Patent Application
20210147889
Material in an ASCII text file is incorporated herein by reference. The text file is named 20-198_sequence_listing_12-17-2020, the file was created on Dec. 17, 2020, and the file contains 8634 bytes.
The present invention relates to microbiological processes for producing a natural compound. In particular, the invention relates to methods of enhancing the concentration of a biosynthesized substance in the culture medium or in the cytoplasm of cultivated archaea and bacteria during the stationary and exponential growth phase.
As used therein, the term “natural compound” refers to substances such as amino and carboxylic acids and their derivatives—e.g. lysine, proline, trimethylglycine (i.e. glycine-betaine), N,N-dimethyl-L-proline (i.e. proline betaine), diamino butyric acid, Nα-acetyl-L-2,4-diaminobutyrate, ectoine (i.e. 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid), hydroxyectoine; sugars and their derivatives—e.g. trehalose, glyceryl glucoside; polyols and their derivatives—e.g. erythritol, xylitol; to name a few.
Some of these substances are also known as so called compatible solutes. Compatible solutes are typically synthesized by extremophilic organisms as protectants to mitigate the detrimental effects caused by the extreme environment, e.g. low or high temperature, high salt concentration, low or high pH value. Extremophilic organisms are, e.g., halophilic, thermophilic, or xerophilic archaea, and bacteria. These can thus be used to obtain compatible solutes. As not being disruptive of enzymatic structure or function, even when accumulated in large quantities, compatible solutes are attractive, e.g., for stabilization of bio-functional proteins. For their water-structuring properties and as cell protectants they are used in cosmetics and health care applications. Accordingly, producer strains and appropriately modified cell lines can be used for large-scale production of compatible solutes in fermenters and bio-reactors. The optimization of producer strains and cell lines is the subject of constant effort.
In view of the above, according to one aspect a method is suggested for increasing the production of a natural compound by a microorganism during an exponential growth and during a stationary phase of a culture of the microorganism, the method comprising:
enhancing a translation of a target mRNA, wherein the target mRNA comprises a transcript of a target gene, wherein the target gene is encoding the natural compound or a key enzyme involved in a biosynthesis of the natural compound in the microorganism, by replacing a wild type (wt) ribosome binding site (RBS) upstream of the target gene with a synthetic RBS, the synthetic RBS possessing a higher affinity towards a 16S rRNA of a ribosome of the microorganism than the wt RBS; and
enhancing a transcription of the target gene by converting an osmotically regulated σ38 promoter upstream of the target gene into a stationary σ38 promoter, wherein the osmotically regulated σ38 promoter comprises a −35 G-element and a −10 element which are separated from each other by a spacer sequence, by deleting the −35 G element.
According to another aspect, a nucleic acid construct is suggested, comprising: a synthetic ribosome binding site (RBS), a stationary σ38 promoter, a σ70 promoter, and a target gene;
the synthetic RBS possessing a higher affinity towards a 16S rRNA of a bacterial ribosome than a wild type (wt) RBS upstream of the target gene;
the stationary σ38 promoter being generated from an osmotically regulated wt σ38 promoter, the osmotically regulated wt σ38 promoter comprising a −35 G-element, by deleting the −35 G-element and/or by replacing the −35 G-element by a nucleotide, the nucleotide being selected from nucleotides others than guanine;
wherein the target gene is encoding a key enzyme, the key enzyme catalyzing a reaction selected from: a phosphorylation/dephosphorylation, a carboxylation/decarboxylation, and an acetylation; the key enzyme being involved in a biosynthesis of a natural compound,
wherein the natural compound is selected from a protein, an amino acid, an derivative of an amino acid, a sugar, and a sugar-polyol.
