Patent Application
20240240209
The present application claims priority to Singapore patent application number 10202104047X filed on 20 Apr. 2021 which is incorporated by reference herein in its entirety.
Genetically engineered microorganisms have been rapidly developed as the workhorse to produce essential molecules at unprecedented yield1. Technological advancement has enabled simultaneous optimization of multiple genes in a single microbial strain. To date, multiple genes can be rapidly assembled and transformed into microbial strains to probe the collective functions of the set of genes. In this manner, many complex and valuable natural products can be produced. However, one important prerequisite is the availability of prior knowledge about the product's biosynthetic pathway. Unfortunately for many industrially important molecules, their biosynthetic pathways are often incomplete with many missing enzymatic reactions.
Cis-α-irone, the principal component of orris root oil, is a good example. Traditionally used in “Queen's water”, orris oil commands a hefty price in the fragrance industry due to its lengthy and inefficient manufacturing process. With a production period spanning over three years, approximately 30-70 mg of natural irone can be produced from 1 kg of fresh iris rhizome. In the 1990s, Gil and co-workers patented an enzymatic process to degrade irone precursors from orris roots with lipoxidase or peroxidase2. The method improved the production rate but was still reliant on plant materials. Furthermore, different species of iris give rise to divergent odor perceptions, because the composition of irone isomers are non-identical3,4 as there are 10 different regio- and stereoisomers of irone5. GC-Olfactory analysis of α-irone mixtures had been performed (
Artificial biosynthetic routes could be designed by leveraging on promiscuous enzymes to overcome the problem of identifying the specific enzyme/s in the natural biosynthetic pathway
In a first aspect of the invention, there is provided a modified enzyme comprising a first substitution of base SEQ ID No. 3 at a position selected from the group consisting of position 200, position 180, position 160, and position 236, wherein if the first substitution is at position 200, the first substitution is selected from the group consisting of phenylalanine, isoleucine, leucine, valine, and tryptophan, wherein if the first substitution is at position 180, the first substitution is selected from the group consisting of alanine, cysteine, glutamic acid, isoleucine, methionine, and valine, wherein if the first substitution is at position 160, the first substitution is selected from the group consisting of alanine, cysteine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, threonine, valine, and tyrosine, wherein if the first substitution at position 236 is selected, the first substitution is selected from the group consisting of cysteine, glutamic acid, histidine, isoleucine, leucine, asparagine, serine, threonine, and valine. More preferably, the first substitution is at position 200, position 180, or position 160 as described above.
Preferably, the modified enzyme comprises a second substitution selected from the group consisting of position 182 and position 180, wherein if the second substitution is at position 182, the second substitution is selected from the group consisting of glutamic acid, threonine, asparagine and glutamine, wherein the second substitution may be selected at position 180 only if the first substitution is not at position 180, and if the second substitution is at position 180, the second substitution is selected from the group consisting of alanine, cysteine. In an embodiment, the first substitution is at position 200 and the second substitution is at position 182.
Preferably, the modified enzyme comprises a third substitution at position 273, wherein the third substitution is selected from the group consisting of valine, isoleucine, and lysine. In an embodiment, the first, second, and third substitutions are at positions 200, 182, and 273 respectively.
Preferably, the modified enzyme comprises a fourth substitution at position 180, wherein the first substitution is not at position 180 and the second substitution if present is not at position 180, and the fourth substitution is alanine or cysteine. In an embodiment, the first, second, third, and fourth substitutions are at positions 200, 182, 273, and 180 respectively.
Preferably, the modified enzyme comprises a fifth substitution at position 202 to a bulkier amino acid. For example, the bulkier amino acid may be selected from the group consisting of leucine, valine, and phenylalanine. In an embodiment, the first, second, third, fourth, and fifth substitutions are at positions 200, 182, 273, 180, and 202 respectively.
Preferably, the modified enzyme comprises a sixth substitution at position 65, wherein the sixth substitution is selected from the group consisting of phenylalanine, leucine, and methionine. In an embodiment, the first, second, third, fourth, fifth and sixth substitutions are at positions 200, 182, 273, 180, 202 and 65 respectively.
Preferably, the modified enzyme comprises a seventh substitution at position 156, wherein the seventh substitution is selected from the group consisting of aspartic acid, alanine, proline, glycine, and serine. In an embodiment, the first, second, third, fourth, fifth and seventh substitutions are at positions 200, 182, 273, 180, 202 and 156 respectively. In an embodiment, the first, second, third, fourth, fifth, sixth, and seventh substitutions are at positions 200, 182, 273, 180, 202, 65 and 156 respectively.
Preferably, the modified enzyme comprises an eighth substitution at position 91, wherein the eighth substitution is proline. In an embodiment, the first, second, third, fourth, fifth, sixth, seventh, and eighth substitutions are at positions 200, 182, 273, 180, 202, 65, 156, and 91 respectively.
Preferably, the modified enzyme comprises a ninth substitution at position 231, wherein the ninth substitution is aspartic acid. In an embodiment, the first, second, third, fourth, fifth, sixth, seventh, eighth, and ninth substitutions are at positions 200, 182, 273, 180, 202, 65, 156, 91, and 231 respectively.
