Patent Application
20230042171
Mogrosides are triterpene-derived specialized secondary metabolites found in the fruit of the Cucurbitaceae family plant Siraitia grosvenorii (a/k/a monkfruit or Luo Han Guo). Their biosynthesis in fruit involves a number of consecutive glycosylations of the aglycone mogrol. The food industry is increasing its use of mogroside fruit extract as a natural non-sugar food sweetener. For example, mogroside V (Mog.V) has a sweetening capacity that is ˜250 times that of sucrose (Kasai et al., Agric Biol Chem (1989)). Moreover, additional health benefits of mogrosides have been revealed in recent studies (Li et al., Chin J Nat Med (2014)).
A variety of factors are promoting a surge in interest in research and commercialization of the mogrosides and monkfruit in general, including, for example, the explosion in popularity of and demand for natural sweeteners; the difficulties in scalable sourcing of other promising natural sweeteners such as rebaudioside M (RebM) from the Stevia plant; the superior taste performance of Mog.V relative to other natural and artificial sweetener products on the market; and the medicinal potential of the plant and fruit.
Purified Mog.V has been approved as a high-intensity sweetening agent in Japan (Jakinovich et al., Journal of Natural Products (1990)) and the extract has gained GRAS status in the USA as a non-nutritive sweetener and flavor enhancer (GRAS 522). Extraction of mogrosides from the fruit can yield a product of varying degrees of purity, often accompanied by undesirable aftertaste. In addition, yields of mogroside from cultivated fruit are limited due to low plant yields and particular cultivation requirements of the plant. Mogrosides are present at about 1% in the fresh fruit and about 4% in the dried fruit (Li H B, et al., 2006). Mog.V is the main component, with a content of 0.5% to 1.4% in the dried fruit. Moreover, purification difficulties limit purity for Mog.V, with commercial products from plant extracts being standardized to about 50% Mog.V. It is highly likely that a pure Mog.V product will achieve greater commercial success than the blend, since it is less likely to have off flavors, will be easier to formulate into products, and has good solubility potential. It is therefore advantageous to be able to produce sweet mogroside compounds via biotechnological processes.
The present invention, in various aspects and embodiments, provides enzymes (including engineered enzymes), microbial strains, and methods for making mogrol and mogrol glycosides (“mogrosides”) using recombinant microbial processes. In other aspects, the invention provides methods for making products, including foods, beverages, and sweeteners (among others), by incorporating the mogrol glycosides produced according to the present disclosure.
In various aspects, the invention provides microbial strains and methods for making mogrol or mogrol glycoside(s). The invention involves a recombinant microbial host cell expressing a heterologous enzyme pathway catalyzing the conversion of isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP) to mogrol or mogrol glycoside(s). The microbial host cell in various embodiments may be prokaryotic (e.g., E. coli) or eukaryotic (e.g., yeast).
In various embodiments, the heterologous enzyme pathway comprises a farnesyl diphosphate synthase (FPPS) and a squalene synthase (SQS), which are recombinantly expressed. In various embodiments, the SQS comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOS: 2 to 16, 166, and 167. In some embodiments, the SQS comprises an amino acid sequence that is at least 70% identical to SQS (SEQ ID NO: 11), which has high activity in E. coli.
In some embodiments, the host cell expresses one or more enzymes that produce mogrol from squalene. For example, the host cell may express one or more squalene epoxidase (SQE) enzymes, one or more triterpenoid cyclases, an epoxide hydrolase (EPH), one or more cytochrome P450 oxidase enzymes (CYP450), a non-heme iron-dependent oxygenases, and a cytochrome P450 reductases (CPR). As shown in
In some embodiments, the heterologous enzyme pathway comprises two squalene epoxidase (SQE) enzymes. For example, the heterologous enzyme pathway may comprise an SQE that produces 2,3-oxidosqualene. In some embodiments, the SQE will produce 2,3:22,23-dioxidosqualene, and this conversion can be catalyzed by the same SQE enzyme, or an enzyme that differs in amino acid sequence by at least one amino acid modification. For example, the squalene epoxidase enzymes may include at least two SQE enzymes each comprising (independently) an amino acid sequence that is at least 70% identical to any one of SEQ ID NOS: 17 to 39, 168 to 170, and 177 to 183.
In some embodiments, at least one SQE comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 39.
In some embodiments, the host cell comprises two squalene epoxidase enzymes that each comprise an amino acid sequence that is at least 70% identical to squalene epoxidase (SEQ ID NO: 39). For example, one of the SQE enzymes may have one or more amino acid modifications that improve specificity or productivity for conversion of 2,3-oxidosqualene to 2,3:22,23 dioxidosqualene, as compared to the enzyme having the amino acid sequence of SEQ ID NO: 39. In some embodiments, the amino acid modifications comprise one or more modifications at positions corresponding to the following positions of SEQ ID NO: 39: 35, 133, 163, 254, 283, 380, and 395. For example, the amino acid at the position corresponding to position 35 of SEQ ID NO: 39 may be arginine (e.g., H35R). The position corresponding to position 133 of SEQ ID NO 39 may be glycine (e.g., N133G). The amino acid at the position corresponding to position 163 of SEQ ID NO: 39 may be alanine (e.g., F163A). The amino acid at the position corresponding to position 254 of SEQ ID NO: 39 may be phenylalanine (e.g., Y254F). The amino acid at the position corresponding to position 283 of SEQ ID NO: 39 may be leucine (e.g., M283L). The amino acid at the position corresponding to position 380 of SEQ ID NO: 39 may be leucine (e.g., V280L). The amino acid at the position corresponding to position 395 of SEQ ID NO: 39 may be tyrosine (e.g., F395Y).
In various embodiments, the heterologous enzyme pathway comprises a triterpene cyclase (TTC) enzyme. In some embodiments, where the microbial cell coexpresses FPPS, along with the SQS, SQE, and one or more triterpene cyclase enzymes, the microbial cell produces 2,3;22,23-dioxidosqualene. The 2,3;22,23-dioxidosqualene may be the substrate for downstream enzymes in the heterologous pathway. In some embodiments, the triterpene cyclase (TTC) comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOS: 40 to 55 and 191 to 193. The TTC in various embodiments comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 40.
In various embodiments, the heterologous enzyme pathway comprises at least two copies of a TTC enzyme gene, or comprises at least two enzymes having triterpene cyclase activity and converting 22,23-dioxidosqualene to 24,25-epoxycucurbitadienol. In such embodiments, product can be pulled to 24,25-epoxycucurbitadienol, with less production of cucurbitadienol. In some embodiments, the heterologous enzyme pathway comprises at least one TTC that comprises an amino acid sequence that is at least 70% identical to one of SEQ ID NO: 191, SEQ ID NO: 192, and SEQ ID NO: 193. For example, when co-expressed with SgCDS, these enzymes demonstrated improved production of 24,25-epoxycucurbitadienol compared to expression of SgCDS alone.
In some embodiments, the heterologous enzyme pathway comprises an epoxide hydrolase (EPH). The EPH may comprise an amino acid sequence that is at least 70% identical to amino acid sequence selected from SEQ ID NOS: 56 to 72, 184 to 190, and 212. In some embodiments, the EPH may employ as a substrate 24,25-epoxycucurbitadienol, for production of 24,25-dihydroxycucurbitadienol
In some embodiments, the heterologous pathway comprises at least one EPH converting 24,25-epoxycucurbitadienol to 24,25-dihydroxycucurbitadienol, the at least one EPH comprising an amino acid sequence that is at least 70% identical to one of: SEQ ID NO: 189, SEQ ID NO: 58, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 190, and SEQ ID NO: 212.
In some embodiments, the heterologous pathway comprises one or more oxidases. The one or more oxidases may be active on cucurbitadienol or oxygenated products thereof as a substrate, adding (collectively) hydroxylations at C11, C24 and 25, thereby producing mogrol. Alternatively or in addition, the heterologous pathway may comprise one or more oxidases that oxidize C11 of C24,25 dihydroxycucurbitadienol to produce mogrol.
