WO2022161914A1

WO2022161914A1 - High temperature fermentation process and microorganisms

Info

Publication number: WO2022161914A1
Application number: PCT/EP2022/051515
Authority: WO
Inventors: Max Fabian FELLE; Oskar Zelder; Erhard BREMER; Tamara HOFFMANN; Christine FRANK
Original assignee: Basf Se
Priority date: 2021-01-26
Filing date: 2022-01-24
Publication date: 2022-08-04

Abstract

The present invention relates to high temperature fermentation of fine chemicals, in particular L-proline and intermediates of L-proline synthesis, and to microorganisms for such fermentation processes. The invention further relates to genes, expression cassettes and vectors for the construction of such microorganisms.

Description

HIGH TEMPERATURE FERMENTATION PROCESS AND MICROORGANISMS

BACKGROUND

The amino acid proline is a useful substrate in various industrial applications. It is thus desirable to provide industrial fermentation processes for the production of this amino acid and/or of intermediates of proline, specifically L-proline, biosynthesis. The metabolic pathway for L-proline production is known per se. Key enzymes and reactions involved in the pathway are described, for example, in Belitzky et al, J. Bacteriology 2001, 4389-4392, which is incorporated herein in its entirety. Generally speaking in Bacillaceae the genes proJ and proB code for a glutamate 5-kinase to convert glutamate into L-glutamyl-5-phosphate (also known as gamma-glutamyl phosphate), a gamma-glutamyl phosphate reductase ProA converts L-glutamyl-5-phosphate to gamma-glutamate-semialdehyde, which in turn spontaneously rearranges to 1-pyrroline-5-carboxylate. The latter substance is converted to proline by a pyrroline-5-carboxylate reductase coded by any of the genes prol and proH. As described above, the proB gene expression and ProB enzyme activity is downregulated in the presence of L-proline.

Industrial fermentative production of proline has proven difficult due to proline product inhibition on translation of the proB gene and ProB enzyme activity. Several attempts have been made to overcome product inhibition, either by mutations on the proB gene or by using the proJ-proA-proH pathway. Other attempts are described for example in WO2018074916 and W02006066758. However, such fermentations are only described for temperatures below 40°C. This is disadvantageous, because stirring as applied in industrial fermentations and metabolic heat generally lead to a higher temperature of fermenter contents, thereby requiring cooling to prevent death of the production microorganism or at least to prevent denaturation of the enzymes involved in proline biosynthesis.

It was thus the object of the present invention to provide materials, methods and uses to produce L-proline with a high yield at elevated temperatures.

BRIEF DESCRIPTION OF THE INVENTION

The invention in particular provides a method of high temperature fermentative production of any of L-glutamyl-5-phosphate, gamma-glutamate-semialdehyde, 1-pyrroline-5-carboxylate or L-proline, comprising the steps

1) providing a microorganism capable of glutamate synthesis at a temperature of 40°C to 70°C,

2) fermentative production of L-glutamyl-5-phosphate, gamma-glutamate-semialdehyde, 1-pyrroline-5-carboxylate and/or proline at a temperature of 40°C to 70°C, wherein the microorganism expresses a proB* gene comprising at least one mutation to remove or reduce inhibition by proline.

The invention also provides an expression cassette, comprising a promoter and, operably linked thereto, a proB*, a proA and a prol gene, wherein the proB* gene comprises at least one mutation to remove or reduce inhibition by proline, and preferably the proA and prol gene sequences are linked by a linker comprising a ribosome binding site. And the invention provides a vector comprising an expression cassette according to the invention.

Furthermore, the invention provides a microorganism for high temperature fermentative production of any of L-glutamyl-5-phosphate, gamma-glutamate-semialdehyde, 1-pyrroline-5- carboxylate or L-proline, wherein the microorganism expresses a proB* gene comprising at least one mutation to remove or reduce inhibition by proline and preferably comprises an expression cassette according or a vector according to the present invention, wherein the microorganism is capable of glutamate synthesis at a temperature of 40°C to 70°C and preferably is selected from the taxonomic family Bacillaceae, preferably from genus Bacillus, Falsibacillus or Mesobacillus, more preferably from any of the genera Bacillus aeolius, Bacillus boroniphilus, Bacillus ciccensis, Bacillus circulans, Bacillus dafuensis, Bacillus foraminis, Bacillus marisflavi, Bacillus novalis, Falsibacillus sp., Mesobacillus jeotgali, Bacillus jeotgali, Bacillus niacini, Bacillus pichinotyi, Bacillus oceanisediminis, Bacillus methanolicus, Bacillus firmus, Bacillus subterraneus.

Also provided by the invention is the use of an expression cassette, a vector or a microorganism of the present invention for the production of any of L-glutamyl-5-phosphate, gamma-glutamate-semialdehyde, 1-pyrroline-5-carboxylate or L-proline at a temperature of 40°C to 70°C.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts L-proline synthesis routes in B. subtilis JH642, B. licheniformis DSM13, and B. methanolicus MGA3. a Schematic overview of the conversion of L-glutamate into L-proline by the anabolic proline biosynthetic enzymes (ProB- ProA- Prol) in B methanolicus MGA3. The exporter for these amino acids remains unknown, b The anabolic pathway (ProB- ProA- Prol) and osmostress responsive (ProJ-ProA-ProH; ProJ-ProAA-ProH) biosynthesis pathways of B. subtilis JH642, B. licheniformis DSM13 and B. methanolicus MGA3 are shown. The anabolic glutamate 5-kinase ProB is feedback inhibited by L-proline. B. methanolicus MGA3 does not contain an osmostress responsive proline biosynthesis pathway.

Figure 2 shows the results of implementation of the osmostress proline biosynthetic proHJAA genes from B. licheniformis into B. subtilis JSB8 and B. methanolicus MGA3. a Genetic organization of the proHJAA osmostress responsive operon from B. licheniformis that is located on the plasmids pCF7 and pCF8. The expression of the genes is regulated by the osmoregulated proHJAA promoter PproHJAA of B. licheniformis. On Plasmid pCF8 the expression is under the control of the mdh promoter Pmdh of B. methanolicus. b L-proline synthesis in B. subtilis JSB8 [(delta)(proHJ::tet)] strain carrying the proHJAA plasmids pCF7, pCF8, and of the empty vector pBV2mp. Cultures were grown in either in SMM in the absence of NaCI (gray bars) or in SMM with 0.5 M NaCI (black bars) at 37° C. When cultures reached an OD578 of approximately 1.5, the cells were harvested and assayed for their concentration of L- proline by HPLC. c B. methanolicus MGA3 carrying the proHJAA gene cluster located on the plasmids pCF7 and pCF8, and the empty vector pBV2mp. Cells were cultivated at 50° C in either in MVcM (grey bars) or MVcM with 0.5 M NaCI (black bars) and were harvested at an OD578 of approximately 1.5. The intracellular L- proline was quantified by HPLC analysis. The data shown were derived from two independently grown cultures and each HPLC analysis was performed twice. Figure 3 shows the design of a synthetic L- proline biosynthesis gene cluster with the anabolic L- proline biosynthetic genes of B. methanolicus MGA3. a Genetic organization of the endogenous L-proline biosynthesis genes proBA and prol of B. methanolicus MGA3. The T- Box mediated transcriptional regulatory mechanism and the post- transcriptional feedback inhibition are indicated. The enzyme- inhibitor- interaction is modulated by a flexible 16- residue loop (indicated as a black line) and it possesses a negative glutamate residue (E142) in the center, b The synthetic L-proline biosynthesis gene cluster. This gene cluster was created by amino acid substitution of the negatively charged L-glutamate against a positively charged L-arginine (R142) by site directed mutagenesis. The pCF22 plasmid possesses the native PproBAI promoter in which the T-box mediated mRNA regulatory device was removed. The proB*AI genes located on the plasmid pCF21 were controlled by the Pmdh promoter. The newly assembled 25 bp intergenic region of proA and prol is shown in c and the ribosome binding site of prol is indicated.

Figure 4 depicts the implementation of the synthetic anabolic proline biosynthetic genes in B. methanolicus MGA3. B. methanolicus MGA3 strain carrying the adapted anabolic L- proline biosynthetic genes proB*AI controlled by the native promoter PproBAI (delta)TBox (pCF22) or by the mdh promoter Pmdh (pCF21). Cultures were cultivated in MVcM at 50° C and were harvested at an OD578 of 1.5. The intracellular (a) and extracellular (b) L-proline content was quantified by HPLC analysis. The data shown were derived from two independently grown cultures and each HPLC measurement was performed twice.

Figure 5 shows a sequence alignment of SEQ ID NO 26 and the sequence according to Uniprot entry I3E8T4_BACMM. Numbers are given according to the position of Uniprot entry I3E8T4_BACMM sequence. The number of asterisks above each amino acid of the I3E8T4_BACMM sequence indicates the degree of conservation, wherein higher number of stars indicate a stronger conservation. Amino acids given below each amino acid of SEQ ID NO. 26 indicate potential substitutions allowable at the respective position, wherein indicates a gap (deletion relative to the I3E8T4_BACMM sequence). The possible substitutions are listed in the order of their respective preference, wherein a more preferred substitution is indicated closer to the respective position in SEQ ID NO. 26.

BRIEF DESCRIPTION OF SEQUENCES

DETAILED DESCRIPTION

The technical teaching of the invention is expressed herein using the means of language, in particular by use of scientific and technical terms. However, the skilled person understands that the means of language, detailed and precise as they may be, can only approximate the full content of the technical teaching, if only because there are multiple ways of expressing a teaching, each necessarily failing to completely express all conceptual connections, as each expression necessarily must come to an end . With this in mind the skilled person understands that the subject matter of the invention is the sum of the individual technical concepts signified herein or expressed, necessarily in a pars-pro-toto way, by the innate constrains of a written description. In particular, the skilled person will understand that the signification of individual technical concepts is done herein as an abbreviation of spelling out each possible combination of concepts as far as technically sensible, such that for example the disclosure of three concepts or embodiments A, B and C are a shorthand notation of the concepts A+B, A+C, B+C, A+B+C. In particular, fallback positions for features are described herein in terms of lists of converging alternatives or instantiations. Unless stated otherwise, the invention described herein comprises any combination of such alternatives. The choice of more or less preferred elements from such lists is part of the invention and is due to the skilled person’s preference for a minimum degree of realization of the advantage or advantages conveyed by the respective features. Such multiple combined instantiations represent the adequately preferred form(s) of the invention.