Preferably, the modified enzyme comprises a tenth substitution selected from the group consisting of alanine at position 244, alanine at position 245, and proline at position 267. More preferably, the modified enzyme comprises at least two of the three, even more preferably all three substitutions.
Preferably, the modified enzyme comprises an eleventh substitution at position 197, wherein the eleventh substitution is arginine.
Preferably, the modified enzyme comprises a twelfth substitution at position 60, wherein the twelfth substitution is selected from the group consisting of valine, lysine, and arginine. The twelfth substitution may be considered as a seventh substitution described above. In an embodiment, the first, second, third, fourth, fifth, sixth and twelfth substitutions are at positions 200, 182, 273, 180, 202, 65, and 60 respectively.
Preferably, the modified enzyme comprises a thirteenth substitution selected from the group consisting of: (i) the thirteenth substitution is at position 11 and selected from histidine, leucine, proline, methionine, valine, and tryptophan; (ii) the thirteenth substitution is at position 12 and selected from lysine, alanine, glycine, and arginine; (iii) the thirteenth substitution is at position 13 and selected from leucine, methionine, glutamine, alanine, and glycine; (iv) the thirteenth substitution is at position 14 and selected from methionine, arginine, glycine, proline, leucine, and threonine; (v) the thirteenth substitution is at position 94 and selected from arginine, and valine; (vi) the thirteenth substitution is at position 95 and selected from isoleucine, cysteine, valine, and leucine; (vii) the thirteenth substitution is at position 107 and selected from serine and glycine; (viii) the thirteenth substitution is at position 123 and selected from asparagine, glutamine, and serine; (ix) the thirteenth substitution is at position 126 and selected from serine and glutamic acid; (x) the thirteenth substitution is at position 127 and selected from glycine and threonine; (xi) the thirteenth substitution is at position 129 and selected from cysteine, lysine, and valine; (xii) the thirteenth substitution is at position 137 and selected from glycine, alanine, aspartic acid, histidine, asparagine, serine, and threonine; (xiii) the thirteenth substitution is at position 159 and is leucine; (xiv) the thirteenth substitution is at position 176 and selected from leucine, valine, lysine, arginine, and tyrosine, and optionally a fourteenth substitution of valine at position 248, with preferably the thirteenth substitution being leucine; (xv) the thirteenth substitution is at position 185 and selected from valine and leucine; (xvi) the thirteenth substitution is at position 190 and selected from glutamine, alanine and proline, and optionally a fifteenth substitution of serine at position 122; (xvii) the thirteenth substitution is at position 191 and selected from serine, valine, leucine, isoleucine, tyrosine, and lysine; (xviii) the thirteenth substitution is at position 192 and selected from serine; (xix) the thirteenth substitution is at position 195 and selected from isoleucine, and valine; (xx) the thirteenth substitution is at position 212 and is leucine; (xxi) the thirteenth substitution is at position 268 and selected from lysine, glutamine, histidine, arginine, and leucine; (xxii) the thirteenth substitution is at position 269 and selected from leucine, glycine, arginine, and tryptophan; and (xxiii) the thirteenth substitution is at position 272 and is alanine. The thirteenth substitution may be considered as a tenth substitution described above. In an embodiment, the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and thirteenth substitutions are at positions 200, 182, 273, 180, 202, 65, 156, 91, 231 and as described above for the thirteenth substitution respectively.
Preferably, the modified enzyme comprises a sequence selected from the group consisting of SEQ ID No. 15, SEQ ID No. 14, SEQ ID No. 13, SEQ ID No. 16, SEQ ID No. 12, SEQ ID No. 11, SEQ ID No. 10, SEQ ID No. 9, SEQ ID No. 8, SEQ ID No. 7, SEQ ID No. 6, SEQ ID No. 5, SEQ ID No. 4, and SEQ ID No. 20 to 100.
Preferably, the modified enzyme comprises a polyhistidine-tag.
In a second aspect of the invention, there is provided a method of producing cis-α-irone, the method comprises providing an enzyme capable of converting psi-ionone to cis-α-irone; and contacting the enzyme and psi-ionone under suitable conditions to produce cis-α-irone.
Preferably, the enzyme is selected from the group consisting of a modified enzyme, SEQ ID No. 1, and SEQ ID No. 3, the modified enzyme being as described above for the first aspect of the invention.
Preferably, contacting the modified enzyme and psi-ionone is done in the presence of an auxiliary enzyme that removes or recycles SAH. More preferably, the auxiliary enzyme is selected from the group consisting of S-adenosylmethionine synthase (MetK), adenosylhomocysteinase (SAH1), 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (mtn), 5-methylthioadenosine/S-adenosylhomocysteine deaminase (mtaD), and halide methyl transferase (HMT).
Preferably, psi-ionone has a maximum concentration of 5 mM.
Preferably, the modified enzyme is provided as a lysate of a cell expressing the modified enzyme or a host cell comprising a plurality of nucleic acid sequences to encode at least one host cell enzyme and a first nucleic acid sequence encoding the modified enzyme, wherein the plurality of enzymes are produced by the host cell to assist in converting glucose or glycerol to psi-ionone.