In some embodiments, at least one oxidase is a cytochrome P450 enzyme. Exemplary cytochrome P450 enzymes comprise an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOS: 73 to 91, 171 to 176, and 194 to 200.
In some embodiments, the microbial host cell expresses a heterologous enzyme pathway comprising a P450 enzyme having activity for oxidation at C11 of C24,25 dihydroxycucurbitadienol, to thereby produce mogrol. For example, in some embodiments, the cytochrome P450 comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NO: 194 and SEQ ID NO: 171.
In various embodiments, the microbial host cell expresses one or more electron transfer proteins selected from a cytochrome P450 reductase (CPR), flavodoxin reductase (FPR) and ferredoxin reductase (FDXR) sufficient to regenerate the one or more oxidases. Exemplary CPR proteins are provided herein as SEQ ID NOS: 92 to 99 and 201.
In some embodiments, the microbial host cell expresses SEQ ID NO: 194 or a derivative thereof, and SEQ ID NO: 98 or a derivative thereof. In some embodiments, the microbial host cell expresses SEQ ID NO: 171 or a derivative thereof, and SEQ ID NO. 201 or a derivative thereof.
In some embodiments, the heterologous enzyme pathway further comprises one or more uridine diphosphate-dependent glycosyltransferase (UGT) enzymes, thereby producing one or more mogrol glycosides. The mogrol glycoside may be pentaglycosylated, hexaglycosylated, or more, in some embodiments. In other embodiments, the mogrol glycoside has two, three, or four glucosylations. The one or more mogrol glycosides may be selected from Mog.II-E, Mog.III, Mog.III-A1, Mog.III-A2, Mog.II, Mog.IV, Mog.IV-A, siamenoside, Mog.V, and Mog.VI. In some embodiments, the host cell produces Mog.V or siamenoside.
In some embodiments, the host cell expresses a UGT enzyme that catalyzes the primary glycosylation of mogrol at C24 and/or C3 hydroxyl groups. In some embodiments, the UGT enzyme catalyzes a branching glycosylation, such as a beta 1,2 and/or beta 1,6 branching glycosylation at the primary C3 and C24 glucosyl groups.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOS: 116 to 165, 202 to 210, 211, and 213 to 218.
For example, in some embodiments, the microbial cell expresses at least four UGT enzymes, resulting in glucosylation of mogrol at the C3 hydroxyl group, the C24 hydroxyl group, as well as a further 1,6 glucosylation at the C3 glucosyl group, and a further 1,6 glucosylation and a further 1,2 glucosylation at the C24 glucosyl group. The product of such glucosylation reactions is Mog.V.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO: 164, 165, 138, 204 to 211, and 213 to 218. In some embodiments, the UGT enzyme is engineered to have higher glycosyltransferase productivity as compared to the wild type enzyme.
In various embodiments, the microbial strain expresses one or more UGT enzymes capable of primary glycosylation at C24 and/or C3 of mogrol. Exemplary UGT enzymes include UGT enzymes comprising: an amino acid sequence that is at least 70% identical to SEQ ID NO: 165, an amino acid sequence that is at least 70% identical to SEQ ID NO: 146, an amino acid sequence that is at least 70% identical to SEQ ID NO. 202, an amino acid sequence that is at least 70% identical to SEQ ID NO: 202, an amino acid sequence that is at least 70% identical to SEQ ID NO: 129, an amino acid sequence that is at least 70% identical to SEQ ID NO: 116, an amino acid sequence that is at least 70% identical to SEQ ID NO: 218, and amino acid sequence that is at least 70% identical to SEQ ID NO: 217.
In various embodiments, the microbial strain expresses one or more UGT enzymes capable of catalyzing a branching glycosylation of one or both primary glycosylations. Such UGT enzymes are summarized in Table 2.
In some embodiments, the microbial host cell has one or more genetic modifications that increase the production of UDP-glucose, the co-factor employed by UGT enzymes.
Mogrol glycosides can be recovered from the microbial culture. For example, mogrol glycosides may be recovered from microbial cells, or in some embodiments, are predominately available in the extracellular media, where they may be recovered or sequestered.
Other aspects and embodiments of the invention will be apparent from the following detailed disclosure.
The present invention, in various aspects and embodiments, provides microbial strains and methods for making mogrol and mogrol glycosides, using recombinant microbial processes. In other aspects, the invention provides methods for making products, including foods, beverages, and sweeteners (among others), by incorporating the mogrol glycosides produced according to the methods described herein. In still other aspects, the invention provides engineered UGT enzymes for glycosylating secondary metabolite substrates, such as mogrol or mogrosides.
As used herein, the terms “terpene or triterpene” are used interchangeably with the terms “terpenoid” or “triterpenoid,” respectively.
In various aspects, the invention provides microbial strains and methods for making the triterpenoid compound mogrol, or glycoside products thereof. The invention provides a recombinant microbial host cell expressing a heterologous enzyme pathway catalyzing the conversion of isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP) to one or more of mogrol or mogroside(s).
The microbial host cell in various embodiments may be prokaryotic or eukaryotic. In some embodiments, the microbial host cell is a bacterium, and which can be optionally 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. Alternatively, the microbial cell may be a yeast cell, such as but not limited to a species of Saccharomyces, Pichia, or Yarrowia, including Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica.
The microbial cell will produce MEP or MVA products, which act as substrates for the heterologous enzyme pathway. 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, which is present in bacteria, 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 host cell expresses or overexpresses 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, the triterpenoid (e.g., squalene, mogrol, or other intermediate described herein) is produced at least in part by metabolic flux through an MEP pathway, and wherein the 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.
The MVA pathway refers to the biosynthetic pathway that converts acetyl-CoA to IPP. The mevalonate pathway, which will be present in yeast, 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 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, the triterpenoid (e.g., mogrol or squalene) is produced at least in part by metabolic flux through an MVA pathway, and wherein the 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 host cell is a bacterial host cell engineered to increase production of IPP and DMAPP from glucose as described in U.S. Pat. Nos. 10,480,015 and 10,662,442, the contents of which are hereby incorporated by reference in their entireties. For example, in some embodiments the host cell overexpresses MEP pathway enzymes, with balanced expression to push/pull carbon flux to IPP and DMAP. In some embodiments, the 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 and/or terpenoid production. In some embodiments, the host cell exhibits higher activity of IspH relative to IspG. 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 various embodiments, the heterologous enzyme pathway comprises a farnesyl diphosphate synthase (FPPS) and a squalene synthase (SQS), which are recombinantly expressed. In various embodiments, the SQS comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOS: 2 to 16, 166, and 167.
By way of non-limiting example, the FPPS may be Saccharomyces cerevisiae farnesyl pyrophosphate synthase (ScFPPS)(SEQ ID NO: 1), or modified variants thereof. Modified variants may comprise an amino acid sequence that is at least 70% identical to SEQ ID NO: 1). For example, the FPPS may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the FPPS comprises an amino acid sequence having from 1 to 20 amino acid modifications or having from 1 to 10 amino acid modifications with respect to SEQ ID NO: 1, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Numerous other FPPS enzymes are known in the art, and may be employed for conversion of IPP and/or DMAPP to farnesyl diphosphate in accordance with this aspect.
