Patent Application
20220290111
The present invention relates to polynucleotides and nucleic acid molecules encoding piperine synthases and to proteins and enzyme functional for converting piperoyl-CoA and piperidine to piperine and/or cis/trans isomers thereof. The invention also relates to a method of producing an amide from an acyl-CoA and an amine, and to a method of producing an amide from a carboxylic acid and an amine.
Piperamides constitute a class of natural compounds, where aromatic or aliphatic acids are linked to primary or secondary amines by an amide bond. Whereas numerous piperamides have been isolated from various members of the Piperaceae, the biosynthesis of these metabolites remained enigmatic. Piperine, the most prominent member of these compounds, is present in high amounts in Piper nigrum (black pepper) and largely responsible for the pungent taste of this economically relevant spice. In addition to the use as a spice, piperine and piperamides have been used as antimicrobial, insecticidal, fungicidal as well as health-promoting agents for all kinds of diseases in the ancient world. Whereas previous publications refer to the identification, the chemical characterization and the properties of piperine and related amides, neither a gene sequence nor a protein sequence has been reported so far (Leistner E and Spenser ID, 1973; Geisler J G and Gross G G, 1990). The latter authors reported a piperine synthase activity (EC 2.3.1.145) in crude protein extracts of black pepper shoots, which was able to convert piperoyl-CoA and piperidine to piperine. Due to the apparent instability of the activity, purification of a protein having the activity was not achieved and the catalytic activity of the instable enzyme preparation was rather low. As a consequence, the biotechnological production of piperine of piperamides has not been possible.
Piperamides are known to interact with the human TRPV-vanilloid receptor (McNamara F N et al., 2006), which is involved in pain perception. In addition, piperine stabilizes other voltage-gated sodium channels (Suzuki T and Yamato S, 2018) and is able to inhibit MDR-1 type ABC-transporters, preventing the export of chemotherapeutic agents from cancer cells and thereby reducing the toxicity and exposure of these compounds to healthy human cells (Li H et al., 2018). Synthetic piperlongumine, originally isolated from Piper longum is known to selectively kill tumor cells and has been proposed as selective acetylcholine esterase inhibitors to be used in the treatment of Alzheimer disease (Wiemann et al., 2017). Therefore, piperine synthase-associated enzymes, if found, could serve as a blueprint to develop and engineer a new type of plant derived natural products as potent drugs, also in combination with corresponding CoA-ligases, cytochrome P450 monooxygenases, acyl-, glycosyl-, or methyltransferases. Further, such enzymes could promote the production of natural piperamides and related compounds under controlled conditions in a safe and sustainable way, in combination with, but independent from, chemical synthesis.
Departing from the prior art, it is an object of the present invention to provide proteins, and polynucleotides encoding them, that are capable of converting piperoyl-CoA and piperidine to piperine. It is also an object of the present invention to provide proteins, preferably pure or purified proteins, capable of converting piperoyl-CoA and piperidine to piperine that are stable, have high catalytic activity, and where activity is clearly defined on the basis of amino acid sequence identity. It is also an object of the present invention to provide a method of producing an amide from an acyl-CoA and an amine, and proteins and enzymes therefor. It is also an object of the present invention to provide products that can be synthesized by the enzymes disclosed herein.
For accomplishing these objects, the invention provides:
LFLTAI at the position corresponding to amino acid residues 42 to 47 in SEQ ID NO: 2,
GLML at the position corresponding to amino acid residues 310 to 313 in SEQ ID NO: 2,
SN at the position corresponding to amino acid residues 365 to 366 in SEQ ID NO: 2, and/or
LID at the position corresponding to amino acid residues 376 to 378 in SEQ ID NO: 2.
The inventors have found enzymes from immature black pepper fruits that catalyze the decisive step in piperine formation from piperoyl CoA and piperidine. Identification of piperine synthase was achieved based on three assumptions conceived by the inventors. First, the biosynthetic enzymes involved in piperine and piperamide formation are highly expressed in maturing fruits. Second, piperine synthase is expressed differentially in fruits, leaves, and flowers, with highest expression anticipated for young fruits, and third are specifically detected in black pepper and related Piperaceae and rely on activated CoA esters.
Based on in-house analysis, data assembly, and interpretation of transcriptome data according to above assumptions from immature fruits of black pepper, and relative transcript abundance, the inventors identified individual polynucleotides and genes involved in the synthesis of piperine and functionally expressed the corresponding cDNAs in Escherichia coli (E. coli) cells. The resulting heterologous enzymes encoded by these genes were capable of synthesizing the natural product piperine from piperoyl-coenzyme A (piperoyl-CoA) and piperidine. The inventors have surprisingly found the affinity of the recombinant enzymes for the amine substrate is somewhat low, whereby initial attempts to detect enzymatic activity had failed. Once this was established, the activity of the enzymes allowed efficient production of piperine. In addition, the promiscuity of the isolated enzymes allowed biosynthesis of various amides such as piperamides that would, otherwise, require organic synthesis (Bauer A et al. 2019; Fregnan A M et al., 2017).
The production of piperine in biotechnological processes through recombinant enzymes can be considered “natural production” and any product derived from such processes are considered as “GRAS” (generally recognized as safe) by the Food and Drug Association (FDA) in the USA. This usually facilitates the approval as a dietary component or its use as a drug, or health, specifically skin care preparations. The proteins provided herein can be used for synthesizing a wide range of amides such as piperamides by, for example, microbial fermentation. Such compounds have potential relevance as precursors for active agents of medicaments and in the treatment of diseases as demonstrated for vitiligo and cancer (Mih{hacek over (a)}il{hacek over (a)} B et al. 2019, Xie Z et al., 2019). In addition, such compounds potentially represent alternative insecticides (Navickiene D H M et al. 2007). Targeted mutagenesis allows further expansion of the substrate spectrum of the enzymes described herein. Combinatorial expression with further, known enzymes allows the use of substrate precursors in the synthesis process and a further refinement of the end product.
The inventors of the present invention have identified the coding sequence and the amino acid sequence of a protein termed PipSynthase2 that has enzymatic activity in converting piperoyl-CoA and piperidine to piperine. The inventors have also identified the coding sequence and the amino acid sequence of a protein termed PipSynthase1 that has enzymatic activity in converting piperoyl-CoA and piperidine to piperine, but also to its configurational isomers isopiperine and chavicine. The coding sequence and amino acid sequence of PipSynthase2 are those given in SEQ ID NOs: 3 and 4, respectively, disclosed herein. The coding sequence and amino acid sequence of PipSynthase1 are those given in SEQ ID NOs: 1 and 2, respectively. The terms “PipSynthase1” and “PipSynthase2” are also used herein for proteins comprising the amino acid sequence of SEQ ID NOs: 2 and 4, respectively, and optionally additional sequence stretches like purification tags (e.g. a His-tag).
The invention provides a polynucleotides of a first and of a second general embodiment. Those of the first general embodiment are described in the following with reference to SEQ ID NO: 1 and 2. Those of the second general embodiment as described in the following with reference to SEQ ID NO: 3 and 4.
Accordingly, the invention provides a polynucleotide the nucleotide sequence of which is:
(a) the nucleotide sequence defined in SEQ ID NO: 1, or
(b) a nucleotide sequence of at least 93% sequence identity to SEQ ID NO: 1, or
(c) a nucleotide sequence encoding an amino acid sequence comprising or consisting of the amino acid sequence defined in SEQ ID NO: 2, or
(d) a nucleotide sequence encoding an amino acid sequence of at least 70% sequence identity to the amino acid sequence defined in SEQ ID NO: 2, or
(e) a nucleotide sequence encoding an amino acid sequence of at least 75% sequence similarity to the amino acid sequence defined in SEQ ID NO: 2, or
(f) a nucleotide sequence encoding an amino acid sequence of from 1 to 138 amino acid substitutions, additions, insertions and/or deletions compared to the amino acid sequence defined in SEQ ID NO: 2, or
(g) a fragment of the nucleotide sequence defined in any one of items (a) to (f), the fragment being at most 42, preferably at most 21, nucleotides shorter due to 5′- and/or 3′-terminal deletions of the nucleotide sequence defined in any one of items (a) to (f).
Also disclosed is a polynucleotide as defined above but further containing one or more introns.
