Patent Application
20220042051
The contents of the electronic sequence listing (Revised_Seq_Listing_36803-289.txt; Size: 1,581,474 bytes; and Date of Creation: Sep. 21, 2021) is herein incorporated by reference in its entirety.
The present invention provides novel methods for the lipoxygenase (LOX)-catalyzed production of aliphatic unsaturated C10-aldehyde compounds from polyunsaturated fatty acid (PUFA) sources. The present invention also relates to the isolation and characterization of novel, preferably bifunctional LOXs from different algae sources and the identification of structurally and/or functionally related LOXs from different bacterial sources. The present invention also relates to the provision of enzyme mutants derived from said newly identified enzymes. A further aspect of the present invention relates to corresponding coding sequences of said enzymes, recombinant vectors, and recombinant host cells suitable for the production of such LOXs and for performing the novel production methods of aliphatic unsaturated C10-aldehyde compounds. Another aspect of the invention relates to the use of particular aldehydes or aldehyde mixtures, as obtained according to the present invention as flavor ingredient or ingredient for food or feed compositions.
The unsaturated C10-aldehydes decadienal and decatrienal are very important ingredients for chicken and citrus flavours. In spite of high production costs and low production volumes, flavorists cannot replace them with other ingredients due to their unique olfactory properties. More than 200 commercial formulas contain C10-aldehydes.
C6 and C9 aldehydes are typically biosynthesised by plant defensive systems through a two-step enzymatic reaction starting from polyunsaturated fatty acids (PUFAs) (see Scheme 1 below). First, LOXs convert fatty acids to fatty acid hydroperoxides (HPOs). Subsequently, hydroperoxide lyases (HPL) break down HPOs into metabolites including aldehydes and alcohols. The production of C6 and C9 ingredients by enzymes from plant extracts or enzymes from overexpressed microbial systems is well known. The industrial routes to manufacture C6 and C9 aldehyde flavour ingredients are relatively mature and the product quality is stable. Consequently, the prices remain lower than for C10 analogs.
In comparison to the C6 and C9 analogues, the industrial process to manufacture C10 aldehyde ingredients is more challenging (see Scheme 1 below, right half). It stats with the 9-LOX catalysed peroxidation of linoleic acid and alpha-linolenic acid. The 9-LOX is obtained from a plant source (potato). Considering that no HPL is available that would cleave the 9-HPO intermediates into C10 fragments, a typical process currently relies instead on thermal degradation of 9-HPO. Overall, the approach has two drawbacks. One is product variation issues due to variations in the quality of the potato extracts from different suppliers, i.e. different yields achieved for each production batch since the enzyme content from potato is different. Another one is the low yield of the thermal cracking step which leads to high production costs.
Alsufyani, T. et al describe in Chemistry and Physics of Lipids 183 (2014) 100-109 several seaweeds including Ulva which could produce decadienals and decatrienals through the conventional LOX/HPL pathway. This prior art document doesn't identify any gene sequence, coding sequence, or protein sequence involved in said bioconversion or any key amino acid residues that determine high LOX activity.
Lee, J. et al provide in Environmental Pollution 227 (2017) 252e262 a review pertaining to algae and bacterial odor problems that have been published over the last five decades. Two Microcystis species (Cyanobacteria) were reported to produce decatrienal. While said prior art has its focus on odorant pollution in water no particular teaching on genes, coding sequences, or protein sequences responsible for said decatrienal formation is provided.
Zhu, Z-J. et al further investigate in Journal of Agriculture and Food Chemistry. (2018) 66(5):1233-1241 the multifunctional LOX, PhLOX from seaweed Pyropia haitanensis (also described by the Chen, Hai-min et al in Algal Research, 12, (2015) 316-327), in the one-step bioconversion of fatty acids to primarily C8-C9 aldehydes based on LOX activity and HPL activity. Said multifunctional LOX is said to show LOX, HPL and allene oxide synthase (AOS) activity. The production of a 2E,4Z-decadienal side product was observed merely by feeding with hydrolyzed fish oil but not with the numerous other testes substrates, like ALA, ARA, EPA and DHA. Decatrienals were not observed. Gamma-linolenic acid was not used as substrates in said prior art. The productivity of said decadienal side product is quite low and not of industrial value.
Zhu, et al describe in PLoS One. (2015) 10(2):e0117351) another multifunctional LOX, PhLOX2, from seaweed Pyropia haitanensis. EPA, ARA, GLA and DHA were investigated as substrates; no production of any unsaturated C10 aldehyde was reported therein.
Chinese Patent Application CN 104293805 describes a multifunctional LOX protein sequence from seaweed Pyropia haitanensis (PhLOX) which was also expressed in E. coli. Said LOX species did not produce decadienals and decatrienals when feeding with fatty acid substrates. It only produces short chain aldehydes
Chinese Patent Application CN 104293837 A describes another multifunctional LOX from seaweed Pyropia haitanensis (PhLOX) which was expressed in E. coli. No evidence for a production for C10-aldehydes, in particular decadienals and decatrienals is provided therein.
WO2008056291 and EP-A-1921134 describe a cyanobacterial LOX, WP_012407347.1, and suggest its use in the production of fatty acid hydroperoxides, however do not provide evidence for the production of unsaturated C10-aldehydes, like decadienal.
Despite of different reports on the biocatalytic synthesis of unsaturated C10-aldehydes, the enzymatic systems described in the prior art still suffer from the problem of low productivity and, consequently, do not provide a suitable basis for the industrial scale production of C10-aldehydes.
The problem to be solved by the present invention is, therefore, the provision of an improved biocatalytic method for the production of unsaturated C10-aldehyde compounds, in particular decadienals and/or decatrienals. Another problem to be solved by the present invention is the provision of novel biocatalysts applicable in the fully biosynthetic production of unsaturated C10-aldehydes, in particular decadienals and/or decatrienals.
The above-mentioned problems could, surprisingly, be solved by providing unique and superior LOXs from new sources. In particular, the present inventors succeeded in isolating novel bi-functional LOXs from the seaweed sources Cladophora oligoclara producing high amounts of decadienals and/or decatrienals from different PUFA substrates. The present inventors also succeeded in isolating a novel bi-functional LOX from the seaweed Ulva fasciata which also produces high amounts of decadienals and/or decatrienals from different PUFA substrates.
On the basis of the sequence information derived from said new LOXs, the present inventors also surprisingly succeeded in the identification of LOXs with the desired catalytic LOX activity from bacterial sources, mainly from cyanobacteria.
On the basis of sequence comparisons between said newly identified enzymes, the present inventors were able to perform a systematic investigation on structure and functionality of suitable bifunctional LOXs showing superior productivity and/or specificity, for unsaturated C10-aldehyde compounds, in particular decadienals and/or decatrienals, more particularly decadienals. Improved productivity was observed for several bacterial LOXs. On the basis of such investigations the inventors were able to further improve LOX productivity in the industrial production of such C10-aldehydes.
The newly identified protein sequences may be functionally expressed in the bacterial hosts like Escherichia coli. Surprisingly, cultures with high cell density could be obtained with improved enzymatic capability for the industrial scale production of said C10-aldehydes. Feeding with specific fatty acids as substrates, such recombinant E. coli hosts are highly productive in different decadienals and/or decatrienals.
The new approach allows the provision of more cost-effective methods for the fully biocatalytic production of decadienals and/or decatrienals.
If required said aldehydes may be converted to suitable derivatives, in particular to corresponding alcohols, by chemical or, in particular, biochemical conversion, for example by applying conventional alcohol dehydrogenase (ADH) enzymes.
Particular PUFAs (PUFA substrates) as specifically referred to herein are selected from the following polyunsaturated omega-3 and omega-6 fatty acids and natural or synthetic mixtures of at least two of them:
Non-limiting examples of particular PUFA mixtures as specifically referred to herein are selected from: fish oil, linseed oil, arachidonic acid oil, linseed oil, evening primrose oil echium oil, micro algae oil and borage oil.
“Lipoxygenase” (LOX) (also designated linoleate: oxygen oxidoreductases, EC 1.13.11.12) constitute a large gene family of non-heme iron-containing fatty acid dioxygenases, which are ubiquitous in plants and animals. LOXs catalyze the regio- and stereospecific dioxygenation of PUFAs containing at least one (1Z,4Z)-pentadiene system. Thus, substrates for LOXs are for example linoleic acid (LA), alpha-linolenic acid (ALA), or arachidonic acid (ARA).
The term “LOX” as used herein specifically refers to such PUFA degrading enzymes which have the ability initiate a dioxygenation step in a suitable chain position of said PUFA molecule which ultimately results in the formation of at least one unsaturated C10-aldehyde fragment, in particular at least one decadienal and/or decatrienals compound, as the result of such oxidative degradation reaction. Said C10 compound(s) may be produced as side product (s) together with other oxidation product(s) of different chain length, for example of shorter chain lengths, as for example C6- or C9 unsaturated aldehydes, particularly however said C10 compound(s) may be produced as predominant product (s), i.e. in an molar excess over other oxidation product of different, for example shorter chain lengths, as for example C6- or C9 unsaturated aldehydes, or more particularly said C10 compound(s) may be produced as the single product species.
The “LOX/HPL pathway” or “LOX/HPL pathway” refers to the classical two-step enzymatic reaction for the oxidative degradation of polyunsaturated fatty acid molecules. First, LOXs (LOX) convert said fatty acids to fatty acid hydroperoxides (HPOs). Subsequently, HPLs (HPL) break down HPOs into metabolites including aldehydes and alcohols.
A “bifunctional” LOX designates herein a single enzyme molecule which shows both LOX and HPL activity required for the oxidative degradation of polyunsaturated fatty acid molecules (irrespective of a particular enzymatic mechanism). In a particular embodiment such bi-functional LOX may shows essentially no AOS activity, and more particularly may be absent of such AOS activity. As shown in the experimental section such bifunctional LOX do not only form fatty acid hydroperoxides intermediates they also show the ability to degrade such fatty acid hydroperoxides compounds if applied as synthetic artificial substrate. A “bifunctional” LOX in particular herein refers to a single enzyme molecule which shows both LOX and HPL activity required for the oxidative degradation of polyunsaturated fatty acid molecules (irrespective of a particular enzymatic mechanism). Thus said bifunctional LOX catalyzes the formation of at least one unsaturated C10-aldehyde fragment, in particular at least one decadienal and/or decatrienals compound, as the result of such oxidative degradation reaction. Said C10 compound(s) may be produced as side product(s) together with other oxidation product(s) of different chain length, for example of shorter chain lengths, as for example C6- or C9 unsaturated aldehydes, particularly however said Cu) compound(s) may be produced as predominant product(s), i.e. in an molar excess over other oxidation product of different, for example shorter chain lengths, as for example C6- or C9 unsaturated aldehydes, or more particularly said C10 compound(s) may be produced as the single product species.
Without being bound to any mechanistic considerations, the HLP activity of a “Bifunctional LOX” of the present invention may be further described as the ability to exclusively or preferentially cleave the hydroperoxides intermediate of the PUFA substrate at the C—C bond on the carboxyl-terminal side relative to its the HOO— group. This distinguishes the present enzymes also from plant derived LOX/HLP enzyme systems, as for example depicted in the above Scheme 1. Starting out from LA or ALA (i.e. C18-PUFAs) a bifunctional LOX of the invention may be considered to encompass both a 9-LOX activity and a 9-HPL activity. As opposed to the prior art 9-HLP of rice plants, the 9-HPL activity of the bifunctional LOX of the present invention, however, results in a cleavage of the hydroperoxides intermediate on the opposite (carboxyl-terminal) side of the HOO— group of the intermediate. For cleavage resulting in a C10-aldehyde an extra double bond in beta-position relative to the HOO-group appears to be favorable or necessary, so that a cleavage of the carbon chain between the C-atom carrying the HOO-group and the carbon atom in alpha-position thereto will occur. As a result of this a C10-aldehyde rather than a C9-aldehyde as in the case of the plant enzyme is produced. This is illustrated below in Scheme 2 with GLA as an example.
As is evident from the above Scheme 2 a “bifunctional LOX” of the present invention, in order to produce an unsaturated C10-aldehyde, utilizes particular PUFA substrates. Essentially, a preferred PUFA substrate should comprise cis-double bonds between omega-9 and 10 carbon atoms (i.e. between position (C-9) and (C-10) in C18 fatty acid and between position (C-11) and (C-12) in C20 fatty acid) as well as between omega 12 and 13 carbon atoms (i.e. between position (C-6) and (C-7) in C18 fatty acid and between position (C-8) and (C-9) in C20 fatty acid). For example, in case of C18 fatty acids those comprising two cis double bonds in an all-cis-6, 9 configuration (cf. GLA and SDA) are preferred substrates, and in case of C20 fatty acids those comprising two cis double bonds an all-cis-8,11 configuration (cf. EPA or ARA) are preferred substrates. These preferred PUFA substrates may also be considered as “reference substrates”. In order to qualify as a “bifunctional LOX of the present invention” it is sufficient if the LOX is able to convert at least one of such “reference substrate” to an unsaturated C10-aldehyde, in particular at least one selected from (2E,4Z)-2,4-decadienal, (2E,4E)-2,4-decadienal, (2E,4Z,7Z)-2,4,7-decatrienal and (2E,4E,7Z)-2,4,7-decatrienal.
An “unsaturated C10-aldehyde” encompasses any mono-, di- or tri-unsaturated linear aliphatic aldehyde having ten carbon atoms in its hydrocarbyl chain. It encompasses such compound in any stereoisomerically pure form or in the form of mixtures of at least two different stereoisomers. Particular, non-limiting examples of such aldehydes are decadienals and decatrienals.
A “decadienal” encompasses such compound in any stereoisomerically pure form or in the form of mixtures of at least two different stereoisomers. Typical examples are 2E,4Z-decadienal and 2E,4E-decadienal and mixtures thereof.
A “decatrienal” encompasses such compound in any stereoisomerically pure form or in the form of mixtures of at least two different stereoisomers. Typical examples are 2E,4Z,7Z-decatrienal, 2E,4E,7Z-decatrienal and mixtures thereof.