Preferably, the host cell enzyme includes at least one of the following: HMG-COA synthase (HmgS), Acetoacetyl-CoA thiolase (AtoB), truncated HMG-COA reductase (tHmgR) or HMG-CoA reductase, mevalonate kinase (MevK), phosphomevalonate kinase (PMK), mevalonate pyrophosphate decarboxylase (PMD), IPP isomerase (Idi), GGPP synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (Crtl), FPP synthase (IspA), and modified OfCCD1 enzyme or OfCCD1 enzyme.
Preferably, at least one of the following conditions are fulfilled: (i) the host cell further comprises a second nucleic acid sequence to encode SAM cycle enzymes; (ii) a third nucleic acid sequence encoding a transcription regulator metJ is absent in the host cell; (iii)
the host cell further comprises at least one T7 promoter sequence; (iv) the host cell is Escherichia coli.
Preferably, the modified enzyme is provided by the host cell and the suitable conditions include a dissolved oxygen content of 30% or less, preferably 1% to 15%, more preferably 2% to 10%.
Preferably, at least 0.1 μg/L of cis-α-irone is produced or a minimum cis-to-trans α-irone ratio of 2 to 5.
In a third aspect of the invention, there is provided a host cell comprising a plurality of nucleic acid sequences to encode enzymes to allow the host cell to convert glucose or glycerol to psi-ionone and a first nucleic acid sequence encoding the modified enzyme according to the first aspect of the invention. Preferably, the enzymes include at least one of the following: HMG-COA synthase (HmgS), Acetoacetyl-CoA thiolase (AtoB), truncated HMG-COA reductase (tHmgR) or HMG-COA reductase, mevalonate kinase (MevK), phosphomevalonate kinase (PMK), mevalonate pyrophosphate decarboxylase (PMD), IPP isomerase (Idi), GGPP synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (Crtl), FPP synthase (IspA), and modified OfCCD1 enzyme or OfCCD1 enzyme. More preferably, most of the enzymes are included, even more preferably all the enzymes are included.
Preferably, at least one of the following conditions is fulfilled: (i) the host cell further comprising a second nucleic acid sequence to encode SAM cycle enzymes; (ii) a third nucleic acid sequence encoding a transcription regulator metJ is absent (in the host cell); (iii) the host cell further comprising at least one T7 promoter sequence; (iv) the host cell is Escherichia coli
In a fourth aspect of the invention, there is provided a method of methylating a hydroxyl group, the method comprising providing an enzyme selected from the group consisting of the modified enzyme according to the first aspect of the invention (preferably SEQ ID No. 8), SEQ ID No. 1 and SEQ ID No. 3, or the host cell according to the third aspect of the invention; and contacting the enzyme or the host cell and a hydroxyl group under suitable conditions to methylate the hydroxyl group. Preferably, the hydroxyl group is an allylic hydroxyl group.
In all aspects of the invention, the modified enzyme comprises, or consists essentially of, or consists of, one or more of the substitution/s or SEQ ID No as described herein in the specification. For example, the modified enzyme may comprise, or consists essentially of, or consists of, the first substitution of SEQ ID No. 2, and optionally any additional substitutions as described. For example, the modified enzyme may comprise, or consists essentially of, or consists of, any one of the SEQ ID No. recited above. In the specific examples described herein, whilst substitutions at certain positions of SEQ ID No. 2 is in combination with other substitutions, it is likely that these substitutions alone would have similar effect on improving the selectivity and/or yield of cis-α-irone.
Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and/or combinations and/or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.
The following detailed description is merely exemplary in nature and is not intended to limit the invention or the application and uses of the invention. Furthermore, there is no intention to be bound by any theory presented in the preceding background of the invention or the following detailed description.
Other technical advantages may become readily apparent to one of ordinary skill in the art after review of the following figures and description.
Figure (
mevalonate kinase (MevK), phosphomevalonate kinase (PMK), mevalonate pyrophosphate decarboxylase (PMD) and IPP isomerase (Idi). The third plasmid carried the lycopene synthesis EBIA module: GGPP synthase (CrtE), phytoene synthase (CrtB), phytoene desaturase (Crtl) and FPP synthase (IspA). The last plasmid carried the modified OfCCD1 enzyme fused with thioredoxin (TofCCD1m)7. Tm1, Tm2, Tm3 are mutated T7 promoters with different strength (Tm1>Tm2>Tm3)15.
Some of the figures are composed of panels which are labelled alphabetically, the panel may be referred to herein as FIG. Xa where X is the Figure number and “a” is the appropriate panel.
The following references provide one of skill with a general definition of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).
Various amino acids are described herein by its full name, and conventional 1-letter and 3-letter abbreviations as is known in the art. The substitution of an amino acid residue in a peptide is describe by the conventional notation, for example Y200F indicates that the 200th amino acid residue (or position) of tyrosine (Y) is substituted by phenylalanine (F).