Further, the invention provides a polynucleotide the nucleotide sequence of which is:
(a′) the nucleotide sequence defined in SEQ ID NO: 3, or
(b′) a nucleotide sequence of at least 93% sequence identity to SEQ ID NO: 3, or
(c′) a nucleotide sequence encoding an amino acid sequence comprising or consisting of the amino acid sequence defined in SEQ ID NO: 4, or
(d′) a nucleotide sequence encoding an amino acid sequence of at least 70% sequence identity to the amino acid sequence defined in SEQ ID NO: 4, or
(e′) a nucleotide sequence encoding an amino acid sequence of at least 75% sequence similarity to the amino acid sequence defined in SEQ ID NO: 4, or
(f′) a nucleotide sequence encoding an amino acid sequence of from 1 to 138 amino acid substitutions, additions, insertions and/or deletions compared to the amino acid sequence defined in SEQ ID NO: 4, or
(g′) a fragment of the nucleotide sequence defined in any one of items (a′) to (f′), the fragment being at most 42, preferably at most 21, nucleotides shorter due to 5′- and/or 3′-terminal deletions of the nucleotide sequence defined in any one of items (a′) to (f′). Also disclosed is a polynucleotide as defined above but further containing one or more introns.
The polynucleotide of the invention is a polymer of nucleotides linked by phosphodiester groups. The polynucleotide may be DNA or RNA or may comprise both ribo- and deoxyribonucleotides. The polynucleotide may further comprise nucleotide moieties having modifications at the pentose moiety, the base and/or the phosphodiester moieties. Preferably, the polynucleotide is DNA or RNA.
Herein, the determination of (nucleotide sequence) and amino acid sequence identities is done using the DNASTAR Lasergene software package, using Clustal V as the default algorithm of the MegAlign tool. Herein, where the protein or the amino acid sequence is defined by a number or numerical range of amino acid substitutions, additions, insertions and/or deletions, these amino acid substitutions, additions, insertions or deletions may be combined, but the given number or numerical range refers to the sum of all amino acid substitutions, additions, insertions and deletions. Among amino acid substitutions, additions, insertions and deletions, amino acid substitutions, additions, and deletions are preferred. The term “insertion” relates to insertions within the amino acid sequence of the reference sequence, i.e. excluding additions at the C- or N-terminal end. The term “addition” means additions at the C- or N-terminal end of the amino acid sequence of the reference sequence. A deletion may be a deletion of a terminal or an internal amino acid residue of a reference sequence. A reference sequence is the sequence of the given SEQ ID NO.
The wording “nucleotide sequence of at least x % sequence identity to SEQ ID NO: y means that the nucleotide sequence has at least the same number of nucleotides as the reference sequence of SEQ ID NO: y and has the indicated minimum sequence identity x over the entire length of SEQ ID NO: y. y is an integer that generically refers to the sequences of the SEQ ID NOs of the invention.
The wording “amino acid sequence of at least x % sequence identity to the amino acid sequence defined in SEQ ID NO: y means that the amino acid sequence has at least the same number of amino acid residues as the reference sequence of SEQ ID NO: y and has the indicated minimum sequence identity x over the entire length of SEQ ID NO: y. The wording “amino acid sequence of at least x % sequence similarity to the amino acid sequence defined in SEQ ID NO: y means that the amino acid sequence has at least the same number of amino acid residues as the reference sequence of SEQ ID NO: y and has the indicated minimum sequence similarity x over the entire length of SEQ ID NO: y.
The invention also provides specific polynucleotides and nucleic acids encoding the proteins and embodiments described in the following section.
The invention further provides a nucleic acid molecule comprising a polynucleotide of the invention. The nucleic acid molecule may be a recombinant construct, a plasmid, or a vector, that are further described below.
The polynucleotides of the invention may encode a protein of the invention, as described in more detail in the following, and may have an enzymatic activity as also described in the following. Generally, a polynucleotide of the first general embodiment encodes a protein of the first general embodiment. Generally, a polynucleotide of the second general embodiment encodes a protein of the second general embodiment.
The invention provides two general classes of proteins, referred to herein as the proteins of the first and second general embodiment. The proteins of the first general embodiment are those defined herein with reference to SEQ ID NO:2. The proteins of the second general embodiment are those defined herein with reference to SEQ ID NO:4.
In the first general embodiment, the invention provides a protein comprising a polypeptide, the amino acid sequence of said polypeptide
(i) is or comprises the amino acid sequence defined in SEQ ID NO: 2, or
(ii) comprises or consists of an amino acid sequence that has at least 70% sequence identity to the amino acid sequence defined in SEQ ID NO: 2, or
(iii) comprises or consists of an amino acid sequence that has at least 75% sequence identity to the amino acid sequence defined in SEQ ID NO: 2, or
(iv) comprises or consists of an amino acid sequence of from 1 to 138 amino acid substitutions, additions, insertions and/or deletions compared to the amino acid sequence defined in SEQ ID NO: 2, or
(v) is a fragment of the amino acid sequence defined in any one of items (i) to (iv), the fragment being at most 14 amino acid residues shorter due to N- and/or C-terminal deletions of the amino acid sequence defined in any one of items (i) to (iv).
Preferably, a protein of the first general embodiment according to items (ii) to (v) has a catalytic activity in converting piperoyl-CoA and piperidine to piperine with a catalytic activity of at least 0.14, preferably at least 0.4, more preferably at least 0.6, even more preferably at least 0.8, and most preferably of at least 2.4 nkat/mg protein at pH=8.0 at 30° C. in 30 mM Tris-HCl, 1 mM dithiothreitol (DTT) with substrate concentrations of 500 μM piperoyl-CoA and 20 mM piperidine.
Throughout this invention, 30 mM Tris-HCl means that the concentration of Tris is 30 mM and the pH is suitably adjusted as indicated with HCl.
In the second general embodiment, the invention provides a protein comprising a polypeptide, the amino acid sequence of said polypeptide
(i′) is or comprises the amino acid sequence defined in SEQ ID NO: 4,
(ii′) comprises or consists of an amino acid sequence that has at least 70% sequence identity to the amino acid sequence defined in SEQ ID NO: 4,
(iii′) or comprises or consists of an amino acid sequence that has at least 75% sequence identity to the amino acid sequence defined in SEQ ID NO: 4, or
(iv′) comprises or consists of an amino acid sequence of from 1 to 138 amino acid substitutions, additions, insertions and/or deletions compared to the amino acid sequence defined in SEQ ID NO: 4, or
(v′) is a fragment of the amino acid sequence defined in any one of items (i′) to (iv′), the fragment being at most 14 amino acid residues shorter due to N- and/or C-terminal deletions of the amino acid sequence defined in any one of items (i′) to (iv′).
Preferably, a protein of the second general embodiment according to items (ii′) to (v′) has a catalytic activity in converting piperoyl-CoA and piperidine to piperine with a catalytic activity of at least 0.14, preferably at least 0.4, more preferably at least 0.6, even more preferably at least 0.8, and most preferably of at least 2.4 nkat/mg protein at pH=8.0 at 30° C. in 30 mM Tris-HCl, 1 mM dithiothreitol (DTT) with substrate concentrations of 500 μM piperoyl-CoA and 20 mM piperidine.
In preferred embodiments of the proteins, the sequence identity is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%; and/or the sequence similarity is at least 85%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%; and/or wherein the number of said amino acid substitutions, additions, insertions and/or deletions is from 1 to 100, preferably from 1 to 70, more preferably from 1 to 40, more preferably from 1 to 20, and most preferably from 1 to 10 compared to the amino acid sequence defined in SEQ ID NO: 2 or 4.
The definition given above in the section on the polynucleotides and nucleic acids regarding the determination of amino acid sequence identities and similarities, and regarding the meaning and preferred embodiments of the substitutions, additions, insertions and/or deletions also apply to the proteins of the invention.
The proteins according to the invention may be classified as BAHD-like transferases (
In a preferred embodiment, the proteins of the invention also comprise the amino acid stretch DWGWG (SEQ ID NO: 6) in their amino acid sequence that is different to the stretch DFGWG (SEQ ID NO: 7) present in closely related proteins depicted in
The protein of the invention may be or comprise a polypeptide comprising the sequence stretch of the HXXXD-motif at the position corresponding to the position of the amino acid residues 168 to 172 in SEQ ID NO: 2 or at the position corresponding to the position of the amino acid residues 168 to 172 in SEQ ID NO: 4. Herein, the term “corresponding to” means that the amino acid sequence of the protein of the invention is aligned to the reference sequence of the respective SEQ ID NO using methods generally used in the art, for example the ClustalW program. A position in a given polypeptide corresponding to an amino acid residue or stretch of a reference sequence is the position or stretch aligning to the reference sequence.