In some embodiments, the SQS comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 11. For example, the SQS may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 11. In some embodiments, the SQS comprises an amino acid sequence having from 1 to 20 amino acid modifications or from 1 to 10 amino acid modifications with respect to SEQ ID NO: 11, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Amino acid modifications may be made to increase expression or stability of the enzyme in the microbial cell, or to increase productivity of the enzyme. As shown in
In some embodiments, the SQS comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 2. For example, the SQS may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 2. In some embodiments, the SQS comprises an amino acid sequence having from 1 to 20 amino acid modifications or from 1 to 10 amino acid modifications with respect to SEQ ID NO: 2, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Amino acid modifications may be made to increase expression or stability of the enzyme in the microbial cell, or to increase productivity of the enzyme. As shown in
In some embodiments, the SQS comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 14. For example, the SQS may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 14. In some embodiments, the SQS comprises an amino acid sequence having from 1 to 20 amino acid modifications or from 1 to 10 amino acid modifications with respect to SEQ ID NO: 14, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Amino acid modifications may be made to increase expression or stability of the enzyme in the microbial cell, or to increase productivity of the enzyme. As shown in
In some embodiments, the SQS comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 16. For example, the SQS may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 16. In some embodiments, the SQS comprises an amino acid sequence having from 1 to 20 amino acid modifications or from 1 to 10 amino acid modifications with respect to SEQ ID NO: 16, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Amino acid modifications may be made to increase expression or stability of the enzyme in the microbial cell, or to increase productivity of the enzyme. As shown in
In some embodiments, the SQS comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 166. For example, the SQS may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 166. In some embodiments, the SQS comprises an amino acid sequence having from 1 to 20 amino acid modifications or from 1 to 10 amino acid modifications with respect to SEQ ID NO: 166, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Amino acid modifications may be made to increase expression or stability of the enzyme in the microbial cell, or to increase productivity of the enzyme. As shown in
In some embodiments, the SQS comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 167. For example, the SQS may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 167. In some embodiments, the SQS comprises an amino acid sequence having from 1 to 20 amino acid modifications or from 1 to 10 amino acid modifications with respect to SEQ ID NO: 167, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Amino acid modifications may be made to increase expression or stability of the enzyme in the microbial cell, or to increase productivity of the enzyme. As shown in
Amino acid modifications to the SQS enzyme can be guided by available enzyme structures and homology models, including those described in Aminfar and Tohidfar, In silico analysis of squalene synthase in Fabaceae family using bioinformatics tools, J. Genetic Engineer. and Biotech. 16 (2018) 739-747. The publicly available crystal structure for HsSQE (PDB entry: 6C6N) may be used to inform amino acid modifications. An alignment between AaSQS and HsSQS is shown in
In some embodiments, the host cell expresses one or more enzymes that produce mogrol from squalene. For example, the host cell may express one or more squalene epoxidase (SQE) enzymes, one or more triterpenoid cyclases, one or more epoxide hydrolase (EPH) enzymes, one or more cytochrome P450 oxidases (CYP450), optionally one or more non-heme iron-dependent oxygenases, and one or more cytochrome P450 reductases (CPR). As shown in
In some embodiments, the heterologous enzyme pathway comprises two squalene epoxidase (SQE) enzymes. For example, the heterologous enzyme pathway may comprise an SQE that produces 2,3-oxidosqualene (intermediate (3) in
In some embodiments, the at least one SQE comprises an amino acid sequence that is at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 39. For example, the SQE enzyme may comprise an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 39, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
As shown in
In some embodiments, the host cell comprises two squalene epoxidase enzymes that each comprise an amino acid sequence that is at least 70% identical to Methylomonas lenta squalene epoxidase (SEQ ID NO: 39). For example, one of the SQE enzymes may have one or more amino acid modifications that improve specificity or productivity for conversion of 2,3-oxidosqualene to 2,3;22,23 dioxidosqualene, as compared to the enzyme having the amino acid sequence of SEQ ID NO: 39. In some embodiments, the amino acid modifications comprise one or more (or in some embodiments, 2, 3, 4, 5, 6, or 7) modifications at positions corresponding to the following positions of SEQ ID NO-39: 35, 133, 163, 254, 283, 380, and 395. For example, the amino acid at the position corresponding to position 35 of SEQ ID NO: 39 may be arginine or lysine (e.g., H35R). The position corresponding to position 133 of SEQ ID NO: 39 may be glycine, alanine, leucine, isoleucine, or valine (e.g., N133G). The amino acid at the position corresponding to position 163 of SEQ ID NO: 39 may be glycine, alanine, leucine, isoleucine, or valine (e.g., F163A). The amino acid at the position corresponding to position 254 of SEQ ID NO. 39 may be phenylalanine, alanine, leucine, isoleucine, or valine (e.g., Y254F). The amino acid at the position corresponding to position 283 of SEQ ID NO: 39 may be alanine, leucine, isoleucine, or valine (e.g., M283L). The amino acid at the position corresponding to position 380 of SEQ ID NO: 39 may be alanine, leucine, or glycine (e.g., V280L). The amino acid at the position corresponding to position 395 of SEQ ID NO 39 may be tyrosine, serine, or threonine (e.g., F395Y). Exemplary SQE enzymes in these embodiments are at least 70%, or at least 80%, or at least 90%, or at least 95% identical to SEQ ID NO: 39, but comprise the following sets of amino acid substitutions. H35R, F163A, M283L, V380L, F395Y; or H35R, N133G, F163A, Y254F, V380L, and F395Y, in each case numbered according to SEQ ID NO: 39. For example, the host cell may express an SQE comprising the amino acid sequence of SEQ. ID NO: 203 (referred to herein as MlSQE A4).
In still other embodiments, the squalene epoxidase comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 168). For example, the SQE may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99%/o identical to SEQ ID NO: 168. In various embodiments, the SQE comprises an amino acid sequence having from 1 to 20 amino acid modifications or from 1 to 10 amino acid modifications with respect to SEQ ID NO. 168, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. As shown in
In some embodiments, the squalene epoxidase comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 169. For example, the SQE may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 169. In various embodiments, the SQE comprises an amino acid sequence having from 1 to 20 amino acid modifications or from 1 to 10 amino acid modifications with respect to SEQ ID NO. 169, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. As shown in
In some embodiments, the squalene epoxidase comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 170. For example, the SQE may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 170. In various embodiments, the SQE comprises an amino acid sequence having from 1 to 20 amino acid modifications or from 1 to 10 amino acid modifications with respect to SEQ ID NO: 170, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. As shown in
Amino acid modifications can be guided by available enzyme structures and homology models, including those described in Padyana A K, et al., Structure and inhibition mechanism of the catalytic domain of human squalene epoxidase, Nat. Comm. (2019) Vol. 10(97): 1-10; or Ruckenstulh et al., Structure-Function Correlations of Two Highly Conserved Motifs in Saccharomyces cerevisiae Squalene Epoxidase, Antimicrob. Agents and Chemo. (2008) Vol. 52(4): 1496-1499.
In various embodiments, the heterologous enzyme pathway comprises a triterpene cyclase (TTC). In some embodiments, where the microbial cell coexpresses FPPS, along with the SQS, SQE, and triterpene cyclase enzymes, the microbial cell produces 2,3;22,23-dioxidosqualene. The 2,3;22,23-dioxidosqualene may be the substrate for downstream enzymes in the heterologous pathway. In some embodiments, the triterpene cyclase (TTC) comprises an amino acid sequence that is at least 70%, or at least 80%, or at least 90%, or at least 95% identical to an amino acid sequence selected from SEQ ID NOS: 40 to 55, 191 to 193, and 219 to 220. The TTC in various embodiments comprises an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, the TTC comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 900%, or at least 95%, or at least 98%, or at least 99%/o identical to SEQ ID NO: 40. For example, the TTC may comprise an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 40, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, the TTC comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 192. For example, the TTC may comprise an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 192, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. The enzyme defined by SEQ ID NO: 192 shows improved specificity toward production of 24,25-epoxycucurbitadienol (
In various embodiments, the heterologous enzyme pathway comprises at least two copies of a TTC enzyme gene, or comprises at least two enzymes having triterpene cyclase activity and converting 22,23-dioxidosqualene to 24,25-epoxycucurbitadienol. In such embodiments, product can be pulled to 24,25-epoxycucurbitadienol, with less production of cucurbitadienol.
In some embodiments, the heterologous enzyme pathway comprises at least one TTC that comprises an amino acid sequence that is at least 70% identical to one of SEQ ID NO: 191, SEQ ID NO: 192, and SEQ ID NO. 193. These enzymes may be optionally co-expressed with SgCDS. These enzymes exhibit high production of 24,25-epoxycucurbitadienol.