As used herein, the articles “a”, “an” and “the” as used with regard to a feature or element include a reference to one or more of the features or elements. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. As used herein, the terms “first,” “second,” and “third,” etc. are used merely as labels, and are not intended to impose numerical requirements on their objects. In particular, the modified enzyme may comprise any combination of the first substitution, the second substitution, the third substitution and so forth, it is not required that the modified enzyme contains all the substitutions. As an example, the modified enzyme may comprise the first substitution and the third substation without requiring the second substitution, other substitution patterns may also be possible as described herein.
The terms “polypeptide” and “protein”, used interchangeably herein, refer to a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude chemical or post-expression modifications of the polypeptides of the invention, although chemical or post-expression modifications of these polypeptides may be included or excluded as specific embodiments. Therefore, for example, modifications to polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Further, polypeptides with these modifications may be specified as individual species to be included or excluded from the present invention. The natural or other chemical modifications, such as those listed in examples above can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of 14 hosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. [See, for instance Creighton, (1993), Posttranslational Covalent Modification of Proteins, W.H. Freeman and Company, New York B. C. Johnson, Ed., Academic Press, New York 1-12; Seifter, et al., (1990) Meth Enzymol 182:626-646; Rattan et al., (1992) Ann NY Acad Sci 663:48-62]. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
As used herein, the terms “recombinant polynucleotide” and “polynucleotide construct” are used interchangeably to refer to linear or circular, purified or isolated polynucleotides that have been artificially designed and which comprise at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their initial natural environment. In particular, these terms mean that the polynucleotide or cDNA is adjacent to “backbone” nucleic acid to which it is not adjacent in its natural environment. Additionally, to be “enriched” the cDNAs will represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid backbone molecules. Backbone molecules according to the present invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest. Preferably, the enriched cDNAs represent 15% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. More preferably, the enriched cDNAs represent 50% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. In a highly preferred embodiment, the enriched cDNAs represent 90% or more (including any number between 90 and 100%, to the thousandth position, e.g., 99.5%) of the number of nucleic acid inserts in the population of recombinant backbone molecules.
The term “recombinant polypeptide” is used herein to refer to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.
The terms “sequence similarity”, “percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Identity is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, CLUSTAL W, FASTDB [Pearson and Lipman, (1988), Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., (1990), J. Mol. Biol. 215(3):403-410; Thompson et al. (1994), Nucleic Acids Res. 22(2):4673-4680; Higgins et al., (1996), Meth. Enzymol. 266:383-402; Altschul et al., (1993), Nature Genetics 3:266-272; Brutlag et al. (1990) Comp. App. Biosci. 6:237-24], the disclosures of which are incorporated by reference in their entireties.
Metabolic engineering has become an attractive method for efficient production of natural products. However, one important pre-requisite is to establish the biosynthetic pathways. Many commercially interesting molecules cannot be biosynthesized as their native biochemical pathways are not fully elucidated. Cis-α-irone, a top-end perfumery molecule, is an example. Retrobiosynthetic pathway design by employing promiscuous enzymes provides a solution. Described herein is a synthetic pathway to produce cis-α-irone with a promiscuous methyltransferase (pMT). By structure-guided enzyme engineering strategies, pMT activity and specificity towards cis-α-irone was improved by greater than 11,000-fold and 700-fold respectively. By incorporating the optimized methyltransferase into engineered microbial cells, approximately 172 mg/L of cis-α-irone was produced from a renewable carbon source in a 5 L bioreactor. This illustrates that integrated retrobiosynthetic pathway design and enzyme engineering can offer novel opportunities to expand the scope of natural molecules that can be biosynthesized.
A total enzymatic synthetic route to produce cis-α-irone from glucose has been developed by identifying and optimizing a promiscuous bifunctional methyltransferase/cyclase enzyme (pMT,
Inferring from knowledge of chemical and biochemical synthesis, it was postulated that psi-ionone can be converted to irone by a bMTC enzyme (artificial biosynthesis route,
To identify a potentially more active enzyme, pMT1 sequence (SEQ ID No. 3) was mined using BLAST against the non-redundant protein sequence database11 and two sequences were identified to have greater than 60% sequence identity to pMT1 (
In solution and in X-ray structure, pMT1 shows a hexameric assembly whose minimal functional unit is a dimer10. pMT1 displays a unique active site formed by amino acid residues from two distinct monomeric chains of the hexamer, with the N-terminus of one monomer covering the catalytic cavity of the other monomer (
Among them, site-saturated mutants of G85 were also included. This is a key residue involved in SAM co-factor binding. Mutating this glycine to the other 19 amino acids significantly reduces the methyltransferase activity. Hence, the activity of the pooled G85 mutants could be an approximate baseline to access the impact of mutation on the activity of pMT: if the pool mutants have lower or similar activities as G85 mutants, the corresponding residues are probably essential for methyltransferase activity and should not be mutated; otherwise, the corresponding residues are potential targets for further analysis to identify the specific beneficial mutation.
The 24 mutant libraries and 1 non-mutated wild-type (WT) pMT1 were transformed into E. coli for pooled-colony reaction. Trans-α-irone and cis-α-irone produced from each pooled mutant library were quantified. The fold change was calculated by dividing the cis-α-irone produced from one reaction with cis-α-irone produced by the G85m reaction. Similarly, cis-α-irone to trans-α-irone ratio (cis-to-trans-α-irone ratio) was also quantified and the fold change was calculated against the G85m control.
