Method for optimizing nucleotide sequence by synonymous codon substitution for expression of amino acid sequence in target organism

Abstract

The present invention relates to a method for optimizing a nucleotide sequence for expression of a predetermined amino acid sequence in at least one target organism. The nucleotide sequence comprises a plurality of base triads, and on at least one change position of the nucleotide sequence, a base triad encoding an amino acid of the predetermined amino acid sequence is replaced by a synonymous base triad encoding the same amino acid of the predetermined amino acid sequence, to optimize the nucleotide sequence for expression in at least one target organism. The altered position comprises a direct successive order of n base triads forming a first codon n-tuple and encoding a sequence portion of n amino acids of the predetermined amino acid sequence, the sequence portion forming an amino acid n-tuple, the amino acid n-tuple is encoded by a predetermined amount of an amino acid n-tuple event in the genome or part thereof of the at least one target organism and / or in the genome or part thereof of a virus capable of infecting the at least one target organism. At least one of the n base triads in the direct successive order is replaced by a synonymous base triad selected to produce a second codon n tuple, the second codon n-tuple has a higher relative codon n-tuple frequency in the genome or part thereof of the at least one target organism and / or in the genome or part thereof of a virus capable of infecting the at least one target organism than the first codon n-tuple based on the amount of amino acid n-tuple events.

Description

[0001] Reference to prior application

[0002] This application claims the priority of German patent application number 102022118459.5 filed on July 22, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application includes an electronic sequence listing in xml format according to the WIPOST.26 standard, of which 21 sequences are included as part of the specification, and their contents are incorporated herein by reference in their entirety. Field of the Invention

[0005] The present invention belongs to the field of producing synthetic nucleotide sequences and using them for producing proteins by introducing these nucleotide sequences into expression systems having suitable host organisms expressing the proteins encoded by the nucleotide sequences. The present invention particularly relates to methods for optimizing nucleotide sequences for expression in predefined host organisms. Background of the Invention

[0007] Heterologous expression systems are very important in the biotechnology of producing recombinant proteins. An "expression system" is a biological system that can carry out protein biosynthesis in a targeted and controlled manner, that is, produce a certain protein according to the template of the nucleotide sequence, that is, "express".

[0008] "Heterologous expression" is understood to mean the expression of a gene or part thereof in a host organism that does not naturally possess the gene or gene fragment. The corresponding nucleotide sequence is introduced into the host organism using genetic or recombinant DNA technology, for example with the aid of a vector or genome editing, and then allowed to proliferate and stimulated to overproduce the protein. Thus, "homologous expression" refers to the expression of a gene or part thereof in a host organism or system from which it originally originated.

[0009] Heterologous protein expression can be carried out in many types of host organisms. The host organism can be, for example, bacteria, fungi, yeast, insect cells, mammalian cells or plant cells. A common problem in heterologous protein expression is the low transcription and translation rate of exogenous nucleotide sequences in specific host organisms (also referred to herein as target organisms). The reason is especially the degradation of the genetic code, which results in that for most of the amino acids established during translation, there are multiple codons with the same meaning, also referred to herein as "synonymous codons" available. A codon is the order of three consecutive nuclear bases of a nucleic acid sequence, i.e., a "base triplet" that can encode amino acids. There are a total of 64 possible codons, of which 61 encode a total of 20 classical protein amino acids, and another 3 encode termination codons.

[0010] Different organisms may use codons at different frequencies for the expression of amino acids, also known as "codon usage" or "codon bias". It also indicates that the codons in the genes of an organism are not randomly arranged, but the observed codon pair frequencies may deviate from the expected product of the respective individual frequencies and are therefore statistically "under-represented" or "over-represented". This context is also called "codon context", which can have an additional impact on translation efficiency.

[0011] Thus, known possibilities for increasing the expression rate are codon optimization by replacing individual codons of the nucleotide sequence to be expressed with synonymous codons having a higher frequency in the target organism (also called "codon optimization" or "codon usage optimization"), and by transferring the frequency of codons of highly expressed genes in the target organism to the target protein (also called "codon adaptation index"). There are also possibilities for codon context optimization, in which the codons of the nucleic acid sequence to be expressed are adapted to the codon context of the target organism by replacing them with over- or under-represented synonymous codons, without changing the encoded amino acid sequence.

[0012] WO 2020 / 024917 A1 discloses a computer-implemented method in which a nucleic acid sequence is optimized for protein expression in a host based, inter alia, on a codon adaptation index and a codon context by using a computer-assisted NSGA-III algorithm.

[0013] A computer-assisted method is known from WO 2004 / 059556 A2, in which a quality function is used to optimize a predetermined nucleic acid sequence for expression in a predetermined target organism. The quality function can take into account, inter alia, codon usage and codon context as quality criteria.

[0014] Another method is proposed in WO 2008 / 000632 A1, in which new coding sequences are generated from a predetermined nucleotide sequence encoding a predetermined amino acid sequence by replacing one or more synonymous codons in multiple repeated steps and are further developed with the help of fitness values ​​that take into account, in particular, the codon context of the host organism.

[0015] From WO 2018 / 104385 A1 a method for determining an optimized nucleotide sequence which encodes a predetermined amino acid sequence and is optimized for expression in a specific target organism is known, wherein a large number of candidate nucleotide sequences are generated and evaluated using a statistical machine learning algorithm.

[0016] Another method for optimizing nucleotide sequences for heterologous protein expression based on the codon context of the target organism is known from WO 2007 / 130650 A2.

[0017] It has been shown that these conventional optimizations are often insufficient to achieve significant increases in protein yields in heterologous systems. Furthermore, known methods are often tailored to the quantitative optimization of transcribed mRNA or protein yields and do not take into account the solubility of the expressed protein, which is crucial for its usability for scientific, medical, pharmaceutical and industrial purposes.

[0018] Therefore, an object of the present invention is to provide an improved method and device for optimizing nucleotide sequences for expressing a predetermined amino acid sequence in a target organism, which can at least partially solve the above-mentioned problems and in particular can increase the proportion of soluble protein in the expression of heterologous proteins.

[0019] This object is achieved by the subject matter of the independent claims. Preferred embodiments of the invention are subject matter of the dependent claims and of the following description. Summary of the invention

[0020] The subject of the present invention is a method for optimizing a nucleotide sequence for expressing a predetermined amino acid sequence in at least one target organism. The expression can in principle be heterologous or homologous. Heterologous expression is preferred.

[0021] The nucleotide sequence comprises a plurality of base triplets, wherein at at least one altered position of the nucleotide sequence, a base triplet encoding an amino acid of the predetermined amino acid sequence is replaced by a synonymous base triplet encoding the same amino acid of the predetermined amino acid sequence to optimize the nucleotide sequence for expression in at least one target organism.

[0022] The altered position according to the invention comprises here a directly consecutive order of n base triplets which form a first codon n-gram and encode a sequence portion of n amino acids of a predetermined amino acid sequence, which sequence portion forms an amino acid n-gram, wherein the amino acid n-gram is encoded by a predetermined number of amino acid n-gram events in the genome or a portion thereof of the at least one target organism and / or in the genome or a portion thereof of a virus capable of infecting the at least one target organism.

[0023] The method according to the invention comprises replacing at least one of the n base triplets from the directly consecutive order of the at least one altered position by a synonymous base triplet, wherein the synonymous base triplet is selected so as to generate a second codon n-gram having a higher relative codon n-gram frequency than the first codon n-gram in the genome or part thereof of the at least one target organism and / or in the genome or part thereof of a virus capable of infecting the at least one target organism based on the amount of amino acid n-gram events.

[0024] Here, n is a natural number greater than or equal to 2 and in particular less than or equal to the total number N of amino acids in the predetermined amino acid sequence.

[0025] The present invention is based on the knowledge that the influence of the direct sequential order of n base triplets (referred to herein as codon n-gram) on the translation efficiency of the subsequent order of n amino acids encoded by said n base triplets (referred to herein as amino acid n-gram) can be represented in a specific target organism by the relative frequency with which the direct sequential order of n base triplets encodes the direct sequential order of n amino acids within the genome of the host organism, referred to herein as relative codon n-gram frequency.

[0026] Without being limited to theoretical considerations, it is hypothesized that the translation process is at least temporarily affected by two or more consecutive base triplets that are simultaneously bound to the ribosome during translation. The inventors have found evidence that codon n-grams with favorable effects are found more frequently in the genome than synonymous codon n-grams with less favorable or unfavorable effects.

[0027] In other words, the inventors have recognized that the ratio of the absolute frequency of codon n-tuples to the absolute frequency of the corresponding amino acid n-tuples encoded by the codon n-tuples is an advantageous measure to quantify the suitability of a predetermined nucleic acid sequence for expression in a specific target organism. The relative frequency can take a value of 0 to 1 or 0% to 100%, wherein if a specific codon n-tuple in the genome of the target organism or a portion thereof is not used to encode the corresponding amino acid n-tuple at all, the relative frequency is equal to 0. If a specific amino acid n-tuple in the genome of the target organism or a portion thereof is only encoded by a specific codon n-tuple, the relative frequency is equal to 1 or 100%. In this way, nucleic acids can be optimized by specifically replacing the codon n-tuples with relatively low frequencies in the genome of a predetermined target organism with codon n-tuples with relatively high frequencies, so that they make the target organism better suitable for expressing a predetermined amino acid sequence.

[0028] If a particular amino acid n-tuple of an amino acid sequence is not present at all in the genome or part thereof of the target organism and / or in the genome or part thereof of a virus, in principle any codon n-tuple encoding the amino acid n-tuple can be selected. For example, related synonymous codon n-tuples can each be assigned a uniform relative frequency of 1 / i, where i is the amount of synonymous codon n-tuples encoding the amino acid n-tuple. Alternatively, the relative frequency of the codon n-tuple encoding the amino acid n-tuple can be assigned to 0. Another possible way is to exclude the altered position and / or the amino acid n-tuple from the optimization.

[0029] It has been shown that for a predetermined target organism, the method according to the invention can be used to increase the yield of heterologous protein expression, in particular soluble proteins, several times compared to previously known methods. In this context, reference is also made to the results of comparative experiments in the following exemplary embodiments.

[0030] In addition, the method according to the present invention can also realize the biosynthesis of proteins that could not or were difficult to heterologously express in the past. In this way, the method according to the present invention causes the improvement of the efficiency and sustainability of the biotechnological protein production for science, medicine and technology or industrial purposes.

[0031] In a preferred embodiment of the method, n is less than or equal to 50, less than or equal to 40, less than or equal to 30, less than or equal to 20 or less than or equal to 10. Particularly preferably, n is selected from the group consisting of n=2, n=3, n=4, n=5, n=6 and any combination thereof. In one embodiment, n=2. In one embodiment, n=3. In one embodiment, n=4. In one embodiment, n=5. In one embodiment, n=6. Particularly preferably, n is greater than or equal to 3. With these n, the nucleotide sequence is advantageously suitable for expression in the target organism.

[0032] As used herein, the term "predetermined" means that the amount of amino acid n-tuple events is predetermined by the genome and / or proteome of the at least one target organism or a portion thereof and / or the genome or a portion thereof of a virus capable of infecting the at least one target organism. Therefore, it should be understood that determining the amount of events encoding a specific amino acid n-tuple in the genome or a portion thereof of the at least one target organism and / or in the genome or a portion thereof of a virus capable of infecting the at least one target organism (hereinafter also referred to as the absolute frequency) is a step that can be performed in the method, but is not necessarily performed. On the contrary, information about the absolute frequency of amino acid n-tuples encoding the genome or a portion thereof of the at least one target organism and / or in the genome or a portion thereof of a virus capable of infecting the at least one target organism can also be included in the method according to the present invention by other means, such as from a database, etc. Of course, it is also possible to determine the amount of events encoding the amino acid n-tuple in the genome or a portion thereof of the at least one target organism and / or in the genome or a portion thereof of a virus capable of infecting the at least one target organism as a method step.

[0033] This also applies in principle to determining the number of occurrences, i.e. the absolute frequency, of a particular codon n-gram in the genome or a part thereof of the at least one target organism and / or in the genome or a part thereof of a virus capable of infecting the at least one target organism, and / or the relative frequency of the codon n-grams according to the invention derived therefrom. For example, the absolute frequency and / or the relative frequency of substantially every possible combination of codon n-grams in the genome or a part thereof of the at least one target organism or in the genome or a part thereof of a virus capable of infecting the at least one target organism can be stored in a database and included in the method according to the invention in the form of database information.

[0034] In the method according to the present invention, the selection of synonymous base triplets is carried out from the perspective of the second codon n-tuple satisfying the conditions of the higher relative codon n-tuple frequency required by the present invention compared with the first codon n-tuple. The selection of synonymous base triplets can therefore particularly include determining and / or evaluating the relative codon n-tuple frequency of the second codon n-tuple. As described above, the term "determining" can be performed, for example, in the form of calculating the relative codon n-tuple frequency of the second codon n-tuple or in the form of data comparison, including database information, etc. Therefore, a preferred method embodiment includes at least the following steps: a) determining at least one change position; b) at least one base triplet of the at least one change position is replaced by a synonymous base triplet; c) determining the relative codon n-tuple frequency of the resulting second codon n-tuple. Preferably, the method also includes step d), i.e. evaluating the relative codon n-tuple frequency of the second codon n-tuple determined in step c), wherein the evaluation, for example, includes comparing with the relative codon n-tuple frequency of the first codon n-tuple and / or can be performed with the aid of target criteria such as minimum values. In this regard, reference is also made to the following description and examples.Steps b) and c) and optionally d) can also be repeated until a second codon n-tuple is generated which has a higher relative codon n-tuple frequency than the first codon n-tuple required according to the invention.

[0035] As indicated above, the amount of amino acid n-gram events determined and / or the relative frequencies of codon n-grams determined are not necessarily based on the complete genome of the at least one target organism or the complete genome of an organism capable of infecting the at least one target organism. On the contrary, in certain method variants, it may be sufficient and advantageous if only a portion of one or more genomes is included in the determined absolute amino acid n-gram frequencies and / or relative codon n-gram frequencies, in particular since most genomes each largely contain non-coding regions, which are less relevant for measuring the suitability of a predetermined nucleic acid sequence for expression in a target organism according to the invention.