According to a further aspect, a microorganism is suggested the microorganism being selected from Bacteria and Archaea, wherein a genome of the microorganism encompasses a nucleotide construct, wherein the nucleotide construct comprises: a synthetic ribosome binding site (RBS), a stationary σ38 promoter, a σ70 promoter, and a target gene;
the synthetic RBS possessing a higher affinity towards a 16S rRNA of a bacterial ribosome than a wild type (wt) RBS upstream of the target gene;
the stationary σ38 promoter being generated from an osmotically regulated wt σ38 promoter, the osmotically regulated wt σ38 promoter comprising a −35 G-element, by deleting the −35 G-element and/or by replacing the −35 G-element by a nucleotide, the nucleotide being selected from nucleotides others than guanine;
wherein the target gene is encoding a key enzyme, the key enzyme catalyzing a reaction selected from: a phosphorylation/dephosphorylation, a carboxylation/decarboxylation, and an acetylation; the key enzyme being involved in a biosynthesis of a natural compound, wherein the natural compound is selected from a protein, an amino acid, an derivative of an amino acid, a sugar, and a sugar-polyol.
According to yet another aspect, a biotechnical process for continuous production of a natural compound by a microorganism is suggested, wherein a culture of the microorganism is maintained in an exponential growth phase or in a stationary phase, wherein a genome of the microorganism encompasses the nucleotide construct as described above, wherein the microorganism is adapted to excrete the natural compound into a culture medium, the process comprising extracting the natural compound from the culture medium.
The continuous production of a given compound is typically preferred in any technology. However, the amount of a compound produced by a given microorganism is highly regulated in response to external factors, e.g. temperature, concentration of certain inorganic ions, ratio of certain inorganic ions, ionic strength, pH-value, nutrients—to name a few. Predominant regulation mechanisms may even differ for different growth phases of the microorganism. Therefore, from an engineering point of view, maintaining an artificial cell culture, e.g. in a bioreactor, in the stationary growth phase, i.e. continuous culture is preferred.
Typically, the synthesis of a natural compound like, e.g., a compatible solute, by a single cell (i.e. biosynthesis) is regulated at different levels. Even though synthesis and catabolism are finally dependent on the cell's genome comprising a DNA sequence, the actual abundance of a certain compound, whether within the cytoplasm or in the external medium, finally depends on the activity of different enzymes. Their abundance and activity, again, depend on the presence and activity of other enzymes, and so forth.
From an engineering point of view, the transcription, i.e. the copying of DNA into mRNA is the first level, where regulation is effectuated. While, for instance, one single operon, i.e. a genomic DNA sequence, controlled by one (or several) promoter(s), suffices for a certain compound (e.g. in a certain strain), for another compound (or another strain) a more complex organization of several gene clusters under the control of different promoters may be possible. Moreover, the initiation of the synthesis of an enzyme initially depends on the binding of RNA polymerase to the DNA strand, i.e. on the very first stage of the so-called transcription. As used herein, a promoter is a regulatory region of DNA located upstream of a gene, the gene encoding the key compound, providing a control point for regulated gene transcription.
Transcription proceeds in the following general steps (cf https://en.wikipedia.org/wiki/Transcription_(biology)):
The strand of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene. If the gene encodes a protein, the transcription produces a so-called messenger RNA (mRNA). The mRNA, in turn, serves as a template for the synthesis of the protein (enzyme) through translation.
On the other hand, the mentioned translation, i.e. the process in which ribosomes in the cytoplasm synthesize proteins after the transcription of DNA to RNA (cf. https://en.wikipedia.org/wiki/Translation_(biology)) also regulates the expression of the compound. The entire process is called gene expression. In translation, the mRNA is decoded by a ribosome to produce a specific amino acid chain, or polypeptide. The ribosome facilitates decoding by inducing the binding of complementary tRNA anticodon sequences to mRNA codons. The tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through the ribosome and is consecutively “read” by the ribosome.
The translation proceeds in three phases:
Therein, the initiation generally starts at a ribosome binding site, or ribosomal binding site (RBS), which is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome. Typically, a RBS in the DNA of Bacteria comprises a purine rich, so called Shine-Dalgarno (SD) sequence. It is generally acknowledged that base pairing between the SD sequence in mRNA and the 3′ end of 16S rRNA is of prime importance for initiation of translation by bacterial ribosomes.