Among the 24 pooled reactions, seven positions displayed at least two-fold improvement in cis-α-irone production as compared to the baseline activity (i.e. G85 mutation), and three out of the seven residues, namely Y200, L180 and R160, gave rise to higher cis-α-irone production as compared to pMT1 (
Subsequently, site-saturated mutagenesis of Y200 was carried out. A Y200F mutation (pMT2, SEQ ID No. 4), improved the soluble expression of the methyltransferase and its total turnover number (TTN) by greater than 3-fold and 12-fold respectively (
Next, site-saturation mutagenesis was performed on L180 and S182 using pMT2 as the new template (Table 7 and
To further improve the enzymatic activity and selectivity of the promising mutant (pMT3), molecular dynamics simulations were carried out using the modelled complexes of the four isomers of α-irone and pMT1 (or pMT3) to identify amino acid residues that could be critical to improve binding of cis-α-irone or decrease binding of trans-α-irone. Visual inspection and free energy calculations were performed. Interestingly, free energy calculations showed a significant change of the free energy profile of an important residue, E153 (Table 1). Table 1 shows the free energy contributions per residue computed from MM/GBSA approaches. The free energy calculations were performed using MD simulations from pMT1/3/4 in complex with each of the four isomers of α-irone. The energy profile change of the catalytic residue E153 is highlighted by showing the unfavorable contributions in bold. pMT1 corresponds to the wild-type whereas pMT3 is the Y200F/S182E mutant and pMT4 the Y200/S182E/L273V mutant. The numbering of the residues corresponds to the X-ray structure (PDB id: 5GM2). The free energy values are given in kcal/mol and correspond to the mean from 100 snapshots extracted uniformly. Indeed, based on the proposed mechanism and crystal structure of pMT110, E153 is the key catalytic residue that extracts the proton from psi-ionone (
1.14
0.11
0.00
0.70
Moreover, since psi-ionone (556.3 Å2) is smaller than pMT1's natural substrate (teleocidin A1, 1129.2 Å2), A202 was mutated to bulkier amino acids (leucine, valine or phenylalanine) based on pMT5 to shrink the binding pocket, in order to increase the affinity of the enzyme for the substrate. Among the new mutants, A202L (pMT6, SEQ ID No. 8) significantly increased the cis-α-irone concentration (
Due to the catalytic role of E153, the mutation of surrounding residues is expected to influence the enzyme selectivity. 18 amino acid residues were identified in the close vicinity of E153. Pooled experiments were carried out again using pMT6 as template and a E153 mutation was included as baseline activity (
Among them, mutations of Y65 showed a drastic decrease in the β-irone production, and C156 mutations displayed the highest amount of cis-α-irone produced (
Q60 was identified as another possible mutation site. Site-saturation mutagenesis of Q60 was performed and the Q60V (SEQ ID No. 21), Q60K (SEQ ID No. 22), and Q60R (SEQ ID No. 23) mutants were found to have increased cis-α-irone production in-vivo (
Table 2 below shows the active site residues (or residues in or near the binding pocket) of pMT1, SaMT, and ScMT. The residues in pMT1 that have been mutated and tested to determine if there is improved activity is indicated in bold. Active site residues in pMT7 which are different from SaMT and ScMT are underlined. In addition, some beneficial mutations may not be found within the binding pocket of pMT1, for example Y65F.
Y
L
V
H
C
G
S
S
107
I
107
I
112
Q
136
Q
151
V
153
E
154
S
154
I
156
C
157
H
160
R
180
L
181
E
182
S
196
F
195
L
200
Y
199
L
199
L
202
A
201
S
203
N
205
P
232
L
235
T
236
M
239
F
238
M
238
L
273
L
272
V
272
T
277
T
279
F
The steady-state kinetic parameters of pMT7 were determined (Table 3). The kcat and Km values for catalyzing psi-ionone were 0.0044 h−1 and 35.2 μM, while those for SAM were 0.0052 h−1 and 54.6 μM. While determining the steady-state kinetics of pMT7, it was noticed that the reaction with purified pMT7 stopped at 6 h before significant conversion had taken place (
To reduce SAH inhibition, we mutated the residues (D135 and T91) that form hydrogen bonding with SAH so that the binding between SAH and the enzyme was weakened (
An alternative strategy to reduce SAH inhibition is to introduce auxiliary enzymes that degrade SAH12 (
With the positive effect of mtaD, in vitro biotransformation by using cell lysate containing overexpressed pMT10 and mtaD was explored. To challenge the enzyme, 10 mg/L (0.05 mM), 100 mg/L (0.5 mM) or 1000 mg/L (5 mM) of psi-ionone was supplemented into the reaction, and 6.4 mg/L (˜0.03 mM), 54 mg/L (˜0.26 mM) and 182 mg/L (˜0.88 mM) of cis-α-irone were produced, respectively after incubating the reaction at 28° C. for 3 days (
To further reduce the amount of β-irone and increase cis-α-irone production, C156 was mutated to aspartic acid instead to provide pMT11 based on pMT10. Compared to pMT10, pMT11 provided improved cis-α-irone production both by the fold change and percentage (
