate pathway or the mevalonic acid-independent pathway refers to the pathway that converts glyceraldehyde-3-phosphate and pyruvate to IPP and DMAPP. The pathway typically involves action of the following enzymes: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (IspC), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), and isopentenyl diphosphate isomerase (IspH). The MEP pathway, and the genes and enzymes that make up the MEP pathway, are described in U.S. Pat. No. 8,512,988, which is hereby incorporated by reference in its entirety. For example, genes that make up the MEP pathway include dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, and ispA. In some embodiments, the microbial host cell expresses or overexpresses of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, or modified variants thereof, which results in the increased production of IPP and DMAPP. In some embodiments, artemisinin, DHAA, or AA is produced at least in part by metabolic flux through an MEP pathway, and wherein the microbial host cell has at least one additional gene copy of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, or modified variants thereof.
In some embodiments, the microbial host cell is engineered to express or overexpress one or more enzymes of the MVA pathway. The MVA pathway refers to the biosynthetic pathway that converts acetyl-CoA to IPP. The mevalonate pathway typically comprises enzymes that catalyze the following steps: (a) condensing two molecules of acetyl-CoA to acetoacetyl-CoA (e.g., by action of acetoacetyl-CoA thiolase); (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoenzymeA (HMG-CoA) (e.g., by action of HMG-CoA synthase (HMGS)); (c) converting HMG-CoA to mevalonate (e.g., by action of HMG-CoA reductase (HMGR)); (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by action of mevalonate kinase (MK)); (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g., by action of phosphomevalonate kinase (PMK)); and (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate (e.g., by action of mevalonate pyrophosphate decarboxylase (MPD)). The MVA pathway, and the genes and enzymes that make up the MVA pathway, are described in U.S. Pat. No. 7,667,017, which is hereby incorporated by reference in its entirety. In some embodiments, the microbial host cell expresses or overexpresses one or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, and MPD or modified variants thereof, which results in the increased production of IPP and DMAPP. In some embodiments, artemisinin, DHAA, or AA is produced at least in part by metabolic flux through an MVA pathway, and wherein the microbial host cell has at least one additional gene copy of one or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, or modified variants thereof.
In some embodiments, the microbial host cell is engineered to increase production of IPP and DMAPP from glucose as described in PCT Application Nos. PCT/US2018/016848 and PCT/US2018/015527, the contents of which are hereby incorporated by reference in their entireties. For example, in some embodiments the microbial host cell overexpresses MEP pathway enzymes, with balanced expression to push/pull carbon flux to IPP and DMAPP. In some embodiments, the microbial host cell is engineered to increase the availability or activity of Fe—S cluster proteins, so as to support higher activity of IspG and IspH, which are Fe—S enzymes. In some embodiments, the host cell is engineered to overexpress IspG and IspH, so as to provide increased carbon flux to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) intermediate, but with balanced expression to prevent accumulation of HMBPP at an amount that reduces cell growth or viability, or at an amount that inhibits MEP pathway flux.
Conversion of IPP and DMAPP precursors to farnesyl diphosphate (FPP) is typically through the action of a farnesyl diphosphate synthase (FPPS). Exemplary FPPS enzymes are disclosed in US 2018/0135081, which is hereby incorporated by reference in its entirety.
In some embodiments, the host cell is engineered to downregulate the ubiquinone biosynthesis pathway, e.g., by reducing the expression or activity of IspB, which uses IPP and FPP substrate.
In some embodiments, the microbial host cell is a bacterium selected from Escherichia spp., Bacillus spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp., and Pseudomonas spp. For example, in some embodiments, the bacterial host cell is a species selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens, or Pseudomonas putida. In some embodiments, the bacterial host cell is E. coli.
In some embodiments, the microbial host cell is a species of Saccharomyces, Pichia, or Yarrowia, including, but not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.
In some aspects, the present invention is related to the method of making AA, DHAA, or artemisinin. This method includes culturing any of the microbial cells as described herein and recovering the AA, DHAA, or artemisinin. In some embodiments, the microbial cell expresses a biosynthetic pathway comprising a heterologous enzyme having an amorphadiene synthase activity (ADS), a heterologous enzyme having an amorphadiene oxidase activity (AO), and a heterologous enzyme having a double bond reductase activity (DBR), as described herein. Cells expressing ADS, AO and DBR enzymes can produce AA, DHAA or artemisinin from a carbon source. In some embodiments, the microbial host cell may further comprise one or more peroxidases capable of converting artemisinic acid (AA) or dihydroartemisinic acid (DHAA) to artemisinin. In some embodiments, the microbial host cell may further comprise one or more alpha-ketoglutarate-dependent dioxygenases capable of converting AA or DHAA to artemisinin. Alternatively, the method described herein further include a step of converting AA or DHAA to artemisinin. In some embodiments, the step converting of DHAA to artemisinin is done photochemically. In some embodiments, the conversion is conducted enzymatically.