As evident, the abundance of any natural compound which is produced in a multi-step biosynthesis by enzymatically transforming e.g. an amino acid precursor by an enzyme cascade into an amino acid derivative is regulated by an even more complex cascade of regulatory mechanisms, since the abundance of each of the transforming enzymes is regulated at the level of transcription and translation as well.
Therefore, the manipulation of the metabolism of a microorganism to generate more efficient producer strains is quite challenging. It is further complicated since, e.g., published gene sequences of respective microorganisms may be incorrect. Furthermore, the assignment of putative initiation codons may be incorrect.
In view of the above, different approaches have been followed to establish enhanced production of compatible solutes, e.g. of ectoine, which is synthesized and accumulated as an osmolyte by several halophilic microorganisms.
In particular, halophilic microorganisms from genera like Bacillus, Brevibacterium, Chromohalobacter, Halobacillus, Halomonas, Marinococcus, Methylomicrobium and transgenic or recombinant microorganisms, e.g. Corynebacterium glutamicum, Escherichia coli, Bacillus subtilis, may be used for the industrial production of compatible solutes, like, e.g. ectoine.
The following concepts have been developed and employed to increase ectoine yield. First of all, disruption of ectoine transport, which leads to over-production and excretion of ectoine to the medium, secondly, mutation of the degradation pathway of ectoine, which further increases ectoine synthesis, and finally, feeding with toxic solute analogues, which facilitates to select for ectoine overproduction and excretion strains.
In contrast to previous concepts, we herein suggest a new approach by combining both transcriptional and translational up-regulation.
To exemplarily study the possibility of improving the production of compatible solutes we choose the halophilic bacterium Halomonas elongata. It amasses compatible solutes in the cytoplasm to achieve an osmotic equilibrium with the environment (Kunte, 2006; Schwibbert et al., 2011). Particularly, the compatible solute synthesized by H. elongata is ectoine, an amino acid derivative of aspartate. This bacterium utilizes three enzymes (EctA, EctB, and EctC) for de novo synthesis of ectoine (Schwibbert et al., 2011). The genes encoding these three enzymes are ectABC, which are transcriptionally regulated by three different promoters.
Upstream of ectA there is a sigma factor σ70 promoter and an osmotically induced σ38-osmo promoter. Upstream of ectC there is a σ54 promoter. σ54-controlled promoters are often involved in transcription of nitrogen-regulated genes. These findings indicate that ectoine synthesis is regulated by salinity (σ38-osmo), growth (σ70), and nitrogen supply (σ54) as described by Schwibbert et al., (2011).
Ectoine is used by the H. elongata cell not only as osmolyte but—depending on life conditions—also as an energy source. In the corresponding catabolism four further enzymes DoeA, DoeB, DoeC and DoeD are involved (cf. Schwibbert et al., 2011).
According to the approach suggested here, firstly the key enzyme involved in the biosynthesis of ectoine has been identified for favorably shifting the equilibrium in the complex metabolic pathway of ectoine towards its enhanced synthesis. It is believed that L-2,4-diaminobutyric acid Nγ-acetyltransferase is the key enzyme in ectoine biosynthesis. This enzyme is encoded by the gene ectA. In the following text, the gene which encodes the key enzyme will generally be called target gene. Enzymes that make a certain metabolic pathway irreversible are considered key enzymes. These enzymes catalyze reactions such as phosphorylation/dephosphorylation, carboxylation/decarboxylation and acetylation.
Thus, according one embodiment, in order to enhance the abundance and thereby the production of such a natural compound like, e.g., ectoine, by the cultivated cells we suggest applying a combinatorial approach of optimizing expression at the level of transcription (mRNA synthesis) and translation (protein synthesis) simultaneously.