The advantage of in vivo biotransformation is that microbial cells can regenerate expensive co-factors rendering the scale-up bioprocess more cost-effective. Moreover, microbial cells possess sophisticated mechanisms to regulate SAH concentration14, so it may alleviate the inhibition of SAH to pMT enzyme. Thus, pMT was incorporated into our psi-ionone producing microbe7,8, to synthesize cis-α-irone from cheap renewable carbon sources (glucose/glycerol) (
Lastly, a single-phase fed-batch process was tested in a 5 L bioreactor. Even though 2O31 ΔmetJ strain produced a higher titer of cis-α-irone, its growth seemed to be impeded during fermentation. Thus, 2O31-pMT10 strain was tested. The optimized bioprocess for α-ionone was applied8, in which the glucose feeding rate was set at 10 g/h and the glucose concentration was kept between 0-5 g/L. Two different dissolved oxygen (DO) levels were tested: 2% and 10%. Under these DO settings, evaporation was kept at minimum which is <0.05%. As shown in
Using E. coli cell lysate overexpressing pMT6, a range of structurally similar terpenoids for methylation and cyclization reaction was assayed (
E. coli BI21-Gold DE3 strain (Stratagene) was used in this study. CRISRP-cas9 mediated gene deletion was carried out to modify the genome of E. coli BL21 strain18. pET11a (Novagen) was used to construct the methyltransferase mutants. For in vivo psi-ionone production, the plasmids were modified p15A plasmids as previously described (Table 5) 7 and is exemplified by SEQ ID Nos. 104 to 117. The genes: pMT1 or TleD from Streptomyces blastmyceticus, SaMT from Streptomyces albireticuli, and ScMT11 from Streptomyces clavuligerus were codon optimized and synthesized by Integrated DNA Technologies. Mtn was amplified from Escherichia coli genome. Unless otherwise noted, all chemicals and reagents were obtained from Sigma-Aldrich.
E. coli BL21-
E. Coli
Mutations were carried out with modified QuikChange™ protocol7. Briefly, overlapping primers were designed, which carried the desired mutation, to amplify the plasmid carrying the pMT gene. 10 μL of polymerase chain reaction (PCR) was carried out to amplify the plasmid by high-fidelity iproof polymerase (BioRad). Subsequently, the template plasmids were removed by treating the PCR reaction mixture directly with 0.5 unit of DpnI enzyme (New England Biolabs) at 37° C. for 3 h. Lastly, 1 μL of PCR reaction mixture was transformed into 20 μL of Stellar competent cells (Clonetech) by heat-shock transformation. The mutation was verified by sequencing.
For the pooled colony screening, the cloning steps were the same as the directed mutagenesis except degenerative primers with NNK or MNN were used to amplify the plasmid. After transformation, all the colonies were combined with 1 ml phosphate buffer saline solution (PBS) and subjected to plasmid extraction. The purified plasmid was then transformed into BL21 cells and plated on agar with ZYM auto-induction media and incubated at 20° C. for 3 days19. The colonies formed were collected in 1 ml PBS, and OD was measured. 12.5*250 μl*OD cells were taken for subsequence reaction with psi-ionone to test methyltransferase activity.
His-tag protein purification was carried out according to the manufacturer's instruction (Cube Biotech). For each enzyme, it was overexpressed in 200 ml of ZYM autoinduction media with 5 mM lactose at 20° C. for 36 hrs. Following that, cells were pelleted and resuspended with 10 ml of His-binding buffer (50 mM Tris, pH 8, 0.5M NaCl and 20 mM imidazole) supplemented with 1 mg/ml lysozyme and 3 U/ml of benzonase nuclease (Merck) and treated for 1 hour (hr) at 20° C., 300 rpm. The cells were further lysed by 2 cycles of freeze-thawing with freezing at −80° C. for 4 hrs and thawing at room temperature for 45 mins. The supernatant (soluble fraction) was obtained by centrifuging at 15,000 g for 30 mins at 4° C. The collected supernatant was then mixed with 1 ml of equilibrated Ni-NTA beads (Cube Biotech) overnight at 4° C. with continuous mixing. The next day, the beads were washed 3 times with 5 ml of His-binding buffer to remove any unbound proteins before eluting bound enzyme 5 times with 0.5 ml of His-elution buffer (50 mM Tris, pH8, 0.5M NaCl, 0.5M imidazole). The eluted protein was concentrated using spin-column with a MW cut-off of 10 kDa (Sartorius). Protein quantification and purity were determined through micro BCA assay (Pierce) and running SDS-PAGE, respectively.
To quantify the protein concentration in soluble fraction of cell extracts, western blot analysis was carried out. Standard SDS-PAGE protocol was performed before transferring the proteins onto nitrocellulose membrane using iBolt2 dry transfer system (Thermofisher). The membrane was then blocked with 20 ml of 5% milk in TBST buffer for 1 hr at room temperature. Following that, it was probed with anti-6×His-tag antibody-HRP (Abcam) in 1% milk at a ratio of 1:2000 overnight at 4° C. The membrane was washed 3 times with 20 ml of TBST buffer before adding in substrate for chemiluminescence detection (Millipore) and imaging using ChemiDoc system (Bio-rad). The chemiluminescence signal was quantified using Image Lab (BioRad). Purified pMT enzyme with known concentration (2.8-90 ng/μl) was used to prepare the standard curve. The concentration of pMT enzyme in cell extract was then quantified against the standard curve.