The term “PUFA” as used herein has to be understood broadly. In particular it encompasses one single “pure” or “essentially pure” type of PUFA molecule (like HTA, ALA, SDA, EPA, LA, GLA, or ARA) or any mixture containing at least two different types of PUFAs. A PUFA substrate also encompasses natural products containing at least one PUFA typein admixture with other natural or synthetic constituents, as for example
a) borage oil (containing elevated proportions of GLA)
b) evening primrose oil (containing elevated proportions of GLA)
c) arachidonic oil (containing elevated proportions of ARA)
d) echium seed oil (containing elevated proportions of SDA
e) fish oil (containing elevated proportions of EPA and DHA)
f) linseed oil (containing elevated proportions of ALA)
g) micro algae oil (containing elevated proportions of DHA)
“Bifunctional LOX Activity” is determined under “standard conditions” as described in the experimental section. In general, the LOX product GLA-HPO and HPL product hexanal, and decadienal were quantified by GC-MS and LC-UV by peak areas. To deduce bifunctional LOX activity to make decadienal, we can calculate the peak area ratio of decadienal to GLA-HPO from the LC-UV data as shown in Table 9.
The terms “biological function,” “function”, “biological activity” or “activity” of a LOX refer to the ability of a LOX as described herein to catalyze the formation of at least one unsaturated C10 aldehyde from at least one type of PUFA molecule.
As used herein, the term “host cell” or “transformed cell” refers to a cell (or organism) altered to harbor at least one nucleic acid molecule, for instance, a recombinant gene encoding a desired protein or nucleic acid sequence which upon transcription yields at least one functional polypeptide of the present invention, i.p. a LOX or bifunctional LOX as defined herein above. The host cell is particularly a bacterial cell, a fungal cell or a plant cell or plants. The host cell may contain a recombinant gene or several genes, as for example organized as an operon, which has been integrated into the nuclear or organelle genomes of the host cell. Alternatively, the host may contain the recombinant gene extra-chromosomally.
The term “organism” refers to any non-human multicellular or unicellular organism such as a plant, or a microorganism. Particularly, a micro-organism is a bacterium, a yeast, an algae or a fungus.
The term “plant” is used interchangeably to include plant cells including plant protoplasts, plant tissues, plant cell tissue cultures giving rise to regenerated plants, or parts of plants, or plant organs such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits and the like. Any plant can be used to carry out the methods of an embodiment herein.
A particular organism or cell is meant to be “capable of producing” an unsaturated C10 aldehyde when it produces such aldehyde naturally or when it does not produce such aldehyde naturally but is transformed to produce such aldehyde with a nucleic acid as described herein. Organisms or cells transformed to produce a higher amount of such aldehyde than the naturally occurring organism or cell are also encompassed by the “organisms or cells capable of producing unsaturated C10 aldehyde”.
For the descriptions herein and the appended claims, the use of “or” means “and/or” unless stated otherwise. Similarly, “comprise”, “comprises”, “comprising”, “include”, “includes”, and “including” are interchangeable and not intended to be limiting.
It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of”.
The terms “purified”, “substantially purified”, and “isolated” as used herein refer to the state of being free of other, dissimilar compounds with which a compound of the invention is normally associated in its natural state, so that the “purified”, “substantially purified”, and “isolated” subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, or at least 50% or 75% of the mass, by weight, of a given sample. In one embodiment, these terms refer to the compound of the invention comprising at least 95, 96, 97, 98, 99 or 100%, of the mass, by weight, of a given sample. As used herein, the terms “purified,” “substantially purified,” and “isolated” when referring to a nucleic acid or protein, or nucleic acids or proteins, also refers to a state of purification or concentration different than that which occurs naturally, for example in an prokaryotic or eukaryotic environment, like, for example in a bacterial or fungal cell, or in the mammalian organism, especially human body. Any degree of purification or concentration greater than that which occurs naturally, including (1) the purification from other associated structures or compounds or (2) the association with structures or compounds to which it is not normally associated in said prokaryotic or eukaryotic environment, are within the meaning of “isolated”. The nucleic acid or protein or classes of nucleic acids or proteins, described herein, may be isolated, or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.
The term “about” indicates a potential variation of ±25% of the stated value, in particular ±15%, ±10%, more particularly ±5%, ±2% or ±1%.
The term “substantially” describes a range of values of from about 80 to 100%, such as, for example, 85-99.9%, in particular 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9% and especially 99 to 99.9%.
“Predominantly” refers to a proportion in the range of above 50%, as for example in the range of 51 to 100%, particularly in the range of 75 to 99.9%, more particularly 85 to 98.5%, like 95 to 99%.
A “main product” in the context of the present invention designates a single compound or a group of at least 2 compounds, like 2, 3, 4, 5 or more, particularly 2 or 3 compounds, which single compound or group of compounds is “predominantly” prepared by a reaction as described herein, and is contained in said reaction in a predominant proportion based on the total amount of the constituents of the product formed by said reaction. Said proportion may be a molar proportion, a weight proportion or, preferably based on chromatographic analytics, an area proportion calculated from the corresponding chromatogram of the reaction products.
A “side product” in the context of the present invention designates a single compound or a group of at least 2 compounds, like 2, 3, 4, 5 or more, particularly 2 or 3 compounds, which single compound or group of compounds is not “predominantly” prepared by a reaction as described herein.
Because of the reversibility of enzymatic reactions, the present invention relates, unless otherwise stated, to the enzymatic or biocatalytic reactions described herein in both directions of reaction.
“Functional mutants” of herein described polypeptides include the “functional equivalents” of such polypeptides as defined below.
The term “stereoisomers” includes in particular conformational isomers.
Included in general are, according to the invention, all “stereoisomeric forms” of the compounds described herein, such as constitutional isomers and, in particular, stereoisomers and mixtures thereof, e.g. optical isomers, or geometric isomers, such as E- and Z-isomers, and combinations thereof. If several asymmetric centers are present in one molecule, the invention encompasses all combinations of different conformations of these asymmetry centers, e.g. enantiomeric pairs
“Stereoselectivity” describes the ability to produce a particular stereoisomer of a compound in a stereoisomerically pure form or to specifically convert a particular stereoisomer in an enzyme catalyzed method as described herein out of a plurality of stereoisomers. More specifically, this means that a product of the invention is enriched with respect to a specific stereoisomer, or an educt may be depleted with respect to a particular stereoisomer. This may be quantified via the purity % ee-parameter calculated according to the formula:
% ee=[XA−XB]/[XA+XB]*100,
wherein XA and XB represent the molar ratio (Molenbruch) of the stereoisomers A and B.
The terms “selectively converting” or “increasing the selectivity” in general means that a particular stereoisomeric form, as for example the E-form, of an unsaturated hydrocarbon, is converted in a higher proportion or amount (compared on a molar basis) than the corresponding other stereoisomeric form, as for example Z-form, either during the entire course of said reaction (i.e. between initiation and termination of the reaction), at a certain point of time of said reaction, or during an “interval” of said reaction. In particular, said selectivity may be observed during an “interval” corresponding 1 to 99%, 2 to 95%, 3 to 90%, 5 to 85%, 10 to 80%, 15 to 75%, 20 to 70%, 25 to 65%, 30 to 60%, or 40 to 50% conversion of the initial amount of the substrate. Said higher proportion or amount may, for example, be expressed in terms of:
each of which preferably being observed relative to a reference method, said reference method being performed under otherwise identical conditions with known chemical or biochemical means.
Generally also comprised in accordance with the invention are all “isomeric forms” of the compounds described herein, such as constitutional isomers and in particular stereoisomers and mixtures of these, such as, for example, optical isomers or geometric isomers, such as E- and Z-isomers, and combinations of these. If several centers of asymmetry are present in a molecule, then the invention comprises all combinations of different conformations of these centers of asymmetry, such as, for example, pairs of enantiomers, or any mixtures of stereoisomeric forms.
“Yield” and/or the “conversion rate” of a reaction according to the invention is determined over a defined period of, for example, 4, 6, 8, 10, 12, 16, 20, 24, 36 or 48 hours, in which the reaction takes place. In particular, the reaction is carried out under precisely defined conditions, for example at “standard conditions” as herein defined.
The different yield parameters (“Yield” or YP/S; “Specific Productivity Yield”; or Space-Time-Yield (STY)) are well known in the art and are determined as described in the literature.
“Yield” and “YP/S” (each expressed in mass of product produced/mass of material consumed) are herein used as synonyms.
The specific productivity-yield describes the amount of a product that is produced per h and L fermentation broth per g of biomass. The amount of wet cell weight stated as WCW describes the quantity of biologically active microorganism in a biochemical reaction. The value is given as g product per g WCW per h (i.e. g/gWCW−1h−1). Alternatively, the quantity of biomass can also be expressed as the amount of dry cell weight stated as DCW. Furthermore, the biomass concentration can be more easily determined by measuring the optical density at 600 nm (OD600) and by using an experimentally determined correlation factor for estimating the corresponding wet cell or dry cell weight, respectively.
The term “fermentative production” or “fermentation” refers to the ability of a microorganism (assisted by enzyme activity contained in or generated by said microorganism) to produce a chemical compound in cell culture utilizing at least one carbon source added to the incubation.
The term “fermentation broth” is understood to mean a liquid, particularly aqueous or aqueous/organic solution which is based on a fermentative process and has not been worked up or has been worked up, for example, as described herein.
An “enzymatically catalyzed” or “biocatalytic” method means that said method is performed under the catalytic action of an enzyme, including enzyme mutants, as herein defined. Thus the method can either be performed in the presence of said enzyme in isolated (purified, enriched) or crude form or in the presence of a cellular system, in particular, natural or recombinant microbial cells containing said enzyme in active form, and having the ability to catalyze the conversion reaction as disclosed herein.
If the present disclosure refers to features, parameters and ranges thereof of different degree of preference (including general, not explicitly preferred features, parameters and ranges thereof) then, unless otherwise stated, any combination of two or more of such features, parameters and ranges thereof, irrespective of their respective degree of preference, is encompassed by the disclosure of the present description.
The present invention relates to the following particular embodiments:
1. A polypeptide which comprises the enzymatic activity of a lipoxygenase, i.p. of a bifunctional LOX, with an amino acid sequence that comprises a consensus sequence pattern selected from SEQ ID NO:54; or comprises at least one partial consensus sequence pattern of SEQ ID NO:54 selected from
The present invention also relates to several groups of polypeptides which comprise the enzymatic activity of a lipoxygenase, i.p. of a bifunctional LOX, and which may not show at least one of the above sequence pattern of embodiments 1, 2 and 3 in an identical manner or which may show a sequence pattern that is similar to at least one of the above pattern but does not completely match therewith.
4. Thus another embodiment of the invention refers to a polypeptide which comprises the enzymatic activity of a lipoxygenase, i.p. of a bifunctional LOX, optionally fulfilling any one of the preceding embodiments, and comprising an amino acid sequence selected from
Thus, the polypeptides of the present embodiment may or may not meet the limitations of anyone of the embodiments 1, 2 and 3.
A first particular group of polypeptides comprises an amino acid sequence selected from SEQ ID NO: 3, 6, 9, 12 or 15; (CoLOXs) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least one of these sequences and retaining said bifunctional LOX activity, and which may not meet the limitations of anyone of the embodiments 1, 2 and 3;
or alternatively selected from:
SEQ ID NO: 3, 6, 9, 12 or 15; (CoLOXs) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least one of these sequences and retaining said bifunctional LOX activity, and which meet the limitations of anyone of the embodiments 1, 2 and 3.
A second particular group of polypeptides comprises an amino acid sequence selected from
SEQ ID NO: 18 (UfLOX2) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto and retaining said bifunctional LOX activity and which may not meet the limitations of anyone of the embodiments 1, 2 and 3; or alternatively selected from:
SEQ ID NO: 18 (UfLOX2) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto and retaining said bifunctional LOX activity and which meet the limitations of anyone of the embodiments 1, 2 and 3;
A third particular group of polypeptides comprises an amino acid sequence selected from SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 (bacterial LOXs) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least one of these sequences and retaining said bifunctional LOX activity, and which may not meet the limitations of anyone of the embodiments 1, 2 and 3; or alternatively selected from:
SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 (bacterial LOXs) and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least one of these sequences and retaining said bifunctional LOX activity, and which meet the limitations of anyone of the embodiments 1, 2 and 3.
A particular subgroup of said third group of polypeptides relates to SEQ ID NO: 20 and 26 and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least one of these sequences and retaining said bifunctional LOX activity.
5. A polypeptide which comprises the enzymatic activity of a lipoxygenase with an amino acid sequence that is selected from SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230232, 234, 236, 238 or 239; and amino acid sequences having at least 40% sequence identity to at least one of said sequences and retaining said enzymatic activity of a lipoxygenase.
A fourth particular group of polypeptides comprising an amino acid sequence selected from SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230232, 234, 236, 238 and 239 and amino acid sequences having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to at least one of said sequences and retaining said bifunctional LOX activity.
6. A polypeptide as defined in anyone of the preceding embodiment having, preferably bifunctional, LOX activity and mutants thereof.
Particular examples of suitable mutants of UfLOX 2 (SEQ ID NO:18) are:
Based on the sequence alignments provided herein (see
Particular examples of suitable mutants of bacterial LOX are:
Single and multiple mutants of anyone of the polypeptides of SEQ ID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50, which mutants retain said enzymatic activity of a lipoxygenase, i.p. bifunctional LOX, which mutants are in particular selected from mutants comprising an amino acid sequence selected from SEQ ID NO: 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290; or encoded by a nucleotide sequences encoding a polypeptide retaining said enzymatic activity of a lipoxygenase, in particular selected from SEQ ID NO: 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287 and 289.
Such bifunctional LOX mutants may show, if compared to the non-mutated parent enzyme, a different profile of features, like for example improved unsaturated C10-aldehyde productivity, different unsaturated C10-aldehyde product profile, different PUFA substrate profile, production of less side products, or combinations thereof;
Provided are also mutants derived from SEQ ID NO: 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288 and 290, and having a degree of sequence identity of least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to the respective native bacterial LOX amino acid sequence, while retaining said mutation profile in said key positions and preferably still showing said modified functional profile. In particular, such single or multiple mutants in key positions may be obtained by performing so-called conservative mutations.