Alternatively or additionally, the amino acid stretch DWGWG may be present, in the polypeptide of a protein of the invention, at the position corresponding to the position of the amino acid residues 383 to 387 in SED ID NO: 2 or the position corresponding to the position of the amino acid residues 387 to 391 in SED ID NO: 4.
Alternatively or additionally, the amino acid stretch FLAT (SEQ ID NO: 18) may also be present of a protein of the invention, at the position corresponding to the position of the amino acid residues 404 to 407 in SEQ ID NO: 2 or the position corresponding to the position of the amino acid residues 408 to 411 in SEQ ID NO: 4.
The protein of the invention may comprise, preferably additionally to the embodiments mentioned in the preceding paragraphs, one or more of the following amino acid residues or amino acid sequence stretches in its polypeptide (in the single-letter amino acid code) at the position corresponding to the indicated position of the reference sequences, as follows:
Y1) L at position 42 in SED ID NO: 2 or at position 43 in SED ID NO: 4
Y2) DVG from position 396 to 398 in SED ID NO: 2 or from position 400 to 402 in SED ID NO: 4,
Y3) FLAT from position 404 to 407 in SED ID NO: 2 or from position 408 to 411 in SED ID NO: 4,
Y4) ML from position 312 to 313 in SED ID NO: 2 or from position 316 to 317 in SED ID NO: 4,
Y5) N at position 366 in SED ID NO: 2 or at position 370 in SED ID NO: 4,
Y6) MOO from position 168 to 172 in SED ID NO: 2 or from position 168 to 172 in SED ID NO: 4,
Y7) DWGWG from position 383 to 387 in SED ID NO: 2 or from position 387 to 391 in SED ID NO: 4,
Y8) F at position 130 in SEQ ID NO: 2 or at position 129 in SEQ ID NO:4, and/or
Y9) K at position 133 in SEQ ID NO: 2 or at position 132 in SEQ ID NO:4.
In a preferred embodiment, the proteins of the first and the second general embodiment comprise all the amino acid residues or amino acid sequence stretches of Y1) to Y9). The amino acid residues from Y1) to Y9) may also be identified from
The amino acid residues or amino acid sequence stretches Y1) to Y9) are preferably conserved in the proteins of the invention. Thus, these conserved amino acid residues allow differentiating the proteins from the first and the second general embodiment from other BADH-like proteins. A sequence alignment as depicted in
The proteins of the first and the second general embodiment may be distinguished from their amino acid sequences. A protein of the first general embodiment preferably has one or more of the following amino acid sequence stretches in its polypeptide (in the single-letter amino acid code) at the position corresponding to the indicated position of SEQ ID NO:2 as the reference sequence:
X1) LFLTAI (SEQ ID NO: 14) from position 42 to 47 in SEQ ID NO: 2,
X2) GLML (SEQ ID NO: 15) from position 310 to 313 in SEQ ID NO: 2,
X3) SN from position 365 to 366 in SEQ ID NO: 2, and/or
X4) LID from position 376 to 378 in SEQ ID NO: 2.
In a preferred embodiment, the protein of the first general embodiment comprises all of X1) to X4). More preferably, one, two or all of these amino acid sequence stretches are absent in proteins of the second general embodiment. The amino acid residues from X1) to X4) can also be identified in
A protein of the second general embodiment preferably has one or more of the following amino acid sequence stretches in its polypeptide (in the single-letter amino acid code) at the position corresponding to the indicated position of SEQ ID NO:4 as the reference sequence:
Z1) LHISGF (SEQ ID NO: 16) from position 43 to 48 in SEQ ID NO: 4,
Z2) SIML (SEQ ID NO: 17) from position 314 to 317 in SEQ ID NO: 4,
Z3) TN of position 369 to 370 in SEQ ID NO: 4, and/or
Z4) LVE at position 380 to 382 in SEQ ID NO: 4.
In a preferred embodiment, the protein of the second general embodiment comprises all amino acid sequence stretches of Z1) to Z4). More preferably, one, two or all of these amino acid sequence stretches are absent in proteins of the first general embodiment. The amino acid residues from Z1) to Z4) can also be identified in
The proteins of the present invention may have a purification tag at its N- or C-terminus for easier purification, for example with a 6×-histidine-tag, although any other purification tag is possible. This purification tag can be removed by commercially available proteases, like Factor Xa in subsequent applications.
The proteins defined herein with respect to SEQ ID NO: 2 and 4 preferably have catalytic activity and are thus enzymes. The catalytic activity is formation of an amide from a coenzyme A-thioester (also referred to herein as “SCoA-ester”, “CoA-ester”, or “acyl-CoA”) and an amine such as a primary or secondary amine. The designations “SCoA” and “CoA” refer to the same coenzyme A moiety and differ merely in that “SCoA” highlights the presence of the sulfur atom derived from the terminal thiol group of coenzyme A.
The recombinant proteins of the invention have a catalytic activity in converting piperoyl-CoA and piperidine to piperine, preferably with a catalytic activity of at least 0.14, preferably at least 0.4, more preferably at least 0.6, even more preferably at least 0.8, and most preferably of at least 2.4 nkat/mg total protein at pH=8.0 at 30° C. in 30 mM Tris-HCl, 1 mM dithiothreitol (DTT), preferably with substrate concentrations of 500 μM piperoyl-CoA and 20 mM piperidine. Most preferably, the protein of the second general embodiment has a catalytic activity of at least 5.0 nkat/mg total protein under the conditions defined in the previous sentence. Example 3 contains a protocol for the determination of the catalytic activity of a protein according to the invention.
The protein of the first general embodiment may also have catalytic activity in converting piperoyl-CoA and piperidine to piperine preferably with a catalytic activity of at least 0.14, preferably at least 0.4, more preferably at least 0.6, even more preferably at least 0.8, and most preferably of at least 2.4 nkat/mg total protein at pH=8.0 at 30° C. in 30 mM Tris-HCl, 1 mM dithiothreitol (DTT), preferably with substrate concentrations of 500 μM piperoyl-CoA and 20 mM piperidine. It simultaneously and preferentially produces from the same reactants the piperine configurational isomers isopiperine and/or chavicine, preferably with a catalytic activity (with respect to both isopiperine and chavicine) of at least 0.56, preferably at least 1.6, more preferably at least 2.4, even more preferably at least 3.2, and most preferably of at least 9.6 nkat/mg total protein at pH=8.0 at 30° C. in 30 mM Tris-HCl, 1 mM dithiothreitol (DTT), preferably with substrate concentrations of 500 μM piperoyl-CoA and 20 mM piperidine. The latter catalytic activities are calculated from the sum of peak areas of isopiperine and chavicine peaks due to species of m/z 286.1 other than peaks due to piperine in a liquid chromatography (LC) trace and UV detection at 330 nm calibrated using known amounts of piperine (detected at 340 nm in LC) and assuming identical absorbance (absorption) coefficients for piperine at 340 nm, and of isopiperine and chavicine (having m/z 286.1) both at 330 nm. An assay for measuring the catalytic activity is described in the Examples and illustrated in
The substrate concentrations given above and elsewhere herein are those at the beginning of a reaction or assay, i.e. initial substrate concentrations.
Standard assays may contain from at least 1 up to 10 μg of protein/50 μl, but may be raised to about 50 μg. A suitable concentration range is from 20-500 μg/ml of the protein of the invention.
As the reaction of the CoA-ester with amine can also take place non-catalyzed, the formation of product is also measured in the absence of protein, but under otherwise identical conditions, and subtracted from that measured in the presence of the protein in order to determine the enzymatic activity of a protein of the invention.
The unit kat is the SI-unit katal, wherein 1 kat is 1 mole per second. 1 nkat is one nanomole per second. The unit nkat/mg total protein is relative to the entire protein present in a sample and is thus dependent on the purity of the protein. For many applications of the proteins of the invention, it is not necessary to use highly purified preparations of the protein of the invention, provided the desired catalytic activity is obtained. The total protein in a sample can be determined using the generally known Bradford protein assay that is also described in the Examples. Usually 10-50 μl of a protein solution are mixed with 950 to 990 μl of Coomassie Brilliant Blue G-250, the Bradford Reagent (Bradford, M M, 1976) incubated for 5 minutes and measured at 595 nm against a blank solution where the protein solution is replaced by buffer. The protein of the first general embodiment may further be functional for converting piperoyl-CoA and piperidine to piperine, but preferentially to isopiperine and chavicine.