As used herein, terms in the singular and the singular forms like "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, use of the term "a nucleic acid" optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term "probe" optionally (and typically) encompasses many similar or identical probe molecules. Also as used herein, the word "comprising" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The term "preferably" is used herein to denote alternative embodiments of the invention which may provide increased advantages compared to less preferred alternatives. Thus, the term "preferably" is not intended to limit the invention.

As used herein, the term "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). The term "comprising" also encompasses the term "consisting of".

The term "about", when used in reference to a measurable value, for example an amount of mass, dose, time, temperature, sequence identity and the like, refers to a variation of ± 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or even 20% of the specified value as well as the specified value. Thus, if a given composition is described as comprising "about 50% X," it is to be understood that, in some embodiments, the composition comprises 50% X whilst in other embodiments it may comprise anywhere from 40% to 60% X (i.e. , 50% ± 10%), and if for example a length of about 20 nucleotides is mentioned, then the length can in some embodiments be 18-22 nucleotides (20nt ± 10%).

Within this description, genes names generally start with a lower case letter. The protein encoded by the gene herein bears the name of the gene with a capitalised first letter. Thus, for example, the proB* gene codes for a ProB protein. Correspondingly, the mentioning of expression of a particular protein is intended to also disclose the presence of a gene coding for said protein and the production of mRNA based on said gene template.

As used herein, the term "gene" refers to a biochemical information which, when materialised in a nucleic acid, can be transcribed into a gene product, i.e. a further nucleic acid, preferably an RNA, and preferably also can be translated into a peptide or polypeptide. The term is thus also used to indicate the section of a nucleic acid resembling said information and to the sequence of such nucleic acid (herein also termed "gene sequence").

Also as used herein, the term "allele" or “variant” refers to a variation of a gene characterized by one or more specific differences in the gene sequence compared to the wild type gene sequence, regardless of the presence of other sequence differences. Alleles or nucleotide sequence variants of the invention have at least, in increasing order of preference, 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide "sequence identity" to the nucleotide sequence of the wild type gene. Correspondingly, where an "allele" refers to the biochemical information for expressing a peptide or polypeptide, the respective nucleic acid sequence of the allele has at least, in increasing order of preference, 30%, 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%-84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid "sequence identity" to the respective wild type peptide or polypeptide.

Mutations or alterations of amino or nucleic acid sequences can be any of substitutions, deletions or insertions; the terms "mutations" or "alterations" also encompass any combination of these. Hereinafter, all three specific ways of mutating are described in more detail by way of reference to amino acid sequence mutations; the corresponding teaching applies to nucleic acid sequences such that "amino acid" is replaced by "nucleotide".

When describing protein sequences, the standard IIIPAC single-letter or three-letter amino acid abbreviations are used. “Substitutions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the substituted amino acid. For example, the substitution of histidine at position 120 with alanine is designated as “His120Ala” or “H120A”.

“Deletions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by "*" or Accordingly, the deletion of glycine at position 150 is designated as "Gly150*", "G150*", "Gly150-" or "G150-". Alternatively, deletions are indicated by e.g. “deletion of D183 and G184”.

“Insertions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the original amino acid and the additional amino acid. For example, an insertion at position 180 of lysine next to glycine would be designated as “Gly180GlyLys” or “G180GK”. When more than one amino acid residue is inserted, such as e.g. a Lys and Ala after Gly180 this may be indicated as: Gly180GlyLysAla or G180GKA. In cases where a substitution and an insertion occur at the same position, this may be indicated as S99SD+S99A or in short S99AD. In cases where an amino acid residue identical to the existing amino acid residue is inserted, it is clear that degeneracy in the nomenclature arises. If for example a glycine is inserted after the glycine in the above example this would be indicated by G180GG.

Variants comprising multiple alterations are separated by “+”, e.g. “Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple alterations may be separated by space or a comma e.g. R170Y G195E or R170Y, G195E respectively.

Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g. “Arg170Tyr, Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Alternatively, different alterations or optional substitutions may be indicated in brackets e.g. Arg170[Tyr, Gly] or Arg170{Tyr, Gly} or in short R170[Y,G] or R170{Y, G}.

Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as a peptide or polypeptide. Preferably such mutations are not pertaining the functional domains of a peptide or polypeptide.

Any references to sequences according to the Uniprot database refer to the sequences as published on 2021-01-15, unless explicitly stated otherwise.

Protein or nucleic acid variants may be defined by their sequence identity when compared to a parent protein or nucleic acid. Sequence identity usually is provided as “% sequence identity” or “% identity”. To determine the percent-identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e. , a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program “NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EBLOSUM62). The preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined. The following example is meant to illustrate two nucleotide sequences, but the same calculations apply to protein sequences:

Seq A: AAGATACTG length: 9 bases

Seq B: GATCTGA length: 7 bases

Hence, the shorter sequence is sequence B.

Producing a pairwise global alignment which is showing both sequences over their complete lengths results in

Seq A :

Seq B :

The “I” symbol in the alignment indicates identical residues (which means bases for DNA or amino acids for proteins). The number of identical residues is 6.

The symbol in the alignment indicates gaps. The number of gaps introduced by alignment within the sequence B is 1. The number of gaps introduced by alignment at borders of sequence B is 2, and at borders of sequence A is 1.

The alignment length showing the aligned sequences over their complete length is 10.

Producing a pairwise alignment which is showing the shorter sequence over its complete length according to the invention consequently results in:

Seq A :

Seq B :

Producing a pairwise alignment which is showing sequence A over its complete length according to the invention consequently results in:

Seq A :

Seq B :

Producing a pairwise alignment which is showing sequence B over its complete length according to the invention consequently results in:

Seq A :

Seq B :

The alignment length showing the shorter sequence over its complete length is 8 (one gap is present which is factored in the alignment length of the shorter sequence).

Accordingly, the alignment length showing sequence A over its complete length would be 9 (meaning sequence A is the sequence of the invention), the alignment length showing sequence B over its complete length would be 8 (meaning sequence B is the sequence of the invention).

After aligning the two sequences, in a second step, an identity value shall be determined from the alignment. Therefore, according to the present description the following calculation of percent-identity applies: %-identity = (identical residues I length of the alignment region which is showing the respective sequence of this invention over its complete length) *100. Thus, sequence identity in relation to comparison of two amino acid sequences according to the invention is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give “%-identity”. According to the example provided above, %- identity is: for sequence A being the sequence of the invention (6 / 9) * 100 = 66.7 %; for sequence B being the sequence of the invention (6 / 8) * 100 = 75%.

The term "hybridisation" as defined herein is a process wherein substantially complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.

The term “stringency” refers to the conditions under which a hybridisation takes place. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20°C below Tm, and high stringency conditions are when the temperature is 10°C below Tm. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore, medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid molecules.

The “Tm” is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16°C up to 32°C below Tm. The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher concentrations, this effect may be ignored). Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7°C for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45°C, though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1°C per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:

DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984): Tm= 81.5°C + 16.6xlog([Na+]{a}) + 0.41x%[G/C{b}] - 500x[L{c}]-1 - 0.61x% formamide DNA-RNA or RNA-RNA hybrids:

Tm= 79.8 + 18.5 (Iog10[Na+]{a}) + 0.58 (%G/C{b}) + 11.8 (%G/C{b})2 - 820/L{c}

• oligo-DNA or oligo-RNAd hybrids: for <20 nucleotides: Tm= 2 ({In}) for 20-35 nucleotides: Tm= 22 + 1.46 ({In} ) wherein:

{a} or for other monovalent cation, but only accurate in the 0.01-0.4 M range

{b} only accurate for %GC in the 30% to 75% range

{c} L = length of duplex in base pairs

{d} Oligo, oligonucleotide

{In} effective length of primer = 2* (no. of G/C)+(no. of A/T)

Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase. For non-related probes, a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68°C to 42°C) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%). The skilled artisan is aware of various parameters which may be altered during hybridisation and which will either maintain or change the stringency conditions.

Besides the hybridisation conditions, specificity of hybridisation typically also depends on the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. A positive hybridisation gives a signal that is at least twice of that of the background. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions.

For example, typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65°C in 1x SSC or at 42°C in 1x SSC and 50% formamide, followed by washing at 65°C in 0.3x SSC. Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50°C in 4x SSC or at 40°C in 6x SSC and 50% formamide, followed by washing at 50°C in 2x SSC. The length of the hybrid is the anticipated length for the hybridising nucleic acid. When nucleic acids of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein. 1xSSC is 0.15M NaCI and 15mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5x Denhardt's reagent, 0.5-1.0% SDS, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate. Another example of high stringency conditions is hybridisation at 65°C in 0.1x SSC comprising 0.1 SDS and optionally 5x Denhardt's reagent, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, followed by the washing at 65°C in 0.3x SSC.

For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).

The term "nucleic acid construct" as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or is synthetic.

The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a polynucleotide.

The term "control sequence" is defined herein to include all sequences affecting the expression of a polynucleotide, including but not limited thereto, the expression of a polynucleotide encoding a polypeptide. Each control sequence may be native or foreign to the polynucleotide or native or foreign to each other. Such control sequences include, but are not limited to, promoter sequence, 5’-UTR (also called leader sequence), ribosomal binding site (RBS, Shine Dalgarno sequence), 3’-UTR, and transcription start and stop sites.

The term "functional linkage" or "operably linked" with respect to regulatory elements, is to be understood as meaning the sequential arrangement of a regulatory element (including but not limited thereto a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (including but not limited thereto a terminator) in such a way that each of the regulatory elements can fulfil its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. For example, a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

A "promoter" or "promoter sequence" is a nucleotide sequence located upstream of a gene on the same strand as the gene that enables that gene's transcription. A promoter is followed by the transcription start site of the gene. A promoter is recognized by RNA polymerase (together with any required transcription factors), which initiates transcription. A functional fragment or functional variant of a promoter is a nucleotide sequence which is recognizable by RNA polymerase, and capable of initiating transcription.