A person of ordinary skill will be able to generate, based on the disclosed particular mutants, such further function mutants. For example, conservative amino acid substitutions in one or more of the mutation positions listed in the subsequent Table may be performed in this respect.
Non-limiting examples of possible conservative amino acid residue substitutions are provided in the subsequent section of the description.
7. The polypeptide of anyone of the embodiments 1 to 6 having the enzymatic activity of a bifunctional LOX and in particular of a combination of LOX and HPL activity.
8. The polypeptide of anyone of the embodiments 1 to 7, comprising the ability of converting at least one polyunsaturated fatty acid (PUFA), in particular selected from omega-3 and omega-6 PUFA, to at least one mono- or polyunsaturated aliphatic aldehyde.
9. The polypeptide of embodiment 8, comprising the ability to convert at least one PUFA to at least one polyunsaturated aliphatic C10-aldeyde.
10. The polypeptide of embodiment 9, comprising the ability to convert at least one PUFA to at least one polyunsaturated aliphatic C10-aldeyde, selected from decadienals and decatrienals, each either in essentially pure stereoisomeric form or in the form of a mixture of at least two stereoisomers, preferably selected from 2E,4Z-decadienal, 2E,4E-decadienal, 2E,4Z,7Z-decatrienal, 2E,4E,7Z-decatrienal and mixtures thereof.
11. The polypeptide of any one of the embodiments 7 to 10, wherein said PUFA is selected from C16-C22—, in particular from C16-C20-PUFAs, more particularly selected from omega-3 C16-C20-PUFAs and omega-6 C16-C20-PUFAs.
12. The polypeptide of embodiment 11, wherein said PUFA is selected from
If performed in vivo, said method comprises prior to step a) introducing into a non-human host organism or cell and optionally stably integrated into the respective genome; one or more nucleic acid molecules encoding one or more polypeptides having the enzyme activities required for performing the respective biocatalytic conversion step or steps.
27. The method of any one of embodiments 25 and 26, wherein step a) is carried out by cultivating a non-human host organism or cell expressing at least one of said polypeptides having the enzymatic activity of a preferably bifunctional LOX in the presence of a PUFA substrate under conditions conducive to the peroxidation and subsequent cleavage of at least one PUFA.
28. The method of embodiment 25, wherein said at least one mono- or polyunsaturated aliphatic aldehyde is selected from decadienals and decatrienals.
29. The method of embodiment 28, wherein said decadienal is selected from 2E,4E-decadienal and 2E,4Z-decadienal and mixtures thereof; and wherein said decatrienal is selected from 2E,4E, 7Z-decatrienal and 2E,4Z,7Z-decatrienal and mixtures thereof.
30. The method of one of the embodiments 25 to 29, wherein said PUFA substrate is an isolated, essentially pure PUFA compound or a natural or synthetic composition comprising at least one PUFA convertible by said preferably bifunctional LOX.
31. The method of embodiment 30, wherein said natural PUFA composition is selected from
Optionally said method of anyone of the preceding embodiments further comprises the processing of the obtained aldehyde to a corresponding derivative using chemical or biocatalytic synthesis or a combination of both. For example, such derivative may be selected from a hydrocarbon, an alcohol, diol, triol, acetal, ketal, acid, ether, amide, ketone, lactone, epoxide, acetate, glycoside and/or an ester.
38. A combination of at least two unsaturated C10-aldehyde isomers, selected from 2E,4Z-decadienal, 2E,4E-decadienal, 2E,4Z,7Z-decatrienal and 2E,4E, 7Z-decatrienal, wherein a particular ratio between 2E,4E-decadienal and 2E,4Z-decadienal is from 3:1 to 1:9 and a particular ratio between 2E,4Z,7Z-decatrienal and 2E,4E, 7Z-decatrienal is from 3:1 to 1:9.
39. The use of a mono- or polyunsaturated aliphatic aldehyde or of a mixture of at least two of such aldehydes, and/or of corresponding conversion products and mixtures thereof as obtained by a method of anyone of the embodiments 25 to 37 or of an isomer combination of embodiment 38 as flavour ingredient for the manufacture of food or feed compositions.
40. A food or feed composition supplemented by at least one flavour ingredient as defined in embodiment 39.
41. The use of a polypeptide which comprises the enzymatic activity of a lipoxygenase as defined in anyone of the claims 1 to 12 or encoded by an nucleotide sequence as defined in anyone of the claims 13 and 14 for preparing an at least one mono- or polyunsaturated aliphatic aldehyde, in particular by a method as defined in anyone of the claims 25 to 37.
In this context the following definitions apply:
The generic terms “polypeptide” or “peptide”, which may be used interchangeably, refer to a natural or synthetic linear chain or sequence of consecutive, peptidically linked amino acid residues, comprising about 10 to up to more than 1.000 residues. Short chain polypeptides with up to 30 residues are also designated as “oligopeptides”.
The term “protein” refers to a macromolecular structure consisting of one or more polypeptides. The amino acid sequence of its polypeptide(s) represents the “primary structure” of the protein. The amino acid sequence also predetermines the “secondary structure” of the protein by the formation of special structural elements, such as alpha-helical and beta-sheet structures formed within a polypeptide chain. The arrangement of a plurality of such secondary structural elements defines the “tertiary structure” or spatial arrangement of the protein. If a protein comprises more than one polypeptide chains said chains are spatially arranged forming the “quaternary structure” of the protein. A correct spacial arrangement or “folding” of the protein is prerequisite of protein function. Denaturation or unfolding destroys protein function. If such destruction is reversible, protein function may be restored by refolding.
A typical protein function referred to herein is an “enzyme function”, i.e. the protein acts as biocatalyst on a substrate, for example a chemical compound, and catalyzes the conversion of said substrate to a product. An enzyme may show a high or low degree of substrate and/or product specificity.
A “polypeptide” referred to herein as having a particular “activity” thus implicitly refers to a correctly folded protein showing the indicated activity, as for example a specific enzyme activity.
Thus, unless otherwise indicated the term “polypeptide” also encompasses the terms “protein” and “enzyme”.
Similarly, the term “polypeptide fragment” encompasses the terms “protein fragment” and “enzyme fragment”.
The term “isolated polypeptide” refers to an amino acid sequence that is removed from its natural environment by any method or combination of methods known in the art and includes recombinant, biochemical and synthetic methods.
“Target peptide” refers to an amino acid sequence which targets a protein, or polypeptide to intracellular organelles, i.e., mitochondria, or plastids, or to the extracellular space (secretion signal peptide). A nucleic acid sequence encoding a target peptide may be fused to the nucleic acid sequence encoding the amino terminal end, e.g., N-terminal end, of the protein or polypeptide, or may be used to replace a native targeting polypeptide.
The present invention also relates to “functional equivalents” (also designated as “analogs” or “functional mutations”) of the polypeptides specifically described herein.
For example, “functional equivalents” refer to polypeptides which, in a test used for determining enzymatic LOX activity display at least a 1 to 10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% higher or lower activity, as that of the polypeptides specifically described herein.
“Functional equivalents”, according to the invention, also cover particular mutants, which, in at least one sequence position of an amino acid sequences stated herein, have an amino acid that is different from that concretely stated one, but nevertheless possess one of the aforementioned biological activities, as for example enzyme activity. “Functional equivalents” thus comprise mutants obtainable by one or more, like 1 to 20, in particular 1 to 15 or 5 to 10 amino acid additions, substitutions, in particular conservative substitutions, deletions and/or inversions, where the stated changes can occur in any sequence position, provided they lead to a mutant with the profile of properties according to the invention. Functional equivalence is in particular also provided if the activity patterns coincide qualitatively between the mutant and the unchanged polypeptide, i.e. if, for example, interaction with the same agonist or antagonist or substrate, however at a different rate, (i.e. expressed by a EC50 or IC50 value or any other parameter suitable in the present technical field) is observed. Examples of suitable (conservative) amino acid substitutions are shown in the following table:
“Functional equivalents” in the above sense are also “precursors” of the polypeptides described herein, as well as “functional derivatives” and “salts” of the polypeptides.
“Precursors” are in that case natural or synthetic precursors of the polypeptides with or without the desired biological activity.
The expression “salts” means salts of carboxyl groups as well as salts of acid addition of amino groups of the protein molecules according to the invention. Salts of carboxyl groups can be produced in a known way and comprise inorganic salts, for example sodium, calcium, ammonium, iron and zinc salts, and salts with organic bases, for example amines, such as triethanolamine, arginine, lysine, piperidine and the like. Salts of acid addition, for example salts with inorganic acids, such as hydrochloric acid or sulfuric acid and salts with organic acids, such as acetic acid and oxalic acid, are also covered by the invention.
“Functional derivatives” of polypeptides according to the invention can also be produced on functional amino acid side groups or at their N-terminal or C-terminal end using known techniques. Such derivatives comprise for example aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups, produced by reaction with acyl groups; or O-acyl derivatives of free hydroxyl groups, produced by reaction with acyl groups.
“Functional equivalents” naturally also comprise polypeptides that can be obtained from other organisms, as well as naturally occurring variants. For example, areas of homologous sequence regions can be established by sequence comparison, and equivalent polypeptides can be determined on the basis of the concrete parameters of the invention.
“Functional equivalents” also comprise “fragments”, like individual domains or sequence motifs, of the polypeptides according to the invention, or N- and or C-terminally truncated forms, which may or may not display the desired biological function. Preferably such “fragments” retain the desired biological function at least qualitatively.
“Functional equivalents” are, moreover, fusion proteins, which have one of the polypeptide sequences stated herein or functional equivalents derived there from and at least one further, functionally different, heterologous sequence in functional N-terminal or C-terminal association (i.e. without substantial mutual functional impairment of the fusion protein parts). Non-limiting examples of these heterologous sequences are e.g. signal peptides, histidine anchors or enzymes.
“Functional equivalents” which are also comprised in accordance with the invention are homologs to the specifically disclosed polypeptides. These have at least 60%, preferably at least 75%, in particular at least 80 or 85%, such as, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, homology (or identity) to one of the specifically disclosed amino acid sequences, calculated by the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988, 2444-2448. A homology or identity, expressed as a percentage, of a homologous polypeptide according to the invention means in particular an identity, expressed as a percentage, of the amino acid residues based on the total length of one of the amino acid sequences described specifically herein.
The identity data, expressed as a percentage, may also be determined with the aid of BLAST alignments, algorithm blastp (protein-protein BLAST), or by applying the Clustal settings specified herein below.
In the case of a possible protein glycosylation, “functional equivalents” according to the invention comprise polypeptides as described herein in deglycosylated or glycosylated form as well as modified forms that can be obtained by altering the glycosylation pattern.
Functional equivalents or homologues of the polypeptides according to the invention can be produced by mutagenesis, e.g. by point mutation, lengthening or shortening of the protein or as described in more detail below.
Functional equivalents or homologs of the polypeptides according to the invention can be identified by screening combinatorial databases of mutants, for example shortening mutants. For example, a variegated database of protein variants can be produced by combinatorial mutagenesis at the nucleic acid level, e.g. by enzymatic ligation of a mixture of synthetic oligonucleotides. There are a great many methods that can be used for the production of databases of potential homologues from a degenerated oligonucleotide sequence. Chemical synthesis of a degenerated gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic gene can then be ligated in a suitable expression vector. The use of a degenerated genome makes it possible to supply all sequences in a mixture, which code for the desired set of potential protein sequences. Methods of synthesis of degenerated oligonucleotides are known to a person skilled in the art.
In the prior art, several techniques are known for the screening of gene products of combinatorial databases, which were produced by point mutations or shortening, and for the screening of cDNA libraries for gene products with a selected property. These techniques can be adapted for the rapid screening of the gene banks that were produced by combinatorial mutagenesis of homologues according to the invention. The techniques most frequently used for the screening of large gene banks, which are based on a high-throughput analysis, comprise cloning of the gene bank in expression vectors that can be replicated, transformation of the suitable cells with the resultant vector database and expression of the combinatorial genes in conditions in which detection of the desired activity facilitates isolation of the vector that codes for the gene whose product was detected. Recursive Ensemble Mutagenesis (REM), a technique that increases the frequency of functional mutants in the databases, can be used in combination with the screening tests, in order to identify homologues.
An embodiment provided herein provides orthologs and paralogs of polypeptides disclosed herein as well as methods for identifying and isolating such orthologs and paralogs. A definition of the terms “ortholog” and “paralog” is given below and applies to amino acid and nucleic acid sequences.
In this context the following definitions apply:
The terms “nucleic acid sequence,” “nucleic acid,” “nucleic acid molecule” and “polynucleotide” are used interchangeably meaning a sequence of nucleotides. A nucleic acid sequence may be a single-stranded or double-stranded deoxyribonucleotide, or ribonucleotide of any length, and include coding and non-coding sequences of a gene, exons, introns, sense and anti-sense complimentary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and/or RNA sequences, synthetic DNA and RNA sequences, fragments, primers and nucleic acid probes. The skilled artisan is aware that the nucleic acid sequences of RNA are identical to the DNA sequences with the difference of thymine (T) being replaced by uracil (U). The term “nucleotide sequence” should also be understood as comprising a polynucleotide molecule or an oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid.
An “isolated nucleic acid” or “isolated nucleic acid sequence” relates to a nucleic acid or nucleic acid sequence that is in an environment different from that in which the nucleic acid or nucleic acid sequence naturally occurs and can include those that are substantially free from contaminating endogenous material.
The term “naturally-occurring” as used herein as applied to a nucleic acid refers to a nucleic acid that is found in a cell of an organism in nature and which has not been intentionally modified by a human in the laboratory.
A “fragment” of a polynucleotide or nucleic acid sequence refers to contiguous nucleotides that are particularly at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp and/or at least 60 bp in length of the polynucleotide of an embodiment herein. Particularly the fragment of a polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 contiguous nucleotides of the polynucleotide of an embodiment herein. Without being limited, the fragment of the polynucleotides herein may be used as a PCR primer, and/or as a probe, or for anti-sense gene silencing or RNAi.
As used herein, the term “hybridization” or hybridizes under certain conditions is intended to describe conditions for hybridization and washes under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions may be such that sequences, which are at least about 70%, such as at least about 80%, and such as at least about 85%, 90%, or 95% identical, remain bound to each other. Definitions of low stringency, moderate, and high stringency hybridization conditions are provided herein below. Appropriate hybridization conditions can also be selected by those skilled in the art with minimal experimentation as exemplified in Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Additionally, stringency conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).