[0036] In a preferred embodiment, the amount of the amino acid n-gram events is derived from a plurality, preferably all, of protein-coding genes and / or proteins of the at least one target organism and / or of a virus capable of infecting the at least one target organism, or is determined with the aid of a plurality of protein-coding genes and / or proteins of the at least one target organism and / or of a virus capable of infecting the at least one target organism. Proteins constitutively expressed by the target organism or proteins with high transient expression or high abundance are particularly suitable for this. Particularly preferably, at least 25%, at least 50%, at least 75%, at least 80%, at least 90% or at least 95% of the coding part of the genome of the target organism is included in the determination of the amount of the amino acid n-gram events.

[0037] Correspondingly, the relative codon n-gram frequency is also derived from the amount of occurrences of the respective first and / or second codon n-grams in a plurality, preferably all protein-coding genes and / or proteins of the at least one target organism and / or of a virus capable of infecting the at least one target organism and / or is determined with the aid of a plurality of protein-coding genes and / or proteins of the at least one target organism and / or of a virus capable of infecting the at least one target organism, based on the amount of amino acid n-gram occurrences. Particularly preferably, at least 25%, at least 50%, at least 75%, at least 80%, at least 90% or at least 95% of the coding part of the genome of the target organism is included in the determination of the relative codon n-gram frequency.

[0038] For the method according to the present invention, the relative frequency of the first and / or second codon n-tuples does not have to be the value calculated by absolute frequency arithmetic in principle. For example, the relative frequency of the first and / or second codon n-tuples can also be expressed or replaced by another probability distribution or another probability measure, such as an interval estimate. For example, therefore, a confidence interval of the success probability of a binomial or multinomial distribution can be determined by random observation, such as in the genome of the at least one target organism or its part and / or in the genome of the virus that can infect the at least one target organism or its part (wherein the observed first and / or second codon n-tuple frequency and / or the observed frequency of all synonymous codon n-tuples encoding the same amino acid n-tuples as the number of successes and the amino acid n-tuple frequency as the number of experiments), such as the confidence level is 95% Clopper-Pearson confidence interval or the simultaneous confidence interval of multiple ratios. The relative codon n-tuple frequency is, for example, represented or replaced by the midpoint of the interval at this time, which represents a more accurate estimate. When the amino acid n-tuple frequency is low and when the relative codon n-tuple frequency is very high or very low, this may be particularly advantageous. Of course, relative codon n-gram frequencies can also be calculated or represented using other values ​​in the interval. In addition to the midpoint of the interval, for example, the minimum value of the interval, particularly for very conservative estimates, or the mean value of the weight function (e.g. -1 / x) on the interval represents the relative codon n-gram frequency.

[0039] Thus, according to one variant of the method according to the invention, it is provided that the at least one altered position comprises a directly consecutive sequence of n base triplets, which form a first codon n-gram and encode a sequence portion of n amino acids of a predetermined amino acid sequence, said sequence portion forming an amino acid n-gram, wherein at least one of the n base triplets of the directly consecutive sequence is replaced by a synonymous base triplet, which is selected with the aid of an estimation function such that a second codon n-gram is generated, which encodes an amino acid n-gram with a greater probability in the genome or a portion thereof of the at least one target organism and / or in the genome or a portion thereof of a virus capable of infecting the at least one target organism than the first codon n-gram. Suitable estimation functions that can be implemented for the method according to the invention are known to the person skilled in the art.

[0040] As used herein, the term "at least one altered position" includes the possibility that the method includes a plurality or a large number of altered positions, wherein in each altered position, at least one of the n base triplets in the directly consecutive order is replaced by a synonymous base triplet, and the synonymous base triplets are selected so that at least a portion of the resulting second codon n-gram has a higher relative codon n-gram frequency than the respective first codon n-gram. Of course, for this purpose, it is also possible to replace a plurality or all of the n base triplets in one or more altered positions with synonymous base triplets, respectively.

[0041] In a preferred embodiment, at least in the altered position having the first codon n-tuple with the lowest relative codon n-tuple frequency of all altered positions, at least one of the base triplets in the directly consecutive order is replaced by a synonymous base triplet, which is selected so that the resulting second codon n-tuple has a higher relative codon n-tuple frequency than the first codon n-tuple. The inventors have recognized that the codon n-tuple with the lowest relative frequency in a nucleotide sequence generally has a limiting effect on the entire translation process, and therefore replacing the first codon n-tuple with a second codon n-tuple with a higher relative frequency has a particularly beneficial effect on translation and folding, and can therefore particularly well improve the expression of soluble proteins in target organisms.

[0042] If there are multiple or a large number of changed positions in the nucleotide sequence, the method according to the invention provides for a possible manner in which the directly consecutive order of n base triplets of at least two changed positions overlaps, wherein the base triplets replaced by synonymous base triplets are simultaneously contained by at least these two changed positions.

[0043] For such embodiment, the method according to the present invention can also be specified that the synonymous base triplet is selected so that in one of the two change positions, the obtained second codon n-tuple has a relative codon n-tuple frequency lower than the first codon n-tuple respectively, and in the other of the two change positions, the obtained second codon n-tuple has a relative codon n-tuple frequency higher than the first codon n-tuple respectively.Alternately or in addition, the method according to the present invention can also be specified for such overlapping change positions, and the obtained second codon n-tuple has a relative codon n-tuple frequency higher than the first codon n-tuple respectively in two change positions.Relative codon n-tuple frequency may also be reduced in two change positions.

[0044] If a value greater than or equal to 2 is chosen for n, then of course it is also possible for the directly consecutive order of n base triplets of more than two change positions to overlap and for base triplets replaced by synonymous base triplets to be included simultaneously by more than two change positions. The synonymous base triplets can then be chosen such that in at least one of the change positions the resulting second codon n-tuple has a lower relative codon n-tuple frequency than the respective first codon n-tuple and in another of the change positions the resulting second codon n-tuple has a higher relative codon n-tuple frequency than the respective first codon n-tuple.

[0045] Surprisingly, the method according to the invention has shown that in the case of a large number of at least partially overlapping altered positions in the nucleotide sequence, it may be necessary and even important to accept a deterioration, i.e. a reduction, in the relative codon n-tuple frequency of the second codon n-tuple relative to the first codon n-tuple in order to achieve an overall improvement in the translation efficiency of the entire nucleotide sequence. This may be the case in particular if altered positions with a first codon n-tuple that is particularly disadvantageous for expression in the target organism overlap with altered positions with a first codon n-tuple that is favorable for expression in the target organism and synonymous base triplets cannot be selected to increase the relative frequency of the two second codon n-tuples.

[0046] For example, the relative codon n-gram frequency of the second codon n-gram compared to the respective first codon n-gram can be reduced in at least about 1%, 5% or 10% and / or at most about 40%, 30% or 20% of the altered positions.

[0047] In this context, the inventors have further recognized that, in order to improve the soluble protein expression rate of a nucleotide sequence in a target organism, it may be significantly more important to increase a particularly low relative codon n-tuple frequency by means of the method according to the invention, to a single or even a majority of particularly high relative codon n-tuple frequencies. Therefore, in a preferred embodiment, the conditions or goals for optimization provided by the method according to the invention are that the relative codon n-tuple frequency of the first codon n-tuple and the relative codon n-tuple frequency of the second codon n-tuple each have a global minimum in the changed position, wherein the global minimum of the second codon n-tuple is greater than the global minimum of the first codon n-tuple.

[0048] In a further preferred embodiment, the synonymous base triplet is selected so that the relative codon n-tuple frequency of the second codon n-tuple reaches the maximum possible minimum, i.e. the maximum possible global minimum, or is at least no more than 50%, preferably no more than 40% or no more than 30%, preferably no more than 20%, and particularly preferably no more than 10% lower than the maximum possible global minimum. Suitable mathematical approximations and / or optimization methods for finding the maximum possible global minimum are known to those skilled in the art. Here, also different from the technical teachings of previously prevailing codon optimization or codon context optimization, the method according to the present invention mainly considers the global optimization of unfavorable change positions in the nucleotide sequence, which can be identified according to the present invention by low codon n-tuple frequencies, rather than considering reaching local maxima in codon usage or codon pair preference. In this way, proteins that were previously difficult or not able to be expressed in exogenous target organisms using traditional optimization methods can be very reliably heterologously expressed.

[0049] Preferably, the synonymous base triplets are selected so that the average value of the relative codon n-gram frequency of the second codon n-gram reaches a maximum value or is at least not more than 50%, preferably not more than 40% or not more than 30%, preferably not more than 20%, particularly preferably not more than 10% lower than the maximum value that can be achieved. In this way, the optimization method can be used to provide nucleotide sequences that are particularly well adapted to expression in the target organism and can therefore be expressed in the target organism particularly reliably and with a high expression rate of soluble protein.

[0050] It should be understood that for the aforementioned embodiments, the determination of the relative frequency of the first codon n-gram is not necessary, since the realization of the already mentioned optimization criteria already indicates that at least a portion of the second codon n-gram has a higher relative codon n-gram frequency compared to the respective first codon n-gram. In other words, the basic condition according to the present invention is that at least one of the n base triplets from the directly consecutive order of the at least one change position is replaced by a synonymous base triplet, which is selected in a targeted manner to produce at least one second codon n-gram, which, when the mentioned criteria are met, inevitably always meets the requirement that it has a higher relative codon n-gram frequency than the first codon n-gram in the genome or part thereof of the at least one target organism and / or in the genome or part thereof of a virus capable of infecting the at least one target organism. However, the relative frequency of the first codon n-gram can in principle be used as a control parameter for implementing the method according to the present invention, or, for example, for comparing an intermediate or final result of the optimization with an initial state. Furthermore, in these embodiments, the relative frequency of the first codon n-gram may not be discussed, since the optimization target is not arranged based on which initial sequence is started from.

[0051] As explained above many times, the inventors have recognized that it is usually more advantageous for the optimization of nucleotide sequences to focus the method more on the optimization of the change position with low relative codon n-gram frequency, rather than on the increase of medium or already high relative codon n-gram frequency. For this purpose, it is provided in a preferred embodiment of the method according to the invention that the mean value comprises the decreasing weight of the relative codon n-gram frequency of the first and second codon n-grams, which is configured so that the high relative codon n-gram frequency has an effect on the mean value or its calculated value that is too low in proportion compared to the lower relative codon n-gram frequency. On the contrary, the increase of low relative codon n-gram frequency, even slight, can have a greater impact on the mean value than the increase of medium or high relative codon n-gram frequency.

[0052] Of course, the method can also be additionally or alternatively designed such that the relative codon n-gram frequencies each reach a maximum value at at least a portion of the changed positions.

[0053] It should be understood that for n base triplets, codon n-tuples and amino acid n-tuples of change positions, natural number n must be the same natural number.If the change position for example comprises the direct successive order of three base triplets, it also encodes three amino acid sequence parts of the predetermined amino acid sequence, i.e. the first and second codon n-tuples are codon 3-tuples and amino acid n-tuples are amino acid 3-tuples respectively.In any case, each change position, for example at least two change positions may be different in quantity n, or each change position, for example at least two change positions n are selected to be different.In addition, it is stipulated that the n of the change position can be changed during the method, i.e. the change position can be amplified or reduced during the method, or the large change position can be divided into a plurality of small change positions, and vice versa.This can for example be advantageous, so that in the nucleotide sequence region of the coding amino acid n-tuple (seldom or not appearing in the genome of the described a target organism or or a plurality of target organisms or viruses or in its part) use a smaller n with more reliable statistics. For example, it may be advantageous that if the relevant amino acid 3-tuple only occurs rarely, or in extreme cases does not occur at all, the nucleotide sequence portion with three codons is optimized by two overlapping n=2 change positions, rather than by n=3 change positions. Another advantage is that, according to the method of the present invention, by using a larger n, it is likely to exclude nucleotide sequences that are harmful to expression in the target organism, such as restriction sites. Since such sequence motifs usually have no basis in the target organism's own genome, that is, the relative codon n-tuple frequency in the genome of the one or more target organisms is close to zero, the method according to the present invention implicitly leads to their systematic exclusion. For example, such harmful sequences with a length of up to 3n-2 (inclusive) have been automatically excluded from the optimized nucleotide sequence when n is greater than or equal to 3.

[0054] In a preferred embodiment, computer-aided optimization method is used to perform in multiple iteration steps to replace the base triplet with a synonymous base triplet. In this way, it is particularly possible to continuously optimize the nucleotide sequence with a large number of overlapping change positions. In the iteration step, at least one of n base triplets can be replaced by a synonymous base triplet in all change positions or only in a part of the change position. Preferably, in each iteration step, at least one of n base triplets is replaced by a synonymous base triplet in a part of the change position. Preferably, at least in a part of the iteration step, particularly preferably in most of the iteration steps or in each iteration step, only a base triplet is replaced by a synonymous base triplet. This base triplet can be included by a change position or a plurality of overlapping change positions. For example, replacing the base triplets with synonymous base triplets can be iterated so frequently until one of the target criteria already mentioned above is achieved, such as the relative codon n-gram frequency of the second codon n-gram has the largest possible minimum value, the largest possible average value, in particular the largest possible weighted average value, or reaches a maximum value or approaches these values ​​to the degree defined above.

[0055] It should be understood that the method may also include one or more iterative steps that deviate from the respective target criteria in these embodiments, particularly wherein the relative frequency of the second codon n-tuple in one or more of the change positions is at least temporarily reduced compared to the first codon n-tuple due to the iterative step, so as to be able to overcome the local maximum of the minimum or average value that hinders the achievement of the target criteria. It is therefore also provided that, in one or more of the change positions, at least one of the n base triplets may be replaced multiple times by different synonymous base triplets until the target criteria is reached. In particular, there is no provision for determining the change position on a specific second codon n-tuple after performing the iterative step.

[0056] Computer-aided optimization method preferably comprises approximation method, particularly simulated cooling method, also referred to as " simulated annealing ".These methods have been proved to be particularly suitable for and conducive to finding the approximate solution of the nucleotide sequence that is best for the target organism with respect to relative codon n-tuple frequency, especially when for example having 30 or more codons with a large amount of overlapping change positions longer nucleotide sequence when its complexity excludes the complete detection of all possible synonymous base triplets and mathematical optimization method.Certainly, other heuristic approximation methods, for example flooding algorithm or genetic algorithm are possible.Other suitable approximation methods are well known to those skilled in the art.In addition, other computer-aided optimization methods, for example the application based on artificial intelligence (AI) is conceivable.