In some aspects and embodiments, the present invention provides enzymatic methods for converting AA or DHAA to artemisinin, including through whole cell, cell lysate, or recombinant enzyme based bioconversion of a fed substrate. In some embodiments the method comprises: culturing a microbial host cell that expresses one or more enzymes described herein for converting AA or DHAA to artemisinin, feeding the culture a substrate selected from one or more of AA and DHAA, and recovering artemisinin from the culture. In other embodiments, the method employs contacting the substrate with a cell lysate of the microbial host cells or purified recombinant enzyme under suitable reaction conditions.
In some embodiments, the methods described herein include a microbial host cell that further expresses one or more alcohol dehydrogenases disclosed herein. In some embodiments, the methods described herein include a microbial host cell that further expresses one or more aldehyde dehydrogenases disclosed herein. In some embodiments, the methods described herein include a microbial host cell that expresses one or more ADS, AO/CPR, DBR, ADH, and ALDH, enzymes disclosed herein.
In some embodiments, the invention provides a microbial host cell that further expresses a heterologous enzyme having an activity for converting AA or DHAA to artemisinin. In some embodiments, the heterologous enzyme has a peroxidase activity and has at least 70% sequence identity to one of SEQ ID NOs: 11 to 200 (as described herein). In some embodiments, the heterologous enzyme is an Artemisia annua alpha-ketoglutarate-dependent dioxygenase, or variant thereof. In some embodiments, the heterologous enzyme having an alpha-ketoglutarate-dependent dioxygenase activity comprises an amino acid sequence that has at least 70%, at least 80%, or at least 90% sequence identity to one of SEQ ID NOs: 201 to 488 (as described herein). In some embodiments, the heterologous enzyme having an alpha-ketoglutarate-dependent dioxygenase activity comprises an amino acid sequence that has at least 70%, at least 80%, or at least 90% sequence identity (as described) to one of SEQ ID NOS: 302, 323, 361, 366, and 369. In some embodiments, the heterologous enzyme having an alpha-ketoglutarate-dependent dioxygenase activity comprises an amino acid sequence that has at least 70%, at least 80%, or at least 90% sequence identity (as described) to one of SEQ ID NOS: 302, 361, and 369.
In some embodiments, the host cell is cultured to produce DHAA or artemisinin. In some embodiments, microbial cells are cultured with carbon substrates (sources) such as C1, C2, C3, C4, C5, and/or C6 carbon substrates. In exemplary embodiments, the carbon source is glucose, sucrose, fructose, xylose, and/or glycerol. Culture conditions are generally selected from aerobic, microaerobic, and anaerobic.
In various embodiments, the host cell is cultured at a temperature between 22° C. and 37° C. While commercial biosynthesis in bacteria such as E. coli can be limited by the temperature at which overexpressed and/or foreign enzymes (e.g., enzymes derived from plants) are stable, recombinant enzymes (including the terpenoid synthase) may be engineered to allow for cultures to be maintained at higher temperatures, resulting in higher yields and higher overall productivity. In some embodiments, the host cell is a bacterial host cell, and culturing is conducted at about 22° C. or greater, about 23° C. or greater, about 24° C. or greater, about 25° C. or greater, about 26° C. or greater, about 27° C. or greater, about 28° C. or greater, about 29° C. or greater, about 30° C. or greater, about 31° C. or greater, about 32° C. or greater, about 33° C. or greater, about 34° C. or greater, about 35° C. or greater, about 36° C. or greater, or about 37° C.
AA, DHAA and/or artemisinin can be extracted from media and/or whole cells, and recovered. In some embodiments, AA, DHAA or artemisinin is recovered in a process comprising aqueous extraction followed by precipitation. AA, DHAA or artemisinin can be quantified by any suitable process, including, for example, liquid chromatography. The desired product can be produced in batch or continuous bioreactor systems.