“Recombinant nucleic acid sequences” are nucleic acid sequences that result from the use of laboratory methods (for example, molecular cloning) to bring together genetic material from more than on source, creating or modifying a nucleic acid sequence that does not occur naturally and would not be otherwise found in biological organisms.
“Recombinant DNA technology” refers to molecular biology procedures to prepare a recombinant nucleic acid sequence as described, for instance, in Laboratory Manuals edited by Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook et al., 1989, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press.
The term “gene” means a DNA sequence comprising a region, which is transcribed into a RNA molecule, e.g., an 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 coding region of cDNA or genomic DNA, introns, exons, and/or a 3′non-translated sequence comprising, e.g., transcription termination sites.
“Polycistronic” refers to nucleic acid molecules, in particular mRNAs, that can encode more than one polypeptide separately within the same nucleic acid molecule
A “chimeric gene” refers to any gene which is not normally found in nature in a species, in particular, a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature. For example the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region. The term “chimeric gene” is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more coding sequences or to an antisense, i.e., reverse complement of the sense strand, or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription). The term “chimeric gene” also includes genes obtained through the combination of portions of one or more coding sequences to produce a new gene.
A “3′ UTR” or “3′ non-translated sequence” (also referred to as “3′ untranslated region,” or “3′end”) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal such as AAUAAA or variants thereof. After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the site of translation, e.g., cytoplasm.
The term “primer” refers to a short nucleic acid sequence that is hybridized to a template nucleic acid sequence and is used for polymerization of a nucleic acid sequence complementary to the template.
The term “selectable marker” refers to any gene which upon expression may be used to select a cell or cells that include the selectable marker. Examples of selectable markers are described below. The skilled artisan will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species.
The invention also relates to nucleic acid sequences that code for polypeptides as defined herein.
In particular, the invention also relates to nucleic acid sequences (single-stranded and double-stranded DNA and RNA sequences, e.g. cDNA, genomic DNA and mRNA), coding for one of the above polypeptides and their functional equivalents, which can be obtained for example using artificial nucleotide analogs.
The invention relates both to isolated nucleic acid molecules, which code for polypeptides according to the invention or biologically active segments thereof, and to nucleic acid fragments, which can be used for example as hybridization probes or primers for identifying or amplifying coding nucleic acids according to the invention.
The present invention also relates to nucleic acids with a certain degree of “identity” to the sequences specifically disclosed herein. “Identity” between two nucleic acids means identity of the nucleotides, in each case over the entire length of the nucleic acid.
The “identity” between two nucleotide sequences (the same applies to peptide or amino acid sequences) is a function of the number of nucleotide residues (or amino acid residues) or that are identical in the two sequences when an alignment of these two sequences has been generated. Identical residues are defined as residues that are the same in the two sequences in a given position of the alignment. The percentage of sequence identity, as used herein, is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100. The optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment. These gaps are then taken into account as non-identical residues for the calculation of the percentage of sequence identity. Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs and for instance publicly available computer programs available on the world wide web.
Particularly, the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) website at ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi, can be used to obtain an optimal alignment of protein or nucleic acid sequences and to calculate the percentage of sequence identity.
In another example the identity may be calculated by means of the Vector NTI Suite 7.1 program of the company Informax (USA) employing the Clustal Method (Higgins D G, Sharp P M. ((1989))) with the following settings:
Multiple Alignment Parameters:
Pairwise Alignment Parameter:
Alternatively the identity may be determined according to Chenna, et al. (2003), the web page: http://www.ebi.ac.uk/Tools/clustalw/index.html# and the following settings
All the nucleic acid sequences mentioned herein (single-stranded and double-stranded DNA and RNA sequences, for example cDNA and mRNA) can be produced in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix. Chemical synthesis of oligonucleotides can, for example, be performed in a known way, by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and filling of gaps by means of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989), see below.
The nucleic acid molecules according to the invention can in addition contain non-translated sequences from the 3′ and/or 5′ end of the coding genetic region.
The invention further relates to the nucleic acid molecules that are complementary to the concretely described nucleotide sequences or a segment thereof.
The nucleotide sequences according to the invention make possible the production of probes and primers that can be used for the identification and/or cloning of homologous sequences in other cellular types and organisms. Such probes or primers generally comprise a nucleotide sequence region which hybridizes under “stringent” conditions (as defined herein elsewhere) on at least about 12, preferably at least about 25, for example about 40, 50 or 75 successive nucleotides of a sense strand of a nucleic acid sequence according to the invention or of a corresponding antisense strand.
“Homologous” sequences include orthologous or paralogous sequences. Methods of identifying orthologs or paralogs including phylogenetic methods, sequence similarity and hybridization methods are known in the art and are described herein.
“Paralogs” result from gene duplication that gives rise to two or more genes with similar sequences and similar functions. Paralogs typically cluster together and are formed by duplications of genes within related plant species. Paralogs are found in groups of similar genes using pair-wise Blast analysis or during phylogenetic analysis of gene families using programs such as CLUSTAL. In paralogs, consensus sequences can be identified characteristic to sequences within related genes and having similar functions of the genes.
“Orthologs”, or orthologous sequences, are sequences similar to each other because they are found in species that descended from a common ancestor. For instance, plant species that have common ancestors are known to contain many enzymes that have similar sequences and functions. The skilled artisan can identify orthologous sequences and predict the functions of the orthologs, for example, by constructing a polygenic tree for a gene family of one species using CLUSTAL or BLAST programs. A method for identifying or confirming similar functions among homologous sequences is by comparing of the transcript profiles in host cells or organisms, such as plants or microorganisms, overexpressing or lacking (in knockouts/knockdowns) related polypeptides. The skilled person will understand that genes having similar transcript profiles, with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or greater than 90% regulated transcripts in common will have similar functions. Homologs, paralogs, orthologs and any other variants of the sequences herein are expected to function in a similar manner by making the host cells, organism such as plants or microorganisms producing LOX proteins.
The term “selectable marker” refers to any gene which upon expression may be used to select a cell or cells that include the selectable marker. Examples of selectable markers are described below. The skilled artisan will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species.
An “isolated” nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid and can moreover be substantially free from other cellular material or culture medium, if it is being produced by recombinant techniques, or can be free from chemical precursors or other chemicals, if it is being synthesized chemically.
A nucleic acid molecule according to the invention can be isolated by means of standard techniques of molecular biology and the sequence information supplied according to the invention. For example, cDNA can be isolated from a suitable cDNA library, using one of the concretely disclosed complete sequences or a segment thereof as hybridization probe and standard hybridization techniques (as described for example in Sambrook, (1989)).
In addition, a nucleic acid molecule comprising one of the disclosed sequences or a segment thereof, can be isolated by the polymerase chain reaction, using the oligonucleotide primers that were constructed on the basis of this sequence. The nucleic acid amplified in this way can be cloned in a suitable vector and can be characterized by DNA sequencing. The oligonucleotides according to the invention can also be produced by standard methods of synthesis, e.g. using an automatic DNA synthesizer.
Nucleic acid sequences according to the invention or derivatives thereof, homologues or parts of these sequences, can for example be isolated by usual hybridization techniques or the PCR technique from other bacteria, e.g. via genomic or cDNA libraries. These DNA sequences hybridize in standard conditions with the sequences according to the invention.
“Hybridize” means the ability of a polynucleotide or oligonucleotide to bind to an almost complementary sequence in standard conditions, whereas nonspecific binding does not occur between non-complementary partners in these conditions. For this, the sequences can be 90-100% complementary. The property of complementary sequences of being able to bind specifically to one another is utilized for example in Northern Blotting or Southern Blotting or in primer binding in PCR or RT-PCR.
Short oligonucleotides of the conserved regions are used advantageously for hybridization. However, it is also possible to use longer fragments of the nucleic acids according to the invention or the complete sequences for the hybridization. These “standard conditions” vary depending on the nucleic acid used (oligonucleotide, longer fragment or complete sequence) or depending on which type of nucleic acid—DNA or RNA—is used for hybridization. For example, the melting temperatures for DNA:DNA hybrids are approx. 10° C. lower than those of DNA:RNA hybrids of the same length.
For example, depending on the particular nucleic acid, standard conditions mean temperatures between 42 and 58° C. in an aqueous buffer solution with a concentration between 0.1 to 5×SSC (1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide, for example 42° C. in 5×SSC, 50% formamide. Advantageously, the hybridization conditions for DNA:DNA hybrids are 0.1×SSC and temperatures between about 20° C. to 45° C., preferably between about 30° C. to 45° C. For DNA:RNA hybrids the hybridization conditions are advantageously 0.1×SSC and temperatures between about 30° C. to 55° C., preferably between about 45° C. to 55° C. These stated temperatures for hybridization are examples of calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G+C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant genetics textbooks, for example Sambrook et al., 1989, and can be calculated using formulae that are known by a person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrids or the G+C content. A person skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds), (1985), Brown (ed) (1991).
“Hybridization” can in particular be carried out under stringent conditions. Such hybridization conditions are for example described in Sambrook (1989), or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
As used herein, the term hybridization or hybridizes under certain conditions is intended to describe conditions for hybridization and washes under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions may be such that sequences, which are at least about 70%, such as at least about 80%, and such as at least about 85%, 90%, or 95% identical, remain bound to each other. Definitions of low stringency, moderate, and high stringency hybridization conditions are provided herein.
Appropriate hybridization conditions can be selected by those skilled in the art with minimal experimentation as exemplified in Ausubel et al. (1995, Current Protocols in Molecular Biology, John Wiley & Sons, sections 2, 4, and 6). Additionally, stringency conditions are described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, chapters 7, 9, and 11).
As used herein, defined conditions of low stringency are as follows. Filters containing DNA are pretreated for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 32P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C. In a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography.
As used herein, defined conditions of moderate stringency are as follows. Filters containing DNA are pretreated for 7 h at 50° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 32P-labeled probe is used. Filters are incubated in hybridization mixture for 30 h at 50° C., and then washed for 1.5 h at 55° C. In a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography.
As used herein, defined conditions of high stringency are as follows. Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. in the prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Washing of filters is done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes.
Other conditions of low, moderate, and high stringency well known in the art (e.g., as employed for cross-species hybridizations) may be used if the above conditions are inappropriate (e.g., as employed for cross-species hybridizations).
A detection kit for nucleic acid sequences encoding a polypeptide of the invention may include primers and/or probes specific for nucleic acid sequences encoding the polypeptide, and an associated protocol to use the primers and/or probes to detect nucleic acid sequences encoding the polypeptide in a sample. Such detection kits may be used to determine whether a plant, organism, microorganism or cell has been modified, i.e., transformed with a sequence encoding the polypeptide.
To test a function of variant DNA sequences according to an embodiment herein, the sequence of interest is operably linked to a selectable or screenable marker gene and expression of said reporter gene is tested in transient expression assays, for example, with microorganisms or with protoplasts or in stably transformed plants.
The invention also relates to derivatives of the concretely disclosed or derivable nucleic acid sequences.
Thus, further nucleic acid sequences according to the invention can be derived from the sequences specifically disclosed herein and can differ from it by one or more, like 1 to 20, in particular 1 to 15 or 5 to 10 additions, substitutions, insertions or deletions of one or several (like for example 1 to 10) nucleotides, and furthermore code for polypeptides with the desired profile of properties.
The invention also encompasses nucleic acid sequences that comprise so-called silent mutations or have been altered, in comparison with a concretely stated sequence, according to the codon usage of a special original or host organism.
According to a particular embodiment of the invention variant nucleic acids may be prepared in order to adapt its nucleotide sequence to a specific expression system. For example, bacterial expression systems are known to more efficiently express polypeptides if amino acids are encoded by particular codons. Due to the degeneracy of the genetic code, more than one codon may encode the same amino acid sequence, multiple nucleic acid sequences can code for the same protein or polypeptide, all these DNA sequences being encompassed by an embodiment herein. Where appropriate, the nucleic acid sequences encoding the polypeptides described herein may be optimized for increased expression in the host cell. For example, nucleic acids of an embodiment herein may be synthesized using codons particular to a host for improved expression.
The invention also encompasses naturally occurring variants, e.g. splicing variants or allelic variants, of the sequences described therein.
Allelic variants may have at least 60% homology at the level of the derived amino acid, preferably at least 80% homology, quite especially preferably at least 90% homology over the entire sequence range (regarding homology at the amino acid level, reference should be made to the details given above for the polypeptides). Advantageously, the homologies can be higher over partial regions of the sequences.
The invention also relates to sequences that can be obtained by conservative nucleotide substitutions (i.e. as a result thereof the amino acid in question is replaced by an amino acid of the same charge, size, polarity and/or solubility).
The invention also relates to the molecules derived from the concretely disclosed nucleic acids by sequence polymorphisms. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents. These natural variations usually produce a variance of 1 to 5% in the nucleotide sequence of a gene. Said polymorphisms may lead to changes in the amino acid sequence of the polypeptides disclosed herein. Allelic variants may also include functional equivalents.
Furthermore, derivatives are also to be understood to be homologs of the nucleic acid sequences according to the invention, for example animal, plant, fungal or bacterial homologs, shortened sequences, single-stranded DNA or RNA of the coding and noncoding DNA sequence. For example, homologs have, at the DNA level, a homology of at least 40%, preferably of at least 60%, especially preferably of at least 70%, quite especially preferably of at least 80% over the entire DNA region given in a sequence specifically disclosed herein.
Moreover, derivatives are to be understood to be, for example, fusions with promoters. The promoters that are added to the stated nucleotide sequences can be modified by at least one nucleotide exchange, at least one insertion, inversion and/or deletion, though without impairing the functionality or efficacy of the promoters. Moreover, the efficacy of the promoters can be increased by altering their sequence or can be exchanged completely with more effective promoters even of organisms of a different genus.
Moreover, a person skilled in the art is familiar with methods for generating functional mutants, that is to say nucleotide sequences which code for a polypeptide with at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyone of amino acid related SEQ ID NOs as disclosed herein and/or encoded by a nucleic acid molecule comprising a nucleotide sequence having at least 70% sequence identity to anyone of the nucleotide related SEQ ID NOs as disclosed herein.