Amino acid modifications may be made to increase expression or stability of the enzyme in the microbial cell, or to increase productivity of the enzyme. Amino acid modifications can be guided by available enzyme structures and homology models, including those described in Itkin M., et al., The biosynthetic pathway of the nonsugar, high-intensity sweetener mogroside V from Siraitia grosvenorii, PNAS (2016) Vol 113(47): E7619-E7628. For example, the CDS may be modeled using the structure of human lanosterol synthase (oxidosqualene cyclase) (PDB 1W6K).
In various embodiments, cucurbitadienol (intermediate 9 in
In some embodiments, the heterologous enzyme pathway comprises at least one epoxide hydrolase (EPH). The EPH may comprise an amino acid sequence that is at least 70% identical to amino acid sequence selected from SEQ ID NOS: 56 to 72, 184 to 190, and 212. In some embodiments, the EPH may employ as a substrate 24,25-epoxycucurbitadienol (intermediate (5) of
In some embodiments, the heterologous pathway comprises at least one EPH enzyme converting 24,25-epoxycucurbitadienol to 24,25-dihydroxycucurbitadienol, the at least one EPH enzyme comprising an amino acid sequence that is at least 70% identical to one of: SEQ ID NO: 189, SEQ ID NO: 58, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 190, and SEQ ID NO: 212. See
In some embodiments, the heterologous pathway comprises one or more oxidases. The one or more oxidases may be active on cucurbitadienol or oxygenated products thereof as a substrate, adding (collectively) hydroxylations at C11, C24 and 25, thereby producing mogrol (see
In some embodiments, at least one oxidase is a cytochrome P450 enzyme. Exemplary cytochrome P450 enzymes comprise an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOS: 73 to 91, 171 to 176, and 194 to 200. In some embodiments, at least one P450 enzyme comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to one of SEQ ID NOS: 73 to 91, 171 to 176, and 194 to 200. For example, at least one cytochrome P450 enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to one of SEQ ID NOS: 73 to 91, 171 to 176, and 194 to 200, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, the microbial host cell expresses a heterologous enzyme pathway comprising a P450 enzyme having activity for oxidation at C11 of C24,25 dihydroxycucurbitadienol, to thereby produce mogrol. For example, in some embodiments, the cytochrome P450 comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NO: 194 and SEQ ID NO: 171. See
In some embodiments, the cytochrome P450 enzyme has at least a portion of its transmembrane region substituted with a heterologous transmembrane region. For example, particularly in embodiments in which the microbial cell is a bacterium, the CYP450 and/or CPR is modified as described in US 2018/0251738, the contents of which are hereby incorporated by reference in their entireties. For example, in some embodiments, the CYP450 enzyme has a deletion of all or part of the wild type P450 N-terminal transmembrane region, and the addition of a transmembrane domain derived from an E. coli or bacterial inner membrane, cytoplasmic C-terminus protein. In some embodiments, the transmembrane domain is a single-pass transmembrane domain. In some embodiments, the transmembrane domain is a multi-pass (e.g., 2, 3, or more transmembrane helices)transmembrane domain. Exemplary transmembrane domains are derived from E. coli zipA or sohB. Alternatively, the P450 enzyme can employ its native transmembrane anchor, or the well-known bovine 17a anchor. See
In some embodiments, the microbial host cell expresses a non-heme iron oxidase. Exemplary non-heme iron oxidases comprise an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOS: 100 to 115. In some embodiments, the non-heme iron oxidase comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to one of SEQ ID NOS: 100 to 115.
In various embodiments, the microbial host cell expresses one or more electron transfer proteins selected from a cytochrome P450 reductase (CPR), flavodoxin reductase (FPR) and ferredoxin reductase (FDXR) sufficient to regenerate the one or more oxidases. Exemplary CPR proteins are provided herein as SEQ ID NOS: 92 to 99 and 201.
In some embodiments, the microbial host cell expresses a cytochrome P450 reductase, and which may comprise an amino acid sequence that is at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to one of SEQ ID NOS: 92 to 99 and 201. For example, in some embodiments, the microbial host cell expresses SEQ ID NO: 194 or a derivative thereof (as described above), and SEQ ID NO: 98 or a derivative thereof (i.e., having at least 70%, at least 80%, or at least 90% sequence identity thereto). In some embodiments, the microbial host cell expresses SEQ ID NO: 171 or a derivative thereof (as described above), and SEQ ID NO: 201 or a derivative thereof (i.e., having at least 70%, at least 80%, or at least 90% sequence identity thereto).
In various embodiments, the heterologous enzyme pathway produces mogrol, which may be an intermediate for downstream enzymes in the heterologous pathway, or in some embodiments is recovered from the culture. Mogrol may be recovered from host cells in some embodiments, and/or can be recovered from the culture media.
In some embodiments, the heterologous enzyme pathway further comprises one or more uridine diphosphate-dependent glycosyltransferase (UGT) enzymes, thereby producing one or more mogrol glycosides (or “mogrosides”). The mogrol glycoside may be pentaglycosylated, hexaglycosylated, or more (e.g., 7, 8, or 9 glycosylations), in some embodiments. In other embodiments, the mogrol glycoside has two, three, or four glucosylations. The one or more mogrol glycosides may be selected from Mog.II-E, Mog.III, Mog.III-A1, Mog.III-A2, Mog.III, Mog.IV, Mog.IV-A, siamenoside, isomog.V, Mog.V, or Mog.VI. In some embodiments, the host cell produces Mog.V or siamenoside.
In some embodiments, the host cell expresses a UGT enzyme that catalyzes the primary glycosylation of mogrol at C24 and/or C3 hydroxyl groups. In some embodiments, the UGT enzyme catalyzes a branching glycosylation, such as a beta 1,2 and/or beta 1,6 branching glycosylation at the primary C3 and C24 glucosyl groups. UGT enzymes observed to catalyze primary glycosylation of C24 and/or C3 hydroxyl groups are summarized in Table 1. UGT enzymes observed to catalyze various branching glycosylation reactions are summarized in Table 2.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOS: 116 to 165, 202 to 210, 211, and 213 to 218. For example, in some embodiments, the UGT enzyme comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to one of SEQ ID NOS: 116 to 165, 202 to 210, 211, and 213 to 218 Thus, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to one of SEQ ID NOS: 116 to 165, 202 to 210, 211, and 212 to 218, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
For example, in some embodiments, the microbial cell expresses at least four UGT enzymes, resulting in glucosylation of mogrol at the C3 hydroxyl group, the C24 hydroxyl group, as well as a further 1,6 glucosylation at the C3 glucosyl group, and a further 1,6 glucosylation and a further 1,2 glucosylation at the C24 glucosyl group. The product of such glucosylation reactions is Mog.V.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence having at least 70% sequence identity to one of SEQ ID NO: 164, 165, 138, 204 to 211, and 213 to 218.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to UGT85C1 (SEQ ID NO: 165). UGT85C1 exhibits primary glycosylation at the C3 and C24 hydroxyl groups. Thus, in some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO. 165. The at least one UGT enzyme may comprise an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 165, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Exemplary amino acid substitutions include substitutions at positions 41 (e.g., L41F or L41Y), 49 (e.g., D49E), and 127 (e.g., C127F or C127Y).