[0057] In a preferred embodiment of the method, the altered positions together comprise at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40% or at least 50% of the base triplets of the nucleotide sequence encoding the amino acids of the predetermined amino acid sequence. Preferably, the altered positions together comprise at least 60%, at least 70% or at least 80%, particularly preferably at least 90% or at least 95% of the base triplets of the nucleotide sequence encoding the amino acids of the predetermined amino acid sequence. In this way, the method according to the invention ensures a particularly reliable optimization of the nucleotide sequence for expression in the target organism. As described above, the altered positions may also contain base triplets that are not replaced by synonymous base triplets, i.e. it is not required that each base triplet comprised by the altered position is replaced by a synonymous base triplet.

[0058] Preferably, synonymous base triplets are selected such that the relative codon n-gram frequency of the second codon n-gram is higher than the respective first codon n-gram in at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or at least 35% of the changed positions, preferably in at least 40% or at least 45%, preferably in at least 50%, at least 55%, at least 60%, at least 65% or at least 70% of the changed positions, and particularly preferably in at least 75%, at least 80%, at least 85% or at least 90% of the changed positions.

[0059] As used herein, the term "at least one target organism" includes the possibility that the nucleotide sequence is optimized for simultaneous expression of a predetermined amino acid sequence in a plurality of different target organisms. The method according to the invention provides, for example, for this purpose that the relative codon n-gram frequency is derived from the amount of codon n-gram events based on the amount of corresponding amino acid n-gram events in the genomes or parts thereof of the plurality of different target organisms. In this way, optimized nucleotide sequences that are more suitable for heterologous expression in various expression systems can be provided by means of the method of the invention. This is not possible by conventional methods, such as methods based on codon usage or codon adaptation index.

[0060] The at least one target organism may in principle be a predetermined host cell or any predetermined organism suitable for expressing the predetermined amino acid sequence. The host cell may be a prokaryotic or eukaryotic host cell. The host cell may be a host cell suitable for cultivation in a liquid or solid medium. Alternatively, the host cell may be a cell that is part of a multicellular tissue or multicellular organism, such as a plant, animal or human, in particular a transgenic one.

[0061] The host cell can be microbial or non-microbial. The microbial host cell can be a bacterium, yeast or fungal cell. Suitable bacterial host cells include gram-positive bacteria and gram-negative bacteria. Examples of suitable bacterial host cells are bacteria and lactic acid bacteria from Bacillus, Actinomycetis, Escherichia, Streptomyces, such as Lactobacillus, Streptococcus, Lactococcus, Oenococcus, Leuconostoc, Pediococcus, Carnobacterium, Propionibacterium, Enterococcus and Bifidobacterium. Particularly preferred are Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Escherichia coli, Streptomyces coelicolor, Streptomyces clavuligerus, Lactobacillus plantarum and Lactococcus lactis, in particular Escherichia coli.

[0062] Alternatively, the host cell may also be a eukaryotic microorganism, such as a yeast or a fungus, in particular a filamentous fungus. Preferred yeasts as host cells belong to the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces and Yarrowia. Particularly preferred Debaromyces host cells are Saccharomyces cerevisiae and Kluyveromyces lactis.

[0063] According to another preferred embodiment, the host cell of the present invention is a cell of a filamentous fungus. Filamentous fungi include all filamentous forms of the subclasses of Fungi and Oomycetes. The characteristic of filamentous fungi is that the hyphae wall is composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth occurs by hyphal elongation, and carbon degradation is obligate aerobic. The filamentous fungi whose strains can be used as host cells in the present invention include strains of Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Chrysosporium, Talaromyces, Thermoascus, Thielavia, Tolypocladium and Trichoderma, etc. Preferred filamentous fungal species are selected from Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Trichoderma reesei and Penicillium chrysogenum. Examples of suitable host strains are known to those skilled in the art.

[0064] Suitable non-microbial host cells are, for example, mammalian host cells such as hamster cells (e.g., Chinese hamster ovary (CHO) cells; baby hamster kidney (BHK) cells), mouse cells, monkey cells or human cells or cell lines such as HeLa or HEK293; insect cells, such as Drosophila cells or lepidopteran cell lines Hi5, Sf21; plant cells such as cells from tobacco, tomato, potato, rapeseed, cabbage, pea, wheat, corn, rice, Taxus species such as Taxus brevifolia, Arabidopsis species such as Arabidopsis thaliana and Nicotiana species such as Nicotiana tabacum. In addition, non-pathogenic Leishmania are also suitable for protein expression. Such non-microbial cells are particularly suitable for the production of mammalian or human proteins for mammalian or human therapy.

[0065] The predetermined amino acid sequence is preferably a protein or part thereof, which is in particular naturally a eukaryotic protein. The method according to the invention has proven to be particularly advantageous for expressing eukaryotic proteins such as insect, plant or mammalian proteins in bacterial, in particular prokaryotic expression systems such as Escherichia coli.

[0066] In the method variants in which nucleotide sequence is optimized for simultaneous expression of specific amino acid sequences in a variety of different target organisms, different target organisms may have very different genome sizes, wherein large genomes are generally encoded with a protein group with a larger amount of amino acid n-tuples compared to smaller genomes. This may cause the relative codon n-tuple frequency ratio to be too high to be affected by the large genome, so the optimization of nucleotide sequence is inevitably more conducive to expression in target organisms with large genomes, rather than in target organisms with small genomes. For this case, according to the method provisions of the present invention, the relative codon n-tuple frequency comprises a weight dependent on genome, which is for example configured to make the size difference of the genome or its part of different target organisms, particularly the different ranges of coding regions at least partially compensated. In this way, it is ensured that the nucleotide sequence optimized according to the present invention is more suitable for expression in different target organisms.

[0067] Surprisingly, it has been shown that the method according to the invention is not only able to significantly increase the quantitative protein yield in heterologous expression systems compared to conventional optimization methods, but in particular also to significantly increase the proportion of soluble protein in the yield. This is a particular advantage of the method according to the invention, since the dissolved form of the protein generally represents the natural biochemically active state, which is particularly important in the case of high-value proteins for scientific, medical, pharmaceutical and biotechnological purposes. Therefore, a special feature of the method according to the invention is that after the optimization of the nucleotide sequence, the amino acid sequence expressed in the at least one target organism has a greater solubility than before the optimization and / or is present in a greater proportion in dissolved form than before the optimization.

[0068] As has been derived from the above description, the optimization of the nucleotide sequence for expression in the at least one target organism can be performed instead of or in addition to the optimization based on the genome or part thereof of the target organism by means of the genome or part thereof of a virus capable of infecting the at least one target organism. In particular, the virus can include a bacteriophage.

[0069] For this reason, it is important to optimize using multiple or a large number of viral genomes and / or its parts, because a single viral genome and / or viral transcriptome does not usually have the required scope, and therefore does not have the necessary statistical significance for effectively optimizing nucleotide sequences by means of the relative codon n-tuple frequency according to the present invention. Therefore, the inventors combine the genomes and / or transcriptomes of various viruses into a kind of "supergenome" or "supertranscriptome", which is used as the basis for determining the relative codon n-tuple frequency. Preferably, the method in these embodiments comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 150 or at least 200 genomes of various viruses or their corresponding parts.

[0070] The inventors have made use of the knowledge that the genomes of viruses or bacteriophages are naturally already optimized for high-throughput protein expression in infected target organisms. In this context, the inventors have recognized the particular advantage that the genomes of viruses or bacteriophages generally have a reduced base complexity, which results in the mRNA transcribed from the nucleotide sequences optimized according to the invention forming little or no secondary structure. Thus, the optimization method according to the invention is clearly superior in practice to other methods for mRNA secondary structure optimization using artificial algorithms. It has been shown that, using the method according to the invention, the expression rate of amino acid sequences that can be expressed with very good solubility in heterologous systems even without optimization can even be significantly increased again by using the optimization according to the invention of the viral genome.

[0071] Finally, the method may include a step for reducing or excluding nucleotide sequences and / or motifs that are present in the nucleotide sequence and / or are randomly generated by replacing the base triplet in the position and that can adversely affect the expression in the target organism. In some embodiments of the method, such unfavorable nucleotide sequences and / or motifs are at least partially removed from the nucleotide sequence. Non-limiting examples of unfavorable nucleotide sequences and / or motifs include cis-acting mRNA unstable motifs, RNA enzyme splicing sites, ribosome binding sites, repeat elements and restriction enzyme recognition sequences. In addition, for example, the GC content and mRNA secondary structure of the nucleotide sequence transcribed can be considered to further improve the nucleotide sequence for expression in the target organism. However, as explained above, a special advantage is that the nucleotide sequences that are harmful to the expression in the target organism have been inherently excluded for the most part by the method according to the present invention, particularly by the length of the codon n-tuple that n is greater than or equal to 3. In this way, in certain embodiments, additional steps such as the optimization of mRNA secondary structure or GC content, mRNA unstable motifs, ribosome binding sites, repeat elements and / or restriction enzyme recognition sequences can be excluded from the method.

[0072] A subject of the present invention is also the use of the nucleotide sequences optimized according to the above-described method for the production of synthetic DNA and / or for the expression of proteins in a target organism.

[0073] Therefore, another theme of the present invention is nucleic acid molecules, particularly isolated nucleic acid molecules, which comprise the optimized nucleotide sequence obtained by one of the methods described herein. Preferably, the nucleic acid is DNA. In other embodiments, a vector comprising nucleic acid molecules, particularly isolated nucleic acid molecules is provided. Using sequence comparison, the nucleic acid molecules optimized according to the present invention can be clearly distinguished from the conventional optimized sequence. In this respect, the following comparative examples can also be referred to.

[0074] Another subject of the present invention is a recombinant host cell which contains the above-described, in particular isolated, nucleic acid molecule or the above-described vector.

[0075] Therefore, the present invention also relates to a method for expressing a protein, in particular a recombinant protein, in a target organism, comprising providing a nucleotide sequence encoding a protein optimized according to the above method. In addition, the method may comprise one or more of the following steps: synthesizing a nucleic acid molecule comprising an optimized nucleic acid sequence; introducing the nucleic acid molecule into a target organism; and culturing the target organism under conditions enabling expression of the protein by the optimized nucleic acid sequence. Preferably, the expression is carried out at least partially at a temperature of less than or equal to 30° C., less than or equal to 25° C. or less than or equal to 20° C. It has been shown that the nucleotide sequences optimized according to the present invention significantly facilitate heterologous protein expression at relatively low temperatures compared to conventionally optimized nucleotide sequences. In this regard, reference may also be made to the following examples. In this way, the method according to the present invention is particularly suitable for the expression of sensitive high-value proteins, while bringing greater sustainability through energy-saving potential.

[0076] Another subject of the invention is a computer program with a program code. The program code of the computer program is arranged to implement the method according to the above description when the computer program is executed on a computer. The computer program may include an interface with a DNA and / or RNA synthesis device.

[0077] A subject of the invention is also a computer-readable storage medium on which the above-mentioned computer program is stored in computer-readable form.

[0078] Finally, another subject of the invention is a device for optimizing and / or producing nucleotide sequences for expressing a predetermined amino acid sequence in at least one target organism. Here, the device has a computing device configured to perform one of the above methods. The device can be in particular a DNA and / or RNA synthesis device, also referred to as a "DNA / RNA synthesizer".

[0079] Furthermore, it should be understood that preferred and advantageous embodiments of the method according to the invention may also relate to other subjects of the invention where applicable. Therefore, the features disclosed above and below in conjunction with the method according to the invention may also relate to other objects of the invention, and vice versa. In case of doubt, the use of the term "or" represents an "and / or" relationship.

[0080] A brief description of the sequence

[0081] SEQ ID NO: 1 Nucleotide sequence of I. sakaiensis PETase (wild-type sequence), encoding amino acids (AS) 28-290;

[0082] SEQ ID NO:2 Nucleotide sequence of I. sakaiensis PETase with double Strep tag at C-terminus (encoding AS28-290) synthesized after conventional optimization of expression in E. coli according to the prior art (reference);

[0083] SEQ ID NO:3 Nucleotide sequence of I. sakaiensis PETase with a double Strep tag at the C-terminus (encoding AS28-290) produced synthetically after sequence optimization for expression in E. coli according to the present invention (n=2);

[0084] SEQ ID NO:4 Nucleotide sequence of I. sakaiensis PETase with a double Strep tag at the C-terminus (encoding AS28-290) produced synthetically after sequence optimization for expression in E. coli according to the present invention (n=3);

[0085] SEQ ID NO:5 Nucleotide sequence of Arabidopsis thaliana OTP86-DYW domain (AS 826-960) (wild-type sequence);

[0086] SEQ ID NO:6 Nucleotide sequence of Arabidopsis thaliana OTP86-DYW domain (AS 826-960) with double Strep tag and tobacco etch virus (TEV) site at the N-terminus, synthesized after conventional optimization for expression in E. coli (reference) according to the prior art;

[0087] SEQ ID NO:7 Nucleotide sequence of Arabidopsis thaliana OTP86-DYW domain (AS826-960) with double Strep tag and tobacco etch virus (TEV) site at the N-terminus, produced synthetically after optimization of expression in E. coli (n=3) according to the present invention;

[0088] SEQ ID NO:8 Nucleotide sequence of Arabidopsis thaliana OTP86-DYW domain (AS 826-960) with double Strep tag and tobacco etch virus (TEV) site at the N-terminus synthesized using 226 phage genomes optimized for expression in E. coli according to the present invention (n=3)

[0089] SEQ ID NO: 9 Citrin protein sequence (encoding AS1-239), as a predetermined amino acid sequence for expression in H. sapiens;

[0090] SEQ ID NO: 10 Citrin nucleotide sequence (encoding AS1-239) with a FLAG tag and a double Strep tag at the N-terminus, and a Kozak sequence (GCCACC) flanked by a 5'-flank according to the prior art, generated synthetically after conventional optimization for expression in H. sapiens (reference);