Depending on the technique used, a person skilled in the art can introduce entirely random or else more directed mutations into genes or else noncoding nucleic acid regions (which are for example important for regulating expression) and subsequently generate genetic libraries. The methods of molecular biology required for this purpose are known to the skilled worker and for example described in Sambrook and Russell, Molecular Cloning. 3rd Edition, Cold Spring Harbor Laboratory Press 2001.
Methods for modifying genes and thus for modifying the polypeptide encoded by them have been known to the skilled worker for a long time, such as, for example
Using so-called directed evolution (described, inter alia, in Reetz M T and Jaeger K-E (1999), Topics Curr Chem 200:31; Zhao H, Moore J C, Volkov A A, Arnold F H (1999), Methods for optimizing industrial polypeptides by directed evolution, In: Demain A L, Davies J E (Ed.) Manual of industrial microbiology and biotechnology. American Society for Microbiology), a skilled worker can produce functional mutants in a directed manner and on a large scale. To this end, in a first step, gene libraries of the respective polypeptides are first produced, for example using the methods given above. The gene libraries are expressed in a suitable way, for example by bacteria or by phage display systems.
The relevant genes of host organisms which express functional mutants with properties that largely correspond to the desired properties can be submitted to another mutation cycle. The steps of the mutation and selection or screening can be repeated iteratively until the present functional mutants have the desired properties to a sufficient extent. Using this iterative procedure, a limited number of mutations, for example 1, 2, 3, 4 or 5 mutations, can be performed in stages and assessed and selected for their influence on the activity in question. The selected mutant can then be submitted to a further mutation step in the same way. In this way, the number of individual mutants to be investigated can be reduced significantly.
The results according to the invention also provide important information relating to structure and sequence of the relevant polypeptides, which is required for generating, in a targeted fashion, further polypeptides with desired modified properties. In particular, it is possible to define so-called “hot spots”, i.e. sequence segments that are potentially suitable for modifying a property by introducing targeted mutations.
Information can also be deduced regarding amino acid sequence positions, in the region of which mutations can be effected that should probably have little effect on the activity, and can be designated as potential “silent mutations”.
In this context the following definitions apply:
“Expression of a gene” encompasses “heterologous expression” and “overexpression” and involves transcription of the gene and translation of the mRNA into a protein. Overexpression refers to the production of the gene product as measured by levels of mRNA, polypeptide and/or enzyme activity in transgenic cells or organisms that exceeds levels of production in non-transformed cells or organisms of a similar genetic background.
“Expression vector” as used herein means a nucleic acid molecule engineered using molecular biology methods and recombinant DNA technology for delivery of foreign or exogenous DNA into a host cell. The expression vector typically includes sequences required for proper transcription of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for an RNA, e.g., an antisense RNA, siRNA and the like.
An “expression vector” as used herein includes any linear or circular recombinant vector including but not limited to viral vectors, bacteriophages and plasmids. The skilled person is capable of selecting a suitable vector according to the expression system. In one embodiment, the expression vector includes the nucleic acid of an embodiment herein operably linked to at least one “regulatory sequence”, which controls transcription, translation, initiation and termination, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosomal binding site and, optionally, including at least one selection marker. Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the nucleic acid of an embodiment herein.
An “expression system” as used herein encompasses any combination of nucleic acid molecules required for the expression of one, or the co-expression of two or more polypeptides either in vivo of a given expression host, or in vitro. The respective coding sequences may either be located on a single nucleic acid molecule or vector, as for example a vector containing multiple cloning sites, or on a polycistronic nucleic acid, or may be distributed over two or more physically distinct vectors. As a particular example there may be mentioned an operon comprising a promotor sequence, one or more operator sequences and one or more structural genes each encoding an enzyme as described herein.
As used herein, the terms “amplifying” and “amplification” refer to the use of any suitable amplification methodology for generating or detecting recombinant of naturally expressed nucleic acid, as described in detail, below. For example, the invention provides methods and reagents (e.g., specific degenerate oligonucleotide primer pairs, oligo dT primer) for amplifying (e.g., by polymerase chain reaction, PCR) naturally expressed (e.g., genomic DNA or mRNA) or recombinant (e.g., cDNA) nucleic acids of the invention in vivo, ex vivo or in vitro.
“Regulatory sequence” refers to a nucleic acid sequence that determines expression level of the nucleic acid sequences of an embodiment herein and is capable of regulating the rate of transcription of the nucleic acid sequence operably linked to the regulatory sequence. Regulatory sequences comprise promoters, enhancers, transcription factors, promoter elements and the like.
A “promoter”, a “nucleic acid with promoter activity” or a “promoter sequence” is understood as meaning, in accordance with the invention, a nucleic acid which, when functionally linked to a nucleic acid to be transcribed, regulates the transcription of said nucleic acid. “Promoter” in particular refers to a nucleic acid sequence that controls the expression of a coding sequence by providing a binding site for RNA polymerase and other factors required for proper transcription including without limitation transcription factor binding sites, repressor and activator protein binding sites. The meaning of the term promoter also includes the term “promoter regulatory sequence”. Promoter regulatory sequences may include upstream and downstream elements that may influences transcription, RNA processing or stability of the associated coding nucleic acid sequence. Promoters include naturally-derived and synthetic sequences. The coding nucleic acid sequences is usually located downstream of the promoter with respect to the direction of the transcription starting at the transcription initiation site.
In this context, a “functional” or “operative” linkage is understood as meaning for example the sequential arrangement of one of the nucleic acids with a regulatory sequence. For example the sequence with promoter activity and of a nucleic acid sequence to be transcribed and optionally further regulatory elements, for example nucleic acid sequences which ensure the transcription of nucleic acids, and for example a terminator, are linked in such a way that each of the regulatory elements can perform its function upon transcription of the nucleic acid sequence. This does not necessarily require a direct linkage in the chemical sense. Genetic control sequences, for example enhancer sequences, can even exert their function on the target sequence from more remote positions or even from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be transcribed is positioned behind (i.e. at the 3′-end of) the promoter sequence so that the two sequences are joined together covalently. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly can be smaller than 200 base pairs, or smaller than 100 base pairs or smaller than 50 base pairs.
In addition to promoters and terminator, the following may be mentioned as examples of other regulatory elements: targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, replication origins and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). The term “constitutive promoter” refers to an unregulated promoter that allows for continual transcription of the nucleic acid sequence it is operably linked to.
As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous. The nucleotide sequence associated with the promoter sequence may be of homologous or heterologous origin with respect to the plant to be transformed. The sequence also may be entirely or partially synthetic. Regardless of the origin, the nucleic acid sequence associated with the promoter sequence will be expressed or silenced in accordance with promoter properties to which it is linked after binding to the polypeptide of an embodiment herein. The associated nucleic acid may code for a protein that is desired to be expressed or suppressed throughout the organism at all times or, alternatively, at a specific time or in specific tissues, cells, or cell compartment. Such nucleotide sequences particularly encode proteins conferring desirable phenotypic traits to the host cells or organism altered or transformed therewith. More particularly, the associated nucleotide sequence leads to the production of the product or products of interest as herein defined in the cell or organism. Particularly, the nucleotide sequence encodes a polypeptide having an enzyme activity as herein defined.
The nucleotide sequence as described herein above may be part of an “expression cassette”. The terms “expression cassette” and “expression construct” are used synonymously. The (preferably recombinant) expression construct contains a nucleotide sequence which encodes a polypeptide according to the invention and which is under genetic control of regulatory nucleic acid sequences.
In a process applied according to the invention, the expression cassette may be part of an “expression vector”, in particular of a recombinant expression vector.
An “expression unit” is understood as meaning, in accordance with the invention, a nucleic acid with expression activity which comprises a promoter as defined herein and, after functional linkage with a nucleic acid to be expressed or a gene, regulates the expression, i.e. the transcription and the translation of said nucleic acid or said gene. It is therefore in this connection also referred to as a “regulatory nucleic acid sequence”. In addition to the promoter, other regulatory elements, for example enhancers, can also be present.
An “expression cassette” or “expression construct” is understood as meaning, in accordance with the invention, an expression unit which is functionally linked to the nucleic acid to be expressed or the gene to be expressed. In contrast to an expression unit, an expression cassette therefore comprises not only nucleic acid sequences which regulate transcription and translation, but also the nucleic acid sequences that are to be expressed as protein as a result of transcription and translation.
The terms “expression” or “overexpression” describe, in the context of the invention, the production or increase in intracellular activity of one or more polypeptides in a microorganism, which are encoded by the corresponding DNA. To this end, it is possible for example to introduce a gene into an organism, replace an existing gene with another gene, increase the copy number of the gene(s), use a strong promoter or use a gene which encodes for a corresponding polypeptide with a high activity; optionally, these measures can be combined.
Preferably such constructs according to the invention comprise a promoter 5′-upstream of the respective coding sequence and a terminator sequence 3′-downstream and optionally other usual regulatory elements, in each case in operative linkage with the coding sequence.
Nucleic acid constructs according to the invention comprise in particular a sequence coding for a polypeptide for example derived from the amino acid related SEQ ID NOs as described therein or the reverse complement thereof, or derivatives and homologs thereof and which have been linked operatively or functionally with one or more regulatory signals, advantageously for controlling, for example increasing, gene expression.
In addition to these regulatory sequences, the natural regulation of these sequences may still be present before the actual structural genes and optionally may have been genetically modified so that the natural regulation has been switched off and expression of the genes has been enhanced. The nucleic acid construct may, however, also be of simpler construction, i.e. no additional regulatory signals have been inserted before the coding sequence and the natural promoter, with its regulation, has not been removed. Instead, the natural regulatory sequence is mutated such that regulation no longer takes place and the gene expression is increased.
A preferred nucleic acid construct advantageously also comprises one or more of the already mentioned “enhancer” sequences in functional linkage with the promoter, which sequences make possible an enhanced expression of the nucleic acid sequence. Additional advantageous sequences may also be inserted at the 3′-end of the DNA sequences, such as further regulatory elements or terminators. One or more copies of the nucleic acids according to the invention may be present in a construct. In the construct, other markers, such as genes which complement auxotrophisms or antibiotic resistances, may also optionally be present so as to select for the construct.
Examples of suitable regulatory sequences are present in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, rhaP (rhaPBAD)SP6, lambda-PR or in the lambda-PL promoter, and these are advantageously employed in Gram-negative bacteria. Further advantageous regulatory sequences are present for example in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. Artificial promoters may also be used for regulation.
For expression in a host organism, the nucleic acid construct is inserted advantageously into a vector such as, for example, a plasmid or a phage, which makes possible optimal expression of the genes in the host. Vectors are also understood as meaning, in addition to plasmids and phages, all the other vectors which are known to the skilled worker, that is to say for example viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids and linear or circular DNA or artificial chromosomes. These vectors are capable of replicating autonomously in the host organism or else chromosomally. These vectors are a further development of the invention. Binary or cpo-integration vectors are also applicable.
Suitable plasmids are, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11 or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL2 or pBB116, in yeasts 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 or in plants pLGV23, pGHlac+, pBIN19, pAK2004 or pDH51. The abovementioned plasmids are a small selection of the plasmids which are possible. Further plasmids are well known to the skilled worker and can be found for example in the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0444904018).
In a further development of the vector, the vector which comprises the nucleic acid construct according to the invention or the nucleic acid according to the invention can advantageously also be introduced into the microorganisms in the form of a linear DNA and integrated into the host organism's genome via heterologous or homologous recombination. This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or the nucleic acid according to the invention.
For optimal expression of heterologous genes in organisms, it is advantageous to modify the nucleic acid sequences to match the specific “codon usage” used in the organism. The “codon usage” can be determined readily by computer evaluations of other, known genes of the organism in question.
An expression cassette according to the invention is generated by fusing a suitable promoter to a suitable coding nucleotide sequence and a terminator or polyadenylation signal. Customary recombination and cloning techniques are used for this purpose, as are described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).
For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which makes possible optimal expression of the genes in the host. Vectors are well known to the skilled worker and can be found for example in “cloning vectors” (Pouwels P. H. et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).
An alternative embodiment of an embodiment herein provides a method to “alter gene expression” in a host cell. For instance, the polynucleotide of an embodiment herein may be enhanced or overexpressed or induced in certain contexts (e.g. upon exposure to certain temperatures or culture conditions) in a host cell or host organism.
Alteration of expression of a polynucleotide provided herein may also result in ectopic expression which is a different expression pattern in an altered and in a control or wild-type organism. Alteration of expression occurs from interactions of polypeptide of an embodiment herein with exogenous or endogenous modulators, or as a result of chemical modification of the polypeptide. The term also refers to an altered expression pattern of the polynucleotide of an embodiment herein which is altered below the detection level or completely suppressed activity. In one embodiment, provided herein is also an isolated, recombinant or synthetic polynucleotide encoding a polypeptide or variant polypeptide provided herein.
In one embodiment, several polypeptide encoding nucleic acid sequences are co-expressed in a single host, particularly under control of different promoters. In another embodiment, several polypeptide encoding nucleic acid sequences can be present on a single transformation vector or be co-transformed at the same time using separate vectors and selecting transformants comprising both chimeric genes. Similarly, one or polypeptide encoding genes may be expressed in a single plant, cell, microorganism or organism together with other chimeric genes.
Depending on the context, the term “host” can mean the wild-type host or a genetically altered, recombinant host or both.
In principle, all prokaryotic or eukaryotic organisms may be considered as host or recombinant host organisms for the nucleic acids or the nucleic acid constructs according to the invention.
Using the vectors according to the invention, recombinant hosts can be produced, which are for example transformed with at least one vector according to the invention and can be used for producing the polypeptides according to the invention. Advantageously, the recombinant constructs according to the invention, described above, are introduced into a suitable host system and expressed. Preferably common cloning and transfection methods, known by a person skilled in the art, are used, for example co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, for expressing the stated nucleic acids in the respective expression system. Suitable systems are described for example in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
Advantageously, microorganisms such as bacteria, fungi or yeasts are used as host organisms. Advantageously, gram-positive or gram-negative bacteria are used, preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, Streptococcaceae or Nocardiaceae, especially preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Lactococcus, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus. The genus and species Escherichia coli is quite especially preferred. Furthermore, other advantageous bacteria are to be found in the group of alpha-Proteobacteria, beta-Proteobacteria or gamma-Proteobacteria. Advantageously also yeasts of families like Saccharomyces or Pichia are suitable hosts.