An "active promoter fragment", "active promoter variant", "functional promoter fragment" or "functional promoter variant" describes a fragment or variant of the nucleotide sequences of a promoter, which still has promoter activity.

A promoter can be an “inducer-dependent promoter” or an “inducer-independent promoter” comprising constitutive promoters or promoters which are under the control of other cellular regulating factors.

The person skilled in the art is capable to select suitable promoters for expressing the proB*- proA-prol genes of the scope of the invention. For example, the polynucleotide encoding the polypeptide of interest is, preferably, operably linked to an “inducer-dependent promoter” or an “inducer-independent promoter.

Preferred promoters are: the mdh promoter of Bacillus methanolicus, the xylose inducible PxylA promoter, the mannitol PmtIA of Bacillus methanolicus, the lactose inducible promoters Pspac and Phyper-spank, SP01 promoters P4, P5, P15, the crylllA promoter from Bacillus thuringiensis (WO9425612), Pveg, PlepA, PserA, PymdA, Pfba, P43 promoters.

An "inducer dependent promoter" is understood herein as a promoter that is increased in its activity to enable transcription of the gene to which the promoter is operably linked upon addition of an "inducer molecule" to the fermentation medium. Thus, for an inducerdependent promoter the presence of the inducer molecule triggers via signal transduction an increase in expression of the gene operably linked to the promoter. The gene expression prior activation by the presence of the inducer molecule does not need to be absent, but can also be present at a low level of basal gene expression that is increased after addition of the inducer molecule. The "inducer molecule" is a molecule which presence in the fermentation medium is capable of affecting an increase in expression of a gene by increasing the activity of an inducer-dependent promoter operably linked to the gene. Preferably the inducer molecule is a carbohydrate or an analogue thereof. In one embodiment, the inducer molecule is a secondary carbon source of the Bacillus cell. In the presence of a mixture of carbohydrates cells selectively take up the carbon source that provide them with the most energy and growth advantage (primary carbon source). Simultaneously, they repress the various functions involved in the catabolism and uptake of the less preferred carbon sources (secondary carbon source). Typically, a primary carbon source for Bacillus is glucose and various other sugars and sugar derivates being used by Bacillus as secondary carbon sources. Secondary carbon sources include e.g. mannose, mannitol, xylose or lactose without being restricted to these. In contrast thereto, the activity of promoters that do not depend on the presence of an inducer molecule (herein called "inducer-independent promoters") are either constitutively active or can be increased regardless of the presence of an inducer molecule that is added to the fermentation medium.

Constitutive promoters are independent of other cellular regulating factors and transcription initiation is dependent on sigma factor A (sigA). The sigA-dependent promoters comprise the sigma factor A specific recognition sites ‘-35’-region and ‘-10’-region.

In a preferred embodiment, the constitutive promoter sequence is selected from the group comprising promoters Pveg, PlepA, PserA, PymdA, Pfba and derivatives thereof with different strength of gene expression (Guiziou et al, (2016): Nucleic Acids Res. 44(15), 7495- 7508), bacteriophage SPO1 promoters P4, P5, P15 (WO15118126), the crylllA promoter from Bacillus thuringiensis (WO9425612), and combinations thereof, or active fragments or variants thereof.

The term "transcription start site" or "transcriptional start site" shall be understood as the location where the transcription starts at the 5’ end of a gene sequence. In prokaryotes the first nucleotide, referred to as +1 is in general an adenosine (A) or guanosine (G) nucleotide. In this context, the terms "sites" and "signal" can be used interchangeably herein.

The term "expression" or "gene expression" means the transcription of a specific gene or specific genes or specific nucleic acid construct. The term "expression" or "gene expression" in particular means the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.

Further optionally the promoter comprises a 5'IITR. This is a transcribed but not translated region downstream of the -1 promoter position. Such untranslated region for example should contain a ribosome binding site to facilitate translation in those cases where the target gene codes for a peptide or polypeptide. With respect to the 5'IITR the invention in particular teaches to combine the promoter of the present invention with a 5'IITR comprising one or more stabilising elements. This way the mRNAs synthesized from the promoter region may be processed to generate mRNA transcript with a stabilizer sequence at the 5' end of the transcript. Preferably such a stabilizer sequence at the 5'end of the mRNA transcripts increases their half-life as described by Hue et al, 1995, Journal of Bacteriology 177: 3465-3471. Suitable mRNA stabilizing elements are those described in

WO08148575, preferably SEQ ID NO. 1 to 5 of W008140615, or fragments of these sequences which maintain the mRNA stabilizing function, and in

W008140615, preferably Bacillus thuringiensis CrylllA mRNA stabilising sequence or bacteriophage SP82 mRNA stabilising sequence, more preferably a mRNA stabilising sequence according to SEQ ID NO. 4 or 5 of WQ08140615, more preferably a modified mRNA stabilising sequence according to SEQ ID NO. 6 of WQ08140615, or fragments of these sequences which maintain the mRNA stabilizing function.

Preferred mRNA stabilizing elements are selected from the group consisting of aprE, grpE, cotG, SP82, RSBgsiB, CrylllA mRNA stabilizing elements, or according to fragments of these sequences which maintain the mRNA stabilizing function. A preferred mRNA stabilizing element is the grpE mRNA stabilizing element (corresponding to SEQ ID NO. 2 of WQ08148575).

The 5'IITR also preferably comprises a modified rib leader sequence located downstream of the promoter and upstream of an ribosome binding site (RBS). In the context of the present invention a rib leader is herewith defined as the leader sequence upstream of the riboflavin biosynthetic genes (rib operon) in a Bacillus cell, more preferably in a Bacillus methanolicus cell. In Bacillus subtilis, the rib operon is known and comprises the genes involved in riboflavin biosynthesis, include ribG (ribD), ribB (ribE), ribA, and ribH genes. Transcription of the riboflavin operon from the rib promoter (Prib) in B. subtilis is controlled by a riboswitch involving an untranslated regulatory leader region (the rib leader) of almost 300 nucleotides located in the 5'-region of the rib operon between the transcription start and the translation start codon of the first gene in the operon, ribG. Suitable rib leader sequences are described in WQ2015/1181296, in particular pages 23-25, incorporated herein by reference. The aforementioned elements of the rib operon and promoter can be transferred and optionally adapted to other microorganisms, e.g. Bacillus methanolicus.

The term "vector" is defined herein as a linear or circular DNA molecule that comprises a polynucleotide that is operably linked to one or more control sequences that provides for the expression of the polynucleotide.

As used herein, the term “plasmid” refers to an extrachromosomal circular DNA. A plasmid is autonomously replicating in the host cell. The term “plasmid” is understood to be extrachromosomal circular DNA and may be autonomously replicating under permissive conditions and it may comprise a polynucleotide that is operably linked to one or more control sequences that provides for the expression of the polynucleotide.

In accordance with the present invention, the replication of a plasmid shall be independent of the replication of the chromosome of the bacterial host cell. For autonomous replication, the plasmid comprises an autonomous replication sequence, i.e. an origin of replication enabling the plasmid to replicate autonomously in the bacterial host cell. Examples of bacterial origins of replication are the origins of replication of plasmids pUB110, pBC16, pE194, pC194, pTB19, pAMB1 , pTA1060, pNW33 permitting replication in Bacillus (see e.g Irla M, Wendisch VF (2016). Front. Microbiol. 7:148). and plasmids pBR322, colE1 , pUC19, pSC101 , pACYC177, and pACYC184 permitting replication in E.°coli (see e.g. Sambrook.J. and Russell, D.W. Molecular cloning. A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001.). The copy number of a plasmid is defined as the average number of plasmids per bacterial cell or per chromosome under normal growth conditions. Moreover, there are different types of replication origins that result in different copy numbers in the bacterial host. The plasmid replicon pBS72 (accession number AY102630.1) and the plasmids pTB19 and derivatives pTB51 , pTB52 confer low copy number with 6 copies and 1 to 8 copies, respectively, within Bacillus cells whereas plasmids pE194 (accession number V01278.1) and pUB110 (accession number M19465.1)/pBC16 (accession number U32369.1) confer low-medium copy number with 14-20 and medium copy number with 30-50 copies per cell, respectively. Plasmid pE194 was analyzed in more detail (Villafane, et al (1987): J.Bacteriol. 169(10), 4822-4829) and several pE194 - cop mutants described having high copy numbers within Bacillus ranging from 85 copies to 202 copies. Moreover, plasmid pE194 is temperature sensitive with stable copy number up to 37°C, however abolished replication above 43°C.

A preferred plasmid comprises replicon pBS72 (accession number AY102630.1) as autonomous replication sequence. In another preferred embodiment, the plasmid comprises the replication origin of pUB110 (accession number M19465.1 )/pBC16 (accession number U32369.1) as autonomous replication sequence. The plasmid can be introduced into the host cell by any method suitable for transferring the plasmid into the cell, for example, by transformation using electroporation, protoplast transformation or conjugation.

As used herein, the term "isolated DNA molecule" refers to a DNA molecule at least partially separated from other molecules normally associated with it in its native or natural state. The term "isolated" preferably refers to a DNA molecule that is at least partially separated from some of the nucleic acids which normally flank the DNA molecule in its native or natural state. Thus, DNA molecules fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques, are considered isolated herein. Such molecules are considered isolated when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules, in that they are not in their native state.

Any number of methods well known to those skilled in the art can be used to isolate and manipulate a polynucleotide, or fragment thereof, as disclosed herein. For example, polymerase chain reaction (PCR) technology can be used to amplify a particular starting polynucleotide molecule and/or to produce variants of the original molecule. Polynucleotide molecules, or fragment thereof, can also be obtained by other techniques, such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer. A polynucleotide can be single-stranded (ss) or double- stranded (ds). "Double-stranded" refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure, generally under physiologically relevant conditions. Embodiments of the method include those wherein the polynucleotide is at least one selected from the group consisting of sense single- stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), a double-stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA; a mixture of polynucleotides of any of these types can be used.