The proteins of the invention not only have catalytic activity for converting piperoyl-CoA and piperidine to piperine, but accept a wide range of other substrates to produce a wide range of different amides, as further described below. The proteins of the first and second general embodiment differ in their substrate specificity such that the protein of the first general embodiment has wider (or less specific) substrate specificity.
Further, the proteins of the invention can be used for producing amides using a wide range of conditions, as also described further below. For example, substrate concentrations tested may be from 10 μM-5 mM of the CoA ester, preferably 20 μM-500 μM of the CoA ester such as piperoyl CoA, and 100 μM to 100 mM amine, preferably 200 μM to 20 mM amine, such as piperidine. A suitable buffer other than Tris-HCl is phosphate buffer. The pH may be from 6.0 to 9.0, preferably from 7.0 to 8.5. The reaction temperature may be from 14 to 35° C., preferably from 20 to 30° C.
The proteins of the first and the second general embodiment generally differ in their catalytic activity for the turnover of CoA-esters and amines. In some reactions, a protein of the first general embodiment may show a higher catalytic activity than a protein of the second general embodiment. In other reactions, a protein of the first general embodiment may show lower catalytic activity than a protein of the second general embodiment. For example, a given protein of the invention may be assigned to the first or the second general embodiment as follows. The catalytic activity of the protein is assayed with
(i) piperoyl-CoA and piperidine as substrates and
(ii) 3,4-methylenedioxycinnamoyl-CoA and piperidine as substrates.
If the catalytic activity in terms of nkat/mg protein is higher in assay (i) than in assay (ii), the protein is generally a protein of the second general embodiment. If the catalytic activity in terms of nkat/mg protein is higher in assay (ii) than in assay (i), the protein is generally a protein of the first general embodiment. Preferably, these assays are performed in aqueous solution at pH=8.0 at 30° C. in 30 mM Tris-HCl and 1 mM dithiothreitol (DTT) with substrate concentrations of 500 μM CoA ester and 20 mM piperidine. These assays and the detection of products may be carried out as described in Example 4. Accordingly, the protein of the first general embodiment has a higher catalytic activity with 3,4-methylenedioxycinnamoyl-CoA and piperidine as substrates than with piperoyl-CoA and piperidine as substrates. The protein of the second general embodiment has a higher catalytic activity with piperoyl-CoA and piperidine as substrates than with 3,4-methylenedioxycinnamoyl-CoA and piperidine as substrates.
In these assays, the sum of all peaks in the LC trace due to species having calculated m/z values of the expected products (e.g. that of piperine in assay (i)) was used for quantification of product, as described in Example 4. In assay (i), the product(s) is quantified by comparison of the peak area of the LC-UV trace at 340 nm with the peak area of the LC-UV trace at 340 nm of (authentic) piperine. The product of assay (ii) is also quantified by comparison of the peak area of the LC-UV trace at 340 nm with that of piperine, i.e. it is assumed that the product(s) of assay (ii) and piperine have the same absorption coefficient at 340 nm. The formation of product in assay (i) and assay (ii) is preferably also measured in the absence of protein, but under otherwise identical conditions, and determined peak areas of species having calculated m/z values of the expected products are subtracted from those measured in the presence of the protein.
These embodiments of the first and second general embodiment may be combined with the embodiments described above of the respective general embodiments.
The invention also provides, in a third general embodiment, a protein, or protein composition comprising said protein, said protein or protein composition having a catalytic activity for converting piperoyl-CoA and piperidine to piperine with a catalytic activity of at least 0.8, preferably at least 2.4, and most preferably at least 5.0 nkat/mg of said protein or protein composition, respectively, at 30° C. in 30 mM Tris-HCl buffer pH 8.0, containing 1 mM dithiothreitol (DTT), with substrate concentrations of 500 μM piperoyl-CoA and 20 mM piperidine. This enzymatic activity is determined as described above, notably as described with respect to the second general embodiment and as exemplified in Example 3. The amount of non-enzymatically formed piperine is subtracted from the amount of piperine determined in the presence of protein.
The protein or protein composition of the third general embodiment may be combined with other embodiments described herein, notably with the embodiments described above for the protein of the second general embodiment. For example, the polynucleotide of the second general embodiment may encode the protein of the third general embodiment.
The invention further provides a recombinant construct or vector containing the polynucleotide of the invention. The recombinant construct may comprise a polynucleotide of the invention such that the latter is expressible in prokaryotic cells or eukaryotic cells or in plants to produce the protein of the invention. For this purpose, the recombinant construct may contain a promoter upstream of the polynucleotide such that the polynucleotide is under the control of the promoter for expression in the cells or the plant. The promoter should be active or functional in the prokaryotic or eukaryotic cells where the polynucleotide should be expressed. The recombinant construct may further comprise other sequences such as regulatory sequences for expression of the polynucleotide, such as a 3′ UTR (3′ non-translated sequence) or a terminator downstream of the polynucleotide of the invention. Both prokaryotic and eukaryotic expression of polynucleotides is known to the skilled person. The recombinant construct may further comprise other desired genes or gene constructs that are desired to be co-expressed with the polynucleotide of the invention.
In one embodiment, the recombinant construct may comprise a further nucleic acid or gene to be expressed in the prokaryotic or eukaryotic cells. Expression of such further gene may be under the control of the same or a different promoter as the promoter used for expressing the polynucleotide of the invention. The recombinant construct may further comprise a selectable marker gene for selecting cells containing the recombinant construct, e.g. during cloning or when transforming the prokaryotic or eukaryotic cells. This further nucleic acid or gene may encode a CoA ligase that is capable of producing a CoA ester (e.g. those of formulae 1, 3, 5, 7, or 9 below) from the corresponding carboxylic acid and Coenzyme A (CoA). The CoA ligase may be, but is not limited to, a piperoyl-CoA ligase (PipCoA ligase) for producing piperoyl-CoA from piperic acid. The CoA ligase can be cloned in a different vector (transformed into the same cells, with a different selection marker) or on the same vector (like petDuet-1, Novagene) as the polynucleotide of the invention under the control of any promoter, e.g. the T7 promoter, to achieve simultaneous functional expression of two genes, preferentially, but not limited to, the combination of PipCoA ligase with the protein of the first or second general embodiment. Such and similar embodiments allow production and the purification of the produced amides from externally supplied carboxylic acids and the amines in a pro- or eukaryotic host, without the expensive and tedious chemical or otherwise separate enzymatic preparation of the CoA ester from the carboxylic acid, exemplified by Eudes A et al. 2016 for other BAHD-types of enzymes expressed in yeast. Additional genes to enhance the production of the CoA-ester, e.g. by providing anaplerotic reactions for more efficient CoA-ester production, can be transformed separately into the same cells on a different vector with a different selection marker than that of the ligase and synthase.
Coupled reaction from carboxylic acid to amide via the CoA-ester has been successfully achieved in the Examples by a combination of PipCoA ligase and PipSyn2 cloned on a petDuet-1 vector (Novagene) transformed into E. coli SoluBL21 cells (Amsbio), induced by IPTG at an OD of 0.4, grown at 25° C. for 16 hours and fed by adding 1 mM piperic acid and 10 mM piperidine, both dissolved in 25% DMSO in water directly to the medium. The supernatant was collected and extracted with ethyl acetate. The ethyl acetate fraction was concentrated to dryness, re-dissolved in 100% methanol, resulting in production of piperine and piperine isomers, as confirmed by LC-MS.
Herein, the term “gene” means a DNA sequence comprising a region (transcribed region), which can be transcribed into an RNA molecule (e.g. a mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5′ leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) such as the polynucleotide of the invention, introns, and a 3′-non-translated sequence comprising e.g. transcription termination sites. For bacterial gene expression, several coding sequences may be combined into an operon. Genes and operons and their use for gene expression in different organisms including prokaryotes and eukaryotes are known to the skilled person.
As used herein, the term “promoter” means a transcription promoter, i.e. a nucleic acid (generally DNA) sequence that is capable of controlling (initiating) transcription in a cell or organism where a polynucleotide is to be expressed. For expression in prokaryotic cells such as in Escherichia choli, promoters generally known from prokaryotic gene expression may be used. Promoters for expression of a nucleic acid in yeast are also generally known such as the galactose-inducible promoter used in the examples.