[0091] SEQ ID NO: 11 Citrin nucleotide sequence (encoding AS1-239) with a FLAG tag and a double Strep tag at the N-terminus, synthesized after sequence optimization (n=3) for expression in H. sapiens according to the present invention, and 5'-flanked by a Kozak sequence (GCCACC);

[0092] SEQ ID NO: 12 Nucleotide sequence of H. sapiens STING1 ER exit protein 1 ("STEEP1") encoding AS1-222 (wild type);

[0093] SEQ ID NO: 13 Nucleotide sequence of H. sapiens STEEP1 (encoding AS1-222) with a FLAG tag and a double Strep tag at the N-terminus, 5'-flanked by a Kozak sequence (GCCACC), generated synthetically after conventional optimization for expression in H. sapiens according to the prior art (reference);

[0094] SEQ ID NO: 14 Nucleotide sequence of H. sapiens STEEP1 (encoding AS1-222) with a FLAG tag and a double Strep tag at the N-terminus, synthesized after sequence optimization for expression in H. sapiens according to the present invention (n=3), and 5'-flanked by a Kozak sequence (GCCACC);

[0095] SEQ ID NO: 15 Nucleotide sequence encoding the H. sapiens nitric oxide synthase interacting protein (NOSIP) of AS1-304 (wild type);

[0096] SEQ ID NO: 16 Nucleotide sequence of H. sapiens NOSIP (encoding AS1-304) with a FLAG tag and a double Strep tag at the N-terminus, generated synthetically after conventional optimization using the codon adaptation index and optimization of the secondary structure of mRNA expressed in H. sapiens (reference) according to the prior art, and 5'-flanked by a Kozak sequence (GCCACC);

[0097] SEQ ID NO: 17 Nucleotide sequence of H. sapiens NOSIP with a FLAG tag and a double Strep tag at the N-terminus (encoding AS1-304), synthesized according to WO 2020 / 024917 A1 after conventional optimization for expression in Homo sapiens (reference) according to the prior art, and 5'-flanked by a Kozak sequence (GCCACC);

[0098] SEQ ID NO: 18 Nucleotide sequence of H. sapiens NOSIP (encoding AS1-304) with a FLAG tag and a double Strep tag at the N-terminus, synthesized after sequence optimization for expression in H. sapiens according to the present invention (n=3), and 5'-flanked by a Kozak sequence (GCCACC);

[0099] SEQ ID NO: 19 protein sequence of EqFP611 (AS1-231), as a predetermined amino acid sequence for expression in S. elongatus;

[0100] SEQ ID NO: 20 Nucleotide sequence of EqFP611 (encoding AS1-231) with a double Strep tag at the N-terminus, flanked by the restriction site NdeI at the 5'-flank and the transcription terminator and KpnI restriction site at the 3'-flank, generated synthetically according to the prior art after conventional optimization for expression in S. elongatus (reference);

[0101] SEQ ID NO:21 Nucleotide sequence of EqFP611 (encoding AS1-231) with a double Strep tag at the N-terminus, 5'-flanked by the restriction site NdeI, and 3'-flanked by the transcription terminator and KpnI restriction site, produced synthetically according to the present invention after optimization of expression in S. elongatus (n=3). BRIEF DESCRIPTION OF THE DRAWINGS

[0103] The present invention is explained in more detail below using embodiments with reference to the accompanying drawings. The present invention should not be limited to the embodiments in any case. It is shown in the drawings:

[0104] Figure 1 A flow chart of a schematic sequence of embodiments of the method according to the invention;

[0105] Figure 2SDS-PAGE (A) and quantitative evaluation (B) of heterologous expression of Ideonella sakaiensis PETase in Escherichia coli at 20°C;

[0106] Figure 3 SDS-PAGE (A) and quantitative evaluation (B) of heterologous expression of Ideonella sakaiensis PETase in Escherichia coli at 30°C;

[0107] Figure 4 Graphic representation of the relative frequencies of the first and second codon triplet in a portion of the wild-type sequence of Arabidopsis thaliana OTP86-DYW (A) and the nucleotide sequence optimized according to the present invention (B);

[0108] Figure 5 SDS-PAGE (A) and quantitative evaluation (B) of Arabidopsis thaliana OTP86-DYW heterologously expressed in Escherichia coli at 17°C;

[0109] Figure 6 Western blot of heterologous expression of Citrin in HeLa cells (A) and quantitative evaluation of Western blot (B) and fluorescence (C);

[0110] Figure 7 Western blot of Citrin heterologous expression in HEK293 cells (A) and quantitative evaluation of Western blot (B) and fluorescence (C);

[0111] Figure 8 Western blot (A) and quantitative evaluation (B) of H. sapiens STEEP1 expression in HeLa cells;

[0112] Fig. 9 Western blot (A) and quantitative evaluation (B) of H. sapiens STEEP1 expression in HEK293 cells;

[0113] Fig.10 Western blot (A) and quantitative evaluation (B) of H. sapiens NOSIP expression in HeLa cells with SEQ ID NO: 16 (reference) and SEQ ID NO: 18;

[0114] Fig.11Western blot (A) and quantitative evaluation (B) of H. sapiens NOSIP expression in HeLa cells with SEQ ID NO: 17 (reference) and SEQ ID NO: 18;

[0115] Fig.12 Western blot (A) and quantitative evaluation (B) of H. sapiens NOSIP expression in HEK293 cells with SEQ ID NO: 16 (reference) and SEQ ID NO: 18.

[0116] Detailed description of embodiments

[0117] The invention is explained in more detail below using embodiments with reference to the accompanying drawings. The invention should not be limited to the embodiments in any case.

[0118] Comparative Example 1: Heterologous Expression of Ideonella sakaiensis PET Hydrolase (PETase) in Escherichia coli

[0119] In a first comparative experiment, the nucleotide sequence of the PETase of I. sakaiensis (which encodes amino acid positions 28-290 (molecular weight 27.9 kDa) (SEQ ID NO: 1) was optimized for heterologous expression in Escherichia coli according to the method of the present invention and according to the method of WO 2020 / 024917A1 (as a reference).

[0120] For the expression of PETase in Escherichia coli (E. coli) as a target organism, three pET28a expression plasmids were purchased from Genscript, which encode the amino acid sequence of PETase with a double Strep tag at the C-terminus under the inducible T7 promoter. The N-terminus of the amino acid sequence is supplemented with Met (start codon) and amino acids Ala and Ser. One of the plasmids each contains a nucleotide sequence encoding PETase with a double Strep tag after optimization by the manufacturer's optimization service according to the method of WO 2020 / 024917A1 (SEQ ID NO: 2), after optimization according to the method of the present invention with n = 2 (SEQ ID NO: 3) and after optimization according to the method of the present invention with n = 3 (SEQ ID NO: 4), wherein for the determination of the relative codon n-tuple frequency according to the present invention, the protein-coding part of the Escherichia coli (E. coli) genome is used as a basis.

[0121] In this article, Figure 1A schematic sequence of an exemplary embodiment of a PET enzyme from I. sakaiensis is shown in a computer-implemented embodiment according to method 100 of the present invention. Block 102 represents the nucleotide sequence to be optimized being input into a computer. Of course, an amino acid sequence can also be input and then translated into a nucleotide sequence to be optimized. In the present embodiment, the entire encoding sequence is continuously divided into change positions, i.e., there is a codon offset between adjacent change positions. In this way, based on the total number of amino acids N of a predetermined amino acid sequence, N-2 codon 3-tuples or N-1 codon 2-tuples are generated. In this embodiment, the change position is therefore determined by inputting the nucleotide sequence to be optimized.

[0122] Block 104 establishes a list of Escherichia coli (E. coli) protein coding genes through a DNA sequence database. A suitable DNA sequence database is, for example, GenBank (Nucleic Acids Research 41, 2013, D36-42). A suitable basis for linking sequence data with genetic and functional information is a reference sequence (RefSeq) database (NCBI Manual, 2nd Edition, Chapter 18: The Reference Sequence (RefSeq) Database, Bethesda (MD), National Center for Biotechnology Information, USA, 2013). On this basis, the absolute frequency of each possible combination of codon n-tuples and each possible combination of amino acid n-tuples in the protein coding gene is then determined, wherein n is n=2 in one embodiment and n=3 in one embodiment.

[0123] Using this information, the quotient of the absolute frequency of each possible codon n-gram and the absolute frequency of the corresponding amino acid n-gram it encodes is determined in block 106 and included in further methods.

[0124] As an optional step, undesirable nucleotide sequences are entered in block 108, such as the TATA box "TATAA" or the ribosome binding site "AGGAGG" known to those skilled in the art, which can affect expression in E. coli. Other undesirable sequence motifs are AAAAAA, TTTTT, AGGAGGT, TATAAA, ATCTGTT, GGAGGT, and GGGTGGT.

[0125] By means of a computer-assisted optimization method using a "simulated annealing" algorithm, base triplets in the altered positions of the wild-type sequence are then successively replaced in a block 110 in a large number of interaction steps 112 until the relative codon n-gram frequencies of all codon n-grams in the nucleotide sequence have the largest possible weighted average while minimizing the number of undesired nucleotide sequences. Only the start codon is excluded from the optimization, although in principle the start codon and / or the stop codon can also be included in the optimization.

[0126] The weight W of a codon n-tuple with relative frequency P is W = -1 / P for P greater than 0.0001 and W = -10000 for P less than or equal to 0.0001. To obtain the weighted average F W , summing up W for all codon n-tuples and then dividing by their number L-(n-1), where L is the number of codons in the nucleotide sequence included in the optimization (here L=864). The weight W=-1 / P is decreasing and, due to very high values ​​for very small P, is lowered by -10000.

[0127] In this embodiment, the weighted average is additionally calculated using the representation of the occurrence of undesirable sequence motifs. For each undesirable sequence motif, a value F is determined. E , which corresponds to the number of undesirable sequence motifs in the nucleotide sequence to be optimized multiplied by -1. In addition, the value F E The weighting can be performed with a single value r, wherein in the present exemplary embodiment r=0.035 is selected for the embodiment with n=2 and r=0.058 is selected for the embodiment with n=3.

[0128] In order to optimize the nucleotide sequence, the "simulated annealing" algorithm was used using the function F = F W +r·F E Find the maximum value of the weighted average so that F W The maximum possible value is obtained through optimization, and at the end of the optimization, the number of undesirable sequences contained in the nucleotide sequence is as small as possible.

[0129] After optimization, the weighted average value of (SEQ ID NO: 3) in the embodiment of n=2 of the present invention is F W =-5.1. A similar comparison was performed to obtain the weighted average F of the relative codon 2-tuple frequencies in the wild type (SEQ ID NO: 1). W =-13.2, and according to the weighted average F in the reference sequence (SEQ ID NO: 2) of WO 2020 / 024917 A1 W = -7.3. After optimization according to the present invention (SEQ ID NO: 4), the weighted average value in the embodiment of n = 3 is FW =-10.4. A similar comparison was performed to obtain the weighted average F of the relative codon 3-tuple frequencies in the wild type (SEQ ID NO: 1). W =-526.8, and according to the weighted average F in the reference sequence (SEQ ID NO: 2) of WO 2020 / 024917 A1 W =-336.4.

[0130] After reaching the target standard, the optimized nucleotide sequences SEQ ID NO: 3 and SEQ ID NO: 4 are output in block 114 and then synthesized accordingly.

[0131] It can be immediately seen from the sequence table that the nucleotide sequence optimized according to the present invention is significantly different from the wild-type sequence even at the nucleotide level. The wild-type sequence (SEQ ID NO: 1) has only 82.3% identical nucleotides compared to the sequence optimized in the n = 2 embodiment of the method according to the present invention (SEQ ID NO: 3), and only 82.9% identical nucleotides compared to the sequence optimized in the n = 3 embodiment of the method according to the present invention (SEQ ID NO: 4). There are also very obvious differences between the nucleotide sequence optimized according to the method of the present invention and the sequence optimized according to the prior art. The sequence optimized as a reference according to WO 2020 / 024917 A1 (SEQ ID NO: 2) has only 89.2% identical nucleotides compared to the sequence optimized in the n = 2 embodiment of the method according to the present invention (SEQ ID NO: 3), and only 84.7% identical nucleotides compared to the sequence optimized in the n = 3 embodiment of the method according to the present invention (SEQ ID NO: 4). From the perspective of the predetermined amino acid sequence, these huge differences are surprising from a professional point of view, and indicate that the optimization method according to the present invention leads to completely different results from the prior art method.

[0132] Escherichia coli (E.coli) strain BL21 is used as expression system (New England Biolabs GmbH, Frankfurt am Main, Germany). For this reason, three different equal portions of each of the expression host were transformed with 0.1 μg DNA by electroporation with one of the expression plasmids, and spread onto the LB agar plate containing 100 mg / mL kanamycin. The plate was incubated overnight at 37 ° C. Pre-cultures were prepared in 50 mL LB substratum containing 100 mg / mL kanamycin by multiple bacterium colonies from each plate, and incubated overnight at 37 ° C on shaking tables at 180 rpm.

[0133] Expression cultures were prepared from 1 mL of each preculture and 99 mL of TB medium. Expression of recombinant PETase in the culture was induced at an OD600 of 0.6 by adding IPTG at a final concentration of 1 mM. Expression was carried out at 20°C for 14 hours in one variant and at 30°C for 5 hours in another variant.

[0134] Before harvesting cells, the OD600 of the expression culture was determined, and the same amount of cells were harvested from each culture to normalize protein yield according to cell mass. The cell pellet was dissolved in 10 mL of buffer A (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM DTT) and ultrasonically broken. The cell lysate was then centrifuged at 20,000 g for one hour to separate insoluble cell components into a precipitate.

[0135] The supernatant with the soluble fraction was mixed in an Eppendorf tube with 200 μL of Streptactin beads (IBA Lifesciences, The beads were washed twice in Eppendorf tubes with 1 mL of buffer A by centrifugation and removal of the supernatant. The bound proteins were eluted with 200 μL of buffer A containing 10 mM desthiobiotin.

[0136] The identity of the protein was verified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The amount of protein in the SDS-PAGE gel bands was quantified using ImageJ software (National Institutes of Health, USA). In addition, the protein concentration in each supernatant was determined using Bradford assay (Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer's instructions.