The term "heterologous" (or exogenous or foreign or recombinant or non-native) polypeptide is defined herein as a polypeptide that is not native to the host cell, a polypeptide native to the host cell in which structural modifications, e.g., deletions, substitutions, and/or insertions, have been made by recombinant DNA techniques to alter the native polypeptide, or a polypeptide native to the host cell whose expression is quantitatively altered or whose expression is directed from a genomic location different from the native host cell as a result of manipulation of the DNA of the host cell by recombinant DNA techniques, e.g., a stronger promoter. Similarly, the term "heterologous" (or exogenous or foreign or recombinant or nonnative) polynucleotide refers to a polynucleotide that is not native to the host cell, a polynucleotide native to the host cell in which structural modifications, e.g., deletions, substitutions, and/or insertions, have been made by recombinant DNA techniques to alter the native polynucleotide, or a polynucleotide native to the host cell whose expression is quantitatively altered as a result of manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques, e.g., a stronger promoter, or a polynucleotide native to the host cell, but integrated not within its natural genetic environment as a result of genetic manipulation by recombinant DNA techniques. With respect to two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous" is used to characterized that the two or more polynucleotide sequences or two or more amino acid sequences are naturally not occurring in the specific combination with each other. In particular, the term "heterologous" when referring to a promoter-gene combination means that the specific combination of promoter and gene is not found in nature. A promotor is heterologous to a gene and vice versa in particular when (a) a promoter, which in a wild type cell is operably linked to a gene A, is now operably linked instead to another gene B, or (b) where a promotor not found in nature is operably linked to a gene, or (c) where a promotor is operably linked to a gene of a sequence not found in nature.

The term "host cell", as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, conjugation, and the like with a nucleic acid construct or expression vector or plasmid. Thus, the term "host cell" includes cells that have the capacity to act as a host or expression vehicle for a newly introduced DNA sequence, in particular for expression of a target gene comprised in said newly introduced DNA sequence. The host cell according to the invention is understood to be prokaryotic and preferably belongs to a genus Gram positive microorganisms. More preferably the host cell belongs to the taxonomic family Bacillaceae, more preferably from the genus Bacillus, and most preferably of the species Bacillus methanolicus.

As used herein, "recombinant" when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation. A gene sequence open reading frame is recombinant if (a) that nucleotide sequence is present in a context other than its natural one, for example by virtue of being (i) cloned into any type of artificial nucleic acid vector or (ii) moved or copied to another location of the original genome, or if (b) the nucleotide sequence is mutagenized such that it differs from the wild type sequence. The term recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid is a recombinant plant.

The term "transgenic" refers to an organism, preferably a plant or part thereof, or a nucleic acid that comprises a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to refer to any cell or cell line the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell. A "recombinant" organism preferably is a "transgenic" organism.

As used herein, "mutagenized" refers to an organism or nucleic acid thereof having alteration(s) in the biomolecular sequence of its native genetic material as compared to the sequence of the genetic material of a corresponding wildtype organism or nucleic acid, wherein the alteration(s) in genetic material were induced and/or selected by human action. Methods of inducing mutations can induce mutations in random positions in the genetic material or can induce mutations in specific locations in the genetic material (i.e. , can be directed mutagenesis techniques), such as by use of a genoplasty technique. In addition to unspecific mutations, according to the invention a nucleic acid can also be mutagenized by using mutagenesis means with a preference or even specificity for a particular site, thereby creating an artificially induced heritable allele according to the present invention. Such means, for example site specific nucleases, including for example zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENS) (Malzahn et al., Cell Biosci, 2017, 7:21) and clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease (CRISPR/Cas) with an engineered crRNA/tracr RNA (for example as a single-guide RNA, or as modified crRNA and tracrRNA molecules which form a dual molecule guide), and methods of using this nucleases to target known genomic locations, are well-known in the art (see reviews by Bortesi and Fischer, 2015, Biotechnology Advances 33: 41-52; and by Chen and Gao, 2014, Plant Cell Rep 33: 575-583, and references within).

The term “disruption” means that a chromosomal region, a gene coding region and/or control sequences of a referenced gene is partially or entirely modified, such as by deletion, insertions and/or substitutions of one or more nucleotides, resulting in the absence (inactivation) or decrease in expression, and/or the absence or decrease of enzymatic activity of the encoded polypeptide. Disruptions can be generated by methods known in the art, e.g. by homologous recombination as demonstrated for Bacillus (e.g. Stahl & Ferrari, J. Bacteriol. 1984, 158,411-418; W02014052630, and WO03095658).

In such methods the disruption is accomplished by homologous recombination using a plasmid that has been constructed to contain the 5’ and 3’ regions flanking the chromosomal region to be deleted. Furthermore, the efficiency can be further increased by combination with e.g. CRISPR/Cas9 technologies as shown by Altenbuchner (Altenbuchner J, Appl Environ Microbiol. 2016 Aug 15;82(17):5421-7).

The term “integration” means the integration of a gene or gene expression cassette into a chromosome. This is accomplished by the methods as described under “disruption” when the gene or gene expression cassette is being placed between the 5’ and 3’ homologous regions.

As used herein, a "genetically modified organism" (GMO) is an organism whose genetic characteristics contain alteration(s) that were produced by human effort causing transfection that results in transformation of a target organism with genetic material from another or "source" organism, or with synthetic or modified-native genetic material, or an organism that is a descendant thereof that retains the inserted genetic material. The source organism can be of a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or from the same type of organism (e.g., a GMO plant can contain genetic material from another plant species, from another variety of the same species or further, optionally mutagenized, copies of genetic material from the same plant species or variety). The term "native" (or wildtype or endogenous) cell or organism and "native" (or wildtype or endogenous) polynucleotide or polypeptide refers to the cell or organism as found in nature and to the polynucleotide or polypeptide in question as found in a cell in its natural form and genetic environment, respectively (i.e., without there being any human intervention).

As used herein, "wildtype" means the typical form of an organism or its genetic material, as it normally occurs, as distinguished from e.g. mutagenized and/or recombinant forms.

Similarly, by "control cell" or "wildtype host cell" is intended a cell that lacks the particular polynucleotide of the invention that are disclosed herein. The use of the term "wildtype" is not, therefore, intended to imply that a host cell lacks recombinant DNA in its genome. Preferably, a wildtype organism is a wild-type type-strain deposited at DSMZ. Preferred wild type strains are Bacillus methanolicus MGA3 (ATCC 53907;Schendel et al (1990) Appl. Environ. Microbiol. 56,4 p. 963-970 ), Bacillus methanolicus PB1 (DSM16454/ATCC51375).

The invention provides a method of high temperature fermentative production of L-proline and/or any of the proline metabolic intermediates L-glutamyl-5-phosphate, gamma- glutamate-semialdehyde and 1-pyrroline-5-carboxylate. According to the invention a microorganism is provided which is capable of glutamate synthesis at a temperature of 40°C to 70°C. Such microorganisms can easily be obtained by screening of a microorganism library. The microorganism expresses, in the method of the present invention, a proB* gene, which is a variant of a proB gene comprising at least one mutation to remove or reduce inhibition by proline. When the microorganism is cultivated under suitable conditions for glutamate synthesis at a temperature of 40°C to 70°C, the microorganism expresses said proB* gene, which in turn converts glutamate into L-glutamyl-5-phosphate. The latter substance can then be further converted to gamma-glutamate-semialdehyde, 1-pyrroline-5- carboxylate and/or proline as desired. Preferably said further conversions are also effected at a temperature of 40°C to 70°C to obtain a production process without requiring a temperature downshift. It has now surprisingly been found that it is possible to change a proB gene such that the encoded proB* protein is no longer inhibited by proline and remains functional at a temperature of at least 40°C, preferably 40-70°C, even more preferably 42- 62°C, even more preferably 48-55°C. This was unexpected, particularly since expression of the analogous ProJ protein, which is per se not inhibited by proline, did not result in proline production at such temperatures. Thus, the unexpected stability of the ProB* enzyme advantageously allows to perform fermentative proline biosynthesis with reduced need for cooling of the fermenter.

The microorganism preferably a) expresses a proAA and/or, more preferably, a proA gene for conversion of L-glutamyl- 5-phosphate to gamma-glutamate-semialdehyde and/or 1-pyrroline-5-carboxylate, and optionally b) expresses a proH and/or, more preferably, a prol gene for conversion of 1-pyrroline-5- carboxylate to proline.

In the method of the present invention the microorganism may express both a ProAA and a ProA enzyme or may express either a ProAA or a ProA enzyme. If only one of these enzymes is expressed, then expression of the ProA enzyme is preferred because it is also expressed together with ProB in wild-type strains. Likewise, the microorganism may also express both a ProH and a Prol enzyme or may express either a ProH or a Prol enzyme. If only one of these enzymes is expressed, then expression of the Prol enzyme is preferred, because it is also expressed together with ProB in wild-type strains. As shown in the examples, providing a proA and a prol gene for expression of the corresponding enzymes allows to produce proline and its intermediates at a temperature of at least 40°C, preferably 40-70°C, even more preferably 42-62°C, even more preferably 48-55°C. The at least one mutation of the proB* gene preferably comprises, in the numbering of SEQ ID NO. 26, a mutation selected from L68E, N133D, E142R, E142K and T144A, more preferably E142R. As described above, WO2018074916 discloses proB gene mutations L68E, N133D, E142A and T144A. However, this publication is not concerned with fermentative production of proline or its intermediates at temperatures of 40°C or higher. The publication also does not disclose the E142R and E142K mutations to the proB gene. Likewise, W02006066758 is concerned with proline production at temperatures including 45°C. However, the publication also does not disclose any of the aforementioned mutations. Thus, it was surprising that a microorganism comprising the mutated proB* gene as described in the present invention would be capable of fermenting proline or its intermediates at elevated temperatures as prescribed by the present invention.