The invention further provides a vector or plasmid comprising a polynucleotide of the invention or the recombinant construct. The vector and plasmid may comprise a selectable marker allowing selecting for the presence of said vector or plasmid in eukaryotic or prokaryotic cells.
The invention also provides a prokaryotic or eukaryotic cell, or a plant, comprising the polynucleotide, nucleic acid molecule, vector, plasmid, or the recombinant construct of the invention.
The prokaryotic cell may be any prokaryotic cell usable for cloning the recombinant construct, plasmid and/or vector, or prokaryotic cell used for expressing the polynucleotide or nucleic acid molecule of the invention. An example of a usable prokaryotic cell is Escherichia coll. Other examples are Bacillus subtilis, Corynebacterium glutamicum, photosynthetic cyanobacteria such as Synechocystis sp. PCC6803, or bacteria that can grow on substrates such as methanol, e.g. Methylobacterium extorquens, Methylobacillus sp., Methylophyllus sp., Ancylobacter sp., Hyphomicrobium sp., Xanthobacter sp., Thiobacillus sp., Mycobacterium sp., Paracococcus sp., Methylophaga sp. and Acidomonas sp. Methods of transforming a construct or vector into prokaryotic cells are well-known to the skilled person.
Examples of eukaryotic cells, cell lines or strains are yeast cells, cell lines or strains such as of genus Saccharomyces (most preferably Saccharomyces cerevisiae) or Schizosaccharomyces (e.g. Schizosaccharomyces pombe), Yarrowia lipolytica, or Pichia pastoris. The yeast cells or strains may be edible. Other fungi such as molds, e.g. Fusarium fujikuroi could also be used in the invention. Yeast cells are the preferred eukaryotic cells or cell lines for practicing the invention. Yeast cells may be transformed according to generally known methods, cf. Borts, Rhona H. (1996) Yeast protocols: Methods in cell and molecular biology. Methods in Molecular Biology (Book 53) Humana Press. Methods for nucleic acid expression in S. pombe, including vectors, promoters, terminators, transformation methods, integration into a target region e.g. using homologous recombination are known in the prior art, e.g. from EP 2 468 860 A1. Examples of microalgae are microalgae such as Chlamydomonas reinhardtii, various species of Scenedesmus (e.g. S. obliquus, S. vacuolatus, S. rubescens), Chlorella vulgaris or C. kessleri, Haematococcus pluvialis, or Dunaliella salina. Methods for nucleic acid expression in C. reinhardtii and other microalgae are known in the art as reviewed (Scaife M A et al., 2015).
In one embodiment, the prokaryotic or eukaryotic cell or the plant further contains a gene encoding an enzyme that can produce a reactant (or substrate) of the reaction catalysed by a protein of the invention. The reactant producible by the enzyme is the CoA-ester as described herein. Such enzymes and genes encoding them are known in the art as coenzyme A ligases, such as fatty acid-CoA ligases and coumaroyl CoA ligases. These ligases produce a CoA-ester from a carboxylic acid in an ATP-dependent manner. Thus, co-expression of the ligase with the protein of the invention in cells allows a coupled enzymatic production of an amide from a carboxylic acid.
A protein according to the invention may be produced using known methods of protein expression, e.g. in a standard protein expression system. For protein production, a polynucleotide encoding the protein of the invention may be expressed in a suitable host organism. Methods usable for producing and purifying the protein have been described in the prior art and any of such methods may be used. An E. coli expression system as generally known in the art may, for example, be used. A preferred bacterial expression system makes use of the Lemo BL21 (DE3) bacterial expression system used in the examples below, based on a rhamnose-inducible promotor of a 2nd gene (pLysC) on a 2nd plasmid to control protein expression by controlling T7 RNA-Polymerase. This system provides the benefit that the protein of the invention remains soluble in the liquid medium after homogenizing the cells in which it is expressed.
Alternatively, any other prokaryotic protein expression system including Corynebacterium glutamicum, Bacillus subtilis may be used. If a eukaryotic protein expression system is used, one or more introns may be inserted in the coding sequence of the protein to prevent toxicity in the bacterial organism used for cloning. Eukaryotic expression systems include but are not limited to Saccharomyces spec., Pichia pastoris, Baculovirus insect cultures, transient and stable plant transformation systems for Nicotiana benthamina and Arabidopsis thaliana.
The protein of the invention can be purified to a desired degree according to generally knowns methods (illustrated in Example 2 and
The process of producing an amide according to the invention comprises reacting an acyl-CoA and an amine in the presence of a protein according to the invention. Since the protein of the invention is not limited to piperoyl-CoA and piperidine as substrates, many different amides can be produced. Some examples of products that can be produced using piperoyl-CoA as the CoA-donor and three different amines are shown in
The amine is preferably a primary or secondary amine, i.e. a compound having a primary or secondary amino group. The amine may even have two or more primary or secondary amino groups. However, in this case, mixtures of amides may be produced. Therefore, it is preferred that the amine has one primary or one secondary amino group. Other functional groups on the amine generally do not abolish reactivity with the protein of the invention, and the invention is not limited in this regard.
The amine may be a C1-C14 amine, preferably a C2-C12 amine, more preferably a C3-C10 amine, and most preferably a C5-C8 amine. The numbers of carbon atoms in the previous sentence refer to the number of all carbon atoms of the amine. In more detail, the primary amine may be a hydrocarbylamine or a heterohydrocarbylamine. The secondary amine may be a di-N-hydrocarbylamine, a N-hydrocarbyl-N-heterohydrocarbyl-amine, or a di-N-heterohydrocarbylamine, such as a di-N—C1-C6-hydrocarbylamine, a N—C1-C6-hydrocarbyl-N—C1-C6-heterohydrocarbyl-amine, or a di-N—C1-C6-heterohydrocarbylamine. The heteroatoms of the heterohydrocarbyl moieties are selected from O, N, and S, and are preferably O. There may be from 1 to 3 heteroatoms per heterohydrocarbyl moiety, preferably 1 or 2, most preferably 1. A preferred heterohydrocarbyl group is a heterohydrocarbyl having one oxygen atom. The hydrocarbyl moieties may be linear or branched, or may be cyclic hydrocarbyl moieties, or may comprise linear, branched, and/or cyclic moieties.
Examples of hydrocarbyl groups are methyl, ethyl, propyl, butyl, pentyl, hexyl, cyclohexyl, and phenyl groups. Examples of heterohydrocarbyl groups are methoxy, ethoxy, methylthio, morpholino, and vanillyl groups.
Examples of amines are the following substituted or unsubstituted amines (the carbon atoms of each of the following examples may be, as described above, C1-C14, preferably C2-C12, more preferably C3-C10, and most preferably C6-C8): alkylamine, di-N-alkylamine, cycloalkyl-amine, heterocycloalkyl-amine, N-alkyl-N-cycloalkyl-amine, cycloalkenylamine, N-alkyl-N-cycloalkenyl-amine, N-cycloalkylalkylamine, N-alkyl-N-cycloalkylalkylamine, aniline, N-alkyl-aniline, aniline, N-cycloalkyl-aniline, N-phenylalkylamine, N-alkyl-N-phenylalkyl-amine. The carbon atom number ranges including the preferred ones given above preferably also apply to these amines and include carbon atoms of any substituents. Preferably, the amine is selected from substituted or unsubstituted C1-C6-alkylamine, di-N—C1-C6-alkylamine, C4-C7-cycloalkyl-amine, C3-C6-heterocycloalkyl-amine, N—C1-C6-alkyl-N—C4-C7-cycloalkyl-amine, C4-C7-cycloalkenylamine, N—C1-C6-alkyl-N—C4-C7-cycloalkenyl-amine, N—C4-C7-cycloalkyl-C1-C6-alkylamine, N—C1-C6-alkyl-N—C4-C7-cycloalkyl-C1-C6-alkylamine, aniline, N—C1-C6-alkyl-aniline, N—C4-C7-cycloalkyl-aniline, N-phenyl-C1-C6-alkylamine, N—C1-C6-alkyl-N-phenyl-C1-C6-alkyl-amine.
In another embodiment, the (secondary) amine is a substituted or unsubstituted cyclic amine. The carbon atom numbers including the preferred ones given above also apply to these amines and include carbon atoms of possible substituents. The cyclic amine group of the unsubstituted cyclic amine or the cyclic amine may be a 4-, 5-, 6, or 7-membered nitrogen-containing ring, preferably a 5- or 6-membered nitrogen-containing ring. The ring may be saturated or unsaturated and may contain an (preferably one) additional heteroatom selected from O, N or S, preferably O. Examples of the cyclic amine are substituted or unsubstituted azetidine, pyrrole, pyrroline, pyrrolidine, imidazole, imidazoline, oxazole, oxazoline, oxazolidine, pyridine, dihydropyridine, piperidine, piperazine, morpholine, and azacycloheptane.