According to exemplary embodiments, in order to improve the expression of the natural compound, e.g. ectoine, the RBS upstream of the target gene, encoding the key enzyme, e.g. ectA, can be determined by analyzing the genome of a promising producer microorganism, e.g. of H. elongata. The RBS in front of ectA differs strongly from the consensus sequence known from literature (AGGAGG; SEQ ID NO 10). In particular, the wt RBS of H. elongata appears a weak binding site for ribosomes, as it substantially deviates from the SD sequence, known for bacteria. Usually, the RBS is rich in guanine G and adenine A and the SD motif is typically AGGAGG. The wt sequence strongly deviates from known motifs, indicating that ectA-mRNA is only weakly translated into the corresponding key enzyme (L-2,4-diaminobutyric acid Nγ-acetyltransferase), which is encoded by the gene downstream of the RBS.
Thus, in order to improve translation, we developed a synthetic RBS (
In addition thereto, enhancing transcription of the target mRNA, which mRNA is a transcript of the target gene, is reached by modifying a natural promoter upstream of the target gene. The target gene encodes the key enzyme which is involved in the biosynthesis of the natural compound.
In particular, according to this embodiment, the σ38 osmo promoter of H. elongata is modified by deleting its −35 G-element. In particular, the nucleotide sequence GCGG (SEQ ID NO 3) is deleted. The −35 G-element is the characteristic motif that distinguishes σ38 osmo promoters from σ38 promoters. σ38 osmo promoters can be changed into σ38 by either deleting the G-element or by replacing it by any non-G containing DNA. The −10 region can remain unchanged as both sigma promoters share the same consensus sequence. If the −10 region deviates strongly from the sigma consensus sequence (CTACACT; SEQ ID NO 5), the strength of sigma promoters can be increased however by changing the −10 region and making it more similar to the −10 consensus motif. (Lee & Gralla, 2001).
According to the suggested approach, we do not just introduce an artificial or viral promoter for strong transcription as promoters of this type often have negative effects on cell health, product quality (e.g. formation of inclusion bodies) and volumetric productivity.
Contrary thereto, as mentioned above, we suggest modifying a wild type (wt) promoter. The wild type σ38-osmo promoter comprises −10 and −35 sequences and in that respect, it resembles σ70 dependent promoters (cf.
Thus, according to an embodiment, we suggest removing the −35 G-element of wt osmo σ38. Surprisingly, deleting the −35 G element causes extending transcription from growth phase into stationary phase and at the same time allows for optimal translation.
Thus, our approach is exemplified by the changes introduced at the ectA site. The σ70 promoter allows for transcription during growth phase and remains untouched. The σ38-osmo promoter is induced by potassium (salt stress). In order to extend transcription, the −35 region (G-box) of the osmotically induced promoter is either removed without any substitution or replaced by nucleotides other than G. This changes the promoter into a stationary phase σ38 promoter. These changes are physiological compatible with the cell and have no effect on growth and increase transcription (cf.
Favorably, the suggested modifications are:
Such combinatorial approach of enhancing the production of a natural compound by a microorganism, comprising up-regulating both at the level of transcription and translation, has not been even envisioned before.
Applying our approach can be used to improve
The methodology is feasible in any bacteria but preferable in extremophilic bacteria where genes are under the dual control of a vegetative σ70 promoter and a stress promoter (e.g. heat or osmo stress promoters) and where the RBS is weak. Favorably, improving transcription and translation as described will only introduce minor changes to the genome of the target organism. Since the changes are minor, the resulting organisms are not regarded as genetically modified (no GMO) and can be used for a wide field of application in industry.
As experimentally demonstrated, combining subtle promoter modifications, which closely resemble the natural occurring promoters in the target organism, with a modification of the RBS lead to a dramatic increase in expression of ectA and subsequently of the entire ectoine production (cf.
Surprisingly, optimizing the RBS alone (leaving the wt promoters unchanged) already improved expression. This has been observed experimentally by a β-Galactosidase assay, based on the expression of the gene lacZ, which encodes the enzyme β-galactosidase in Halomonas elongata. This enzyme cleaves lactose, a disaccharide, into glucose and galactose. The enzyme activity can easily be quantified (cf.