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 164, which exhibits activity for adding branching glycosylations, both 1-2 and 1-6 branching glycosylations. In various embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 164. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 164, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Exemplary amino acid substitutions are shown in Table 3. Exemplary amino acid substitutions include substitutions at one or more positions selected from 150 (e.g., S150F, S150Y), 147 (e.g., T147L, T147V, T147I, and T147A), 207 (e.g., N207K or N207R), 270 (e.g., K270E or K270D), 281 (V281L or V281I), 354 (e.g., L354V or L354I), 13 (e.g., L13F or L13Y), 32 (T32A or T32G or T32L), and 101 (K101A or K101G), with respect to SEQ ID NO: 164. An exemplary engineered UGT enzyme comprises the amino acid substitutions T147L and N207K, with respect to SEQ ID NO: 164.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 138, which exhibits an activity to catalyze 1-6 branching glycosylations. In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 138. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 138, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 204, which catalyzes 1-6 branching glycosylation, particularly at the C3 primary glucosylation. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 204. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 204, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 205, which catalyzes 1-6 branching glycosylation, including at both the C3 and C24 primary glucosylations. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 205. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 205, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 206, which catalyzes 1-2 and 1-6 branching glycosylations. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 206. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 206, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 207, which catalyzes 1-6 branching glycosylations of the primary glucosylations. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 207. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 207, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 208, which catalyzes 1-2 and 1-6 branching glycosylations. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 208. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 208, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 209, which catalyzes 1-6 branching glycosylations of the primary glucosylations. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 209. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 209, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 210), which catalyzes 1-6 branching glycosylations of the primary glucosylations. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 210. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 210, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70%/identical to SEQ ID NO: 211, which catalyzes 1-2 branching glycosylation of the C24 primary glucosylation. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 211. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 210, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 213, which catalyzes 1-6 branching glycosylation of the primary glucosylation at C24. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 213. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 213, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 214, which catalyzes primary glucosylation at C24. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 214. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 214, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 215, which catalyzes 1-6 branching glucosylation at C24. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 215. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 215, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In still other embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 146, which provides for glucosylation of the C24 hydroxyl of mogrol or Mog.IE. In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO. 146. In some embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 or from 1 to 10 amino acid modifications with respect to SEQ ID NO: 146, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Amino acid modifications may be made to increase expression or stability of the enzyme in the microbial cell, or to increase productivity of the enzyme for particular substrates.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 202, which catalyzes primary glycosylation at the C3 and C24 hydroxyl. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 202. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 202, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 218, which catalyzes primary glycosylation at the C24 hydroxyl. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 218. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 218, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 217, which catalyzes primary glycosylation at the C24 hydroxyl. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 217. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 217, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Exemplary amino acid substitutions include substitutions at one or more positions (with respect to SEQ ID NO: 17) selected from 74 (e.g., A74E or A74D), 91 (I91F or I91Y), 101 (e.g., H101P), 241 (e.g., Q241E or Q241D), and 436 (e.g., I436L or I436A). In some embodiments, the UGT enzyme comprises the following amino acid substitutions with respect to SEQ ID NO: 217: A74E, 191F, and H101P.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 216, which catalyzes primary glycosylation at the C24 hydroxyl. For example, at least one UGT enzyme may comprise an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 216. In exemplary embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 216, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 117, SEQ ID NO: 210, or SEQ ID NO: 122. For example, the enzyme defined by SEQ ID NO: 117 catalyzes branching glycosylations. In some embodiments, at least one UGT enzyme comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to SEQ ID NO: 117, SEQ ID NO: 210, or SEQ ID NO: 122. In some embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 amino acid modifications with respect to SEQ ID NO: 117, 210, or 122, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions.
In some embodiments, the microbial cell expresses at least one UGT enzyme capable of catalyzing beta 1,2 addition of a glucose molecule to at least the C24 glucosyl group (e.g., of Mog. IVA). Exemplary UGT enzymes in accordance with these embodiments include SEQ ID NO: 117, SEQ ID NO:147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, or SEQ ID NO: 163, or derivatives thereof. Derivatives include enzymes comprising amino acid sequence that are least 70% identical to one or more of SEQ ID NO: 117, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, and SEQ ID NO: 163. In some embodiments, the UGT enzyme catalyzing beta 1,2 addition of a glucose molecule to at least the C24 glucosyl group comprises an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% identical to one or more of SEQ ID NO: 117, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO. 150, and SEQ ID NO: 163. In some embodiments, at least one UGT enzyme comprises an amino acid sequence having from 1 to 20 or having from 1 to 10 amino acid modifications with respect to SEQ ID NO. 117, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, and SEQ ID NO: 163, the amino acid modifications being independently selected from amino acid substitutions, deletions, and insertions. Amino acid modifications may be made to increase expression or stability of the enzyme in the microbial cell, or to increase productivity of the enzyme for particular substrates.
In some embodiments, at least one UGT enzyme is a circular permutant of a wild-type UGT enzyme, optionally having amino acid substitutions, deletions, and/or insertions with respect to the corresponding position of the wild-type enzyme. Circular permutants can provide novel and desirable substrate specificities, product profiles, and reaction kinetics over the wild-type enzymes. A circular permutant retains the same basic fold of the parent enzyme, but has a different position of the N-terminus (e.g., “cut-site”), with the original N- and C-termini connected, optionally by a linking sequence. For example, in the circular permutants, the N-terminal Methionine is positioned at a site in the protein other than the natural N-terminus. UGT circular permutants are described in US 2017/0332673, which is hereby incorporated by reference in its entirety. In some embodiments, at least one UGT enzyme is a circular permutant of a UGT enzyme described herein, such as but not limited to SEQ ID NO: 146, SEQ ID NO: 164, or SEQ ID NO: 165, SEQ ID NO: 117, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 163, SEQ ID NO: 202, SEQ ID NO: 216, SEQ ID NO: 217, and SEQ ID NO: 218. In some embodiments, the circular permutant further has one or more amino acid modifications (e.g., amino acid substitutions, deletions, and/or insertions) with respect to the parent UGT enzyme. In these embodiments, the circular permutant will have at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 98% identity to the parent enzyme, when the corresponding amino acid sequences are aligned (i.e., without regard to the new N-terminus of the circular permutant). An exemplary circular permutant for use according to some embodiments is SEQ ID NO: 206.
In some embodiments, the microbial host cell expresses at least three UGT enzymes: a first UGT enzyme catalyzing primary glycosylation at the C24 hydroxyl of mogrol, a second UGT enzyme catalyzing primary glycosylation at the C3 hydroxyl of mogrol, and a third UGT enzyme catalyzing one or more branching glycosylation reactions. In some embodiments, the microbial host cell expresses one or two UGT enzymes catalyzing beta 1,2 and/or beta 1,6 branching glycosylations of the C3 and/or C24 primary glycosylations. For example, the UGT enzymes may comprise three or four UGT enzymes selected from:
SEQ ID NO: 165 or a derivative thereof;
SEQ ID NO: 146 or a derivative thereof;
SEQ ID NO: 214 or a derivative thereof;
SEQ ID NO: 129 or a derivative thereof;
SEQ ID NO: 164 or a derivative thereof;
SEQ ID NO: 116 or a derivative thereof;
SEQ ID NO: 202 or a derivative thereof;
SEQ ID NO: 218 or a derivative thereof;
SEQ ID NO: 217 or a derivative thereof;
SEQ ID NO: 138 or a derivative thereof;
SEQ ID NO: 204 or a derivative thereof;
SEQ ID NO: 205 or a derivative thereof;
SEQ ID NO: 207 or a derivative thereof;
SEQ ID NO: 208 or a derivative thereof;
SEQ ID NO: 209 or a derivative thereof;
SEQ ID NO: 11 or a derivative thereof;
SEQ ID NO: 215 or a derivative thereof;
SEQ ID NO: 213 or a derivative thereof;
SEQ ID NO: 206 or a derivative thereof;
SEQ ID NO: 122 or a derivative thereof; and
SEQ ID NO: 210) or a derivative thereof. Derivatives have sequence identity to the reference enzyme as described herein.
In some embodiments, the microbial host cell has one or more genetic modifications that increase the production of UDP-glucose, the co-factor employed by UGT enzymes. These genetic modifications may include one or more, or two or more (or all) of ΔgalE, ΔgalT, ΔgalK, ΔgalM, ΔushA, Δagp, Δpgm, duplication of E. coli galU, expression of Bacillus subtilis UGPA, and expression of Bifidobacterium adolescentis SPL.
Mogrol glycosides can be recovered from the microbial culture. For example, mogrol glycosides may be recovered from microbial cells, or in some embodiments, are predominately available in the extracellular media, where they may be recovered or sequestered.