Possible substituents are C1-C6-hydrocarbyl or C1-C6-heterohydrocarbyl. In more detail, the substituents may be C1-C6-alkyl, C1-C6-alkoxy, C3-C7-cycloalkyl, C1-C6-alkoxy, hydroxy, hydroxy-C1-C6-alkyl, halo (F, C1, Br, I), sulfhydryl, oxo, C3-C6-aryl, C3-C6-heteroaryl (preferably containing one heteroatom selected from O, N, or S, preferably O), or two substituents on adjacent carbon atoms together form a five- or six-membered ring. The five- or six-membered ring may be saturated or unsaturated and may contain a heteroatom selected from O, N, and S, preferably O.
Specific examples of amines are piperidine, pyrrolidine, isobutylamine, benzylamine, 4-aminomethylpiperidine, vanillylamine, and 1,7-diamino-n-heptane.
The acyl-CoA-ester of the invention may be a C1-C20 acyl-CoA ester, preferably a C3-C18 acyl-CoA ester, more preferably a C4-C16 acyl-CoA ester, and more preferably a C6-C14 ester. As is clear for the skilled person, these carbon atom number ranges relate to the carbon atom numbers of the acyl group. The acyl moiety may be a hydrocarbylcarbonyl or a heterohydrocarbylcarbonyl group, wherein the heterohydrocarbyl group comprises from 1 to 3 heteroatoms selected from O, N, and S, preferably selected from O and S, and even more preferably 1 or 2 heteroatoms selected from O and S, preferably O.
The acyl group may be a substituted or unsubstituted alkylcarbonyl, alkenylcarbonyl such as (E)-8-methyl-6-nonenoyl, alkadienylcarbonyl carbonyl, alkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl, alkylcycloalkylcarbonyl, cycloalkylalkylcarbonyl, cycloalkylalkenylcarbonyl, cycloalkylalkadienylcarbonyl, alkylcycloalkylalkylcarbonyl group, alkylcycloalkylalkenylcarbonyl, alkylcycloalkylalkadienylcarbonyl, arylcarbonyl, alkylarylcarbonyl, arylalkylcarbonyl such as 5-phenylpentanoyl, arylalkenylcarbonyl, arylalkadienylcarbonyl, alkylarylalkylcarbonyl, alkylarylalkenylcarbonyl, or alkylarylalkadienylcarbonyl group. The carbon atom numbers given above for the acyl group of the acyl-CoA esters preferably also apply to these groups. In the case of substituted groups, the carbon atom numbers given above for the acyl groups of the acyl-CoA esters preferably apply to these groups including carbon atoms of substituents. A preferred alkadienyl moiety is a butadienyl moiety.
Possible substituents are the same as those given above in the context of the amine.
In one embodiment, the amine is a C1-C14 amine and the CoA-ester is a C1-C20 acyl-CoA ester. In another embodiment, the amine is C3-C10 amine and the CoA-ester is a C3-C18 acyl-CoA ester. In another embodiment, the amine is C5-C8 amine and the CoA-ester is a C4-C16 acyl-CoA ester or, preferably, a C6-C14 acyl-CoA ester.
In another embodiment, the acyl-CoA is a compound of the following general formula (1):
preferably of the following general formula (3):
wherein R1 is a C1-C17 hydrocarbyl group (preferably a C1-C15 hydrocarbyl group) or C2-C16 heterohydrocarbyl group (preferably a C2-C12 heterohydrocarbyl group, more preferably a C2-C10 heterohydrocarbyl group), and SCoA is a CoA group (reciting the S merely highlights the terminal sulfur atom of the CoA group). As above, the heterohydrocarbyl group contains from 1 to 3 heteroatoms selected from O, N, and S, preferably selected from O and S, and even more preferably 1 or 2 heteroatoms selected from O and S, preferably O. As is generally known, the wiggly line in the compound of formula (1) and in the formulae below is a single bond and indicates that the stereochemistry of the double bond is not defined.
Products of the reaction involving compounds of formulae (1) and (3) are the compounds of the following formulae (2) and (4), respectively:
wherein R1 is as defined above, and NR2R3 is the amino group derived from the amine used in the invention and described above. In more detail, R2 is a hydrogen atom, a hydrocarbyl group as described above, or a heterohydrocarbyl group as described above; R3 is a hydrocarbyl group as described above or a heterohydrocarbyl group as described above; or R2 and R3 together form with the N-atom to which they are bound an optionally substituted 4-, 5-, 6, or 7-membered nitrogen-containing ring, preferably a 5- or 6-membered nitrogen-containing ring, as described above. The ring may be saturated or unsaturated and may contain an (preferably one) additional heteroatom selected from O, N or S, preferably O. Optional substituents are as defined above. The group NR2R3 is, including optional substituents, preferably a C1-C14—NR2R3-group, more preferably a C2-C12—NR2R3-group, more preferably a C3-C10—NR2R3-group, and most preferably a C5-C8—NR2R3-group.
Examples of R1 are C1-C17 substituted or unsubstituted alkyl, cycloalkyl, alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkylalkyl group, aryl, alkylaryl, arylalkyl, alkylarylalkyl group. Possible substituents are those given above.
In a more preferred embodiment, the acyl-CoA is a compound of the following general formula (5):
preferably the acyl-CoA is a compound of the following general formula (7):
wherein R4 is a C1-C15 hydrocarbyl group (preferably a C2-C10 hydrocarbyl group, more preferably a C2-C8 hydrocarbyl group) or C2-C14 heterohydrocarbyl group (preferably a C2-C10 heterohydrocarbyl group, more preferably a C2-C8 heterohydrocarbyl group). As above, the heterohydrocarbyl group contains from 1 to 3 heteroatoms selected from 0, N, and S, preferably selected from O and S, and even more preferably 1 or 2 heteroatoms selected from O and S, preferably O. SCoA is a CoA group.
Products of the reaction involving compounds of formula (5) and (7) are the compounds of the following formulae (6) and (8), respectively, (wherein R2 and R3 as defined above):
R4 is as described above. Examples of R4 are C1-C15 substituted or unsubstituted alkyl, cycloalkyl, alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkylalkyl group, aryl, alkylaryl, alkenylaryl, arylalkyl, alkylarylalkyl, and alkenylarylalkyl groups. Possible substituents are those given above. R2 and R3 are as defined above.
In a more preferred embodiment, the acyl-CoA is a compound of the following general formula (9):
wherein n is an integer of from 1 to 4, preferably 1 or 2, and multiple groups R5 may be the same or different, and R5 is selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, hydroxy, or hydroxy C1-C6 alkyl, and sulfhydryl, or two R5 at adjacent carbon atoms of the phenyl ring together form a five- or six-membered ring, such as a 3,4-methylene dioxy group. SCoA is a CoA group.
Products of the reaction involving compounds of formula (9) are compounds of the following formula (10):
R5 is as described above. Also, R2 and R3 are as defined above.
In preferred specific embodiments, the acyl-CoA is piperoyl-CoA or 3,4-methylenedioxy cinnamoyl-CoA and the primary or secondary amine is selected from the group consisting of piperidine, pyrrolidine, isobutylamine, benzylamine, 4-aminomethylpiperidine, vanillylamine, and 1,7-diamino-n-heptane, preferably piperidine. In another preferred embodiment, the acyl-CoA is (E)-8-methyl-6-nonenoyl-CoA, the amine is vanillylamine, and the amide product is capsaicin.
The method of the invention may be conducted in a cell-free system or in the presence of, or in, prokaryotic or eukaryotic cells (as described above) expressing the protein of the invention.
In a cell-free system, the method of producing an amide from an acyl-CoA and an amine is generally conducted in an aqueous medium, preferably a buffered aqueous medium. The pH of the medium may be between pH 6.0 and 9.0, preferably between pH 7.0 and 8.5. Generally known buffer substances that buffer at the selected pH may be used, such as Tris. The aqueous medium may be further supplied by a water-soluble polyol such as ethylene glycol or glycerol in a concentration of from 1 to 20 weight-%, preferably from 5 to 15 weight-%. Glycerol is preferred as the polyol.