In order to make the ectoine synthesis process more efficient, the H. elongata strain KB2.13 was used. This strain synthesizes more ectoine than the wild-type strain and excretes ectoine into the medium (ΔteaABC, ΔdoeA). Strain KB2.13 not only produces more ectoine than H. elongata DSM 2581T but also reduces expenses during industrial processes (Grammann et al., 2002; Kunte et al., 2014).
In order improve the translation in comparison to the wild type, a synthetic ribosomal binding site (synthetic RBS) upstream of the target gene ectA was designed using the library calculator software (Espah Borujeni et al., 2014; Salis et al., 2009). In case of the used H. elongata, the target gene ectA comprises the start codon ATG.
The natural RBS located upstream of ectA comprises the sequence (SEQ ID NO 11):
(cf.
The designed nucleotide sequence of the synthetic RBS comprises the sequence (SEQ ID NO 2):
(cf.
The synthetic RBS sequence was transcriptionally fused with the reporter gene lacZ encoding the enzyme β-Galactosidase, cloned in the vector pBBR1MCS (Kovach et al., 1995), and transferred to the H. elongata cells via E. coli ST18 or E. coli 517-1 mediated conjugation (Grammann et al. 2002; Schwibbert et al. 2011). Bacterial conjugation is the transfer of genetic material such as plasmids by cell to cell contact via a bridge like connection (here: E. coli to H. elongata). Conjugation has the advantage over other transfer methods that it is very efficient in transferring DNA and allows transfer of large amounts of DNA. For further quantification, the same procedure described in the second embodiment was used (see below).
In order to further enhance the ectoine production, even at the level of transcription, the osmotically regulated σ38 promoter of the wild type was edited by removing the −35 G-element (cf.
In particular, the new σ38 promoter as shown in
The sequence of the σ38-osmo promoter of H. elongata comprises a sequence (SEQ ID NO 12): GCGGCCTGGGGAGTGGGCTATAAT. The G-element GCGG (SEQ ID NO 3) at −35 and the −10 sequence are written in bold. The spacer that separates the −35 and −10 region is written in cursive. As indicated above σ38-osmo regulates the transcription of ectA, and hence the biosynthesis of ectoine during the growth phase under osmotic stress.
In contrats thereto, the sequence of the stationary σ38 comprises CTACACT (SEQ ID NO 5) as shown in
The synthetic RBS sequence was transcriptionally fused with the reporter gene lacZ encoding the enzyme β-Galactosidase, cloned in the vector pBBR1MCS (Kovach et al., 1995), and transferred to H. elongata via E. coli mediated conjugation. The same was done for the stationary σ38 promoter (
Therein, Φ refers to the fusion sequence of the specified RBS with the lacZ gene. To determine the activity of the new promoter and synthetic RBS, β-Galactosidase assays (Sambrook & Russell, 2001) were performed in MM63 media (Larsen et al 1987), at 30° C., and 155 rpm. A total of four replicates were used per treatment. The assays revealed that the synthetic RBS and stationary promoter σ38-stationary enhances the expression of gene lacZ by a factor of approximately 20 (
In particular,
After determining the activity of the different promoters and RBS, the wild type promoter σ38-osmo and the natural RBS were removed from the chromosome of H. elongata KB2.13 and replaced by the stationary promoter σ38-stationary and the synthetic RBS. The resulting strain was named H. elongata bEH30.
Then, ectoine synthesis was measured and quantified via HPLC in the KB2.13 strain and bEH30 strain. For this, both strains were cultured at 33° C., 250 rpm in 20 mL MOPS-buffered minimal medium. Growth and glucose consumption of both strains were identical. Ectoine is excreted to the medium and was measured by HPLC every hour during a period of 10 h. Introducing a stationary promoter σ38-stationary and a synthetic RBS upstream of ectA in H. elongata increased ectoine production. The newly developed strain bEH30 (
In particular,
As shown above, in order to improve ectoine production, the osmo σ38 promoter) (σ38-osmo) and the natural RBS located upstream of ectA were exchanged for a stationary σ38 promoter (σ38-stationary) and a synthetic RBS, respectively. The methods described allow for enhanced gene expression by a synthetic ribosomal binding site (RBS) in combination with an optimized σ38 promoter for increased synthesis of proteins, amino acids, amino acid derivatives, sugars and sugar-polyols in extremophilic Bacteria and Archaea.