In some embodiments, the microbial host cells and methods disclosed herein are suitable for commercial production of AA, DHAA, and/or artemisinin, that is, the microbial host cells and methods are productive at commercial scale. In some embodiments, the size of the culture is at least about 100 L, at least about 200 L, at least about 500 L, at least about 1,000 L, at least about 10,000 L, at least about 100,000 L, or at least about 1,000,000 L. In some embodiment, the culturing may be conducted in batch culture, continuous culture, or semi-continuous culture.
In some aspects, the present disclosure provides methods for making a product comprising artemisinin, including a pharmaceutical product. In some embodiments, the method comprises producing artemisinin as described herein through microbial culture, recovering artemisinin, and incorporating the artemisinin into the pharmaceutical product.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like.
As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 10% in either direction (greater than or less than) of the number.
Artemisinin is a sesquiterpene lactone (
The amorphadiene precursor farnesyl diphosphate (FPP) is a terpenoid, and can be produced by biosynthetic fermentation processes, using microbial strains that produce high levels of MEP pathway products along with heterologous expression of artemisinin biosynthesis enzymes.
For example, in bacteria such as E. coli, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) can be produced from glucose or other carbon source, and which can be converted to farnesyl diphosphate (FPP) by recombinant farnesyl diphosphate synthase (FPPS). FPP is converted through a cyclization reaction to amorphadiene by recombinant expression of ADS.
Using an E. coli background strain that produces high levels of the MEP pathway products IPP and DMAPP (see US 2018/0245103 and US 2018/0216137, which are hereby incorporated by reference), several mutants of Artemisia annua amorphadiene synthases were screened by co-expression with FPPS. Fermentation was performed in 96 well plates for 48 hours. Several mutant enzymes, as shown in
Artemisia annua AO was engineered to conduct multiple oxygenations of AD, to produce the AA. AaAO was engineered to delete a portion of the transmembrane domain, with the addition of a membrane anchor derived from E. coli yhhm. See, US 2018/0251738, which is hereby incorporated by reference. A panel of mutations to the AO were screened for their ability to catalyze multiple oxygenation events. Mutants were screened by fermentation in 96-well plates for 48 hours.
A second round of AO mutations were screened for their ability to produce artemisinic acid from AD (
To support production of A-CHO, AaADH was engineered for activity in E. coli. A panel of mutations to AaADH were screened for their ability to improve production of artemisinic aldehyde. Mutants were screened by fermentation in 96-well plates for 48 hours.
To produce dihydroartemisinic Acid (DHAA) in microbial cells, candidate double bond reductase (DBR) enzymes from Artemisia absinthium and Artemisia annua where screened by co-expression in E. coli with ADS, AO2, AaCPR, ADH, and ALDH. Fermentation was performed in 96-well plates for 48 hours.
Co-expression of A. absinthium DBR2 along with ADS, AO2, AaCPR, ADH, and ALDH produces DHAA (
To support production of DHAA, A. annua DBR2 was engineered for activity in E. coli. A panel of mutations to DBR2 were screened for their ability to improve production of DHAA. Mutants were screened in two separate 96-well plate fermentation assays for 48 hours. Assay 1 and assay 2 differ by the organic solvent used as a culture overlay.
To produce artemisinin in microbial cells, candidate non-heme Fe(II) α-ketoglutarate-dependent dioxygenase enzymes from Artemisia annua were screened in an AA-producing E. coli strain (co-expressing ADS, AO2, AaCPR, AaADH1, and ALDH) or a DHAA-producing E. coli strain (co-expressing ADS, AO2, AaCPR, AaADH1, ALDH, and DBR2). Fermentation was performed in 96-well plates for 48 hours.
Artemisia annua amorphadiene synthase (ADS, wild-type)
Artemisia annua amorphadiene synthase (ADS1, engineered
Artemisia annua amorphadiene oxidase (AO, wild-type)
Artemisia annua amorphadiene oxidase (AO2, engineered
VYKFATRSKSTKKSLPEPWRLPIIGHMHHLIGTTPHR
Artemisia annua AaCPR
Artemisia annua alcohol dehydrogenase (AaADH, wild-type)
Artemisia annua alcohol dehydrogenase (AaADH1, engineered
Artemisia annua aldehyde dehydrogenase (AaALDH, wild-type)
Artemisia annua double-bond reductase (AaDBR2) (wild-type)
Artemisia annua peroxidase 1 (AAO45182.1)
Artemisia annu peroxidase 1 (PWA70787.1)
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| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/047694 | 8/26/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63071630 | Aug 2020 | US |