The buffered solution in the cell-free system also contains a reducing agent, preferentially 1-2 mM DTT to prevent dimerization and oxidation of the enzymes, specifically during longer incubation times. The term “longer” refers to a time frame longer than 5 minutes
The method may be carried out in a method (e.g. a batch method), wherein the protein of the invention is mixed with the aqueous medium containing the acyl-CoA and the amine. The starting concentrations of the acyl-CoA may be from 10 μM-50 mM, preferably from 50 μM-20 mM, more preferably 100 μM-5 mM. The starting concentrations of the amine may be from 100 μM to 100 mM amine, preferably 500 μM to 50 mM, more preferably from 2 to 20 mM.
The protein is added in an amount to give a desired turnover of the substrates to product and is thus not particularly limited. Reactions may contain the protein in a concentration range from 1 μg/ml to 1 mg/ml, preferably from 5 μg/ml to 500 μg/ml, more preferably from 10 μg/ml to 200 μg/ml.
The amide product may be isolated from the aqueous medium as follows. For example, protein components of the reaction system may be precipitated e.g. using trichloroacetic acid, or formic acid together with acetonitrile or ethanol in a 50 to 50 ratio to prevent simultaneous precipitation of the hydrophobic products. This Stop-mix of organic solvent and acid is premade and added to the reaction system and mixed. The protein is removed e.g. by centrifugation or filtration. The supernatant contains the amide reaction product that may be purified by liquid chromatography (LC) as described in the experimental section.
A standard analytical procedure to isolate the amide can be achieved by preparative HPLC, where the reaction mix is applied to a C18 or C8 reversed phase-(RP) column (available from several suppliers, e.g. Macherey and Nagel, Waters, or Agilent) and the products are separated from buffer, residual substrates, and enzyme by a gradient of increased organic solvent, either methanol or acetonitrile, while individual fractions are collected. The product isolated in one such fraction is concentrated preferentially by freeze-drying, but evaporation of the solvent to dryness may also be possible.
An alternative suitable approach for larger scale production of a desired amide is by standard solid phase extraction cartridges or columns on a reversed phase-C-8 or C-18 matrix or a silica matrix, depending on the product and provided by several manufacturers (Waters, Agilent, Merck). In this case the supernatant of the said protein reaction may be applied in a single batch to the matrix, more hydrophilic substrates elute and the product, such as piperoylpyrrolidine, is batch-eluted with the desired concentration of an organic solvent, preferentially 80-100% methanol or acetonitrile and concentrated as said, by evaporation of the organic solvent, alternatively by adding water to 50% final concentration and subsequent freeze-drying. More precise elution conditions are dependent on the matrix and the properties of the amide produced and require prior testing. Specific conditions need to be optimized for individual products, specifically in preparative large scale processes.
The acyl-CoA for use in the process of the invention may be generated chemically from the coenzyme A and the corresponding carboxylic acid. The carboxylic acid is first activated e.g. by N-hydroxysuccinimide/carbodiimide-based activation of the carboxylic acid, followed by reaction with coenzyme A, which is an established procedure, see Stöckigt and Zenk, 1975.
It is also possible to generate the CoA-ester in situ in an enzymatic reaction from a carboxylic acid and coenzyme A in the presence of a CoA-ligase and a suitable energy source such as ATP. One example is PipCoA-ligase which was isolated from black pepper (Piper nigrum) and can produce piperoyl CoA and 5-phenylpentanoyl CoA from the corresponding acids. Both compounds can serve as CoA-donors in the in vitro reactions using the proteins of the inventions (see Table 1). Individual CoA-esters can be purified by chromatography on silica gel and/or solid phase extraction cartridges.
The invention also provides a process of producing an amide from a carboxylic acid and an amine that is a primary or secondary amine, comprising incubating the carboxylic acid and the amine in the presence of a CoA ligase, coenzyme A, and a protein according to the invention, optionally followed by isolating the amide produced. The amine may be as defined above, and the carboxylic acid may be a compound having the CoA moiety of any of the CoA-esters defined above replaced by a hydroxyl moiety.
The process of the invention is preferably carried out in the presence of, or in, prokaryotic or eukaryotic cells expressing the protein of the invention. The cells may further express a CoA-ligase for generating the acyl-CoA in said cells or in the reaction medium. The process of the invention may be carried within prokaryotic or eukaryotic cells. Alternatively, the protein of the invention (and optionally further enzymes required for the process) may be expressed with a tag that targets the protein to the secretory pathway to secret the protein into the medium. In this case, the process can be carried out in the medium in the presence of the cells that provide the protein(s).
The cells may be cultured in a suitable culture medium to allow the process of the invention to take place. Suitable culture media for various cells are known in the art. If the process is performed in eukaryotic cells, the cells may be a transgenic and contain the polynucleotide of the invention stably and expressibly incorporated in a chromosome. The eukaryotic cells may further contain, as the case requires, any of the other genes mentioned above. Usable cells are those listed above.
After the product has been produced, the product may be isolated and purified. If the product is secreted, it can be purified by solid phase extraction on a suitable matrix, reversed phase or silica, after the cells have been separated by filtration or by centrifugation. Isolation of the product may alternatively be performed from the cells homogenizing the cells containing the product, for example in the presence of an aqueous buffer. Again, insoluble debris or cells may be removed. The amide product may be purified from the aqueous buffer by liquid chromatography (LS) or solid phase extraction as done in the experimental section.
To identify piperine synthase and piperamide related genes we monitored piperine formation during fruit development of P. nigrum in greenhouse grown black pepper plants over several months. Drupes of several plants were marked and piperine amounts were quantified by HPLC-MS and UV-detection respectively, based on a standard of commercially available piperine. From this time course, it was evident that in our greenhouse grown plants piperine accumulation started after a lag-phase of roughly 20 days after anthesis and peaked approximately three months after anthesis at levels of 2.5% piperine calculated per fresh weight. No significant increase was observed during later stages of fruit development. Therefore, our focus turned to a time frame between 20-50 days post anthesis, a few days before the maximum slope of piperine increase was observed. For the differential RNA-Seq approach two time periods were selected, a period between 20-30 day post anthesis, where piperine formation just started and a 2nd period between 40-50 days post anthesis based on the assumption that specific transcript levels preceded accumulation of biosynthetic product formation. Spadices with flowers as well as young leaves served as additional negative controls in the differential RNA-Seq approach. In both organs, no or very low piperine signals were detected by LC-MS. From the differential screening, it was evident, that individual organs show unique transcript profiles and contain numerous acyltransferase sequence candidates.
Candidate gene sequences were selected based on i) relative transcript abundance in young fruits of black pepper plants (Piper nigrum) in comparison to leaves and flowers in an RNA-seq experiment and ii) sequence identity to putative BAHD-signature sequences in various databases. This RNA-seq data set was based on plants and plant tissue grown at the IPB greenhouse and has been uniquely designed and developed at the Leibniz Institute of Plant Biochemistry and was a requirement to identify the nucleotide and protein sequences of PipSyn1 and PipSyn2. Full length sequences of the candidate genes were obtained from assembled sequence contigs. One candidate gene, PipSynthase 1 (also PipSyn1), revealed an open reading frame of 1383 nucleotides (SEQ ID NO: 1) and the other candidate gene, PipSynthase2 (also PipSyn2), an open reading frame of 1386 nucleotides (SEQ ID NO: 3). Sequences were assembled from fragments obtained by Illumina High Throughput Sequencing of the transcriptome of black pepper according to Haas B J et al., 2013.