In view of the above, according to an embodiment, a method for permanent production of Nγ-acetyl-L-2,4-diaminobutyrate and/or ectoine by Halomonas elongata during an exponential growth and during a stationary phase of a cultivated strain of Halomonas elongata is suggested. This method comprises the steps of:
and
Herein, the first four steps effectuate an enhanced translation of the genetic information related to the biosynthesis of ectoine and at least of Nγ-acetyl-L-2,4-diaminobutyrate, and comprise an up-regulation of their biosynthesis at the level of translation. The next four steps effectuate an enhanced transcription and hence, comprise up-regulating the biosynthesis of the mentioned compounds at the level of transcription.
As can be easily contemplated, the suggested approach is not only applicable to halophilic microorganisms, like, e.g., Halomonas elongata, but can also be adapted to other halophilic Bacteria and Archaea, or other biotechnically cultivated cells, wherein the cultivated cells are modified to have available genetic information concerning the production of a natural compound of interest. In particular, the genome of the cultivated cell contains at least a nucleotide sequence comprising the described above stationary σ38 promoter (σ38-stationary) and a synthetic RBS upstream of a target gene, which encodes either the natural compound of interest or a key enzyme, wherein the key enzyme is involved in the biosynthesis of the natural compound of interest. Advantageously, the genome of the cultivated cell further comprises, between the stationary σ38 promoter and the synthetic RBS a σ70 promoter.
In view of the above, the target gene (key gene) may also be selected to contain a start codon which is different from ATG, (such as GTG, TTG or CTG), depending on the natural compound of interest.
According to further embodiments, we herewith suggest:
Thus, according to an embodiment (1), a method for increased and permanent production of a natural compound by a microorganism during an exponential growth and during a stationary phase of a culture of the microorganism is suggested. The method comprises: enhancing a translation of a target mRNA, wherein the target mRNA comprises a transcript of a target gene, wherein the target gene is encoding the natural compound or a key enzyme involved in a biosynthesis of the natural compound in the microorganism, by replacing a wild type (wt) ribosome binding site (RBS) upstream of the target gene with a synthetic RBS, the synthetic RBS possessing a higher affinity towards a 16S rRNA of a ribosome of the microorganism than the wt RBS; and enhancing a transcription of the target gene by converting an osmotically regulated σ38 promoter upstream of the target gene into a stationary σ38 promoter, wherein the osmotically regulated σ38 promoter comprises a −35 G-element and a −10 element which are separated from each other by a spacer sequence, by deleting the −35 G element.
According to an embodiment (2), the mentioned −35 G element of the osmotically regulated wt σ38 promoter is deleted by replacing it with nucleotides others than guanine (G).
According to an embodiment (3), the method of embodiment (1) or (2) further comprises: cultivating the microorganism, wherein a genome of the cultivated microorganism encompasses in a downstream direction the stationary σ38 promoter, a σ70 promoter, the synthetic RBS, and the target gene.
According to an embodiment (4), in the method according to any of embodiments 1-3 the natural compound is selected from a protein, an amino acid, an derivative of an amino acid, a sugar, and a sugar-polyol.
According to an embodiment (5), in the method according to any of embodiments 1-4 the microorganism is a halophilic microorganism and the natural compound is selected from a compatible solute, the compatible solute being selected from a sugar, a sugar-polyol, an amino acid and an amino acid derivative.
According to an embodiment (6), in the method according to embodiment (4) the natural compound is selected from Nγ-acetyl-L-2,4-diaminobutyrate, ectoine, and hydroxyectoine.
According to an embodiment (7), in the method according to embodiment (6) the microorganism is Halomonas elongata and the key enzyme is L-2,4-diaminobutyric acid Nγ-acetyltransferase.