The proB gene as such, without any mutations which turn it into a proB* gene with removed or reduced inhibition by proline, is known to the skilled person from various Uniprot database entries, e.g. the latest sequences obtainable on 2021-01-15 by any of the Uniprot identifiers I3E8T4_BACMM, A0A3S0UGE6_9BACI, A0A179T832_9BACI, A0A3E2JKU5_9BACI, N0AV01_9BACI, A0A268EB23_9BACI, A0A2N5MML6_9BACI, A0A2C1 KGE9_9BACI, A0A0C2VP83_9BACL, A0A327RXP8_9BACI, A0A372LRL1_9BACI, A0A1 L3MTX2_9BACI, AOA2N5I938_9BACI, A0A2N6R9K9_9BACI, A0A0M1NXA4_9BACI, A0A431VVE3_9BACI, A0A2X4W281_BACLE, A0A398BHV3_9BACI, A0A0K9GDP0_9BACI, A0A0M2SZ36_9BACI, A0A2N5G6N4_9BACI, A0A4V2RE31_9BACI, A0A1H7VYR9_9BACI, A0A429X148_9BACI, A0A2N0Z2R3_9BACI, A0A3S4QA65_9BACI, W4F2Q0_9BACL, A0A0J1IQ11_BACCI, A0A0K9GV64_9BACI, A0A0M8Q3Y5_9BACI, A0A268IKE8_9BACI, A0A3S2U6S2_9BACI, A0A4P6UWT8_9BACL, A0A177ZZA3_9BACI, A0A3M8H6E3_9BACI, A0A1S2QUS4_9BACI, A0A432LHX8_9BACI, A0A3N9UJF9_9BACI, A0A3S0HE62_9BACI, A0A084H220_BACID, PROB1_BACLD, A0A285SCN6_9BACI, A0A1 H0TD68_9BACI, A0A380C728_SPOPA, A0A0A8JL90_BACSX, A0A1V2SB74_9BACI, A0A4R1 E5C2_9BACI, A0A0A3J2B2_9BACI, A0A2V3W0M7_9BACI, AOA127VXM1_SPOPS, A0A075JKV9_9BACI, A0A3N6BS40_9BACL, G2TNRO_BACCO, A0A0A3HXY4_9BACI, F9DNJ8_9BACL, A0A345PF29_9BACI, A0A2W7MJD1_9BACI, A0A0W7Y848_9BACI, AOAOA3IOV3_9BACI, PROB_BACSU, A0A135WAS1_9BACL, A0A494Z7X8_9BACI, A8FCC4_BACP2, A0A090IST9_9BACI, A0A1X7GET7_9BACI, A0A0K0G9B1_9FIRM, A0A087N1B9_9BACI, A0A3L7K166_9BACI, A0A2D1SRK3_9BACL, A0A2N0ZIQ1_9BACI, A0A268J4S9_9BACL, A0A433RVH1_9BACL, A0A0M5JMM2_9BACI, A0A396SQE4_9BACI, A0A398BD72_9BACI, A0A1C0Y7H5_9BACL, A0A495A306_9BACI, F2F5T3_SOLSS, A0A0A3J3Y1_9BACI, A0A2S1H0B8_9BACI, A0A0X1 RVJ2_9BACL, A0A150YM98_9BACI, A0A1S2ME71_9BACI, A0A498DB05_9BACI, A0A024QF11_9BACI, A0A0H4NZG2_9BACI, A0A3R9N7H4_9BACI, A0A385NW37_9BACI, A0A4V1LG95_9BACI, A0A0L0QT04_VIRPA, A0A1S2LUH2_9BACI, A0A0V8JES0_9BACI, C3BKH6_9BACI, PROB_BACAN, A0A165Z8Z1_9BACI, A0A1H1BZK2_9BACI, A0A0J6FRX0_9BACI, A0A1S2LJJ9_9BACI, A0A2U3AF62_9BACL, A0A248TNK1_9BACI, K6DK88_BACAZ, PROB_BACCR, A0A327YQR0_9BACI, PROB_GEOKA, A0A0A1MK25_9BACI, A0A4R1QJK5_9BACI, A0A2A8S658_9BACI, A0A2C1ZFW6_9BACI, A0A366XTL2_9BACI, A0A2U3ANA7_9BACL, PROB_GEOSW, F5L5J4_CALTT, A0A1G8G7J5_9BACI, A0A0U4WD61_9BACL, A0A0X8CZV9_9BACL, A0A1H4F0U7_9BACI, X0RR96_9BACI, E6U0V6_BACCJ, AOAOMOGE75_SPOGL, PROB_BACHD, W4PYC8_9BACI, A0A160F8C0_9BACI, A0A3M8P872_9BACL, A0A1H3NWB4_9BACI, N4W9N6_9BACI, A0A1Q9PP54_9BACI, A0A4R2P460_9BACL, A0A4V1W845_9BACL, A0A1G7YCW8_9BACI, PROBJDCEIH, A0A094WMA1_BACAG, A0A0D6ZE59_9BACI, A0A1 H0AHR6_9BACI, K6DBJ6_9BACI, A0A368XBE8_9BACI, A0A0D1YF85_ANEMI, A0A2U1 K7X7_9BACI, A0A111S491_9BACI, A0A0B0IQ22_9BACI, A0A223KWW2_9BACI, A0A1E5LJE1_9BACI, A0A371S4A8_9BACI, A0A3R9QIG9_9BACI, A0A4Q3X6R7_9BACI, A0A1M7PLP2_9BACI, A0A0A5GGA1_9BACI, A0A419V658_9BACL, A0A1X9MA40_9BACI, A0A3T0HYJ0_9BACI, A0A077J5S0_9BACI, A0A1S2QZ12_9BACI, A0A2A8IJR5_9BACI, A0A1 N6NEU4_9BACI, W1SP14_9BACI, V6IWL0_9BACL, A0A3R9NZK2_9BACI, AOA1I2ACGO_9BACI, A0A0U1NYT1_9BACI, AOA1V2A7I4_9BACI, A0A268KC07_9BACI, A0A317KYZ1_9BACI, W4QHJ6_9BACI, A0A261QLJ9_9BACI, A0A1H9LG19_9BACI, A0A1Q2L302_9BACL, A0A177L380_9BACI, AOA1I4L7Z3_9BACI, A0A1H9VS23_9BACI, B7GJH2_ANOFW, A0A0U1QR99_9BACL, A0A0C2XXC0_BACBA, A0A160F1D0_9BACI, A0A0K9HBA6_9BACI, A0A4Q4JEW6_9BACL, A0A114IV37_9BACI, A0A0J5QVG7_9BACI, A0A4Q4IJH7_9BACL, A0A0U1KNI3_9BACI, A0A0M0KVJ1_9BACI, D3FZ73_BACPE, W4QQC6_BACA3, A0A366EI27_9BACI, A0A098EG35_9BACL, A0A0F5HUX3_9BACI, K0IYE8_AMPXN, AOA3A9E8W2_9CLOT, A0A4Q1SW67_9BACL, AOA1H2Q6I5_9BACI, A0A3A9KY88_9BACI, A0A419SF73_9BACL, M7P5Z4_9BACL, A0A2P8HX60_9BACI, A0A0N0Z815_9BACI, A0A2V3WT02_9BACI, A0A221M7G6_9BACI, AOA1I6T37O_9BACI, A0A4R6U781_9BACI, A0A1G6H744_9BACI, L0EFK8_THECK, PROB_BACSK and PR0B_THET2. Further proB genes can be obtained by hybridization, under stringent hybridization conditions, of a nucleic acid comprising a putative proB gene to a probe specific for a proB gene, preferably for a probe specific to any of the sequences of the aforementioned Uniprot identifiers and/or SEQ ID NO. 26. Preferably, the proB* gene codes for a polypeptide comprising an amino acid sequence at least 40% identical to SEQ ID NO. 26. This condition is fulfilled for each sequence referenced by one of the aforementioned Uniprot identifiers. The sequence SEQ ID NO. 26 herein is an artificial sequence created regardless of metabolic activity. Instead, the sequence serves as a template to isolate or identify a proB gene which, in a second step, is mutated into a proB* gene to remove or reduce inhibition by proline, preferably by introducing any of the aforementioned mutations, more preferably the mutation E142R or E142K. Thus, the proB* gene preferably has a sequence according to any of the aforementioned Uniprot identifiers mutated to remove or reduce inhibition by proline, preferably by introducing any of the aforementioned mutations, more preferably the mutation E142R or E142K. Preferably, the proB gene sequence is 55- 80% identical to SEQ ID NO. 26, more preferably 60-80% identical to SEQ ID NO. 26, more preferably 65-80% identical to SEQ ID NO. 26, more preferably 70-80% identical to SEQ ID NO. 26, more preferably 72-80% identical to SEQ ID NO. 26, more preferably 73-79% identical to SEQ ID NO. 26, more preferably 74-79% identical to SEQ ID NO. 26. As shown in the examples, the gene of Uniprot identifier I3E8T4_BACMM with an E142R exchange codes for a ProB* enzyme functional at temperatures of 40-70°C to fermentatively produce proline.

Further preferably, except for the at least one mutation to remove or reduce inhibition by proline, the sequence of the proB* gene differs from SEQ ID NO. 26 only by a) conservative substitutions according to the following table, wherein higher numbers in brackets indicate higher degree of preference:

b) substitutions only by an amino acid according to figure 5, and c) insertions only before position 1 or immediately after any of the following positions in SEQ ID NO. 26: S91 , G202, V228, S260, G320, S342, R347, V357, L359

Preferably, the proB* gene is 55-80% identical to SEQ ID NO. 26, more preferably 60-80% identical to SEQ ID NO. 26, more preferably 65-80% identical to SEQ ID NO. 26, more preferably 70-80% identical to SEQ ID NO. 26, more preferably 72-80% identical to SEQ ID NO. 26, more preferably 73-79% identical to SEQ ID NO. 26, more preferably 74-79% identical to SEQ ID NO. 26, and any insertion occurs only before position 1 or immediately after any of the following positions in SEQ ID NO. 26: S91, G202, V228, S260, G320, S342, R347, V357, L359, and each substitution relative to SEQ ID NO. 26 is a conservative substitution or a substitution allowed at the corresponding position according to figure 5, wherein at positions 68, 133, 142 and 144 according to SEQ ID NO. 26 the following substitutions are also allowed: L68E, N133D, E142R, E142K and T144A, more preferably the proB* gene comprises at position 142 according to SEQ ID NO. 26 an amino acid selected from R and K, most preferably R.