[0137] The results are as follows Figure 2 and Figure 3 shown. Figure 2A shows an image of SDS-PAGE analysis of expression at 20°C. Lane 1 contains a size marker, lane 2 contains the expression product of the plasmid inserted with SEQ ID NO: 2 (reference), lane 3 contains the expression product of the plasmid inserted with SEQ ID NO: 3, and lane 4 contains the expression product of the plasmid inserted with SEQ ID NO: 4. The band at 30.4 kDa, which corresponds to the molecular weight of the protein including the double Strep tag, proves that all three plasmids successfully expressed the PETase. However, it can be clearly seen from the intensity of the band that the plasmid with the nucleotide sequence SEQ ID NO: 3 optimized according to the present invention (where n=2 was selected for optimization) produces a significantly increased protein yield compared to the plasmid with the conventionally optimized nucleotide sequence SEQ ID NO: 2. The plasmid with the nucleotide sequence SEQ ID NO: 4 optimized according to the present invention (where n=3 was selected for optimization) generally produces the highest yield of recombinant PETase.

[0138] Figure 2 The bar graph shown in B shows the relative protein yield of soluble PETase depending on the nucleotide sequence used in each case, wherein the quantitatively determined protein amounts were each normalized to the protein amount from a reference experiment with SEQ ID NO: 2. The shaded bars show the quantitative results using SDS-PAGE, the white bars show the quantitative results using Bradford assay. The quantification shows that the optimization of the PETase nucleotide sequence according to the invention with n=2 doubles the soluble protein yield compared to the reference optimization, while the optimization according to the invention with n=3 even increases the protein yield to about six times.

[0139] Figure 3 A shows an SDS-PAGE analysis image of expression at 30°C. Lane 1 contains a size marker, lane 2 contains the expression product of the plasmid inserted with SEQ ID NO:2 (reference), and lane 3 contains the expression product of the plasmid inserted with SEQ ID NO:4. Here, the band at 30.4 kDa proves that both plasmids also successfully expressed PETase. Based on the band intensity, it can be seen that the plasmid with the nucleotide sequence SEQ ID NO:4 optimized according to the present invention produces a higher protein yield than the plasmid with the reference sequence SEQ ID NO:2.

[0140] Figure 3B shows again the relative protein yield of soluble PETase depending on the nucleotide sequence used in each case, wherein the quantitatively determined protein amounts were normalized to the protein amounts of the reference experiment with SEQ ID NO: 2. The shaded columns show the quantitative results using SDS-PAGE, the white columns show the quantitative results using the Bradford assay. The quantitative analysis shows that the expression of the PETase using the nucleotide sequence SEQ ID NO: 4 optimized according to the present invention is more than doubled compared to the sequence optimized according to WO 2020 / 024917 A1.

[0141] Such a significant improvement in the heterologous protein expression rate caused by the method for optimizing protein encoding nucleotide sequences according to the present invention compared to the current state of the art was unexpected by a person skilled in the art. Surprisingly, even the lowest parameter n=2 specified led to an increase in expression rate of almost 200% compared to the prior art, thus clearly demonstrating the superiority of the optimization method according to the present invention compared to established concepts.

[0142] Comparative Example 2: Heterologous Expression of Arabidopsis thaliana OTP86-DYW in Escherichia coli

[0143] In another comparative experiment, the nucleotide sequence of the OTP86-DYW domain at amino acid positions 826-960 of Arabidopsis thaliana (SEQ ID NO: 5) was optimized for heterologous expression in Escherichia coli according to the method of the present invention and according to the method of WO 2020 / 024917 A1 (as a reference). The OTP86-DYW domain is a sensitive plant protein that is known to be difficult to express in heterologous systems.

[0144] To express the OTP86-DYW domain in Escherichia coli as the target organism, three pET41 expression plasmids were cloned, which contained the amino acid sequence 826-960 of the OTP86-DYW domain with a double Strep tag and a TEV protease cleavage site as an insert under an inducible T7 promoter. Met (start codon) and Gly were also supplemented to the N-terminus of the amino acid sequence. The insert was purchased from Genscript. One of the plasmids, after optimization by the manufacturer's optimization service according to the method of WO 2020 / 024917A1 (SEQ ID NO: 6), after optimization according to the method of the invention with parameter n=3 (SEQ ID NO: 7) (wherein for determining the relative codon 3-tuple frequency according to the invention, the coding part of the Escherichia coli (E. coli) genome is taken as the basis), and after optimization according to the method of the invention with n=3 (SEQ ID NO: 8) (wherein for determining the relative codon 3-tuple frequency according to the invention, the coding part of the genome of the following virus or bacteriophage capable of infecting E. coli is taken as the basis) contains the OTP86-DYW encoding nucleotide sequence:

[0145] Enterobacteria phage 13a, Enterobacteria phage 285P, Enterobacteria phage 933W, Enterobacteria phage 9g, Enterobacteria phage BA14, Enterobacteria phage BP-4795, Enterobacteria phage Bp7, Enterobacteria phage EcoDS1, Enterobacteria phage G4, Enterobacteria phage GA, Enterobacteria phage GEC-3S, Enterobacteria phage HK106, Enterobacteria phage HK140, Enterobacteria phage HK225, Enterobacteria phage ID2 Moscow / ID / 2001, Enterobacteria phage IME08, Enterobacteria phage IME10, Enterobacteria phage If1, Enterobacteria phage Ike, Enterobacteria phage J8-65, Enterobacteria phage JS10, Enterobacteria phage JenK1, Enterobacteria phage JenP1, Enterobacteria phage JenP2, Enterobacteria phage K1F, Enterobacteria phage M, Enterobacteria phage MS2, Enterobacteria phage MX1, Enterobacteria phage P4, Enterobacteria phage P88, Enterobacteria phage PRD1, Enterobacteria phage Phi1, Enterobacteria phage RB27, Enterobacteria phage RB49, Enterobacteria phage RB51, Enterobacteria phage RB68, Enterobacteria phage Bacteriophage RB69, Enterobacteriaceae phage SP, Enterobacteriaceae phage ST104, Enterobacteriaceae phage Sf101, Enterobacteriaceae phage SfI, Enterobacteriaceae phage SfV, Enterobacteriaceae phage St-1, Enterobacteriaceae phage T3, Enterobacteriaceae phage T7, Enterobacteriaceae phage UAB_Phi20, Enterobacteriaceae phage UAB_Phi78, Enterobacteriaceae phage VT2-Sakai, Enterobacteriaceae phage VT2phi_272, Enterobacteriaceae phage WA13, Enterobacteriaceae phage YYZ-2008, Enterobacteriaceae phage alpha3, Enterobacteriaceae phage cdtI, Enterobacteriaceae phage fd, Enterobacteriaceae phage fiAA91-ss, Enterobacteriaceae phage mEp043 c-1, Enterobacteriaceae phage mEp235, Enterobacteriaceae phage mEp237, Enterobacteriaceae phage mEp460, Enterobacteriaceae phage phi80, Enterobacteriaceae phage phi92, Enterobacteriaceae phage phiEcoM-GJ1, Enterobacteriaceae phage phiP27, Enterobacteriaceae phage vB_EcoM_VR5, Enterobacteriaceae phage vB_EcoP_ACG-C91, Enterobacteriaceae phage vB_EcoS_NBD2, Enterobacteriaceae phage vB_EcoS_Rogue1, Enterobacteriaceae phage vB_KleM-RaK2, Escherichia coli phage (Escherichia phage)121Q, E. coli phage 172-1, E. coli phage 4MG, E. coli phage 64795_ec1, E. coli phage ADB-2, E. coli phage APCEc01, E. coli phage AR1, E. coli phage Av-05,Klebsiella globulin Bp4, Klebsiella globulin CAjan, Klebsiella globulin CICC80001, Klebsiella globulin D108, Klebsiella globulin EB49, Klebsiella globulin EC6, Klebsiella globulin ECBP1, Klebsiella globulin ECBP2, Klebsiella globulin ECBP5, Klebsiella globulin ECML-117, Klebsiella globulin ECML-134, Klebsiella globulin EC ML-4, E. coli EK99P-1, E. coli Envy, E. coli FFH2, E. coli FV3, E. coli Gluttony, E. coli HK446, E. coli HK542, E. coli HK544, E. coli HK578, E. coli HK629, E. coli HK630, E. coli H K633, Klebsiella globulin HK639, Klebsiella globulin HK75, Klebsiella globulin HX01, Klebsiella globulin HY01, Klebsiella globulin HY02, Klebsiella globulin HY03, Klebsiella globulin IME11, Klebsiella globulin JES2013, Klebsiella globulin JH2, Klebsiella globulin JS98, Klebsiella globulin JSE, Klebsiella globulin K1-dep(1), Klebsiella globulin K1-dep(4), Klebsiella globulin KBNP1711, Klebsiella globulin LM33_P1, Klebsiella globulin Lw1, Klebsiella globulin MX01, Klebsiella globulin Min27, Klebsiella globulin NJ01, Klebsiella globulin P13374, Klebsiella globulin P483, Klebsiella globulin P694, Klebsiella globulin PA2, Klebsiella globulin PBECO 4, Klebsiella globulin PE3-1, Klebsiella globulin PhaxI, Klebsiella globulin Pollock, Klebsiella globulin QL01, Klebsiella globulin RB3, Klebsiella globulin SUSP1, Klebsiella globulin SUSP2, Klebsiella globulin Seurat, Klebsiella globulin Stx2 II, L. largeum bacterium TL-2011b, L. largeum bacterium TL-2011c, L. largeum bacterium UFV-AREG1, L. largeum bacterium V5, large Bacterium bacillus major WG01, Bacterium major bacterium YD-2008.s, Bacillus major e4 / 1c, Bacillus major ime09, Bacillus major mE p234, E. coli bacterium mEpX1, E. coli bacterium mEpX2, E. coli bacterium phAPEC8, E. coli bacterium phi191, E. coli bacterium phiK, E. coli bacterium phiKT, E. coli bacterium phiV10, E. coli bacterium pro147, E. coli bacterium pro483,Escherichia coli phage slur01, Escherichia coli phage slur02, Escherichia coli phage slur05, Escherichia coli phage slur14, Escherichia coli phage slur16, Escherichia coli phage vB_EcoM-UFV13, Escherichia coli phage vB_EcoM-VpaE1, Escherichia coli phage vB_EcoM-ep3, Escherichia coli phage vB_EcoM_112, Escherichia coli phage vB_EcoM_ACG-C40, Escherichia coli phage vB_EcoM_AYO145A, Escherichia coli phage vB_EcoM_Alf5, Escherichia coli phage vB_EcoM_E CO1230-10, Escherichia coli phage vB_EcoM_JS09, Escherichia coli phage vB_EcoM_PhAPEC2, Escherichia coli phage vB_EcoM_VR20, Escherichia coli phage vB_EcoM_VR25, Escherichia coli phage vB_EcoM_VR26, Escherichia coli phage vB_EcoM_VR7, Escherichia coli phage vB_EcoP_24B, Escherichia coli phage vB_EcoP_G7C, Escherichia coli phage vB_EcoP_GA2A, Escherichia coli phage vB_EcoP_PhAPEC5, Escherichia coli phage vB_EcoP_PhAPE C7, Escherichia coli phage vB_EcoP_SU10, Escherichia coli phage vB_EcoS_AHP42, Escherichia coli phage vB_EcoS_AHS24, Escherichia coli phage vB_EcoS_AKS96, Escherichia coli phage vB_EcoS_FFH1, Escherichia coli phage vB_Eco_ACG-M12, Escherichia coli phage wV7, Escherichia coli phage wV8, Escherichia coli virus 186, Escherichia coli virus AKFV33, Escherichia coli virus CBA120, Escherichia coli virus DT57C, Escherichia coli virus EPS7, Escherichia coli virus HK022, Escherichia coli virus HK97, E. coli virus I22, E. coli virus JL1, E. coli virus K1-5, E. coli virus K1E, E. coli virus Lambda, E. coli virus M13, E. coli virus Mu, E. coli virus N15, E. coli virus N4, E. coli virus P1, E. coli virus P2, E. coli virus RB16, E. coli virus RB32, E. coli virus Rtp, E. coli virus SSL2009a, E. coli virus T1, E. coli virus T4, E. coli virus T5, E. coli virus TLS, E. coli virus Wphi, E. coli virus phiEco32, E. coli virus phiX174. The viral genome was retrieved from the Refseq database on May 31, 2019.

[0146] In addition, the experimental implementation of sequence optimization according to the present invention is carried out as described in Comparative Example 1. For the optimization based on phage genome (its cumulative size is significantly smaller than E. coli genome), the relative codon 3-tuple frequency P is calculated as the midpoint of the Clopper-Pearson confidence interval, wherein the confidence level is 95%. In addition, the undesirable sequence motifs in the present embodiment are AAAAAA, TTTTT, AGGAGGT, TATAAA, ATTCGTT, GGAGGT, GGTGGT, CCATGG and AAGCTT, and the number of optimizations based on the E. coli genome and based on the phage genome is weighted by r=0.06. In the present embodiment, the number L of codons in the nucleotide sequence included in the optimization is 501, because the start codon and the subsequent glycine codon are not included in the optimization. Expression is carried out at 17°C, wherein buffer A does not contain DTT.

[0147] After optimization, the weighted average value of (SEQ ID NO: 7) in the embodiment of n=3 based on the E. coli genome according to the present invention is F W =-7.1. A similar comparison was calculated to obtain the weighted average F based on the relative codon 3-tuple frequencies in the wild type (SEQ ID NO: 5) of the E. coli genome. W =-2533.5, and for the F according to the reference sequence (SEQ ID NO: 6) of WO 2020 / 024917 A1 W = -451.4. The weighted average F of (SEQ ID NO: 8) after optimization according to the present invention in an embodiment where n=3 is based on a viral or bacteriophage genome capable of infecting E. coli is W =-9.2. A similar comparison was performed to obtain the weighted average F based on the relative codon 3-tuple frequencies in the wild type (SEQ ID NO: 5) of the viral or phage genome. W =-56.8, and according to the reference sequence of WO 2020 / 024917 (SEQ ID NO: 6) is F W =-37.2.