Alternatively, entire plants or plant cells may serve as natural or recombinant host. As non-limiting examples the following plants or cells derived therefrom may be mentioned the genera Nicotiana, in particular Nicotiana benthamiana and Nicotiana tabacum (tobacco); as well as Arabidopsis, in particular Arabidopsis thaliana.
Depending on the host organism, the organisms used in the method according to the invention are grown or cultured in a manner known by a person skilled in the art. Culture can be batchwise, semi-batchwise or continuous. Nutrients can be present at the beginning of fermentation or can be supplied later, semicontinuously or continuously. This is also described in more detail below.
The invention further relates to methods for recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultured, optionally the expression of the polypeptides is induced by applying at least one inducer inducing gene expression and the expressed polypeptides are isolated from the culture. The polypeptides can also be produced in this way on an industrial scale, if desired.
The microorganisms produced according to the invention can be cultured continuously or discontinuously in the batch method or in the fed-batch method or repeated fed-batch method. A summary of known cultivation methods can be found in the textbook by Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must suitably meet the requirements of the respective strains. Descriptions of culture media for various microorganisms are given in the manual “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D. C., USA, 1981).
These media usable according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.
Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Very good carbon sources are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It can also be advantageous to add mixtures of different carbon sources. Other possible carbon sources are oils and fats, for example soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids, for example palmitic acid, stearic acid or linoleic acid, alcohols, for example glycerol, methanol or ethanol and organic acids, for example acetic acid or lactic acid.
Nitrogen sources are usually organic or inorganic nitrogen compounds or materials that contain these compounds. Examples of nitrogen sources comprise ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources, such as corn-steep liquor, soya flour, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used alone or as a mixture.
Inorganic salt compounds that can be present in the media comprise the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
Inorganic sulfur-containing compounds, for example sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, as well as organic sulfur compounds, such as mercaptans and thiols, can be used as the sulfur source.
Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
Chelating agents can be added to the medium, in order to keep the metal ions in solution. Especially suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
The fermentation media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often originate from the components of complex media, such as yeast extract, molasses, corn-steep liquor and the like. Moreover, suitable precursors can be added to the culture medium. The exact composition of the compounds in the medium is strongly dependent on the respective experiment and is decided for each specific case individually. Information on media optimization can be found in the textbook “Applied Microbiol. Physiology, A Practical Approach” (Ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) p. 53-73, ISBN 0199635773). Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.
All components of the medium are sterilized, either by heat (20 min at 1.5 bar and 121° C.) or by sterile filtration. The components can either be sterilized together, or separately if necessary. All components of the medium can be present at the start of culture or can be added either continuously or batchwise.
The culture temperature is normally between 15° C. and 45° C., preferably 25° C. to 40° C. and can be varied or kept constant during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, for example fatty acid polyglycol esters, can be used for controlling foaming. To maintain the stability of plasmids, suitable selective substances, for example antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, for example ambient air, are fed into the culture. The temperature of the culture is normally in the range from 20° C. to 45° C. The culture is continued until a maximum of the desired product has formed. This target is normally reached within 10 hours to 160 hours.
The fermentation broth is then processed further. Depending on requirements, the biomass can be removed from the fermentation broth completely or partially by separation techniques, for example centrifugation, filtration, decanting or a combination of these methods or can be left in it completely.
If the polypeptides are not secreted in the culture medium, the cells can also be lysed and the product can be obtained from the lysate by known methods for isolation of proteins. The cells can optionally be disrupted with high-frequency ultrasound, high pressure, for example in a French press, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by means of homogenizers or by a combination of several of the aforementioned methods.
The polypeptides can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), such as Q-sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and with other usual techniques such as ultrafiltration, crystallization, salting-out, dialysis and native gel electrophoresis. Suitable methods are described for example in Cooper, T. G., Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, N.Y. or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
For isolating the recombinant protein, it can be advantageous to use vector systems or oligonucleotides, which lengthen the cDNA by defined nucleotide sequences and therefore code for altered polypeptides or fusion proteins, which for example serve for easier purification. Suitable modifications of this type are for example so-called “tags” functioning as anchors, for example the modification known as hexa-histidine anchor or epitopes that can be recognized as antigens of antibodies (described for example in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). These anchors can serve for attaching the proteins to a solid carrier, for example a polymer matrix, which can for example be used as packing in a chromatography column, or can be used on a microtiter plate or on some other carrier.
At the same time these anchors can also be used for recognition of the proteins. For recognition of the proteins, it is moreover also possible to use usual markers, such as fluorescent dyes, enzyme markers, which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors for derivatization of the proteins.
The enzymes or polypeptides according to the invention can be used free or immobilized in the method described herein. An immobilized enzyme is an enzyme that is fixed to an inert carrier. Suitable carrier materials and the enzymes immobilized thereon are known from EP-A-1149849, EP-A-1069183 and DE-OS 100193773 and from the references cited therein. Reference is made in this respect to the disclosure of these documents in their entirety. Suitable carrier materials include for example clays, clay minerals, such as kaolinite, diatomaceous earth, perlite, silica, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger materials, synthetic polymers, such as polystyrene, acrylic resins, phenol formaldehyde resins, polyurethanes and polyolefins, such as polyethylene and polypropylene. For making the supported enzymes, the carrier materials are usually employed in a finely-divided, particulate form, porous forms being preferred. The particle size of the carrier material is usually not more than 5 mm, in particular not more than 2 mm (particle-size distribution curve). Similarly, when using dehydrogenase as whole-cell catalyst, a free or immobilized form can be selected. Carrier materials are e.g. Ca-alginate, and carrageenan. Enzymes as well as cells can also be crosslinked directly with glutaraldehyde (cross-linking to CLEAs). Corresponding and other immobilization techniques are described for example in J. Lalonde and A. Margolin “Immobilization of Enzymes” in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Further information on biotransformations and bioreactors for carrying out methods according to the invention are also given for example in Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol 3, Chapter 17, VCH, Weinheim.
The reaction of the present invention may be performed under in vivo or in vitro conditions.
The at least one polypeptide/enzyme which is present during a method of the invention or an individual step of a multistep-method as defined herein above, can be present in living cells naturally or recombinantly producing the enzyme or enzymes, in harvested cells. i.e. under in vivo conditions, or, in dead cells, in permeabilized cells, in crude cell extracts, in purified extracts, or in essentially pure or completely pure form, i.e. under in vitro conditions. The at least one enzyme may be present in solution or as an enzyme immobilized on a carrier. One or several enzymes may simultaneously be present in soluble and/or immobilised form.
The methods according to the invention can be performed in common reactors, which are known to those skilled in the art, and in different ranges of scale, e.g. from a laboratory scale (few millilitres to dozens of litres of reaction volume) to an industrial scale (several litres to thousands of cubic meters of reaction volume). If the polypeptide is used in a form encapsulated by non-living, optionally permeabilized cells, in the form of a more or less purified cell extract or in purified form, a chemical reactor can be used. The chemical reactor usually allows controlling the amount of the at least one enzyme, the amount of the at least one substrate, the pH, the temperature and the circulation of the reaction medium. When the at least one polypeptide/enzyme is present in living cells, the process will be a fermentation. In this case the biocatalytic production will take place in a bioreactor (fermenter), where parameters necessary for suitable living conditions for the living cells (e.g. culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, and the like) can be controlled. Those skilled in the art are familiar with chemical reactors or bioreactors, e.g. with procedures for up-scaling chemical or biotechnological methods from laboratory scale to industrial scale, or for optimizing process parameters, which are also extensively described in the literature (for biotechnological methods see e.g. Crueger and Crueger, Biotechnologie—Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, München, Wien, 1984).
Cells containing the at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or radiofrequency pulses, French presses, or chemical means, such as hypotonic media, lytic enzymes and detergents present in the medium, or combination of such methods. Examples for detergents are digitonin, n-dodecylmaltoside, octylglycoside, Triton® X-100, Tween® 20, deoxycholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propansulfonate), Nonidet® P40 (Ethylphenolpoly(ethyleneglycolether), and the like.
Instead of living cells biomass of non-living cells containing the required biocatalyst(s) may be applied of the biotransformation reactions of the invention as well.
If the at least one enzyme is immobilised, it is attached to an inert carrier as described above.
The conversion reaction can be carried out batch wise, semi-batch wise or continuously. Reactants (and optionally nutrients) can be supplied at the start of reaction or can be supplied subsequently, either semi-continuously or continuously.
The reaction of the invention, depending on the particular reaction type, may be performed in an aqueous, aqueous-organic or non-aqueous reaction medium.
An aqueous or aqueous-organic medium may contain a suitable buffer in order to adjust the pH to a value in the range of 5 to 11, like 6 to 10.
In an aqueous-organic medium an organic solvent miscible, partly miscible or immiscible with water may be applied. Non-limiting examples of suitable organic solvents are listed below. Further examples are mono- or polyhydric, aromatic or aliphatic alcohols, in particular polyhydric aliphatic alcohols like glycerol.
The non-aqueous medium may contain is substantially free of water, i.e. will contain less that about 1 wt.-% or 0.5 wt.-% of water.
Biocatalytic methods may also be performed in an organic non-aqueous medium. As suitable organic solvents there may be mentioned aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; aromatic carbohydrates, like benzene, toluene, xylenes, chlorobenzene or dichlorobenzene, aliphatic acyclic and ethers, like diethylether, methyl-tert-butylether, ethyl-tert.-butylether, dipropylether, diisopropylether, dibutylether; or mixtures thereof.
The concentration of the reactants/substrates may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied. For example, the initial substrate concentration may be in the 0.1 to 0.5 M, as for example 10 to 100 mM.
The reaction temperature may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied. For example, the reaction may be performed at a temperature in a range of from 0 to 70° C., as for example 20 to 50 or 25 to 40° C. Examples for reaction temperatures are about 30° C., about 35° C., about 37° C., about 40° C., about 45° C., about 50° C., about 55° C. and about 60° C.
The process may proceed until equilibrium between the substrate and then product(s) is achieved, but may be stopped earlier. Usual process times are in the range from 1 minute to 25 hours, in particular 10 min to 6 hours, as for example in the range from 1 hour to 4 hours, in particular 1.5 hours to 3.5 hours. These parameters are non-limiting examples of suitable process conditions.
If the host is a transgenic plant, optimal growth conditions can be provided, such as optimal light, water and nutrient conditions, for example.
The methodology of the present invention can further include a step of recovering an end or intermediate product, optionally in stereoisomerically or enantiomerically substantially pure form. The term “recovering” includes extracting, harvesting, isolating or purifying the compound from culture or reaction media. Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like. Identity and purity of the isolated product may be determined by known techniques, like High Performance Liquid Chromatography (HPLC), gas chromatography (GC), Spektroskopy (like IR, UV, NMR), Colouring methods, TLC, NIRS, enzymatic or microbial assays. (see for example: Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 1127-32; und Schmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, S. 89-90, S. 521-540, S. 540-547, S. 559-566, 575-581 und S. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.)
The unsaturated C10 aldehydes compound produced in any of the method described herein can be converted to derivatives such as, but not limited to hydrocarbons, esters, amides, glycosides, ethers, epoxides, ketons, alcohols, diols, acetals or ketals. The unsaturated C10 aldehyde derivatives can be obtained by a chemical method such as, but not limited to oxidation, reduction, alkylation, acylation and/or rearrangement. Alternatively, the unsaturated C10 aldehyde derivatives can be obtained using a biochemical method by contacting the unsaturated C10 aldehyde with an enzyme such as, but not limited to an oxidoreductase, a monooxygenase, a dioxygenase, a transferase. The biochemical conversion can be performed in-vitro using isolated enzymes, enzymes from lysed cells or in-vivo using whole cells.
The invention also relates to methods for the fermentative production of unsaturated C10 aldehydes.
A fermentation as used according to the present invention can, for example, be performed in stirred fermenters, bubble columns and loop reactors. A comprehensive overview of the possible method types including stirrer types and geometric designs can be found in “Chmiel: Bioprozesstechnik: Einführung in die Bioverfahrenstechnik, Band 1”. In the process of the invention, typical variants available are the following variants known to those skilled in the art or explained, for example, in “Chmiel, Hammes and Bailey: Biochemical Engineering”, such as batch, fed-batch, repeated fed-batch or else continuous fermentation with and without recycling of the biomass. Depending on the production strain, sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen or appropriate gas mixtures may be effected in order to achieve good yield (YP/S).
The culture medium that is to be used must satisfy the requirements of the particular strains in an appropriate manner. Descriptions of culture media for various microorganisms are given in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D. C., USA, 1981).
These media that can be used according to the invention may comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and/or trace elements.
Preferred sources of carbon are sugars, such as mono-, di- or polysaccharides. Very good sources of carbon are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products from sugar refining. It may also be advantageous to add mixtures of various sources of carbon. Other possible sources of carbon are oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid or linoleic acid, alcohols such as glycerol, methanol or ethanol and organic acids such as acetic acid or lactic acid.
Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials containing these compounds. Examples of sources of nitrogen include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex sources of nitrogen, such as corn-steep liquor, soybean flour, soy-bean protein, yeast extract, meat extract and others. The sources of nitrogen can be used separately or as a mixture.
Inorganic salt compounds that may be present in the media comprise the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
Inorganic sulfur-containing compounds, for example sulfates, sulfites, di-thionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols, can be used as sources of sulfur.
Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used as sources of phosphorus.
Chelating agents can be added to the medium, in order to keep the metal ions in solution. Especially suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
The fermentation media used according to the invention may also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often come from complex components of the media, such as yeast extract, molasses, corn-steep liquor and the like. In addition, suitable precursors can be added to the culture medium. The precise composition of the compounds in the medium is strongly dependent on the particular experiment and must be decided individually for each specific case. Information on media optimization can be found in the textbook “Applied Microbiol. Physiology, A Practical Approach” (1997) Growing media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) etc.