Such preferred proB* genes have a high similarity to the proB* gene explored in the examples. They are thus even more likely than other gene sequences to lead, when expressed to a ProB* enzyme without or with reduced inhibition by proline compared to the proB gene according to Uniprot identifier I3E8T4_BACMM.

According to the invention the microorganism is preferably selected from the taxonomic family Bacillaceae, preferably from genus Bacillus, Falsibacillus or Mesobacillus, more preferably from any of the genera Bacillus aeolius, Bacillus boroniphilus, Bacillus ciccensis, Bacillus circulans, Bacillus dafuensis, Bacillus foraminis, Bacillus marisflavi, Bacillus novalis, Falsibacillus sp., Mesobacillus jeotgali, Bacillus jeotgali, Bacillus niacini, Bacillus pichinotyi, Bacillus oceanisediminis, Bacillus methanolicus, Bacillus firmus, Bacillus subterraneus, more preferably Bacillus methanolicus. Strains of these microorganism species, and in particular Bacillus methanolicus, advantageously allow for a fermentative production of proline according to the present invention at temperatures of 40-70°C.

Preferably the microorganism is capable of fermenting methanol for producing glutamate. Such microorganism, e.g. Bacillus methanolicus, is easy to handle in fermentations because methanol is a substrate which is easy to handle and to dose to a fermentation broth. Furthermore, methanol is a liquid under normal fermentation conditions and does not need to be dissolved in water, which creates additional problems for sterilization of containers.

According to the invention a) the proA gene preferably codes for a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO. 24, and/or b) the prol gene preferably codes for a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO. 25.

The proA gene as such is known to the skilled person from various Uniprot database entries, e.g. the sequences latest obtainable on 2021-01-15 by any of the Uniprot identifiers I3E8T3_BACMM, N0ASY7_9BACI, A0A0M1 NYK6_9BACI, A0A2N5G6M1_9BACI, A0A0C2RVM6_9BACL, AOA2N5I991_9BACI, A0A433HRY1_9BACI, A0A398BPE5_9BACI, A0A327RVQ8_9BACI, A0A268EB44_9BACI, A0A3E2JLG4_9BACI, A0A1 H7VXJ2_9BACI, A0A179T7F6_9BACI, A0A0M2SVZ7_9BACI, A0A2N5MMM2_9BACI, A0A2C1 KGH2_9BACI, A0A372LQF6_9BACI, A0A2N6R9K4_9BACI, A0A2X4WEF7_BACLE, A0A084H221_BACID, A0A4R2BLL5_9BACI, A0A431VV48_9BACI, A0A1V2SBM6_9BACI, A0A0K0GAB8_9FIRM, A0A1L3MTT6_9BACI, A0A495A2Y1_9BACI, A0A3S5EDA1_9BACI, W4F3F8_9BACL, A0A1H0TBW1_9BACI, A0A3L7K5Z9_9BACI, A0A268IKF4_9BACI, A0A0K9GCK4_9BACI, A0A075JMR5_9BACI, A0A0A8JMC7_BACSX, A0A429X117_9BACI, A0A177ZW98_9BACI, AOA127VXUO_SPOPS, A0A345PF30_9BACI, A0A2N0Z2P3_9BACI, A0A498DD74_9BACI, A0A2W7MLC5_9BACI, A0A0L0QTD3_VIRPA, AOAOA3I4A6_9BACI, A0A494Z7Y1_9BACI, A0A090ITX7_9BACI, A0A135WAK5_9BACL, A0A3S2UTR1_9BACI, AOAOM9XOI7_9BACI, A0A3S0KAI5_9BACI, A0A0K9GV46_9BACI, A0A1S2QW71_9BACI, A0A3R9M110_9BACI, A0A268J4V4_9BACL, A0A3N6A9V1_9BACL, G2TNR1_BACCO, A0A380C922_SPOPA, A0A024QFU6_9BACI, A0A0J1 LFZ6_BACCI, A0A3S0QS05_9BACI, A0A285SI56_9BACI, A0A4R1 E2T6_9BACI, A0A398B889_9BACI, A0A3A9FH08_9CLOT, A0A2D1SRJ6_9BACL, A0A0A3IAF5_9BACI, A0A0A3IYT2_9BACI, PROA1_BACLD, PROA_BACSU, A0A2N0ZIS9_9BACI, A0A0W7Y875_9BACI, A0A396SBH2_9BACI, A0A3M8H6Q2_9BACI, A0A3N9UJH9_9BACI, A0A2V3VXN3_9BACI, A8FCC5_BACP2, A0A087N1 B8_9BACI, A0A0A3IRK5_9BACI, A0A2U3ANF6_9BACL, A0A433RVE3_9BACL, A0A2S1 H6M6_9BACI, A0A1X7GEI5_9BACI, F9DNJ7_9BACL, A0A0X1 RZN3_9BACL, C3BKH7_9BACI, A0A1C0Y7K5_9BACL, F2F5T4_SOLSS, A0A2A8S655_9BACI, A0A0H4P2T4_9BACI, A0A0M4FI20_9BACI, A0A248TNS3_9BACI, PROAJDCEIH, A0A2U3AF46_9BACL, A0A165Z8X7_9BACI, A0A368X953_9BACI, A0A0A1MUW2_9BACI, A0A317KZU0_9BACI, PROA_BACAN, A0A0V8JEJ3_9BACI, PROA_BACHD, A0A1 H3NVC2_9BACI, K6D7Q9_BACAZ, A0A111S4G4_9BACI, A0A150YMD1_9BACI, A0A2U1 K8A9_9BACI, A0A0A5GAE4_9BACI, A0A2P8H1 M5_9BACL, A0A0B4RFV9_9BACL, A0A1M7PLG9_9BACI, A0A0M0GFL1_SPOGL, A0A4Q3X6E2_9BACI, A0A094YUB6_BACAO, A0A371S4A3_9BACI, A0A223KWR0_9BACI, F5L5J3_CALTT, A0A1S2LKI6_9BACI, AOA1 I6T359_9BACI, A0A0D6ZGQ1_9BACI, X0RTC3_9BACI, N4WCP3_9BACI, A0A1G7YCT9_9BACI, A0A365L7W0_9BACL, AOA1 I4L822_9BACI, A0A1Q2L463_9BACL, A0A385NUH5_9BACI, AOA1 I2AD26_9BACI, A0A1S2LX78_9BACI, A0A4R2P6H0_9BACL, A0A3M8P9M2_9BACL, A0A4V1 LG92_9BACI, A0A0B4RDI7_9BACL, A0A0J5TRL1_9BACI, A0A1 H9LGU8_9BACI, A0A1 H1 BZE8_9BACI, A0A098EHJ9_9BACL, A0A0U2XHN4_9BACL, A0A0B0ILM4_9BACI, W4QGM7_9BACI, A0A1S2MED0_9BACI, A0A366EJQ4_9BACI, A0A428MYE7_9BACI, A0A2V3WFC2_9BACI, A0A0U1 KNJ1_9BACI, A8FE26_BACP2, A0A1G6H752_9BACI, E6U0V5_BACCJ, A0A077J2W7_9BACI, A0A327YP08_9BACI, A0A4R1QIH6_9BACI, PROA_GEOKA, A0A1H8GU32_9BACI, A0A1X9M7P7_9BACI, W4QPE1_BACA3, A0A4R6U6Q4_9BACI, A0A1V2A833_9BACI, A0A3Q9QSG6_9BACI, PROA_ANOFW, A0A1Q9PPE4_9BACI, A0A1H2Q5C9_9BACI, D3FZ72_BACPE, A0A1G8G7U8_9BACI, A0A0N1 KHI7_9BACI, A0A4R3KKP3_9BACI, A0A177L394_9BACI, A0A0U1 NYS9_9BACI, K0J7J3_AMPXN, V6J4G1_9BACL, A0A1 E5LJF8_9BACI, K6DAW1_9BACI, A0A167SZ84_9BACI, A0A2C1ZET9_9BACI, A0A268KC34_9BACI, A0A4Q4IJ42_9BACL, A0A261QLK2_9BACI, A0A4Q4JEQ3_9BACL, A0A0C2YBN8_BACBA, A0A1 N6NES2_9BACI, A0A0F5HTZ6_9BACI, A0A1S2QYW9_9BACI, A0A1 B8UTL4_9BACL, AOA1 I4IUT6_9BACI, PROA_GEOSW, A0A3R9QFR1_9BACI, A0A2T6C8A4_9BACL, A0A2A8IL53_9BACI, A0A109QI32_9BACL, A0A4Q1SV65_9BACL, A0A1U9K432_9BACL, A0A1H0AG32_9BACI, A0A0U2VYP9_9BACL, A0A0U1QR89_9BACL, W1SI45_9BACI, A0A0K9H5F9_9BACI, A0A0D1VKE8_ANEMI, A0A3G3JWQ7_9BACL, A0A1V4H857_9BACL, A0A1U9K4N9_9BACL, A0A1G6IXF0_9BACL, A0A4Q4INL8_9BACL, A0A090YD67_PAEMA and A0A089N739_9BACL. Further proA genes can be obtained by hybridization, under stringent hybridization conditions, of a nucleic acid comprising a putative proA gene to a probe specific for a proA gene, preferably for a probe specific to any of the sequences of the aforementioned Uniprot identifiers and/or SEQ ID NO. 24. Preferably, the proA gene codes for a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO. 24. This condition is fulfilled for each sequence referenced by one of the aforementioned Uniprot identifiers. The sequence SEQ ID NO. 24 herein is the sequence accessible under Uniprot identifier I3E8T3_BACMM. Preferably, the proA gene sequence is 55-80% identical to SEQ ID NO. 24, more preferably 60-80% identical to SEQ ID NO. 24, more preferably 65- 80% identical to SEQ ID NO. 24, more preferably 70-80% identical to SEQ ID NO. 24, more preferably 72-80% identical to SEQ ID NO. 24, more preferably 73-79% identical to SEQ ID NO. 24, more preferably 74-79% identical to SEQ ID NO. 24.