In various embodiments, the reaction is performed in a microbial cell, and UGT enzymes are recombinantly expressed in the cell. In some embodiments, mogrol is produced in the cell by a heterologous mogrol synthesis pathway, as described herein. In other embodiments, mogrol or mogrol glycosides (such as a monkfruit extract) are fed to the cells for glycosylation. In still other embodiments, the reaction is performed in vitro using purified UGT enzyme, partially purified UGT enzyme, or recombinant cell lysates.
As described herein, the microbial host cell can be prokaryotic or eukaryotic, and is optionally a bacterium selected from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis, Vibrio natriegens, or Pseudomonas putida. In some embodiments, the microbial cell is a yeast selected from a species of Saccharomyces, Pichia, or Yarrowia, including Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica. In some embodiments, the microbial host cell is E. coli.
The bacterial host cell is cultured to produce the triterpenoid product (e.g., mogroside). In some embodiments, carbon substrates such as C1, C2, C3, C4, C5, and/or C6 carbon substrates are employed for the production phase. 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 bacterial host cell may be 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 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 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.
In some embodiments, the bacterial host cells are further suitable for commercial production, 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, or at least about 10,000 L, or at least about 100,000 L, or at least about 500,000 L, or at least about 600,000 L. In an embodiment, the culturing may be conducted in batch culture, continuous culture, or semi-continuous culture.
In various embodiments, methods further include recovering the product from the cell culture or from cell lysates. In some embodiments, the culture produces at least about 100 mg/L, or at least about 200 mg/L, or at least about 500 mg/L, or at least about 1 g/L, or at least about 2 g/L, or at least about 5 g/L, or at least about 10 g/L, or at least about 20 g/L, or at least about 30 g/L, or at least about 40 g/L of the terpenoid or terpenoid glycoside product.
In some embodiments, the production of indole (including prenylated indole) is used as a surrogate marker for terpenoid production, and/or the accumulation of indole in the culture is controlled to increase production. For example, in various embodiments, accumulation of indole in the culture is controlled to below about 100 mg/L, or below about 75 mg/L, or below about 50 mg/L, or below about 25 mg/L, or below about 10 mg/L. The accumulation of indole can be controlled by balancing protein expression and activity using the multivariate modular approach as described in U.S. Pat. No. 8,927,241 (which is hereby incorporated by reference), and/or is controlled by chemical means.
Other markers for efficient production of terpene and terpenoids, include accumulation of DOX or ME in the culture media. Generally, the bacterial strains may be engineered to accumulate less of these chemical species, which accumulate in the culture at less than about 5 g/L, or less than about 4 g/L, or less than about 3 g/L, or less than about 2 g/L, or less than about 1 g/L, or less than about 500 mg/L, or less than about 100 mg/L.
The optimization of terpene or terpenoid production by manipulation of MEP pathway genes, as well as manipulation of the upstream and downstream pathways, is not expected to be a simple linear or additive process. Rather, through combinatorial analysis, optimization is achieved through balancing components of the MEP pathway, as well as upstream and downstream pathways. Indole (including prenylated indole) accumulation and MEP metabolite accumulation (e.g., DOX, ME, MEcPP, and/or farnesol) in the culture can be used as surrogate markers to guide this process.
For example, in some embodiments, the bacterial strain has at least one additional copy of dxs and idi expressed as an operon/module; or dxs, ispD, ispF, and idi expressed as an operon or module (either on a plasmid or integrated into the genome), with additional MEP pathway complementation described herein to improve MEP carbon. For example, the bacterial strain may have a further copy of dxr, and ispG and/or ispH, optionally with a further copy of ispE and/or idi, with expressions of these genes tuned to increase MEP carbon and/or improve terpene or terpenoid titer. In various embodiments, the bacterial strain has a further copy of at least dxr, ispE, ispG and ispH, optionally with a further copy of idi, with expressions of these genes tuned to increase MEP carbon and/or improve terpene or terpenoid titer.
Manipulation of the expression of genes and/or proteins, including gene modules, can be achieved through various methods. For example, expression of the genes or operons can be regulated through selection of promoters, such as inducible or constitutive promoters, with different strengths (e.g., strong, intermediate, or weak). Several non-limiting examples of promoters of different strengths include Trc, T5 and T7. Additionally, expression of genes or operons can be regulated through manipulation of the copy number of the gene or operon in the cell. In some embodiments, expression of genes or operons can be regulated through manipulating the order of the genes within a module, where the genes transcribed first are generally expressed at a higher level. In some embodiments, expression of genes or operons is regulated through integration of one or more genes or operons into the chromosome.
Optimization of protein expression can also be achieved through selection of appropriate promoters and ribosomal binding sites. In some embodiments, this may include the selection of high-copy number plasmids, or single-, low- or medium-copy number plasmids. The step of transcription termination can also be targeted for regulation of gene expression, through the introduction or elimination of structures such as stem-loops.
Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA. The heterologous DNA is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
In some embodiments, endogenous genes are edited, as opposed to gene complementation. Editing can modify endogenous promoters, ribosomal binding sequences, or other expression control sequences, and/or in some embodiments modifies trans-acting and/or cis-acting factors in gene regulation. Genome editing can take place using CRISPR/Cas genome editing techniques, or similar techniques employing zinc finger nucleases and TALENs. In some embodiments, the endogenous genes are replaced by homologous recombination.
In some embodiments, genes are overexpressed at least in part by controlling gene copy number. While gene copy number can be conveniently controlled using plasmids with varying copy number, gene duplication and chromosomal integration can also be employed. For example, a process for genetically stable tandem gene duplication is described in US 2011/0236927, which is hereby incorporated by reference in its entirety.
The terpene or terpenoid product can be recovered by any suitable process. For example, the aqueous phase can be recovered, and/or the whole cell biomass can be recovered, for further processing. The production of the desired product can be determined and/or quantified, for example, by gas chromatography (e.g., GC-MS). The desired product can be produced in batch or continuous bioreactor systems.
The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, 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 polynucleotide searches can be performed with the BLASTN program, score=100, word length=12.
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.
“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:
(1) hydrophobic: Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro; and
(6) aromatic: Trp, Tyr, Phe.
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 Gin; (iv) Lys and Arg; and (v) Tyr and Phe.
As used herein, “non-conservative substitutions” 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.
Modifications of enzymes as described herein can include conservative and/or non-conservative mutations. In some embodiments, an Alanine is substituted or inserted at position 2, to increase stability.
In some embodiments “rational design” is involved in constructing specific mutations in enzymes. Rational design refers to incorporating knowledge of the enzyme, or related enzymes, such as its reaction thermodynamics and kinetics, its three dimensional structure, its active site(s), its substrate(s) and/or the interaction between the enzyme and substrate, into the design of the specific mutation. Based on a rational design approach, mutations can be created in an enzyme which can then be screened for increased production of a terpene or terpenoid relative to control levels. In some embodiments, mutations can be rationally designed based on homology modeling. As used herein, “homology modeling” refers to the process of constructing an atomic resolution model of one protein from its amino acid sequence and a three-dimensional structure of a related homologous protein.
In other aspects, the invention provides a method for making a product comprising a mogrol glycoside. The method comprises producing a mogrol glycoside in accordance with this disclosure, and incorporating the mogrol glycoside into a product. In some embodiments, the mogrol glycoside is siamenoside, Mog.V, Mog.VI, or Isomog.V. In some embodiments, the product is a sweetener composition, flavoring composition, food, beverage, chewing gum, texturant, pharmaceutical composition, tobacco product, nutraceutical composition, or oral hygiene composition.
The product may be a sweetener composition comprising a blend of artificial and/or natural sweeteners. For example, the composition may further comprise one or more of a steviol glycoside, aspartame, and neotame. Exemplary steviol glycosides comprises one or more of RebM, RebB, RebD, RebA, RebE, and RebI.
Non-limiting examples of flavors for which the products can be used in combination include lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, bitter almond, cola, cinnamon, sugar, cotton candy and vanilla flavors. Non-limiting examples of other food ingredients include flavors, acidulants, and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilizers, thickeners and gelling agents.