[0148] The sequence listing also clearly shows that there are already significant differences at the nucleotide level between the wild-type nucleotide sequence and the nucleotide sequence optimized according to the method of the present invention. For example, the wild-type sequence (SEQ ID NO: 5) has only 72.7% identical nucleotides compared to the optimized sequence (SEQ ID NO: 7) from the n=3 embodiment of the method of the present invention (wherein the coding part of the E. coli genome is used as the basis for determining the relative codon 3-tuple frequency according to the present invention), and has only 76.9% identical nucleotides compared to the optimized sequence (SEQ ID NO: 8) from the n=3 embodiment of the method of the present invention (wherein the coding part of the genome of a virus or bacteriophage capable of infecting E. coli is used as the basis for determining the relative codon 3-tuple frequency according to the present invention).

[0149] There are also very obvious differences between the nucleotide sequences optimized according to the method of the present invention and the sequences optimized according to the prior art. The sequence optimized according to the prior art as a reference according to WO 2020 / 024917 A1 (SEQ ID NO: 6) has only 85.2% identical nucleotides compared to the sequence optimized according to the method of the present invention using the Escherichia coli (E. coli) genome (SEQ ID NO: 7), and has only 74.6% identical nucleotides compared to the optimized sequence (SEQ ID NO: 8) using the genome of a virus or bacteriophage capable of infecting E. coli (E. coli) according to the method of the present invention.

[0150] Figure 4 The relative frequencies of the first codon 3-tuple in the wild-type sequence SEQ ID NO:5 (A) of OTP86-DYW and the second codon 3-tuple in the nucleotide sequence SEQ ID NO:7 (B) optimized according to the present invention, which correspond to the sequence portion of the nucleotide position 211-330 in the sequence list, are shown. The sequence portion shown contains codon numbers 71 to 110 of OTP86-DYW. Each of three adjacent codons forms a change position with a codon 3-tuple, wherein the sequence portion shown is continuously divided into 38 change positions or codon 3-tuples, which are referred to herein as n1 to n2. 38 . There is a shift of one codon between successive change positions. In the figure, for each codon triplet (x-axis) the corresponding relative frequency (expressed in percentage) (y-axis) is specified by a horizontal line, by which the respective codon triplet encodes the corresponding amino acid triplet in the protein coding gene of E. coli (E. coli). The vertical lines each show the span of the relative frequencies of all codon triplet considered for a particular change position and encoding the corresponding amino acid n-tuple of the change position.

[0151] It can be seen that the method according to the invention leads to a significant increase in the relative codon 3-tuple frequency in most of the positions shown for the changes. 25 In addition, in each of the altered positions shown, at least one of the base triplets was replaced by a synonymous base triplet; in most cases, two or three base triplets were replaced by synonymous base triplets in the altered positions to optimally increase the relative frequency of the second codon triplet. It can also be seen that only in a portion of the altered positions, the second codon triplet each corresponds to the codon triplet with the highest relative frequency in E. coli. In addition, for example, in the altered position n 35 In the present invention, the second codon 3-tuple is formed, and its relative frequency is lower than the original first codon 3-tuple, so that the relative frequency of the more critical first codon 3-tuple in other change positions can be improved. In this way, the maximum possible weighted average of relative codon 3-tuple frequencies can be obtained.

[0152] The results of related heterologous expression in Escherichia coli (E. coli) are as follows Figure 5 shown. Figure 5 A shows an SDS-PAGE analysis image of expression at 17°C. Lane 1 contains a size marker, lane 2 contains the expression product of the plasmid inserted with SEQ ID NO:6 (reference), lane 3 contains the expression product of the plasmid inserted with SEQ ID NO:7, and lane 4 contains the expression product of the plasmid inserted with SEQ ID NO:8. The band at 19.5 kDa proves that all three plasmids successfully expressed the OTP86-DYW domain. Comparison of the band intensities has shown that the plasmids with the nucleotide sequences SEQ ID NO:7 and SEQ ID NO:8 optimized according to the present invention produce significantly higher protein yields of the recombinant OTP86-DYW domain than the plasmid with the conventional optimized nucleotide sequence SEQ ID NO:6.

[0153] Figure 5 The bar graph shown in B shows the relative protein yield of the soluble OTP86-DYW domain depending on the nucleotide sequence used in each case, wherein the quantitatively determined protein amounts were each normalized to the protein amount from a reference experiment with SEQ ID NO: 6. The shaded bars show the quantitative results using SDS-PAGE, the white bars show the quantitative results using photometric UV absorption measurement (Nanodrop) at 260 and 280 nm. Quantification demonstrated that the optimization of the nucleotide sequence according to the invention with parameter n=3 based on the transcriptome of E. coli resulted in almost three times the soluble protein yield compared to the reference optimization, while the protein yield of the optimization according to the invention with parameter n=3 based on the genome of a virus or bacteriophage was almost doubled.

[0154] Compared with the current prior art, such a significant improvement in the heterologous protein expression rate using the method for optimizing protein encoding nucleotide sequences according to the present invention is unexpected by those skilled in the art.

[0155] Comparative Example 3: Heterologous expression of citrin in H. sapiens (HeLa) cell culture

[0156] In another comparative experiment, the nucleotide sequence of the fluorescent protein Citrin (SEQ ID NO: 9) was optimized for heterologous expression in human Hela cells as a target organism according to the method of the present invention and according to a conventional method based on codon adaptation index and local mRNA secondary structure optimization (as a reference). Citrin is a variant of the green fluorescent protein (GFP) from Aquaeoria victoria, which is commonly used in reporter gene detection and fluorescence microscopy.

[0157] In order to express Citrin in HeLa cells, two pTwist CMV expression plasmids were purchased from Twist Bioscience (San Francisco, CA, USA), which encode the amino acid sequence of Citrin with a FLAG tag and a double Strep tag at the N-terminus under a constitutive cytomegalovirus promoter. The N-terminus of the amino acid sequence is supplemented with Met (start codon) and the amino acid Ala. A Kozak sequence is inserted before the start codon. One of the plasmids contains the Citrin encoding nucleotide sequence (SEQ ID NO: 10) with FLAG and double Strep tags after optimization by the manufacturer's method as a reference. The other plasmid contains the Citrin encoding nucleotide sequence (SEQ ID NO: 11) with FLAG and double Strep tags after optimization according to the method of the present invention (n = 3), wherein in order to determine the relative codon n-gram frequency according to the present invention, the protein-encoding portion of the H. sapiens genome is used as a basis.

[0158] In addition, the experimental implementation of sequence optimization according to the present invention was carried out basically as described in Comparative Example 1. Similar to the optimization of E. coli as a host, for H. sapiens, as an optional step in block 108, an undesirable sequence motif such as the TATA box TATAAA is input, which is known to those skilled in the art to affect expression in H. sapiens. Other undesirable sequence motifs are ATTTA, GCCACC, GCCGCC, AATAAA and ATTAAA, each of which is weighted by r=0.62. The number of codons L included in the optimization in the nucleotide sequence in this embodiment is 819, and the Kozak sequence at the 5'-end is not optimized.

[0159] It can be immediately seen from the sequence listing that the nucleotide sequence optimized according to the invention (SEQ ID NO: 11) is already significantly different from the sequence optimized according to the prior art (SEQ ID NO: 10) even at the nucleotide level. The sequence optimized according to the prior art (SEQ ID NO: 10) has only 81.4% of identical nucleotides compared to the sequence optimized according to the method of the invention in the embodiment with n=3 (SEQ ID NO: 11). These huge differences are surprising from a professional point of view in view of the predetermined amino acid sequence and indicate that the optimization method according to the invention also leads to completely different results for the optimization of mammalian cells (here H. sapiens) than the methods according to the current prior art.

[0160] After optimization, the weighted average value of (SEQ ID NO: 11) in the embodiment of the present invention where n=3 is F W =-9.6. A similar comparison was performed to obtain the weighted average F of the relative codon 3-tuple frequency of the reference sequence (SEQ ID NO: 10). W =-41.8.

[0161] To express Citrin, HeLa cells were transferred to 6-well plates containing DMEM high glucose medium (Biowest SAS, Nuaillé, France) containing 10% FCS (Biochrom AG-Berlin, Germany) and 1% penicillin / streptomycin (Biowest) 24 hours before transfection. Transfection was performed with 2 μg of plasmid and Rotifect (Carl Roth GmbH, Karlsruhe, Germany) according to the manufacturer's instructions. 70 hours after transfection, the medium was removed, and the cells were washed with 1 mL of ice-cold phosphate-buffered saline (PBS) and resuspended in RIPA lysis buffer. 6x SDS loading buffer was added to the lysate and separated by size on a 15% SDS polyacrylamide gel. The protein samples on the gel were then transferred to a nitrocellulose membrane using Western blotting. The nonspecific binding sites of the membrane were blocked with 2% BSA, and the membrane was incubated overnight with a primary antibody against the FLAG-tagged expressed target protein or the housekeeping gene GAPDH (loading control). The membrane was washed with TBS Tween and incubated with a secondary antibody for rabbit (FLAG) or mouse (GAPDH) coupled with horseradish peroxidase (HRP). Proteins were visualized using an ECL kit (Pierce, Waltham, MA, USA), and bands were quantified using ImageQuantTL (Cytiva, Marlborough, MA, USA). In order to compare and evaluate protein expression, the band intensity of Citrin was established in a respective relationship with the band intensity of the loading control GAPDH, so that the amount of protein applied was normalized relative to the amount of cells.

[0162] In addition, the cell lysate was centrifuged at 13000 g for two minutes and the fluorescence of Citrin in the supernatant was measured in triplicate in a Tecan Spark plate reader at an excitation wavelength of 516 nm and an emission wavelength of 529 nm. The fluorescence intensity was also related to the intensity of the respective band of the loading control (GAPDH) to take into account the different cell densities of the cultures.

[0163] The results are as follows Figure 6 shown. Figure 6A shows a Western blot image of staining FLAG-tagged Citrin with HRP-coupled secondary antibody and staining of loading control GAPDH with HRP-coupled secondary antibody. Lane 1 contains the expression product of the plasmid inserted with SEQ ID NO: 10 (reference), and lane 2 contains the expression product of the plasmid inserted with SEQ ID NO: 11 optimized according to the present invention. The bands stained with the specific HRP-coupled secondary antibody prove that both plasmids successfully expressed Citrin. However, it can be clearly seen from the band intensity that the plasmid with the nucleotide sequence SEQ ID NO: 11 optimized according to the present invention leads to a significantly increased protein yield compared to the plasmid with the conventionally optimized nucleotide sequence SEQ ID NO: 11.

[0164] Figure 6 The bar graph shown in B shows the relative protein yield of Citrin depending on the nucleotide sequence used in each case, wherein the quantitatively determined protein amounts were each normalized based on the amount of cells using the loading control GAPDH as internal standard and normalized based on the amount of protein from the reference experiment with SEQ ID NO: 10. The shaded bars show the quantitative results based on the intensity of the bands from the Western blot. The quantification shows that the optimization of the Citrin nucleotide sequence according to the invention with n=3 increases the protein yield by more than three times compared to the conventionally optimized sequence.

[0165] exist Figure 6 The bar graph shown in C shows the relative Citrin fluorescence according to the nucleotide sequence used in each case. Citrin fluorescence is a measure of the proportion of dissolved functional protein. Figure 6 B, the fluorescence was normalized according to the amount of cells and standardized according to the fluorescence from the reference experiment with SEQ ID NO: 10. Quantification of fluorescence showed that the optimization of the Citrin nucleotide sequence according to the present invention with n=3 increased the yield of dissolved functional protein to about 4.7 times. When viewed together with the results of Western blotting, it is therefore clear that the optimization of the nucleotide sequence of the fluorescent protein Citrin according to the present invention resulted in significantly more (51%) soluble protein than conventional optimization.

[0166] Compared with the current state of the art, such a significant improvement in the heterologous expression rate of soluble proteins in mammalian cell culture using the method for optimizing protein encoding nucleotide sequences according to the present invention was unexpected by a person skilled in the art. This clearly demonstrates the superiority of the optimization method according to the present invention over the established concepts of eukaryotic expression systems.

[0167] Comparative Example 4: Heterologous expression of Citrin in Homo sapiens (HEK293) cell culture

[0168] In another comparative experiment, the two optimized Citrin nucleotide sequences used in Comparative Example 3 were expressed in HEK293 cells. In addition, the experiment was basically carried out as described in Comparative Example 3.

[0169] The results are as follows Figure 7 shown. Figure 7 A shows a Western blot image of FLAG-tagged Citrin stained with an HRP-coupled secondary antibody and a loading control GAPDH stained with an HRP-coupled secondary antibody. Lane 1 contains the expression product of the plasmid inserted with SEQ ID NO: 10 (reference), and lane 2 contains the expression product of the plasmid inserted with SEQ ID NO: 11 optimized according to the present invention. The bands stained with the specific HRP-coupled secondary antibody prove that both plasmids also successfully expressed Citrin in HEK293 cells. It can be clearly seen from the band intensity that the plasmid with the nucleotide sequence SEQ ID NO: 11 optimized according to the present invention leads to a significant increase in protein production compared to the plasmid with the conventionally optimized nucleotide sequence SEQ ID NO: 10.

[0170] Figure 7 The bar graph shown in B shows the relative protein yield of Citrin depending on the nucleotide sequence used in each case, wherein the quantitatively determined protein amounts each used the loading control GAPDH as internal standard and were normalized based on the cell amount and standardized to the protein amount from the reference experiment with SEQ ID NO: 10. The shaded bars show the quantitative results based on the band intensity of the Western blot. The quantification shows that the optimization of the Citrin nucleotide sequence according to the invention with n=3 increases the protein yield by about 75%.

[0171] Figure 7 The bar graph shown in C shows the relative Citrin fluorescence depending on the nucleotide sequence used in each case. Citrin fluorescence is a measure of the proportion of dissolved functional protein. Figure 7 B, the fluorescence is normalized according to the cell amount and standardized according to the fluorescence from the reference experiment with SEQ ID NO: 10. The shaded bar shows the quantitative results by means of Citrin fluorescence, which reflects functional and soluble proteins. Quantitatively, it is shown that the optimization of the Citrin nucleotide sequence of n=3 according to the present invention increases the soluble protein yield to about 2.3 times. When observed together with the results of Western blotting, it is therefore obvious that the optimization of the nucleotide sequence of the fluorescent protein Citrin according to the present invention leads to significantly more (32%) soluble protein than conventional optimization.