The prol gene as such is known to the skilled person from various Uniprot database entries, e.g. the sequences latest obtainable on 2021-01-15 by any of the Uniprot identifiers I3EA78_BACMM, A0A2N5H601_9BACI, A0A0A8XDW4_9BACI, A0A0M0XBE8_9BACI, N0AXR1_9BACI, A0A0D6ZDG9_9BACI, A0A2N5G6P4_9BACI, K6DNF1_9BACI, A0A077J1S7_9BACI, AOA1I6BHP9_9BACI, A0A268KB56_9BACI, W1SBU0_9BACI, A0A0M0GB59_SPQGL, A0A1G9QQH9_9BACI, A0A3T0I1 L0_9BACI, A0A2C1Z1C1_9BACI, A0A2A8IGD2_9BACI, A0A098F2Z1_9BACI, A0A2N0ZCI4_9BACI, A0A4R2BBP4_9BACI, A0A0M2SG62_9BACI, A0A1S2R3K8_9BACI, A0A0M4FUT7_9BACI, A0A385NV82_9BACI, A0A417YZ05_9BACI, A0A248TLS6_9BACI, A0A0U1 P1A5_9BACI, A0A268IJ00_9BACI, A0A1Q9Q0K2_9BACI, A0A398B7S6_9BACI, A0A431WI67_9BACI, U5LDC4_9BACI, A0A372L9L2_9BACI, A0A3D8GRC9_9BACI, AOA2N5IO65_9BACI, A0A0J1 IMB9_BACCI, A0A2N0YX79_9BACI, A0A0M1NZ01_9BACI, A0A0K9H761_9BACI, A0A433HE11_9BACI, A0A429XD49_9BACI, A0A0C2TUL2_BACBA, A0A1 L8ZM00_9BACI, A0A1V2SBC1_9BACI, A0A398BLB9_9BACI, A0A2N5MKC0_9BACI, A0A150KMB8_9BACI, A0A261QGP2_9BACI, A0A0Q3TJ67_9BACI, A0A3S4QFI8_9BACI, A0A3E2JUB9_9BACI, A0A0K9GB50_9BACI, A0A2N6RN20_9BACI, A0A1H0WHB7_9BACI, A0A285CLU8_9BACI, A0A2N5M1K4_9BACI, A0A1B9AQU1_9BACI, A0A2C1 KDP1_9BACI, A0A0C2VJV8_9BACL, A0A160F2H9_9BACI, A0A0F5HVX8_9BACI, A0A371SH89_9BACI, A0A0K9GVN4_9BACI, A0A0H4P0S3_9BACI, A7GSF8_BACCN, A0A0M0L044_9BACI, A0A2S5GE58_9BACL, A0A073K3S7_9BACI, A0A428J729_9BACI, A0A223KSF1_9BACI, Q818V7_BACCR, A0A4R5XUP8_9BACL, AOA1IOC4A3_9BACI, C3BPD3_9BACI, A0A176JB31_9BACI, A0A161Y402_9BACI, A0A2X4WC02_BACLE, A0A327YMU1_9BACI, A0A4R1QHJ8_9BACI, A0A0C2RVW5_9BACL, C5D425_GEOSW, A0A2A8S736_9BACI, A0A268E067_9BACI, B7GHJ7_ANOFW, A0A1L3MQ88_9BACI, A0A160F7X8_9BACI, K6CSA3_BACAZ, A0A366XW65_9BACI, A0A1S2QT20_9BACI, A0A179T327_9BACI, A0A1G9A8E5_9BACI, A0A0F7RCD8_BACAN, A0A1 E5LKB5_9BACI, A0A0N0E9S0_9BACI, D5DRP5_BACMQ, P5CR2_BACSU, A0A1S2ME41_9BACI, A0A178A579_9BACI, A0A327S710_9BACI, A0A1V2A4R1_9BACI, A0A084GW14_BACID, Q5KXHO_GEOKA, A0A2U1K721_9BACI, A0A4Q0VRA0_9BACI, A0A0V8JR74_9BACI, G2TKY2_BACCO, A0A268IYY4_9BACL, A0A0M4FQL3_9BACI, A8FEW1_BACP2, A0A0J6CTC7_9BACI, A0A1N6PEJ6_9BACI, Q62T44_BACLD, A0A0B5AS40_9BACL, A0A177L081_9BACI, A0A0A3INK4_9BACI, A0A396SAL9_9BACI, A0A498D4J6_9BACI, A0A4P6USS1_9BACL, A0A1S2LUF4_9BACI, A0A075JKS9_9BACI, A0A4R3NCW4_9BACI, A0A024Q5L5_9BACI, A0A1 H0XNV9_9BACI, A0A0A3HWH9_9BACI, A0A1S2LJW1_9BACI, F2F104_SQLSS, A0A113ZXY6_9BACL, A0A317L3L6_9BACI, A0A221M826_9BACI, A0A075RB74_BRELA, V6M5P5_9BACL, A0A0U5B4Y1_9BACL, A0A143HB55_9BACL, A0A0Q3TP23_9BACI, A0A4R1 E0H6_9BACI, A0A285SCF6_9BACI, A0A1X7D1C3_9BACI, A0A4Q1SU74_9BACL, A0A0A3J2R0_9BACI, A0A2D1SSN4_9BACL, W9AGM5_9BACI, A0A494YYF3_9BACI, A0A1G8G7F4_9BACI, A0A0D1VDR8_ANEMI, A0A3S0R8H8_9BACI, A0A3D9SGB3_9BACL, A0A4R1YER7_9BACI, A0A0N0S3P3_9BACI, A0A1 J6WSD7_9BACI and AOAOA3IOW1_9BACI. Further prol genes can be obtained by hybridization, under stringent hybridization conditions, of a nucleic acid comprising a putative prol gene to a probe specific for a prol gene, preferably for a probe specific to any of the sequences of the aforementioned Uniprot identifiers and/or SEQ ID NO. 25. Preferably, the prol gene codes for a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO. 25. This condition is fulfilled for each sequence referenced by one of the aforementioned Uniprot identifiers. The sequence SEQ ID NO. 25 herein is the sequence accessible under Uniprot identifier I3EA78_BACMM. Preferably, the prol gene sequence is 55-80% identical to SEQ ID NO. 25, more preferably 60-80% identical to SEQ ID NO. 25, more preferably 65-80% identical to SEQ ID NO. 25, more preferably 70-80% identical to SEQ ID NO. 25, more preferably 72- 80% identical to SEQ ID NO. 25, more preferably 73-79% identical to SEQ ID NO. 25, more preferably 74-79% identical to SEQ ID NO. 25.

Further preferably the proB* gene is functionally linked to a promoter which allows expression of the proB* gene without proline-directed repression. For example, in wild type Bacillus methanolicus the proB gene is operably linked to a promoter comprising a T-box for inhibition of transcription in the presence of proline-laden tRNA. Thus, by removing repression of proB* transcription, expression of the proB* gene is improved. This, in turn, allows to increase fermentation yield of proline and/or an intermediate of the glutamateproline biosynthesis pathway. Preferred promoters are indicated above. Preferred promoters have been described above.

According to the invention it is preferred that the proB*, proA and preferably, if present, also the prol gene are comprised in a single operon. Such expression cassette structure allows for an easy cloning and regulation of the genes of the operon. Furthermore, as the activity of each of the enzymes coded by proB*, proA and prol is required or preferred, as described above, providing these genes in a single operon ensures that transcripts of all genes are present in the microorganism at least in the amount awarded by the promoter driving proB* expression.

The proA and prol gene sequences preferably are linked by a linker comprising a ribosome binding site. For example, in wild type Bacillus methanolicus the proB and proA genes already form a single operon. It has now been found that by providing an additional ribosome binding site between the proA and prol genes, expression of the prol gene is advantageously increased. It is preferred that the linker has a length of 6-60 nucleotides, counting after the adjacent stop codon and ending immediately in front of the adjacent start codon. For example, the linker in figure 3 has a length of 24 nucleotides. Preferably, the linker has a length of 10-35 nucleotides, more preferably 19-28 nucleotides.

The invention correspondingly also provides an expression cassette, comprising a promoter and, operably linked thereto, a proB*, a proA and a prol gene, wherein the proB* gene comprises at least one mutation to remove or reduce inhibition by proline, and preferably the proA and prol gene sequences are linked by a linker comprising a ribosome binding site. The expression cassette thus allows for easy cloning of the genes used according to the present invention, and also allows for an easy expression of the genes. In particular, the expression cassette allows to fermentatively produce proline or its intermediates in the glutamate-proline metabolic pathway in high yield at temperatures of 40-70°C as described above. Preferably the proB* gene is operably linked to a heterologous promoter. As described above, the promoter preferably allows expression of the proB* gene without proline-directed repression. However, the native promoter of the proBA operon is downregulated in the presence of proline-laden tRNA. Thus, by exchanging the promoter for a non-proline- downregulated promoter or by removing the proline-tRNA binding site (T-box), expression of the proB* gene is advantageously increased compared to the Bacillus methanolicus wild type, thereby allowing to obtain a higher yield of proline or its intermediates in the glutamateproline metabolic pathway at temperatures of 40-70°C as described above. Preferred promoters are described above.

The invention also provides an isolated nucleic acid or vector comprising an expression cassette of the present invention. The isolated nucleic acid and the vector facilitate construction of the proB* gene, the construction of the proB*A or proB*AI operon as described above and/or the transformation of a microorganism with the expression cassette of the present invention.

Correspondingly the invention provides a microorganism for high temperature fermentative production of any of L-glutamyl-5-phosphate, gamma-glutamate-semialdehyde, 1-pyrroline-5- carboxylate or L-proline, wherein the microorganism expresses a proB* gene comprising at least one mutation to remove or reduce inhibition by proline and preferably comprises an expression cassette or a vector of the present invention, wherein the microorganism is capable of glutamate synthesis at a temperature of 40°C to 70°C and preferably is selected from the taxonomic family Bacillaceae, preferably from genus Bacillus, Falsibacillus or Mesobacillus, more preferably from any of the genera Bacillus aeolius, Bacillus boroniphilus, Bacillus ciccensis, Bacillus circulans, Bacillus dafuensis, Bacillus foraminis, Bacillus marisflavi, Bacillus novalis, Falsibacillus sp., Mesobacillus jeotgali, Bacillus jeotgali, Bacillus niacini, Bacillus pichinotyi, Bacillus oceanisediminis, Bacillus methanolicus, Bacillus firmus, Bacillus subterraneus.