Following enzyme purification, a 100 μl enzymatic reaction was setup with a final concentration of 0.5 mg/ml of enzyme, 40 mg/L psi-ionone, 0.2 mM SAM, 100 mM Tris (pH 7), 10 mM MgCl2 and 15 mM NaCl. The reaction was done in 0.2 ml PCR strips and incubated at 28° C., 1200 rpm overnight. Alongside, a control without enzyme (with 40% glycerol only) was included as negative control. The next day, cis-α-irone was extracted with 100 μl of ethyl acetate and 50 μl organic layer taken for GCMS analysis. For enzyme characterization, similar enzymatic reaction was performed, with varying concentration of psi-ionone (2-40 mg/L) and SAM (10-100 μM), and the reaction was stopped after 2 h incubation at 28° C. to ensure the enzymatic conversion is still within linear range.
For each pMT mutant, it was transformed into BL21 and grown in autoinduction media with 5 mM lactose at 20° C. for 36 hrs. Then, cells were pelleted and concentrated 10 times by adding 1 ml of PBS. The OD600 was measured and 40*600 OD*ul cells were transferred into a new 1.5 ml tube. The cells were spun down and resuspended in 600 μl of lysis buffer (1×DNase buffer, 150 mM NaCl, 0.5% glycerol, 1 mg/ml lysozyme and 20U of DNasel). The cells were incubated for 2 hrs at 20° C. with shaking at 300 rpm. Following that, 2 cycles of freeze-thaw was done to completely lyse the cells. At the end of the 2nd thawing, the cell lysate was used to setup a 1 ml reaction in 2 ml GC vials. The reaction consisted of 50 mM Tris (pH7), 10 mM MgCl2, 60 mM NaCl, 0.2 mM SAM and 10 mg/L of psi-ionone and caryophyllene each. To this, 500 μl of cell lysate was added while the remaining 100 μl was used for SDS-PAGE to analyze the total and soluble protein expression. The reaction was incubated for 2 days at 28° C., 300 rpm prior to extraction of cis-α-irone with 500 μl hexane. From this, 20 μl of organic layer was taken into 180 μl hexane for GCMS analysis.
Biotransformation of Glucose/Glycerol into Cis-α-Irone
The four plasmids were transformed into E. coli BL21 (DE3), ΔaroA, ΔaroB, ΔaroC, ΔserC or E. coli BL21 (DE3), ΔaroA, ΔaroB, ΔaroC, ΔserC, ΔmetJ strains and plated on Agar plate containing LB media (10 g, tryptone. 5 g, yeast extract. 10 g, NaCl) supplemented with appropriate antibiotics (100 mg/L ampicillin, 34 mg/L chloramphenicol, 50 mg/L kanamycin, and 100 mg/L spectinomycin). One colony was picked and inoculate into LB media with antibiotics. 1% of overnight culture was inoculated into 1 ml fresh auto-inducing chemically defined media. The chemical media contains carbon source solution: 0.5 g/L glucose, 10 g/L glycerol; inducer: 30 mM lactose and base media: 2 g/L ammonium sulfate, 4.2 g/L KH2PO4, 11.24 g/L K2HPO4, 1.7 g/L citric acid, 0.5 g/L MgSO4, and 10 mL/L trace element solution. The trace element solution (100×) contained 0.25 g/L CoCl2·6H2O, 1.5 g/L MnSO4·4H2O, 0.15 g/L CuSO4·2H2O, 0.3 g/L H3BO3, 0.25 g/L Na2MoO4·2H2O, 0.8 g/L Zn(CH3COO)2, 5 g/L Fe(III) citrate, and 0.84 g/L ethylenediaminetetra-acetic acid (EDTA) at pH 8.0. The culture was grown at 28° C. for 3 days and the products were extracted with 1 ml hexane for GCMS analysis. An example of a fed-batch process for α-ionone Is briefly described, overnight culture was inoculated into 2 L chemical media comprising the base media and 5 g/L glucose, at 37° C. and 30% DO. pH was controlled at 7 with base solution (14% ammonia and 0.5 M NaOH). Feed media (500 g/L glucose and 5 g/L MgSO4) was added into bioreactor once OD600 reached 5, at a rate of 7.15-25.75 g/h glucose for about 4-5 h until OD600 reached ˜30-40. Subsequently, 0.1 mM IPTG was added to induce the production, and feeding rate was kept at a constant rate of 10 g/h glucose, and the temperature was reduced to 30° C. DO was adjusted to 2% or 10%. Feeding was stopped at 113 h when 2 L feeding media was finished, but the culture was further incubated for 24 h. To capture the evaporated products, the exhaust was connected to 25 ml sunflower oil.