[0172] Using the method for optimizing protein encoding nucleotide sequences according to the present invention, such a significant improvement in heterologous expression rate in HEK293 cells confirms the results of Comparative Example 3 in another target organism and means the general applicability of the method according to the present invention in mammalian cell culture. This makes the superiority of the optimization method according to the present invention over the established concept of eukaryotic expression systems obvious.

[0173] Comparative Example 5: Expression of H. sapiens STING1 ERexitprotein 1 (STEEP1) in Hela cell culture

[0174] In another comparative experiment, according to the method of the present invention and the conventional method as reference according to Comparative Example 3, the nucleotide sequence of the H. sapiens protein STEEP1 was optimized for expression in HeLa cells. STEEP1 is a human protein present in the endoplasmic reticulum membrane. Mutations in STEEP1 are the culprit for many diseases. H. sapiens proteins are usually difficult to express in host systems.

[0175] In order to express STEEP1 in HeLa cells as target organism, two pTwist CMV expression plasmids were purchased from Twist Bioscience, which encode the amino acid sequence of STEEP1 with a FLAG tag at the N-terminus and followed by a double Strep tag under a constitutive cytomegalovirus promoter. The N-terminus of the amino acid sequence is supplemented with Met (start codon) and the amino acid Ala. A Kozak sequence is inserted before the start codon. One of the plasmids contains the STEEP1 encoding nucleotide sequence with FLAG and double Strep tags after routine optimization by the manufacturer (SEQ ID NO: 13). The other plasmid contains the STEEP1 encoding nucleotide sequence with FLAG and double Strep tags after optimization according to the method of the present invention with n=3 (SEQ ID NO: 14), wherein for the determination of the relative codon n-gram frequency according to the present invention, the protein-encoding part of the H. sapiens genome is used as a basis.

[0176] In addition, the experimental implementation of the sequence optimization according to the present invention was carried out basically as described in Comparative Example 3, starting from the wild-type sequence of STEEP1 from H. sapiens (SEQ ID NO: 12) and taking into account the undesirable sequences mentioned in Comparative Example 3, the respective values ​​were weighted with r = 0.60. The number of codons L included in the optimization in the nucleotide sequence in this example was 762; the Kozak sequence at the 5'-end was not optimized.

[0177] The sequence listing shows significant differences between the wild-type nucleotide sequence (SEQ ID NO: 12) and the nucleotide sequence optimized according to the method of the present invention (SEQ ID NO: 14). For example, the wild-type sequence has only 81.6% identical nucleotides compared to the optimized sequence in the embodiment of the method of the present invention in n=3.

[0178] It can also be directly seen from the sequence table that the nucleotide sequence optimized according to the present invention (SEQ ID NO: 14) is also significantly different from the sequence optimized according to the prior art (SEQ ID NO: 13) at the nucleotide level. For example, the sequence optimized according to the prior art (SEQ ID NO: 13) has only 77.3% identical nucleotides compared to the sequence optimized according to the method of the present invention in the embodiment of n=3 (SEQ ID NO: 14). In view of the predetermined amino acid sequence, these huge differences are surprising from a professional point of view and indicate that the optimization method according to the present invention also leads to completely different results for optimization in mammalian cells (here, Homo sapiens) than the method according to the current prior art.

[0179] After optimization, the weighted average value of (SEQ ID NO: 14) in the embodiment of the present invention where n=3 is F W =-9.8. A similar comparison was performed to obtain the weighted average F of the relative codon 3-tuple frequency in the wild type (SEQ ID NO: 12). W =-36.9 and for the reference sequence (SEQ ID NO: 13) the F W =87.1.

[0180] The expression experiment was also carried out basically as described in Comparative Example 3. The results are shown in Figure 8 shown. Figure 8 A shows an image of a Western blot stained with HRP-conjugated secondary antibody for FLAG-tagged STEEP1 and with HRP-conjugated secondary antibody for loading control GAPDH. Lane 1 contains the expression product of the plasmid inserted with SEQ ID NO: 13 (reference), and lane 2 contains the expression product of the plasmid with SEQ ID NO: 14 optimized according to the present invention. The bands stained with the specific HRP-conjugated secondary antibody prove that STEEP1 is successfully expressed in both plasmids. However, it can be clearly seen from the band intensity that the plasmid with the nucleotide sequence SEQ ID NO: 14 optimized according to the present invention leads to a significantly increased protein yield compared to the plasmid with the conventionally optimized nucleotide sequence SEQ ID NO: 13.

[0181] exist Figure 8In the bar graph shown in B, the relative protein yield of STEEP1 is shown depending on the nucleotide sequence used in each case, wherein the quantitatively determined protein amounts were normalized to the cell amount using a loading control as described above and based on the protein amount from a reference experiment with SEQ ID NO: 13. The hatched bar shows the quantitative results of the band intensity by means of Western blot. The quantification shows that the optimization of the STEEP1 nucleotide sequence according to the invention with n=3 increases the protein yield by more than 75% compared to the conventional optimization.

[0182] Such a significant improvement in the expression rate of membrane-bound human proteins using the method for optimizing protein-coding nucleotide sequences according to the present invention compared to the current state of the art was unexpected by a person skilled in the art. This clearly demonstrates the superiority of the optimization method according to the present invention over the established concepts of eukaryotic expression systems.

[0183] Comparative Example 6: Expression of H. sapiens STING1 ER exit protein 1 (STEEP1) in HEK293 cell culture

[0184] In another comparative experiment, the two optimized STEEP1 nucleotide sequences used in Comparative Example 5 were expressed in HEK293 cells. Otherwise, the experiment was performed essentially as described in Comparative Example 5.

[0185] The results are as follows Fig. 9 shown. Fig. 9 A shows an image of a Western blot stained with HRP-conjugated secondary antibody for FLAG-tagged STEEP1 and with HRP-conjugated secondary antibody for loading control GAPDH. Lane 1 contains the expression product of the plasmid inserted with SEQ ID NO: 13 (reference), and lane 2 contains the expression product of the plasmid inserted with SEQ ID NO: 14 optimized according to the present invention. As demonstrated by the bands stained with the specific HRP-conjugated secondary antibody, both plasmids also successfully expressed STEEP1 in HEK293 cells. From the intensity of the bands, it can already be seen that the plasmid with the nucleotide sequence SEQ ID NO: 14 optimized according to the present invention leads to a significant increase in protein production compared to the plasmid with the conventionally optimized nucleotide sequence SEQ ID NO: 13.

[0186] exist Fig. 9In the bar graph shown in B, the relative protein yield of STEEP1 is shown depending on the nucleotide sequence used in each case, wherein the quantitatively determined protein amounts were normalized to the cell amount using a loading control as described above and based on the protein amount from a reference experiment with SEQ ID NO: 13. The hatched bar shows the quantitative results of the band intensity by means of Western blot. The quantification shows that the optimization of the STEEP1 nucleotide sequence according to the invention with n=3 increased the protein yield by 22% compared to the expression of the conventionally optimized sequence.

[0187] These results confirm that the expression rate of STEEP1 in HEK293 cells was significantly increased using the method for optimizing protein-coding nucleotide sequences according to the present invention, and emphasize the general applicability of the method according to the present invention for protein expression in mammalian cells. This once again demonstrates the superiority of the optimization method according to the present invention over the established concept of eukaryotic expression systems.

[0188] Comparative Example 7: Expression of H. sapiens nitric oxide synthase interacting protein (NOSIP) in Hela cell culture

[0189] In another comparative experiment in a eukaryotic expression system, the nucleotide sequence of the H. sapiens protein NOSIP was optimized for expression in HeLa according to the method of the present invention and according to the prior art as a reference. Reference optimization was performed in a variant according to Comparative Example 3 and in a second variant according to WO 2020 / 024917 A1. NOSIP regulates the activity and localization of nitrite synthase, thereby regulating the production of nitrite, which is essential for the development of the human brain, eyes and face.

[0190] In order to express NOSIP in HeLa cells as a target organism, three pTwist CMV expression plasmids were purchased from Twist Bioscience, which encode the amino acid sequence of NOSIP with a FLAG tag at the N-terminus and followed by a double Strep tag under a constitutive cytomegalovirus promoter. The N-terminus of the amino acid sequence is supplemented with Met (start codon) and the amino acid Ala. The Kozak sequence is inserted before the start codon. One of the plasmids contains the NOSIP encoding nucleotide sequence with FLAG and double Strep tags (SEQ ID NO: 16) optimized by the manufacturer's optimization service according to the prior art (as a reference). The second plasmid contains the NOSIP encoding nucleotide sequence with FLAG and double Strep tags optimized according to WO 2020 / 024917A1 as a further reference (SEQ ID NO: 17). The third plasmid contains the NOSIP encoding nucleotide sequence (SEQ ID NO: 18) with FLAG and double Strep tags after optimization with n=3 according to the method of the present invention, wherein in order to determine the relative codon n-gram frequency according to the present invention, the protein-encoding part of the H. sapiens genome is used as a basis.

[0191] In addition, the experimental implementation of the sequence optimization according to the present invention was carried out essentially as described in Comparative Example 3, starting from the NOSIP wild-type sequence (SEQ ID NO: 15) from Homo sapiens (H. sapiens) and taking into account the undesirable sequences mentioned in Comparative Example 3, the respective numbers of which were weighted by r = 0.64. The number of codons L included in the optimization in the nucleotide sequence in this example was 1008; the Kozak sequence at the 5'-end was not optimized.

[0192] The sequence listing also clearly shows that there are already significant differences at the nucleotide level between the wild-type nucleotide sequence and the nucleotide sequence optimized according to the method of the present invention. The wild-type sequence (SEQ ID NO: 15) has only 86.0% identical nucleotides compared to the sequence (SEQ ID NO: 18) optimized according to the method of the present invention in the embodiment of n=3.

[0193] It can also be seen from the sequence table that the nucleotide sequence optimized according to the present invention (SEQ ID NO: 18) is also significantly different from the sequence optimized according to the prior art (SEQ ID NO: 16, SEQ ID NO: 17) at the nucleotide level. The sequence optimized according to the prior art (SEQ ID NO: 16) has only 76.3% identical nucleotides compared to the sequence optimized according to the method of the present invention in the embodiment of n = 3 (SEQ ID NO: 18). The sequence optimized according to the prior art of WO 2020 / 024917 A1 (SEQ ID NO: 17) has only 85.2% identical nucleotides compared to the sequence optimized according to the method of the present invention in the embodiment of n = 3 (SEQ ID NO: 18). These huge differences show again that the optimization method according to the present invention also leads to completely different results for optimization in mammalian cells (here, Homo sapiens (H. sapiens)) compared to the current prior art method.

[0194] After optimization, the weighted average value of (SEQ ID NO: 18) in the embodiment of the present invention where n=3 is F W =-13.1. A similar comparison was performed to obtain the weighted average F of the relative codon 3-tuple frequency in the wild type (SEQ ID NO: 15). W =-39.1, for reference sequence SEQ ID NO: 16 W =-39.1, for reference sequence SEQ ID NO: 17 W =-36.3.

[0195] In addition, the expression experiment was performed essentially as described in Comparative Example 3. Fig.10 and 11 shown.

[0196] Fig.10 A comparison between SEQ ID NO: 16 and SEQ ID NO: 18 is shown. Fig.10 A shows an image of a Western blot of FLAG-tagged NOSIP stained with an HRP-coupled secondary antibody and a loading control GAPDH stained with an HRP-coupled secondary antibody. Lane 1 contains the expression product of a plasmid inserted with SEQ ID NO: 16 (reference), and lane 2 contains the expression product of a plasmid inserted with SEQ ID NO: 18 optimized according to the present invention. As evidenced by the bands stained with a specific HRP-coupled secondary antibody, both plasmids successfully expressed NOSIP, but the band intensity showed that significantly better protein yields were achieved by expression of the nucleotide sequence SEQ ID NO: 18 optimized according to the present invention compared to the plasmid with the conventionally optimized nucleotide sequence SEQ ID NO: 16.

[0197] exist Fig.10 In the bar graph shown in B, the relative protein yield of NOSIP depending on the nucleotide sequence used in each case is shown, wherein the quantitatively determined protein amount is first normalized according to the cell amount using the loading control GAPDH as described above, and then based on the protein amount from the reference experiment with SEQ ID NO: 16. The hatched bar shows the quantitative results of the band intensity by western blot. Quantitatively showing that the optimization of the NOSIP nucleotide sequence of n=3 according to the present invention can increase protein yield to 31 times compared to conventional optimization.

[0198] Compared with the current state of the art, such a significant improvement in the expression rate of membrane-bound human proteins using the method for optimizing protein-encoding nucleotide sequences according to the present invention far exceeds the expectations of those skilled in the art. This once again clearly demonstrates the superiority of the optimization method according to the present invention over the established concepts of eukaryotic expression systems.

[0199] Fig.11 A comparison between SEQ ID NO: 17 and SEQ ID NO: 18 is shown. Fig.11 A shows an image of a protein blot of FLAG-tagged NOSIP stained with an HRP-conjugated secondary antibody and a loading control GAPDH stained with an HRP-conjugated secondary antibody. Lane 1 contains the expression product of a plasmid having the reference sequence SEQ ID NO:17 optimized according to WO 2020 / 024917 A1, and lane 2 contains the expression product of a plasmid having the sequence SEQ ID NO:18 optimized according to the present invention. The bands stained with the specific HRP-conjugated secondary antibody prove that the two plasmids successfully expressed NOSIP. It can be seen from the band intensity that the plasmid having the nucleotide sequence SEQ ID NO:18 optimized according to the present invention results in an increase in protein production compared to the plasmid having the conventionally optimized nucleotide sequence SEQ ID NO:17.