Mogrol glycosides obtained according to this invention may be incorporated as a high intensity natural sweetener in foodstuffs, beverages, pharmaceutical compositions, cosmetics, chewing gums, table top products, cereals, dairy products, toothpastes and other oral cavity compositions, etc.
Mogrol glycosides obtained according to this invention can be used in combination with various physiologically active substances or functional ingredients. Functional ingredients generally are classified into categories such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, plant sterols and stanols (phytosterols and phytostanols), polyols; prebiotics, probiotics; phytoestrogens; soy protein; sulfides/thiols; amino acids; proteins; vitamins; and minerals. Functional ingredients also may be classified based on their health benefits, such as cardiovascular, cholesterol-reducing, and anti-inflammatory.
Mogrol glycosides obtained according to this invention may be applied as a high intensity sweetener to produce zero calorie, reduced calorie or diabetic beverages and food products with improved taste characteristics. It may also be used in drinks, foodstuffs, pharmaceuticals, and other products in which sugar cannot be used. In addition, highly purified target mogrol glycoside(s), particularly, Mog.V, Mog.VI, or Isomog.V, can be used as a sweetener not only for drinks, foodstuffs, and other products dedicated for human consumption, but also in animal feed and fodder with improved characteristics.
Examples of products in which mogrol glycoside(s) may be used as a sweetening compound include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce: powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products: seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products, frozen food products; preserved seaweed; preserved meat; tobacco: medicinal products; and many others.
During the manufacturing of products such as foodstuffs, drinks, pharmaceuticals, cosmetics, table top products, and chewing gum, the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods may be used.
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.
The biosynthesis of mogrosides in fruit involves a number of consecutive glycosylations of the aglycone mogrol to the final sweet products, including mogroside V (Mog.V). Mog.V has a sweetening capacity that is about 250 times that of sucrose (Kasai et al., Agric Biol Chem (1989)). Mogrosides are reported to have health benefits as well (Li et al., Chin J Nat Med (2014)).
A variety of factors are promoting a surge in interest in mogrosides and monkfruit in general, including an explosion in demand for natural sweeteners, difficulties in scalable sourcing of the current lead natural sweetener, rebaudioside M (RebM) from the Stevia plant, the superior taste performance of Mog.V relative to other natural and artificial sweetener products on the market, and the medicinal potential of the plant and fruit.
Purified Mog.V has been approved as a high-intensity sweetening agent in Japan (Jakinovich et al., Journal of Natural Products (1990)) and the extract has gained GRAS status in the USA as a non-nutritive sweetener and flavor enhancer (GRAS 522). Extraction of mogrosides from the fruit can yield a product of varying degrees of purity, often accompanied by undesirable aftertaste. In addition, yields of mogroside from cultivated fruit are limited due to low plant yields and particular cultivation requirements of the plant. Mogrosides are present at ˜1% in the fresh fruit and ˜4% in the dried fruit. Mog.V is the main component, with a content of 0.5%-1.4% in the dried fruit. Moreover, purification difficulties limit purity for Mog.V, with commercial products from plant extracts being standardized to ˜50% Mog.V. A pure Mog.V product is desirable to avoid off flavors, and will be easier to formulate into products, since Mog.V has good solubility potential. It is therefore advantageous to produce sweet mogroside compounds, such as but not limited to Mog.V, via biotechnological processes.
Mogrosides can be produced by biosynthetic fermentation processes, as illustrated in
The pathway can alternatively proceed by cyclization of (3) to produce cucurbitadienol (9), followed by epoxidation to (5), or multiple hydroxylations of cucurbitadienol to 24,25-dihydroxycucurbitadienol (6), or to mogrol (7).
While biosynthetic enzymes from monkfruit (Siraitia grosvenorii) have been identified for production of mogrol (See, WO 2016/038617 and US 2015/0322473, which are hereby incorporated by reference in their entireties), many of these enzymes lack the productivity or physical properties desired for overexpression in microbial hosts, particularly for fermentation approaches that operate at higher temperatures than the natural climate of the plant. Accordingly, alternative or engineered enzymes are desired to improve production of mogrol using microbial fermentation, with mogrol acting as the substrate for glucosylation to produce Mog.V or other target mogroside.
Using an E. coli 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), and with overexpression of ScFPPS, enzymes were screened for their ability to convert FPP to squalene (SQS activity), as well epoxidation of squalene to produce 2,3-oxidosqualene (SQE activity). The 2,3-oxidosqualene intermediate can by cyclized by a triterpene cyclase, such as CDS from Siraitia grosvenorii. As demonstrated in
As shown in
Mogrol was used as a substrate for in vitro glucosylation reactions with candidate UGT enzymes, to identify candidate enzymes that provide efficient glucosylation of mogrol to Mog.V. Reactions were carried out in 50 mM Tris-HCl buffer (pH 7.0) containing beta-mercaptoethanol (5 mM), magnesium chloride (400 uM), substrate (200 uM), UDP-glucose (5 mM), and a phosphatase (1 U). Results are shown in
A summary of observed primary glycosylation reactions at C3 and C24 hydroxyls of mogrol are provided in Table 1. Specifically, 0.2 mM mogrol was fed to cells expressing various UGT enzymes. Reactions were incubated at 37° C. for 48 hrs. Products were quantified by LCMS/MS with authentic standards of each compound.
A summary of branched glycosylation reactions are provided in Table 2. 0.2 mM Mog.IIE or Mog.IE was fed to cells expressing various UGT enzymes. Reactions were incubated at 37° C. for 48 hr. Products were quantified by LC-MS/MS with authentic standards of each compound. “Indirect” evidence means that consumption of substrate was observed.
An exemplary E. coli strain producing Mog.V was created by expressing the following enzymes in an K E. coli strain engineered to produce high levels of MEP pathway products: SQS (SEQ ID NO: 11), SQE (SEQ ID NO: 39), SQE A4 (SEQ ID NO: 203), TTC (SEQ ID NO: 40), EPH (SEQ ID NO: 189), .sohB_CppCYP (SEQ ID NO: 199), AtUGT73C3 (SEQ ID NO: 202), UGT85C1 (SEQ ID NO: 165), and UGT94-289-1 (SEQ ID NO: 122). Production of Mog.V is demonstrated in
Biosynthesis enzymes can be further engineered for expression and activity in microbial cells, using known structures and primary sequences.
The UGT enzyme of SEQ ID NO: 164 was engineered for improved glycosylation activity. Various amino acid substitutions were made to the enzyme, as informed by in silico analysis. The following amino acid substitutions in Table 3 were tested for further glycosylation of mog.IIE.