All components of the medium are sterilized, either by heating (20 min at 1.5 bar and 121° C.) or by sterile filtration. The components can be sterilized either together, or if necessary separately. All the components of the medium can be present at the start of growing, or optionally can be added continuously or by batch feed.
The temperature of the culture is normally between 15° C. and 45° C., preferably 25° C. to 40° C. and can be kept constant or can be varied during the experiment. The pH value of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH value for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, e.g. fatty acid polyglycol esters, can be used for controlling foaming. To maintain the stability of plasmids, suitable substances with selective action, e.g. antibiotics, can be added to the medium. Oxygen or oxygen-containing gas mixtures, e.g. the ambient air, are fed into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20° C. to 45° C. Culture is continued until a maximum of the desired product has formed. This is normally achieved within 1 hour to 160 hours.
The methodology of the present invention can further include a step of recovering said one or more unsaturated C10 aldehydes.
The term “recovering” includes extracting, harvesting, isolating or purifying the compound from culture media. Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like.
Before the intended isolation the biomass of the broth can be removed. Processes for removing the biomass are known to those skilled in the art, for example filtration, sedimentation and flotation. Consequently, the biomass can be removed, for example, with centrifuges, separators, decanters, filters or in flotation apparatus. For maximum recovery of the product of value, washing of the biomass is often advisable, for example in the form of a diafiltration. The selection of the method is dependent upon the biomass content in the fermenter broth and the properties of the biomass, and also the interaction of the biomass with the product of value.
In one embodiment, the fermentation broth can be sterilized or pasteurized. In a further embodiment, the fermentation broth is concentrated. Depending on the requirement, this concentration can be done batch wise or continuously. The pressure and temperature range should be selected such that firstly no product damage occurs, and secondly minimal use of apparatus and energy is necessary. The skillful selection of pressure and temperature levels for a multistage evaporation in particular enables saving of energy.
The following examples are illustrative only and are not meant to limit the scope of invention as set forth in the Summary, Description or in the Claims.
The numerous possible variations that will become immediately evident to a person skilled in the art after heaving considered the disclosure provided herein also fall within the scope of the invention.
Unless otherwise stated, all chemical and biochemical materials and microorganisms or cells employed herein are commercially available products.
Unless otherwise specified, recombinant proteins are cloned and expressed by standard methods, such as, for example, as described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
Functional Expression of Lipoxygenase
The coding sequences of lipoxygenase (LOX) were optimized by following the genetic codon frequency of E. coli, synthesized and then subcloned into the pETDuet-1 (Novagen, Merck KGaA, Germany) plasmid for subsequent expression in E. coli. BL21 E. coli cells (Tiangen, China) were transformed with the plasmids pETDuet-LOX. The transformed cells were selected on LB-agar plates containing Ampicillin (50 μg/mL final). Single colonies were used to inoculate 25 mL liquid LB medium containing Ampicillin (50 μg/mL final). Cultures were incubated at 37° C. and 200 rpm shaking. After 4 hours incubation, the cultures were cooled down to 20° C. for 1.5 hour and IPTG (0.016 mM final) was added to induce protein expression. To express proteins the cultures were incubated for another 16 hours at 20° C. and 200 rpm shaking. The cultures were spin down and resuspended in 3 mL of reaction buffer (25 mM Tris-HCl pH7.5) followed by a sonication process to make protein solution, respectively. The protein solution was transferred into a 20 mL SPME vial, 30 μL fatty acid substrate and 10 μL internal standard (80 ppm alpha-ionone in ethanol) were added into the vial. After 10 min incubation, the SPME-GC-MS method described below was used for analysis of decadienals and decatrienals.
Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (SPME-GC-MS)
The reaction mixture was concentrated on a solid phase microextraction (SPME) fiber assembly polydimethylsiloxane/carboxen/divinylbenzene (57329-U, SUPELCO). The extraction was performed in headspace mode at 40° C. for 20 min. After extraction, the SPME fiber was introduced into the GC-MS inlet and maintained at 250° C. for 5 min, and the products were analyzed on an Agilent 6890 series GC system equipped with a DB1-ms column 30 m×0.25 mm×0.25 μm film thickness (P/N 122-0132, J&W scientific Inc., Folsom, Calif.) and coupled with a 5975 series mass spectrometer (Agilent, US). The carrier gas was helium at a constant flow of 0.7 mL/min. Injection was in splitless mode with the injector temperature set at 250° C. The oven temperature was programmed from 50° C. (5 min hold) to 250° C. at 15° C./min (5 min hold). Identification of products was based on mass spectra and retention indices as well as respective product standards.
Liquid Chromatography Coupled to UV Detection and Mass Spectrometry (LC-UV/MS)
200 μL of reaction mixture was diluted with 800 μL acetonitrile and then put on ice for 30 min. Filtration with 0.2 μL regenerated cellulose membrane (5190-5108, Agilent) was applied to remove the protein precipitation from the mixture. 1 μL of sample was injected to LC for the quantification of decadienal as well as side products.
Plant materials of Ulva fasciata (sample ID: PA-2017-0012) were collected from Nanao, Guangdong Province, China. One gram of smashed sample was put into a 20 mL vial for further SPME-GC-MS analysis.
To determine whether U. fasciata contained decadienals or decatrienals, fresh samples were analyzed by SPME-GC-MS as described in the Methods section.
One gram of smashed U. fasciata sample was put into a 20 mL vial with 3 mL Tris-HCl buffer (pH=7.5). 30 μL fatty acid substrate (30 μL LA, ALA, GLA, EPA, ARA, borage oil hydrolysate, arachidonic oil hydrolysate, linseed oil hydrolysate or fish oil hydrolysate in 1 ml ethanol respectively) and 10 μL internal standard (80 ppm alpha-ionone in ethanol) were added into the vial for incubation. After 10 min incubation at RT, the SPME-GC-MS method described in the method section was used for analysis of decadienals and decatrienals.
GC-MS analysis revealed that there were limited amounts of 2E,4Z-decadienal (retention time 13.0 min) and 2E,4E-decadienal (retention time 13.25 min) (
fasciata
Total RNA of U. fasciata was extracted using the RNeasy Plant Mini Kit (Qiagen, Germany). The total RNA sample was processed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina (NEB, USA) and TruSeq PE Cluster Kit (Illumina, USA) and then sequenced on Illumina HiSeq 2500 System. An amount of 38 million of paired-end reads of 2×150 bp was generated. The reads were processed using the Trinity (http://trinityrnaseq.sf.net/) software and 91564 transcripts with an N50 of 2262 were obtained. The obtained transcripts were translated into protein sequences and then functionally annotated by searching the NCBI non-redundant protein sequence database using the tblastx algorithm. One candidate protein sequence of LOX was mined by Pfam search and relative expression level.
The total RNA sample of U. fasciata was first reverse transcribed into cDNA using SMARTer™ RACE cDNA Amplification Kit (Clontech, Takara, Japan). The products were then used as the template for gene cloning. The coding sequence of UfLOX2 (SEQ ID NO:18) was amplified from the cDNA by using forward primer (5′-TCGTCCAACAGGTTCTCTT-3′) (SEQ ID NO:57) and reverse primer (5′-TTCTTTCCACTCACCGCCA-3′) (SEQ ID NO:58).
The coding sequence of UfLOX2 was optimized by following the genetic codon frequency of E. coli, synthesized and then subcloned into the pETDuet-1 plasmid for subsequent expression in E. coli. The following codon optimized sequences were applied: UfLOX2 (SEQ ID NO:17) and plasmid pETDuet-UfLOX2 was obtained.
Functional expression of the gene was performed as described above in the Methods section to yield protein solution. The enzymatic activity of the UfLOX2 was evaluated as described below:
a) UfLOX2 (SEQ ID NO:18) was tested by feeding with fatty acid substrate including gamma-linolenic acid (GLA), alpha-linolenic acid (ALA), linoleic acid (LA) and arachidonic acid (ARA) as below:
The protein solution (3 mL) from E. coli which contain UfLOX2 was put into a 20 mL SPME vial, 30 μL fatty acid substrate (30 μL LA, ALA, GLA, EPA, ARA, borage oil, arachidonic oil, linseed oil or fish oil in 1 mL ethanol respectively) and 10 μL internal standard (80 ppm alpha-ionone in ethanol) were added into the vial for incubation. After 10 min at RT, the SPME-GC-MS method described in the method section was used for analysis of decadienals and decatrienals.
UfLOX2 showed capability to produce decadienals (retention time 12.60 and 12.80 min) when feeding with specific substrates (Table 2)
b) To prove the lyase activity for UfLOX2, feeding experiments with fatty acid hydroperoxide was performed.
To test the HPL activity, UfLOX2 was produced in E. coli and cell lysates that contain UfLOX2 were prepared for testing its HPL activity. One aliquot of UfLOX2 was feed with GLA as a positive control of making decadienal. A second and third aliquot of UfLOX2 was denatured (boiled at 100° C. for 20 min) and feed with GLA or GLA hydroperoxide (GLA-HPO) as negative control to exclude UfLOX2 functionality to make decadienal and to show the conversion of GLA-HPO to decadienal in a non-UfLOX2 manner, respectively. A fourth aliquot of UfLOX2 was feed with GLA hydroperoxide (GLA-HPO) to prove its HPL activity in comparison with the third aliquot (i.e. non-UfLOX2 conversion of GLA-HPO to decadienal). In addition, the buffer for making UfLOX2 aliquots was also set as a negative control to show the non-UfLOX2 conversion of GLA-HPO to decadienal.
To prepare the GLA hydroperoxide (GLA-HPO) intermediate, 50 mL of UfLOX2 protein solution was incubated with 0.5 mL GLA (60 mg/mL) and stored at room temperature for 10 min. The reaction mixture was then loaded on a HLB column (Waters. US Part No. 186000118). The column was eluted with 10 mL of methanol to get GLA-HPO. After incubation for 1 hour, the reaction mixture was checked with LC-MS.
The results are summarized in Table 3 below.
Plant materials of Cladophora oligoclada (sample ID: AVLH2012-011) were collected from Qingdao, Shandong Province, China. One gram of smashed sample was put into a 20 mL vial for further SPME-GC-MS analysis.
Identification of peaks was based on comparison of their mass spectra and retention indices with those in internal libraries. GC-MS analysis revealed four main components in C. oligoclada as showed in Table 4 and
Fresh sample from C. oligoclada was extracted by MiniBest plant RNA extraction kit to yield total RNA by following protocol I provided by the kit (Cat. #9769 v201309 Da, Takara, Japan). The total RNA sample was processed using the TruSeq PE Cluster Kit (Illumina, USA) and then sequenced on an Illumina MiSeq System. An amount of 14 million of paired-end reads of 2×251 bp was generated. The reads were processed using the Trinity (http://trinityrnaseq.sf.net/) software and 225917 transcripts with an N50 of 676 were obtained. The obtained transcripts were translated into protein sequences and then functionally annotated by searching the NCBI non-redundant protein sequence database using the tblastx algorithm. One candidate protein sequence of LOX was mined by Pfam search and relative expression level.
The total RNA sample C. oligoclada (sample ID: PA-2017-0028) was first reverse transcribed into cDNA using SMARTer™ RACE cDNA Amplification Kit (Clontech Takara, Japan). The products were then used as the template for gene cloning. By using forward primer (5′-CTCTCTCTCTTTCTCTCTGTTCT-3′) (SEQ ID NO:55) and reverse primer (5′-CTCGTTCCCTTACCGTCT-3′) (SEQ ID NO:56) several coding sequences of LOX were amplified from the cDNA, designated CoLOX-3 (SEQ ID NO:3) (and its variants) CoLOX-0317 (SEQ ID NO:6), CoLOX-19 (SEQ ID NO:9), CoLOX-22 (SEQ ID NO:12) and CoLOX-d4 (SEQ ID NO:15).
The nucleic acid sequences of CoLOX-3 and its variants CoLOX-0317, CoLOX-19, CoLOX-22 and CoLOX-d4 were codon optimized by following the genetic codon frequency of E. coli, synthesized and then subcloned into the pETDuet-1 (Novagen, Merck KGaA, Germany) between NdeI and KpnI sites, respectively, for subsequent expression in E. coli. The following codon optimized sequences were applied: CoLOX-3 (SEQ ID NO:2), CoLOX-0317 (SEQ ID NO:5), CoLOX-19 (SEQ ID NO:8), CoLOX-22 (SEQ ID NO:11) and CoLOX-d4 (SEQ ID NO:14), and the following plasmids were prepared: pETDuet-CoLOX-3, pETDuet-CoLOX-0317, pETDuet-CoLOX-19, pETDuet-CoLOX-22 and pETDuet-CoLOX-d4. Functional expression of the genes was performed as described above in the Methods section. The cultures were spin down and resuspended in 3 mL of buffer (25 mM Tris-HCl pH7.5, 0.2 mM CaCl2) followed by a sonication step to make the respective protein solution.
The crude protein solutions (3 mL) of CoLOX-3, CoLOX-0317, CoLOX-19, CoLOX-22 and CoLOX-d4 were put into a 20 mL SPME vial, respectively, 30 μL fatty acid substrate (30 μL LA, ALA, GLA, EPA, ARA borage oil, arachidonic oil, linseed oil or fish oil in 1 ml ethanol respectively) and 10 μL internal standard (80 ppm alpha-ionone in ethanol) were added into each of the vial for incubation. After 10 min at RT, the SPME-GC-MS method described in the methods section was used for analysis of decadienals and decatrienals. A mixture of buffer plus fatty acid plus internal standard was used as control.
All five proteins showed capability to produce decadienals and/or decatrienals when feeding with specific substrates (see Table 5 and 6 below and
Due to its activity of producing decadienals and decatrienals, UfLOX2 was used to search for more LOXs from GenBank by using BLASTP 2.8.0+ (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A total of 188 LOXs were found by this approach, in which 181 LOXs are from cyanobacteria, 5 LOXs are from proteobacteria, and 2 LOXs are from planctomycetes, with sequence identity of less than 42% to UfLOX2. 16 LOXs were selected as example for a relatively higher sequence identity to UfLOX2 and being representative for their own homologs, as listed in Table 7. Two known LOXs from red algae were listed and used for comparison. The residual 83 LOXs with a relatively higher identity to UfLOX2 were listed in the attached sequence listing as SEQ ID NO: 75 to 239 (amino acid and nucleic acid sequences. The start codons, where necessary, were set as ATG.