[0200] exist Fig.11 In the bar graph shown in B, the relative protein yield of NOSIP depending on the nucleotide sequence used in each case is shown, wherein the quantitatively determined protein amount is first normalized according to the cell amount using the loading control GAPDH as described above, and then based on the protein amount from the reference experiment with SEQ ID NO: 17. The hatched bar shows the quantitative results of the band intensity by means of Western blot. Quantitatively showing that the optimization of the NOSIP nucleotide sequence of n=3 according to the present invention increases protein yield by more than 60%.

[0201] This demonstrates the superiority of the optimization method according to the invention even for eukaryotic expression systems compared to the technical teachings of the current prior art (here WO 2020 / 024917 A1).

[0202] Comparative Example 8: Expression of NOSIP in HEK293 cell culture

[0203] In another comparative experiment, the two optimized NOSIP nucleotide sequences SEQ ID NO: 16 (reference) and SEQ ID NO: 18 used in Comparative Example 7 were expressed in HEK293 cells. Otherwise, the experiment was performed essentially as described in Comparative Example 7.

[0204] The results are as follows Fig.12 shown. Fig.12 A shows an image of a western blot of NOSIP stained with a HRP-coupled secondary antibody to FLAG tags and GAPDH stained with a HRP-coupled secondary antibody to control loading. Lane 1 contains the expression product of a plasmid with conventionally optimized SEQ ID NO: 16 (reference), and lane 2 contains the expression product of a plasmid with SEQ ID NO: 18 optimized according to the present invention. As evidenced by the bands stained with a specific HRP-coupled secondary antibody, NOSIP can also be successfully expressed in two plasmids in HEK293 cells, wherein the band intensity shows significantly better expression of the nucleotide sequence SEQ ID NO: 18 optimized according to the present invention.

[0205] exist Fig.12 In the bar graph shown in B, the relative protein yield of NOSIP depending on the nucleotide sequence used in each case is shown, wherein the quantitatively determined protein amounts are first normalized according to the cell amount using the loading control GAPDH as described above and then based on the protein amount from the reference experiment with SEQ ID NO: 16. The hatched bar shows the quantitative results of the band intensity by means of Western blot. Quantitatively, the optimization of the NOSIP nucleotide sequence according to the invention with n=3 also increases the protein yield in the HEK293 expression system by about four times compared to conventional sequence optimization.

[0206] Here, as in the case of comparative experiments 4 and 6, a significant improvement in the expression rate in HEK293 cells was confirmed using the method for optimizing protein encoding nucleotide sequences according to the present invention. This shows the general applicability of the method according to the present invention in mammalian cell cultures. This once again confirms the superiority of the optimization method according to the present invention over the established optimization concepts for eukaryotic expression systems.

[0207] Comparative Example 9: Heterologous expression of eqFP611 in the cyanobacterium Synechococcus elongatus

[0208] In another comparative experiment, the nucleotide sequence of the fluorescent protein eqFP611 was optimized for heterologous expression in S. elongatus according to the method of the present invention and according to the method of WO 2020 / 024917A1 as a reference. eqFP611 is a red fluorescent protein (RFP) from entacmaea quadricolor, which is commonly used in reporter gene assays and fluorescence microscopy.

[0209] In order to express eqFP611 in S. elongatus as a target organism, two pSyn-6 expression plasmids were purchased from Genscript (Thermo Fisher, Waltham, MA, USA), which encode the amino acid sequence of eqFP611 with a double Strep tag at the N-terminus under the constitutive psbA1 promoter. One of the plasmids contains the eqFP611 encoding nucleotide sequence with a double Strep tag optimized according to WO 2020 / 024917A1 (SEQ ID NO: 20). The other plasmid contains the eqFP611 encoding nucleotide sequence with a double Strep tag optimized according to the method of n=3 of the present invention (SEQ ID NO: 21), wherein in order to determine the relative codon n-tuple frequency according to the present invention, the protein-encoding part of the S. elongatus genome is used as a basis.

[0210] The optimization method is carried out as described in Comparative Example 1. Similar to the optimization of Escherichia coli (E.coli) as the host, for S.elongatus, as an optional step in block 108, an undesirable sequence motif such as ribosome binding site AGGAGG is input, which is known to those skilled in the art to affect expression in S.elongatus. Other undesirable sequence motifs are GGGGGG, CGCGCG, ATTTA, CATATG, GGTACC and GTCGAC. The number of undesirable sequence motifs is weighted by r=0.62. The number of codons L included in the optimization in the nucleotide sequence in the present embodiment is 768; terminator sequences and terminal restriction sites are not optimized.

[0211] The sequence listing shows that for heterologous expression of eqFP611 in S. elongatus, the nucleotide sequence optimized according to the invention (SEQ ID NO: 21) differs significantly from the sequence optimized according to the prior art (SEQ ID NO: 20) at the nucleotide level. For example, the sequence optimized according to the prior art (SEQ ID NO: 20) has only 89.8% identical nucleotides compared to the sequence optimized according to the method of the invention in the embodiment of n=3 (SEQ ID NO: 21). These huge differences are surprising from a professional point of view in view of the predetermined amino acid sequence and indicate that the optimization method according to the invention also leads to completely different results for optimization in cyanobacteria (here S. elongatus) than the optimization method according to the current prior art.

[0212] After optimization, the weighted average value of (SEQ ID NO: 21) in the embodiment of the present invention where n=3 is F W =-8.7. A similar comparison was performed to obtain the weighted average F of the relative codon 3-tuple frequency of the reference sequence (SEQ ID NO: 20) of WO 2020 / 024917 A1: W =-496.2.

[0213] For expression in elongated cyanobacteria (S.elongatus), the manufacturer's protocol of "GeneArt algae protein expression system" (Thermo Fisher) was followed. Further processing of cyanobacterial biomass was performed as described in Comparative Example 1 for Escherichia coli (E.coli). The expressed eqFP611 protein was purified by Streptactin and quantified by SDS-PAGE. In addition, the fractions of eqFP611 cell lysates were centrifuged for two minutes at 13000g, and the eqFP611 fluorescence in the supernatant was measured in triplicate in a Tecan Spark plate reader at an excitation wavelength of 559nm and an emission wavelength of 611nm.

[0214] As a result of this experiment, a similarly high increase in the yield of soluble proteins can be expected also for cyanobacteria by the method according to the invention, as already described in comparative examples 1 to 8 for bacterial and eukaryotic expression systems.

[0215] The invention is not restricted to the description based on the exemplary embodiments. On the contrary, the invention covers every new feature and every combination of features, which in particular includes every combination of features in the claims and the description, even if this feature or combination of features itself is not explicitly specified in the claims, the description or the exemplary embodiments.

Claims

1. A method for optimizing a nucleotide sequence for expression of a predetermined amino acid sequence in at least one target organism, wherein the nucleotide sequence comprises a plurality of base triplets and at at least one altered position of the nucleotide sequence, a base triplet encoding an amino acid of the predetermined amino acid sequence is replaced by a synonymous base triplet encoding the same amino acid of the predetermined amino acid sequence, so as to optimize the nucleotide sequence for expression in the at least one target organism, It is characterized in that The at least one altered position comprises a directly consecutive order of n base triplets which form a first codon n-gram and encode a sequence portion of n amino acids of the predetermined amino acid sequence, the sequence portion forming an amino acid n-gram which is encoded in the genome or a portion thereof of the at least one target organism and / or in the genome or a portion thereof of a virus capable of infecting the at least one target organism by a predetermined number of amino acid n-gram events, and at least one of the n base triplets in the directly consecutive order is replaced by a synonymous base triplet selected to generate a second codon n-gram having a higher relative codon n-gram frequency in the genome or a portion thereof of the at least one target organism and / or in the genome or a portion thereof of a virus capable of infecting the at least one target organism than the first codon n-gram based on the amount of amino acid n-gram events, Wherein n is a natural number greater than or equal to 2 and less than or equal to the total number N of amino acids in the predetermined amino acid sequence.

2. The method according to claim 1, characterized in that n is less than or equal to 50, less than or equal to 40, less than or equal to 30, less than or equal to 20, or less than or equal to 10.

3. The method according to any one of claims 1 or 2, characterized in that n is selected from n=2, n=3, n=4, n=5 and any combination thereof.

4. The method according to any one of claims 1 to 3, characterized in that The amount of amino acid n-gram events is determined with the aid of and / or produced from a plurality of protein encoding genes and / or proteins of the at least one target organism and / or a virus capable of infecting the at least one target organism.

5. The method according to any one of claims 1 to 4, characterized in that The relative codon n-gram frequency is determined and / or generated by the amount of events of the respective first and / or second codon n-grams in a plurality of protein-coding genes of the at least one target organism and / or a virus capable of infecting the at least one target organism, wherein the amount of events of the first and / or second codon n-grams is based on the amount of the amino acid n-gram events.

6. The method according to any one of claims 1 to 5, characterized in that The following steps are involved: a) determining the at least one changed position; b) replacing said at least one base triplet at said at least one altered position with a synonymous base triplet; c) determining the relative codon n-gram frequencies of the resulting second codon n-grams.

7. The method according to any one of claims 1 to 6, characterized in that The at least one altered position is a plurality of altered positions, wherein in each of the altered positions at least one of the n base triplets in the directly consecutive order is replaced by a synonymous base triplet, wherein the synonymous base triplets are selected such that at least a portion of the resulting second codon n-grams have a higher relative codon n-gram frequency than the respective first codon n-grams.

8. The method according to claim 7, characterized in that In the changed position of the first codon n-gram having the lowest relative codon n-gram frequency, at least one of the n base triplets in the directly consecutive order is replaced by a synonymous base triplet, which is selected so that the resulting second codon n-gram has a higher relative codon n-gram frequency than the first codon n-gram.

9. The method according to any one of claims 7 or 8, characterized in that The directly consecutive order of n base triplets of at least two altered positions overlap, wherein the base triplets replaced by the synonymous base triplets are simultaneously contained by the two altered positions.

10. The method according to claim 9, characterized in that The synonymous base triplets are selected such that in one of the two changed positions, the resulting second codon n-gram has a lower relative codon n-gram frequency than the respective first codon n-gram, while in the other of the two changed positions, the resulting second codon n-gram has a higher relative codon n-gram frequency than the respective first codon n-gram.

11. The method according to any one of claims 7 to 10, characterized in that The relative codon n-tuple frequency of the first codon n-tuple and the relative codon n-tuple frequency of the second codon n-tuple each have a global minimum in the changed position, wherein the global minimum of the second codon n-tuple is greater than the global minimum of the first codon n-tuple.

12. The method according to any one of claims 7 to 11, characterized in that The synonymous base triplets are selected such that the relative codon n-gram frequency of the second codon n-gram is at or no less than 50% below the maximum possible minimum.

13. The method according to any one of claims 7 to 12, characterized in that The synonymous base triplets are selected so that the average value of the relative codon n-gram frequency of the second codon n-gram reaches a maximum value or is no more than 50% lower than the maximum value that can be achieved.

14. The method according to claim 13, characterized in that The average comprises decreasing weights for relative codon n-gram frequencies, configured such that high relative codon n-gram frequencies have a numerically disproportionately lower impact on the average than lower relative codon n-gram frequencies.

15. The method according to any one of claims 7 to 14, characterized in that At least two of the change positions differ in the number n, or different n is selected for at least two of the change positions.

16. The method according to any one of claims 7 to 15, characterized in that A computer-assisted optimization method was used to replace base triplets with synonymous base triplets in multiple iterative steps.

17. The method according to claim 16, characterized in that The computer-aided optimization method includes an approximation method and / or a simulated cooling method (simulated annealing method).

18. The method according to any one of claims 7 to 17, characterized in that The altered positions together comprise at least 50% of the base triplets of the nucleotide sequence encoding the amino acids of the predetermined amino acid sequence.

19. The method according to any one of claims 1 to 18, characterized in that The at least one target organism is a plurality of different target organisms, wherein the relative codon n-gram frequencies comprise genome-dependent weights configured such that differences in size of genomes or parts thereof of different target organisms are at least partially compensated.

20. The method according to any one of claims 1 to 19, characterized in that After optimization of the nucleotide sequence, the amino acid sequence expressed in the at least one target organism has a greater solubility than before optimization and / or is present in a greater proportion in soluble form than before optimization.

21. A method for optimizing a nucleotide sequence for expression of a predetermined amino acid sequence in at least one target organism, wherein the nucleotide sequence comprises a plurality of base triplets and at at least one altered position of the nucleotide sequence, a base triplet encoding an amino acid of the predetermined amino acid sequence is replaced by a synonymous base triplet encoding the same amino acid of the predetermined amino acid sequence, to optimize the nucleotide sequence for expression in the at least one target organism, It is characterized in that The at least one altered position comprises a directly consecutive order of n base triplets which form a first codon n-gram and encode a sequence portion of n amino acids of the predetermined amino acid sequence, the sequence portion forming an amino acid n-gram, and at least one of the n base triplets in the directly consecutive order is replaced by a synonymous base triplet, which is selected with the aid of an estimation function in such a way that a second codon n-gram is generated, which codes for an amino acid n-gram with a higher probability in the genome or a part thereof of the at least one target organism and / or in the genome or a part thereof of a virus capable of infecting the at least one target organism than the first codon n-gram, Wherein n is a natural number greater than or equal to 2 and less than or equal to the total number N of amino acids in the predetermined amino acid sequence.

22. Use of a nucleotide sequence optimized according to the method of any one of claims 1 to 21 for producing synthetic DNA and / or for expressing a protein in at least one target organism.

23. A computer program having a program code, characterized in that The program code is configured to implement the method according to any one of claims 1 to 21 when the computer program is executed on a computer.

24. Computer-readable storage medium having a computer program according to claim 23 stored in a computer-readable form.

25. Apparatus for optimizing and / or producing nucleotide sequences for expressing a predetermined amino acid sequence in at least one target organism, comprising a computing device configured to implement the method according to any one of claims 1 to 21.

26. A method for producing a protein, comprising providing a nucleotide sequence which has been optimized according to the method of any one of claims 1 to 21 for expression of the protein in at least one target organism, The protein is expressed in the at least one target organism.

27. A nucleic acid molecule comprising a nucleotide sequence obtained by the method according to any one of claims 1 to 21.

28. A vector comprising the nucleic acid molecule according to claim 27.

29. A recombinant host cell comprising the nucleic acid molecule according to claim 27 or the vector according to claim 28.

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