For the mutant pMT activity screening, the reactants and products were analyzed by head space solid phase microextraction (HS-SPME) coupled with an Agilent 5977B gas chromatography (GC) system equipped with Agilent DB-5 ms column (30 m×250 μm×0.25 μm) and mass spectrometry (MS) with high efficiency source. The reaction mixtures were carried out in SPME vials which were incubated at 60° C. for 20 mins to allow the release of volatile compounds. The absorbent fiber (50/30 μm divinylbenzol/carboxen/polydimethyl-SPME fiber, SUPELCO) was then exposed to the headspace of the vial for 20 mins. The extracted analytes were desorbed at the GC inlet at 250° C. for 1 min and injected into GC with a split ratio of 200:1. The GC oven temperature increased from 50° C. to 140° C. at a rate of 10° C./min and held at 140° C. for 10 min. Subsequently, the temperature was increased to 320° C. at a rate of 60° C./min and held at 320° C. for 2 mins. The concentration of psi-ionone and irone were calculated by interpolating with a standard curve prepared by synthetic standards.
To determine the purified enzyme kinetics and in vivo production, the reaction mixture was extracted with 0.5× or 1× volume of ethyl acetate and the organic phase was diluted appropriately before subjected to GCMS analysis. The samples were analyzed on an Agilent Intuvo 9000 GC system equipped with Agilent DB-WAX Ultra Inert Intuvo GC column (30 m×250 μm×0.25 μm) and Agilent 5977B mass spectrometry with high efficiency source. 1 μl organic phase was injected at the split ratio 10:1 at 250° C. The oven temperature was held at 50° ° C. for 1 min and increased to 200° C. at a rate of 40° C./min and held at 200° C. for 3 min. Subsequently, the temperature increased to 230° C. at a rate of 40° C./min and held at 230° ° C. for 5 mins. The concentration of psi-ionone and irone were calculated by interpolating with a standard curve prepared by synthetic standards.
The three-dimensional structure of pMT1 in complex with the cofactor S-adenosyl-I-homocysteine with the substrate teleocidin A1 was retrieved from the PDB database (PDB id: 5GM2). One dimeric unit was used as template for modelling pMT1 (wild-type) in complex with the four α-irone isomers (1R5R and 1S5S for the trans-α-irone and 1S5R and 1R5S for the cis-α-irone). The α-irones were first built and gradually relaxed using the Avogadro software version 1.1.120. They were then manually docked into pMT1 active site using the bound substrate teleocidin A1 as template from crystallographic structure. pMT3 (Y200F, S282E) and pMT4 (Y200F, S282E, L273V) were modelled using Modeller 9.1921 and 3D models with lowest DOPE score were kept for further analyses.
The corresponding complexes of pMT3 or pMT4 with the four α-irones were built by superposing pMT1 complexes.
MD simulations were performed using the AMBER ff14S force-field22 for enzymes and GAFF23 for the ligands using pmemd.CUDA of AMBER16 software24. The partial charges for the ligand were computed using AM1-BCC method25 from antechamber. The system was protonated using propka webserver to set the experimental pH of 7. MD simulation parameters were the same as previously described26. In all stages, the sulphur of SAH was kept close to the irone isomer by setting a distance constraint lower than 5 Å and using a force constant of 25 kcal mol-1 Å-2.
The MD simulations were carried out for a total of 5 ns for all complexes. MM/PBSA calculations were performed using MMPBSA.py software and default parameters for Poisson-Boltzmann as described by Miller et al.27
Streptomyces
blastmyceticus
Taken together, eight mutations were introduced to pMT1 and improved both activity and product-selectivity by greater than 11,000-fold and 700-fold respectively (Tables 4 and 6). Wild-type pMT1 converted psi-ionone to predominantly trans-α-irone, which is not olfactive. The improved mutant pMT7 converted psi-ionone to more than 90% cis-α-irone, which has the finest, iris-like notes5. By further modulating the SAH binding pocket residues, pMT10 with 10-fold improvement of catalytic efficiency was obtained (Table 3), and produced approximately 182 mg/L of cis-α-irone from psi-ionone. By incorporating pMT10 into metabolically engineered Escherichia coli, ˜172 mg/L cis-α-irone was produced from glucose in a fed-batch process. As compared to natural extraction, our current bioprocess is 7,200 to 33,000 fold higher in yield for the same land size and duration (Table 9), paving a way towards sustainable bioproduction of the premium perfume molecule—cis-α-irone.
Protein sequences (the sequences used are codon-optimized with a N-terminal 6×his-tag) and DNA sequences used herein. Other polyhistidine tags may be used as desired and at the C-terminal.
SEQ ID Numbers 4 to 16 and 20 to 100 are mutations introduced to SEQ ID No. 3 (pMT1) and the codon optimized sequence may be derived from SEQ ID No. 19. SEQ ID No. 101 to 114 set out example of sequences that may be used in the plasmid for in vivo production of cis-α-irone in the following order respectively: HmgS, AtoB, tHmgR, MevK, PMK, PMD, Idi, crtE, crtB, Crtl, ispA, TOfCCD1m, MetK, and mtn.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202104047X | Apr 2021 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2022/050235 | 4/20/2022 | WO |