Ulva fasciata
Cladophora oligoclada
Pyropia haitanensis
Pyropia haitanensis
Microcystis aeruginosa
Microcoleus vaginatus
Oscillatoria nigro-viridis
Crinalium epipsammum
Dolichospermum circinale
Aphanizomenon flos-aquae
Anabaena sp. AL09
Aphanizomenon flos-aquae
Nostoc punctiforme
Calothrix brevissima
Mastigocoleus testarum
Nostoc calcicola
Aliterella atlantica
Scytonema sp. HK-05
Nostoc sp. ‘Peltigera malacea
cyanobiont’ DB3992
Nitrosococcus watsonii
aCoLOX-3 of present invention;
bUfLOX2 of present invention;
cAFQ59981.1 (PhLOX) was described for example by Jechan Lee et al., Environmental Pollution 227 (2017) 252-262;
dAGN54275.1 (PhLOX2) was described in Zhujun Zhu et al., PLoS One. (2015) 10(2): e0117351.
The amino acid sequence identity and the number of different residues are summarized in Table 8. The upper right block shows the number of unmatched amino acids, the lower left block shows the sequence identity. The sequence identities between the bacterial LOXs and UfLOX2 range from 32 to 42%. The sequence identities between the bacterial LOXs and CoLOX-3 range from 13 to 16%. The sequence identities between the bacterial LOXs and the red algae LOXs are less than 15%.
The coding sequences of the bifunctional LOXs were optimized by following the genetic codon frequency of E. coli, synthesized and then subcloned into the pETDuet-1 plasmid for subsequent expression in E. coli.
Functional expression of the mined LOXs was performed as described above in the Methods section. The different LOX proteins expressed by E. coli were released by sonication in 25 mM Tris-HCl buffer (pH7.5) to deliver LOX protein solution, respectively. Each LOX protein solution was transferred into a 20 mL SPME vial, 30 μL of GLA and 10 μL of internal standard were added into the vial. After 10 min incubation, SPME-GC-MS was used for analysis of decadienals, decatrienals and hexanal, and LC-UV was used for analysis of decadienals, decatrienals and the GLA-HPO (intermediate between gamma-linolenic acid and decadienals). SPME-GC-MS was performed as described in the Methods section above. GC-MS analysis revealed 2E,4Z-decadienal (retention time 13.0 min), 2E,4E-decadienal (retention time 13.25) and hexanal in the reactions for each LOX but with different levels. LC-UV revealed 2E,4Z-decadienal (retention time 6.61 min at 280 nm), 2E,4E-decadienal (retention time 6.62 min at 280 nm) and GLA-HPO (retention time 6.90 min at 235 nm).
The selectivity, bifunctionality and productivity of LOXs for the decadienal end product from the GLA substrate were calculated and shown in Table 9 below (UfLOX2 and CoLOX-3 were involved for comparison). The selectivity can be deduced by calculating the peak area ratio of decadienal (C10) to hexanal (C6). The productivity can be deduced from the peak area of decadienal. The bifunctionality can be deduced by calculating the peak area ratio of decadienal (C10) to GLA-HPO (intermediate). In this comparison, UfLOX2 remains the best bifunctional LOX, followed by cyanobacterial bifunctional LOX WP_002738122.1 (from Microcystis aeruginosa) and WP_015204462.1 (from Crinalium epipsammum). There are still some cyanobacterial LOXs with similar activity compared to CoLOX-3, e.g. WP_039200563.1, WP_073641301.1.
High performance LOXs, UfLOX2 and WP_002738122.1 and WP_015204462.1 were compared with the other less active LOXs in an alignment view (see
The importance of some of the identified conserved residues by mutagenesis studies was investigated. The results are summarized in Table 10.
1)Double mutation in positions 134 and 136
2)Numbering relates to SEQ ID NO: 18
In a first series of mutagenesis studies, some UfLOX2 mutants showed reduced activity, see in
Based on these date the following may be concluded:
The residues identified in Experiment 1 were introduced into several bacterial LOXs with several other residues that are conserved in bacterial LOXs to improve productivity. The designed sequences are as shown in Table 11.
The coding sequences of the mutants of bacterial LOXs were optimized by following the genetic codon frequency of E. coli, synthesized and then subcloned into the pETDuet-1 plasmid for subsequent expression in E. coli.
Functional expression of the mutants of bacterial LOXs was performed as described above in the Methods section. The different LOX proteins expressed by E. coli were released by sonication in 25 mM Tris-HCl buffer (pH7.5) to deliver LOX protein solution, respectively. Each LOX protein solution was transferred into a 20 mL SPME vial, 30 μL of GLA and 104 of internal standard were added into the vial. After 10 min incubation, LC-UV was used for analysis of decadienals. The productivity of LOX mutants for the decadienal end product were calculated and shown in
Previous studies indicated that five essential conserved amino acid residues in the active site are involved in the binding of cofactors as described by Toralf Senger, et al., J. Biol. Chem. 2005, 280:7588-7596 (residues cited therein as His-585, His-590, His-774, Asn-778 and Ile-899). Both iron and manganese were reported to be the cofactors as described by Alexandra Andreou, et al., J. Biol. Chem. 2010. The algal LOXs and the bacterial LOXs also have these five conservative residues as shown in said alignment in
In the case of making decadienal by using UfLOX2 and gamma-linolenic acid, the molar yield for total decadienal (including 2E,4Z-decadienal and 2E,4E-decadienal) is approx. 30-40% based on quantification by LC-UV/MS with external calibration as described above in the Methods section. However, the overall percentage for decadienal, based total volatiles is above 90%.
To obtain information of other downstream side products, UfLOX2 was produced in E. coli. Cell lysates (20 ml) that contain UfLOX2 were fed with GLA at room temperature. 200 μl sample aliquots were picked up and mixed with 800 μl acetonitrile for further LC-UV/MS analysis as described above in the Methods section. Nine side product (see Table 12) were proposed based on the observed mass spectra as well as comparison with literature.
All the publications mentioned in this application are incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
Cladophora oligoclada
Cladophora oligoclada
Cladophora oligoclada
Cladophora oligoclada
Cladophora oligoclada
Cladophora oligoclada
Cladophora oligoclada
Cladophora oligoclada
Cladophora oligoclada
Cladophora oligoclada
Ulva fasciata
Ulva fasciata
Microcystis aeruginosa
Microcoleus vaginatus
Oscillatoria nigro-viridis
Crinalium epipsammum
Dolichospermum circinale
Anabaena sp. AL09
Aphanizomenon flos-aquae LD13
Aphanizomenon flos-aquae
Nostoc punctiforme
Mastigocoleus testarum
Nostoc calcicola
Calothrix brevissima
Nostoc sp. ‘Peltigera malacea
cyanobiont’ DB3992
Aliterella atlantica
Scytonema sp. HK-05
Nitrosococcus watsonii
Microcystis aeruginosa
Microcoleus vaginatus
Oscillatoria nigro-viridis
Crinalium epipsammum
Dolichospermum circinale
Anabaena sp. AL09
Aphanizomenon flos-aquae LD13
Aphanizomenon flos-aquae
Nostoc punctiforme
Mastigocoleus testarum
Nostoc calcicola
Calothrix brevissima
Nostoc sp. ‘Peltigera malacea
cyanobiont’ DB3992
Aliterella atlantica
Scytonema sp. HK-05
Nitrosococcus watsonii
Microcystis sp. 0824
Microcystis sp. 0824
Acaryochloris sp. RCC1774
Acaryochloris sp. RCC1774
Anabaena sp. WA102
Anabaena sp. WA102
Dolichospermum circinale
Dolichospermum circinale
Anabaena sp. CRKS33
Anabaena sp. CRKS33
Anabaena sp. LE011-02
Anabaena sp. LE011-02
Anabaena sp. AL93
Anabaena sp. AL93
Aphanizomenon flos-aquae
Aphanizomenon flos-aquae
Anabaena sp. WA113
Anabaena sp. WA113
Anabaena sp. 90
Anabaena sp. 90
Aphanizomenon flos-aquae
Aphanizomenon flos-aquae
Nostoc sp. ATCC 53789
Nostoc sp. ATCC 53789
Nostoc linckia
Nostoc linckia
Nostoc sp. ATCC 43529
Nostoc sp. ATCC 43529
Mastigocladopsis repens
Mastigocladopsis repens
Aliterella atlantica CENA595
Aliterella atlantica CENA595
Synechococcus sp. PCC 7336
Synechococcus sp. PCC 7336
Nostoc flagelliforme
Nostoc flagelliforme
Nostoc punctiforme NIES-2108
Nostoc punctiforme NIES-2108
Nostoc sp. ‘Peltigera membranacea
cyanobiont’ 210A
Nostoc sp. ‘Peltigera membranacea
cyanobiont’ 210A
Nostoc sp. ‘Lobaria pulmonaria
Nostoc sp. ‘Lobaria pulmonaria
Cyanosarcina burmensis
Cyanosarcina burmensis
Pseudanabaena sp. PCC 6802
Pseudanabaena sp. PCC 6802
Nostoc cycadae
Nostoc cycadae
Nostoc sp. HK-01
Nostoc sp. HK-01
Anabaenopsis circularis
Anabaenopsis circularis
Pleurocapsa sp. PCC 7319
Pleurocapsa sp. PCC 7319
Nostoc sp. MBR 210
Nostoc sp. MBR 210
Nostoc piscinale
Nostoc piscinale
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis flos-aquae DF17
Microcystis flos-aquae DF17
Microcystis aeruginosa TA09
Microcystis aeruginosa TA09
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa NIES-843
Microcystis aeruginosa NIES-843
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Microcystis aeruginosa
Gammaproteobacteria bacterium
Gammaproteobacteria bacterium
Chamaesiphon polymorphus
Chamaesiphon polymorphus
Nostoc linckia
Nostoc linckia
Cyanothece sp. PCC 8801
Cyanothece sp. PCC 8801
Neosynechococcus sphagnicola
Neosynechococcus sphagnicola
Cyanothece sp. PCC 8802
Cyanothece sp. PCC 8802
Pseudanabaena sp. SR411
Pseudanabaena sp. SR411
Pseudanabaena frigida
Pseudanabaena frigida
Ahniella affigens
Ahniella affigens
Pseudanabaena sp. ABRG5-3
Pseudanabaena sp. ABRG5-3
Pseudanabaena sp. ‘Roaring Creek’
Pseudanabaena sp. ‘Roaring Creek’
Pseudanabaena biceps
Pseudanabaena biceps
Leptolyngbya sp. PCC 7376
Leptolyngbya sp. PCC 7376
Nodularia spumigena
Nodularia spumigena
Calothrix sp.
Calothrix sp.
Nostoc sp. PCC 7524
Nostoc sp. PCC 7524
Nostoc sp. ‘Peltigera membranacea
cyanobiont’ 210A
Nostoc sp. ‘Peltigera membranacea
cyanobiont’ 210A
Acaryochloris marina
Acaryochloris marina
Rivularia sp. PCC 7116
Rivularia sp. PCC 7116
Tolypothrix bouteillei
Tolypothrix bouteillei
Leptolyngbya sp. PCC 7375
Leptolyngbya sp. PCC 7375
Nostoc sp. ‘Peltigera malacea
cyanobiont’ DB3992
Nostoc sp. ‘Peltigera malacea
cyanobiont’ DB3992
Nostocales
Nostocales
Nostoc punctiforme NIES-2108
Nostoc punctiforme NIES-2108
Gemmata sp. SH-PL17
Gemmata sp. SH-PL17
Pseudanabaena sp. BC1403
Pseudanabaena sp. BC1403
Leptolyngbya sp. Heron Island J
Leptolyngbya sp. Heron Island J
Calothrix sp. NIES-4101
Calothrix sp. NIES-4101
Nodularia spumigena
Nodularia spumigena
Nodularia spumigena
Nodularia spumigena
Acaryochloris sp. CCMEE 5410
Acaryochloris sp. CCMEE 5410
Nostoc sp. CENA543
Nostoc sp. CENA543
Limnothrix rosea
Limnothrix rosea
Tolypothrix bouteillei
Tolypothrix bouteillei
Acaryochloris marina
Acaryochloris marina
Tolypothrix bouteillei
Tolypothrix bouteillei
Calothrix parasitica
Calothrix parasitica
Acaryochloris sp. RCC1774
Calothrix parasitica
Acaryochloris sp. CCMEE 5410
Acaryochloris sp. CCMEE 5410
Cyanothece sp. PCC 8801
Encompassed within the general disclosure of the present description is any coding nucleic acid described herein without a 5′-terminal start codon triplet or with an artificial or natural start codon triplet.
WxxAKxCxQxADxNHxExxxHxxxTHxVMxPxAxxTxxxLxxNHPxxxLLxPHxxFMLxxNxLxxxxxxxxxGxx
xxQxxYxxxxxNMPxAxYx
xxx = amino acids that are locate in a key long helix close to the reaction center
xxx = amino acids that are locate in a key shorter helix close to the reaction center
= amino acids that are locate in a key long helix close to the reaction center
xxxxxxxxxxxxxxxxxxxxPxxxxx
xxQxxYxxxxxNMPxAxYxxxx
xxxxxxxxxxxxxxxxxxxQxxLxxxxxxIxxxNxxRxxxYxxxxxxxxxNSIxx
xxx = amino acids that locate in a key long helix close to the reaction center
xxx = amino acids that locate in a key shorter helix close to the reaction center
= amino acids that locate in a key long helix close to the reaction center
xPxAxxxxxxxxxxxHPxxxxLxxHxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
QxxYxxxxxNxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
xxx = amino acids that locate in a key long helix close to the reaction center
= amino acids that locate in a key long helix close to the reaction center
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/110960 | Oct 2018 | CN | national |
This application is a U.S. National Phase application of International Patent Application No. PCT/EP2019/078370, filed Oct. 18, 2019, which claims the benefit of priority to International Patent Application No. PCT/CN2018/110960, filed Oct. 19, 2018, the entire contents of each of which are hereby incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/078370 | 10/18/2019 | WO | 00 |