According to an embodiment (8), in the method according to embodiment (7) the synthetic RBS comprises the nucleotide sequence CTAAGGAGAC (SEQ ID NO 1).
According to an embodiment (9), in the method according to embodiment (8) the synthetic RBS comprises the nucleotide sequence
According to an embodiment (10), in the method according to embodiment (9) the −35 G-element of the osmotically regulated σ38 promoter comprises the nucleotide sequence GCGG (SEQ ID NO 3), wherein converting the stationary regulated σ38 promoter into the stationary σ38 promoter comprises replacing GCGG (SEQ ID NO 3) with AAAT (SEQ ID NO 4).
According to an embodiment (11), in the method according to embodiment (10) the nucleotide sequence of the stationary σ38 promoter comprises the nucleotide sequence
According to an embodiment (12), in the method according to embodiment (11) the nucleotide sequence of the stationary σ38 promoter comprises the nucleotide sequence
According to an embodiment (13), a nucleotide construct is suggested, wherein the nucleotide construct comprises: a synthetic ribosome binding site (RBS), a stationary σ38 promoter, a σ70 promoter, and a target gene; wherein the synthetic RBS upstream of the target gene possesses a higher affinity towards a 16S rRNA of a bacterial ribosome than a wild type (wt) RBS towards the 16S rRNA of the bacterial ribosome; wherein the stationary σ38 promoter is generated starting from an osmotically regulated wt σ38 promoter, the osmotically regulated wt σ38 promoter comprising a −35 G-element, by deleting the −35 G-element and/or by replacing the −35 G-element by a nucleotide, wherein the nucleotide is selected from nucleotides others than guanine (G); wherein the target gene is encoding a key enzyme, the key enzyme catalyzing a reaction selected from: a phosphorylation/dephosphorylation, a carboxylation/decarboxylation, and an acetylation; the key enzyme being involved in a biosynthesis of a natural compound, wherein the natural compound is selected from a protein, an amino acid, a derivative of an amino acid, a sugar, and a sugar-polyol.
According to an embodiment (14), in the nucleotide construct according to embodiment (13) the −35 G-element of the osmotically regulated wt σ38 promoter comprises the nucleotide sequence GCGG (SEQ ID NO 3), wherein in the stationary σ38 promoter the nucleotide sequence GCGG (SEQ ID NO 3) is replaced by the nucleotide sequence AAAT (SEQ ID NO 4).
According to an embodiment (15), a microorganism is suggested, wherein the microorganism is selected from Bacteria and Archaea and a genome of the microorganism encompasses the nucleotide construct according to any of embodiments (13) and (14).
According to an embodiment (16), the microorganism according to embodiment (15) is selected from Bacteria, wherein a strain of the Bacteria is selected from: a Bacillus, a Bacillus subtilis, a Brevibacterium, a Chromohalobacter, a Corynebacterium glutamicum, an Escherichia coli, a Halobacillus, a Halomonas, a Halomonas elongata, a Marinococcus, and a Methylomicrobium.
According to an embodiment (17), a biotechnical process for continuous production of a natural compound by a microorganism is suggested, wherein a culture of the microorganism is maintained in an exponential growth phase or in a stationary phase, wherein a genome of the microorganism encompasses the nucleotide construct according to claim 13 or 14, wherein the microorganism is adapted to excrete the natural compound into a culture medium, wherein the process comprises extracting the natural compound from the culture medium.
According to an embodiment (18), in the process according to embodiment (17) the microorganism is Halomonas elongata, and the natural compound is selected from: Nγ-acetyl-L-2,4-diaminobutyrate, ectoine, and hydroxyectoine.
Although various exemplary embodiments of the invention have been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made which will achieve some of the advantages of the invention without departing from the spirit and scope of the invention. It will be obvious to those reasonably skilled in the art that other components performing the same functions may be suitably substituted. It should be mentioned that features explained with reference to a specific figure may be combined with features of other figures, even in those cases in which this has not explicitly been mentioned. Such modifications to the inventive concept are intended to be covered by the appended claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/055077 | 3/1/2018 | WO | 00 |