An engineered UGT enzyme based on SEQ ID NO: 164 was prepared having substitutions T147L and N207K. The bioconversion of Mog.IIE to further glycosylated products is shown in
The UGT enzyme of SEQ ID NO: 165 was engineered for improved glycosylation activity. The following amino acid substitutions were identified as improving bioconversion of Mog.IA to Mog.IIE (Table 4):
An engineered UGT enzyme based on 85C1 was prepared having substitutions L41F, D49E, and C127F. The bioconversion of Mog.IA to Mog.IIE is shown in
The UGT enzyme of SEQ ID NO: 217 (UGT73F24) was engineered for improved glycosylation activity. The following amino acid substitutions were identified as improving bioconversion of Mog.IE to Mog.IIE with UGT73F24 (Table 5):
An engineered UGT enzyme based on UGT73F24 was prepared having substitutions A74E, 19F, and H101P. The bioconversion of Mog.IE to Mog.IIE is shown in
Saccharomyces cerevisiae FPPS
Siraitia grosvenorii SQSa
Siraitia grosvenorii SQSb
Cucumis sativus
Cucumis melo
Cucumis melo
Cucurbita moschata
Sechium edule
Panax quinquefolius
Malus domestica
Artemisia annua
Glycine soja
Diospyros kaki
Euphorbia lathyris
Camellia oleifera
Eleutherococcus senticosus
Flavobacteriales bacterium
Bacteroidetes bacterium
Siraitia grosvenorii SQE1
Siraitia grosvenorii SQE2
Momordica charantia
Cucurbita maxima
Cucurbita moschata
Cucurbita moschata
Cucumis sativus
Cucurais melo
Cucurbits maxima
Ziziphus jujube
Morus alba
Juglans regia (JrSQE1)
Cucumis melo
Cucumis sativus
Juglans regia (JrSQE2)
Theobroma cacao
Cucurbita moschata
Phaseolus vulgaris
Hevea brasiliensis
Sorghum bicolor
Zea mays
Medicago sativa
Methylomonas lenta
Bathymodiolus azoricus Endosymbiont
Methyloprofundus sediment
Methylomicrobium buryatense
Ononis spinosa SQE1
Ononis spinosa SOE2
Medicago truncatula SQE1
Medicago truncatula SQE2
Hypholoma sublateritium SQE
Hypholoma sublateritium SQE2
Hypholoma sublateritium SQE3
Siraitia grosvenorii CDS
Momordica charantia
Cucurbita maxima
Citrullus colocynthis (CcCDS1)
Citrullus colocynthis (CcCDS2)
Cucurbita moschata
Cucumis sativus
Cucumis melo
Citrullus lanatus subsp. vulgaris
Theobroma cacao
Ziziphus jujube
Prunus avium
Brassica napus
Spinacia oleracea
Trigonella foenum-graecum
Ricinus communis
Pisum sativum cycloartenol synthase (PsCAS_mut)
Cucurbita pepo subsp. pepo cycloartenol synthase (CpCAS mut)
Zostera marina cycloartenol synthase (ZmCAS mut)
Artemisia annua (AaCASmut)
Citrullus colocynthis (CcCDS2)
Siraitia grosvenorii EPH1 (SgEPH1)
Siraitia grosvenorii EPH2 (SqEPH2)
Siraitia grosvenorii EPH3 (SgEPH3)
Momordica charantia
Cucurbita moschata
Cucurbita maxima
Prunus persica
Morus notabilis
Ricinus communis
Citrus unshiu
Hevea brasiliensis
Handroanthus impetiginosus
Coffea canephora
Punica granatum
Arabidopsis lyrata subsp. lyrata
Rhinolophus sinicus
Gossypium raimondii (GrEPH)
Gossypium hirsutum (GhEPH)
Siraitia grosnevorii (SgEPH4)
Cucumis melo (CmEPH1)
Cucumis melo (CmEPH2)
Trema orientals (ToEPH)
Arachis hypogaea (AhEPH)
Mycobacterium tuberculosis (MtEPH)
Siraitia grosvenorii CYP87D18
Cucumis melo
Cucurbita maxima
Cucumis sativus
Cucurbita moschata
Prunus avium
Populus trichocarpa
Primus persica
Populus euphratica
Juglans regia
Pyrus x bretschneideri
Morus notabilis
Jatropha curcas (JcP450.1)
Hevea brasiliensis
Jatropha curcas (JcP450.2)
Chenopodium quinoa
Spinacia oleracea
Manihot esculenta
Olea europaea var. sylvestris
Cucurbita pepo subsp. pepo
Capsella rubella CYP705A38
Brassica rapa CYP705A37v2
Siraitia qrosvenorii
Camelina sativa
Raphanus sativus
Cucumis sativus (CsCYP87D20)
Cucumis sativus (sohB_CsCYP87D20)
Cucumis sativus (zipA_CsCYP87D20)
Cucumis sativus (CsCYP87D20_mut)
Cucumis sativus (sohB_CsCYP87D20_mut)
Cucurbita pepo subsp. pepo (sohB_CppCYP)
Cucurbita pepo subsp. pepo (17alpha_CppCYP)
Siraitia grosvenorii (CYP1798)
Stevia rebaudiana (SrCPR1)
Arabidopsis thaliana CPR1 (AtCPR1)
Arabidopsis thaliana CPR2 (AtCPR2)
Arabidopsis thaliana (AtCPR3)
Stevia rebaudiana CPR2 (SrCPR2)
Stevia rebaudiana CPR3 (SrCPR3)
Artemisia annua CPR (AaCPR)
Camptotheca acuminate CaCPR
Acetobacter pasteurianus subsp. ascendens (ApGA2ox)
Cucurbita maxima (CmGA2ox)
Dendrobium catenatum (DcGA3ox)
Cucurbita maxima (CmGA20ox)
Agapanthus praecox subsp. orientalis (ApoGA20ox)
Arabidopsis thaliana (AtFH)
Chrysosplenium americanum (CaF6H)
Datura stramonium (DsH6H)
Arabidopsis thaliana (AtH6DH)
Solanuni lycopersicum (S1F35H)
Catharanthus roseus (crD4Hlike)
Zea mays (ZmBX6)
Hordeum vulgare subsp. vulgare (HVIDS2)
Hordeum vulgare subsp. vulgare (HvIDS3)
Siraitia grosvenorii UGT720-269-1
Siraitia grosvenorii UGT94-289-3
Siraitia grosvenorii UGT74-345-2
Siraitia grosvenorii UGT75-281-2
Siraitia grosvenorii UGT720-269-4
Siraitia grosvenorii UGT94-289-2
Siraitia grosvenorii UGT94-289-1
Momordica charantia 1 (McUGT1)
Momordica charantia 2 (McUGT2)
Momordica charantia 3 (McUGT3)
Momordica charantia 4 (McUGT4)
Momordica charantia 5 (MCUGT5)
Cucumis sativus
Cucurbita maxima 1 (CmaUGTl)
Cucurbita maxima 2 (CmaUGT2)
Cucurbita maxima 3 (CmaUGT3)
Cucurbita moschata 1 (CmoUGT1)
Cucurbita moschata 2 (CmoUGT2)
Cucurbita moschata 3 (CmoUGT3)
Prunus persica
Theobronia cacao
Corchorus capsularis
Ziziphus jujube
Vitis vinifera
Juglans regia
Hevea brasiliensis
Manihot esculenta
Cephalotus follicularis
Stevia rebaudiana UGT74G1
Stevia rebaudiana UGT76G1
Stevia rebaudiana UGT85C2
Stevia rebaudiana UGT91D1
Stevia rebaudiana UGT91D2e
Arabidopsis thaliana AAN72025.1
Arabidopsis thaliana AAF87256.1
Columba livia CIUGTI
Haemophilus ducreyi LgtE Q9L875
Neisseria gonorrhoeae Q5F735
Rhizobium meliloti (strain 1021) ExoM P33695
Rhizobium radiobacter Q44418
Streptococcus agalactiae cpsI 087183
Streptococcus pneumoniae cps33 054611
Coffea arabica (CaUGT_1, 6)
Stevia rebaudiana UGT85C1
Arabidopsis thaliana AtUCT7303
Hordeum vulgare subsp. Vulgare HVUGT_B1
Hordeum vulgare subsp. Vulgare HVUGT_B3
Coffea canephora (CCUGT_1, 6) (207)
Coffea eugenioides (CeUGT_1, 6) (208)
Coffea eugenioides (CeUGT 1, 6.2) (209)
Siraitia grosvenorii (SgUGT94-289-3.2) (210)
Oryza sativa (OsJUGT 1, 6)
Panax ginseng (PsUGT94_B1)
Stevia rebaudiana (SrUGT73E1, with optional His tag)
Oryza sativa (OsUGT1-2)
Camelina sativa (XP_010516905.1)
Glycyrrhiza uralensis (UGT73F24)
Glycyrrhiza uralensis (UGT73033)
This application claims priority to, and the benefit of, U.S. Provisional Application No. 63/085,557 filed Sep. 30, 2020, U.S. Provisional Application No. 63/075,631 filed Sep. 8, 2020, and U.S. Provisional Application No. 62/948,657 filed Dec. 16, 2019, the disclosures of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/065285 | 12/16/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63075631 | Sep 2020 | US | |
| 63085557 | Sep 2020 | US | |
| 62948657 | Dec 2019 | US |