The nucleotide sequence of SEQ ID NO: 1 (PipSynthase1) was in silico-translated into the amino acid sequence of SEQ ID NO: 2 and the nucleotide sequence of SEQ ID NO: 3 (PipSynthase2) was in silico-translated to the amino acid sequence of SEQ ID NO: 4. The amino acid sequences of SEQ ID NO: 2 and 4 were then BLASTed against protein databases to find similar amino acid sequences. The identified protein sequences of SEQ ID NO: 2 and 4 were found to have some similarity to BAHD-type transferases and the results are displayed in the dendrogram in
Both SEQ ID NO: 2 and SEQ ID NO: 4 contain an HXXXD-motif consistent with the family of BAHD-type acyltransferases. The histidine is required for the abstraction of a proton as the initial step of the reaction mechanism and considered to be characteristic for all BAHD-like transferases. In addition, several other structural motifs of BAHD-type transferases are present. The overall amino acid sequence identity to the closest sequence which is annotated as a benzoyl-benzoate transferase from Clarkia brewerii (Onagraceae) was about 42%, whereas the sequence identity between SEQ ID NO: 2 and SEQ ID NO: 4 is 62% at the amino acid level and 69% at the nucleotide sequence level. Other similar proteins of the BAHD-family in that sub-cluster, which are not functionally characterized, show sequence identities of 40%, and the sequence identities in other sub-clusters are between 15-25% (
Full length nucleotide sequences of SEQ ID NO:1 and SEQ ID NO: 3 were amplified from cDNA extracted from young black pepper fruits by Phusion (Thermofischer Scientific) polymerase using the primers:
subsequently A-tailed by Flexi DNA-polymerase (Promega) to add A-overhangs and then cloned into the pGem-T easy cloning vector (Promega). This vector was then used to transform E. coli DH10B blue cells. The transformed cells were plated on liquid broth agar, LB, (Duchefa) supplemented with 50 μg/ml ampicillin and grown over night at 37° C. For each construct, a single colony was picked to inoculate into liquid media culture, LB high salt (Duchefa), supplied with 50 μg ml−1 ampicillin and grown over night at 37° C. After harvest and plasmid isolation, the cloned sequences were verified by sequencing. The verified sequences were excised by the restriction enzymes NdeI and BamHI according to the manufacturer's protocol and cloned into NdeI/BamHI restriction sites of the protein expression vector pet16b (Novagen), followed by ligation with T4 DNA-ligase (Thermofischer Scientific).
The ligated expression vector was then used to transform Lemo BL21 (DE3) cells (New England Biolabs) according to the manufacturer's protocol. These cells served as the protein expression system. The cells were plated on LB agar supplemented with 50 μg/ml ampicillin and grown over night at 37° C. A single bacterial colony for each of the expression constructs comprising SEQ ID NO: 1 or SEQ ID NO: 3 was picked to inoculate a 25 ml liquid pre-culture in LB medium containing 50 μg/ml ampicillin and grown overnight at 30° C. An 8 ml aliquot of the pre-culture was then used to inoculate the 400 ml-liquid main-culture containing 50 μg/ml ampicillin and 0.4 mM rhamnose. At a cell density of 0.6 OD600, recombinant protein expression was induced by addition of 1 mM IPTG, and the culture was grown for 18 to 22 h at 25° C.
The Lemo BL21 (DE3) bacterial expression system used herein is based on a rhamnose-inducible promotor of a 2nd gene (pLysC) on a 2nd plasmid to control protein expression by controlling T7 RNA-Polymerase, which provides the transcriptional control of the sequence to be expressed. This system allowed tunable expression of proteins encoded by SEQ ID NO: 1 and SEQ NO: 3 at lower amounts than conventional expression system, like BL21DE cells (Novagen, NEB) or M15Rep4 cells (Qiagen), but provided the benefit that large parts of the recombinant proteins can be extracted as soluble recombinant proteins after bacteria are collected from liquid culture and are extracted by sonication (info by the manufacturer and verified by our experimental approach).
After cultivation, the cells of the Lemo BL21 (DE3) expression system were harvested by centrifugation at 4000 g for 10 min at 4° C. Pellets were resolved in 50 ml volume buffer (20 mM Tris-HCl, pH 7.5, containing 100 mM NaCl and 15% glycerol) and disrupted by sonification on ice. The homogenate was centrifuged at 10,000 g for 10 min at 4° C. to pellet the debris. The supernatant was tested for PipSynthase activity by a standard enzyme activity test with 20 μg of crude protein, instead of 2-5 μg of purified protein, and product formation was analyzed by LC-MS as reported and illustrated in Example. 3. The supernatant was then applied to a 1 ml prepacked Ni-NTA column (Macherey-Nagel). This is possible, since the pET16b vector contains an N-terminal protease cleavable His-Tag cloned in frame with the protein according to SEQ ID NO: 1 and 2. His-tagged PipSynthases were eluted with 300 mM imidazole, 20 mM Tris-HCl, pH 7.5, 100 mM NaCl and 15% glycerol. The purified proteins were then desalted into 30 mM Tris-HCl pH 8.0, 1 mM DTT, 10% glycerol and stored at −80° C. and remained stable over several freeze/thaw cycles. The purified protein of PipSynthase1 and 2 was visualized on SDS-PAGE gels (
Standard assays are routine for a person of average skill in the art.
Enzyme activity test with regard to piperine formation in case of PipSynthase 2.
A standard enzyme assay contains:
10 μl enzyme solution in 30 mM Tris/HCl buffer pH 8.0 (10% glycerol, 1 mM DTT)
1 μg-5 μg protein, amount determined according to Bradford
10 μl piperidine, 100 mM (in water)
10 μl 5 mM DTT in water
10 μl piperoyl-CoA (PipCoA) 2.5 mM in 25% DMSO/75% water
Mix by vortex, incubate on ice for 5 minutes and centrifugation for 1 min at 20,000 g to precipitate any debris or insoluble material e.g. residual protein.
Remove 50 μl of supernatant for analysis of products (amides) by LC-MS.
Injected to an RP-HPLC column are 10 μl (out of 50 μl).
Quantification was performed based on a commercially available piperine standard. The peak area of the peak due to piperine in the LC trace was determined. Peaks due to possible isomerization products of piperine were neglected for the determination of enzyme activity of piperine formation. Product formation was observed without protein and under otherwise identical conditions (
Protein amounts were determined photometrically by the Bradford assay of combining a defined volume, preferentially 10 μl of the (partly) purified PipSyn1 and PipSyn2 enzyme and 990 μl of Bradford reagent, and calculating the resulting absorbance at λ=595 nm based on a standard curve made with a serial dilution of commercially available Bovine Serum Albumin fraction V in water.
This Example provides in Table 1 substrates which were tested for enzyme activity with both enzymes. Activity data were calculated from the UV-(PDA) area in the range of 320-370 nm. The data are supported by LC-MS measurements of corresponding m/z values recorded in positive ionization mode and based on calculated expected masses. Mass accuracy of Waters QDA-detector is +/−0.2 Dalton. Contrary to the procedure of Example 3, for the determination of the activity of the reaction of piperoyl-CoA and piperidine as substrates, the sum of all peaks in the LC trace due to species having calculated m/z values of the expected products was used for quantification of product, assuming the same ionization efficiency in positive ionization mode as in case of piperine for all molecule species.
Production of piperine in a fermentation process using E. coli was achieved by combining the sequences of said PipCoA ligase and said PipSyn2, both cloned on a petDuet-1 vector (Novagene) transformed into E. coli SoluBL21 cells (Amsbio), induced by IPTG at an OD of 0.4, grown at 25° C. for 16 hours and fed with substrates by adding 1 mM piperic acid and 10 mM piperidine, both dissolved in 25% DMSO (final concentrations) directly to 1 ml of the said E. coli cell suspension. After 30 min, the cells were centrifuged, the supernatant was collected, and extracted with 100% ethyl acetate. The ethyl acetate fraction was concentrated to dryness, redissolved in 100% methanol and checked by LC-MS, as said, resulting in the production of piperine and piperine isomers.
A standard analytical procedure to isolate the amide in small quantities from enzyme assays in a cell free system can be achieved by preparative HPLC, where the reaction mix is applied to a C18 or C8 reversed phase-(RP) column (available from several suppliers, e.g. Macherey and Nagel, Waters, or Agilent) and the products are separated from buffer, residual substrates and enzyme by a gradient of increased organic solvent, either methanol or acetonitrile, while individual fractions are collected. The product isolated in one such fraction is concentrated by freeze drying and evaporation of the solvent to dryness. This is a routine procedure and can be achieved by any researcher provided with the corresponding LC and freeze drying equipment.
An alternative approach for larger scale production of the desired amide is by standard solid phase extraction on a RP-matrix (or a silica matrix), depending on the product with the solid phase provided by several manufacturers (Waters, Agilent, Merck), where the supernatant of the protein reaction is applied in a single batch to the solid phase matrix where hydrophilic impurities are washed in aqueous solutions from the matrix of increasing hydrophobicity, usually from 50-100% methanol or acetonitrile and concentrated, preferentially by freeze drying. Exact elution conditions are dependent on the matrix and the properties of the amide and can be determined by testing. Specific conditions are preferably optimized for individual products, specifically in preparative large scale processes.
annuum
| Number | Date | Country | Kind |
|---|---|---|---|
| 19195598.8 | Sep 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/060165 | 9/4/2020 | WO |