As described above, such microorganism is advantageously capable of fermentative production of proline or its intermediates in the glutamate-proline metabolic pathway at temperatures of 40-70°C in high yield.

As described above, the microorganism preferably comprises, for the production of gamma- glutamate-semialdehyde, 1-pyrroline-5-carboxylate or L-proline, one or more preferably heterologous proA and/or prol gene copies and/or one or more proA and/or prol gene operably linked to a heterologous promoter.

And the invention provides the use of an expression cassette, a vector or a microorganism of the present invention for the production of any of L-glutamyl-5-phosphate, gamma-glutamate- semialdehyde, 1-pyrroline-5-carboxylate or L-proline at a temperature of 40°C to 70°C. Correspondingly the invention also teaches the use of an expression cassette or a vector comprising, a proB* gene comprising at least one mutation to remove or reduce inhibition by proline and preferably comprises an expression cassette or a vector of the present invention, a proA gene and/or a prol gene, preferably both a proA and a prol gene, wherein the proB* gene and the at least one proA/prol gene, preferably the proA and the prol gene, are comprised in a single expression cassette under control of a) a promoter heterologous to the proBA promoter, or a functional fragment of the proBA promoter, b) an inducer-dependent promoter or c) a constitutive promoter for i) the transformation of a microorganism capable of glutamate synthesis at a temperature of 40°C to 70°C to fermentatively produce any of L-glutamyl-5-phosphate, gamma- glutamate-semialdehyde, 1-pyrroline-5-carboxylate or L-proline at a temperature of 40- 70°C, more preferably 42-62°C, even more preferably 48-55°C, and/orii) fermentative production any of L-glutamyl-5-phosphate, gamma-glutamate- semialdehyde, 1-pyrroline-5-carboxylate or L-proline at a temperature of 40-70°C, more preferably 42-62°C, even more preferably 48-55°C, in a a microorganism capable of glutamate synthesis at a temperature of 40°C to 70°C.

The invention is hereinafter further illustrated by way of the following non-limiting examples.

EXAMPLES

General description of materials and methods used in the examples

Unless otherwise stated the following experiments have been performed by applying standard equipment, methods, chemicals, and biochemicals as used in genetic engineering, molecular biology and fermentative production of chemical compounds by cultivation of microorganisms. See also Sambrook et al. (Sambrook.J. and Russell, D.W. Molecular cloning. A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001) and Chmiel et al. (Bioprocesstechnik 1. Einfuhrung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991).

Bacterial strains

The methylotrophic strain B. methanolicus MGA3 strain was used (Schendel, F. Hanson, R. S. (1990). L-lysine production at 50°C by mutants of a newly isolated and characterized methylotrophic Bacillus sp. Applied and Environmental Microbiology, 56(4), 963-970). The B. subtilis JH642 strain (trpC2 pheA1 ; BGSC 1A96) is a derivative from the B. subtilis 168 strain (trpC2; BGSC 1A1), and the B. subtilis JSB8 ((delta-proHJ::tet) strain is a derivate of B. subtilis JH642 lacking in osmoadaptive proline biosynthesis (Brill, J., Bremer, E. (2011). Osmotically controlled synthesis of the compatible solute proline is critical for cellular defense of Bacillus subtilis against high osmolarity. Journal of Bacteriology, 193(19), 5335-5346). The proline auxotrophic E. coli strain MG165548 (proB::tn5), a derivative of the E. coli wild type strain MG1655 with non-functional proB gene (Miller, J. H. 1992. A Short course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The B. licheniformis DSM3 strain was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

Bacterial strains, media, and growth conditions

The B. subtilis strain JH642 was routinely maintained and propagated on LB agar plates or cultured in LB liquid medium at 37°C. B. methanolicus MGA3 was propagated on SOB agar plates or cultured in SOB liquid medium at 50°C. Spizizen’s minimal medium (SMM) with 0.5% glucose as a carbon source, a solution of trace elements (Harwood, C. R., & Archibald, A. (1990). Growth, maintenance and general techniques, p 1-26. In Harwood CR, Cutting SM (ed) Molecular biological methods for Bacillus. John Wiley & Sons, Chichester, United Kingdom), and L- tryptophan (20 mg/ liter) and L- phenylalanine (18 mg/ liter) was used as defined medium for the growth of the wild type strain B. subtilis JH642. For growth of B. methanolicus MGA3, the minimal medium MVcM supplemented with methanol (200mM), d- Biotin (0.1 mg/ liter), and vitamin B12 (0.01 mg/ liter) was used (Brautaset, T., Ellingsen, T. E. (2004). Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus. Journal of Bacteriology, 186(5), 1229-1238).

Bacterial growth was spectrophotometrically monitored at a wavelength 578nm (OD578). For quantification of proline and glutamate, cultures were inoculated from exponentially growing culture in 20 ml prewarmed minimal media in 250 ml Erlenmeyer flasks to an OD578nm of 0.1 and were grown at 37°C (B. subtilis JH642), or at 50°C (B. methanolicus MGA3) in a shaking water bath set at 220 rpm. The osmolarity of the defined minimal medium was increased by addition from a 5M NaCI stock solution to the final concentration indicated in the individual experiments.

Transformation of naturally competent B. subtilis

Naturally competent B. subtilis cells were made competent and DNA transformed into B. subtilis cells according to the method of Spizizen (Anagnostopoulos.C. and Spizizen.J. (1961). J. Bacteriol. 81 , 741-746). For auxotrophic strains L-tryptophan (20 mg/liter), and L- phenylalanine (18 mg/liter) was added to the growth media.

Electrocompetent Bacillus methanolicus cells and electroporation

Preparation of electrocompetent B. methanolicus MGA3 cells and transformation of DNA into B. methanolicus MGA3 strain is performed via electroporation and performed as essentially described by Jakobsen et al. (0yvind M. Jakobsen, Trygve Brautaset; Journal of Bacteriology Apr 2006, 188 (8) 3063-3072).

Plasmid Isolation

Plasmid DNA was isolated from Bacillus and E. coli cells by standard molecular biology methods described in (Sambrook.J. and Russell, D.W. Molecular cloning. A laboratory manual, 3rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2001) or the alkaline lysis method (Birnboim, H. C., Doly, J. (1979). Nucleic Acids Res 7(6): 1513-1523). Bacillus cells were in comparison to E. coli treated with 10 mg/ml lysozyme for 30 min at 37°C prior to cell lysis.

Quantification of proline

Quantification of proline using Ninhydrin

The intracellular proline content of B. subtilis was measured by a colorimetric assay that detect proline as a colored proline- ninhydrin complex that can be quantified by measuring the absorption at 480 nm (Bates, L., Waldren, R., & Teare, I.; 1973 Rapid determination of free proline for water-stress studies. Plant Soil, 39, 205-207). B. subtilis were grown to an OD578 of approximately 1.0 with the desired osmotic conditions. 8.0 ml of the cell culture was pelleted by centrifugation (4000 g, 10 min, 25°C). B. subtilis cells were extracted and analyzed according to the protocol developed by Bates et al. Intracellular proline contents were calculated using a volume for a B. subtilis cell 0.67 pl per 1 OD578.

Quantification of proline and glutamate using HPLC analysis

For quantitative HPLC analysis of glutamate and proline, B. methanolicus cells were cultivated in MVcM media. Cultures were cultivated until OD578 of approximately 1.0 -1.5 and were collected by centrifugation. The supernatant was separated from the pellet and stored at -20°C. The cell pellets were dried, and the cell dry weight (CDW) was determined. Cell extracts were prepared as previously described (Kuhlmann, Bremer, E.; 2002, Osmotically Regulated Synthesis of the Compatible Solute Ectoine in Bacillus pasteurii and Related Bacillus spp. 68(2), 772-783). The extracts, respectively the supernatant containing the proline and glutamate were derivatized with OPA and FMOC. HPLC analysis was performed as described by Kromer et al (Kromer, Wittmann, C.; 2005, In vivo quantification of intracellular amino acids and intermediates of the methionine pathway in Corynebacterium glutamicum. Analytical Biochemistry, 340(1), 171-173) using the HPLC system Agilent 1100 (Waldbronn, Germany). Glutamate and proline detection were performed by using a fluorescence detector (Agilent, Waldbronn, Germany). Glutamate was quantified at an excitation wavelength of 340nm and emission wavelength of 450nm, and proline measurements were performed at an excitation of 266nm and emission wavelength of 305nm.

Plasmids

Plasmid pCF6

To construct a promoterless Bacillus/E. coli shuttle vector, the mdh promoter from B. methanolicus of the vector pBV2mp (Irla, M., Wendisch, V. F.; 2016, Genome-based genetic tool development for Bacillus methanolicus: Theta-and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production. Frontiers in Microbiology, 7(SEP), 1-13) was removed by using restriction enzymes generating compatible overhangs (Xbal and Spel), resulting in fragments 1 046 bp and 6 736 bp. The 6 736 bp fragment was religated using a T4 DNA ligase (Thermo Fisher Scientific Inc., Waltham, MA, USA). The reaction mixture was transformed into competent E. coli DH10B cells (Invitrogen). The resulting promoterless Bacillus/E. coli shuttle vector was named pCF6.

Plasmid pCF7

For the construction of a plasmid-based proHJAA osmoadaptive proline biosynthetic system, the proHJAA gene cluster of B. licheniformis (Seq ID NO.1) including the native promoter PproHJAA (SEQ ID NO.2) was amplified by PCR from chromosomal DNA of B. licheniformis DSM13 using the oligonucleotides SEQ ID NO.3 and SEQ ID NO. 4. The PCR product and the linearized vector pCF6 (with restriction endonuclease Kpnl) were introduced into the Gibson assembly mixture (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs) following transformation into competent E. coli DH10B cells (Invitrogen). The correct plasmid was recovered and named pCF7.

Plasmid pCF8

The proHJAA region (SEQ ID NO. 1) including the native promoter PproHJAA of Bacillus licheniformis (SEQ ID NO.2) was PCR-amplified with oligonucleotides SEQ ID NO. 5 and SEQ ID NO. 6 using chromosomal DNA of B. licheniformis DSM13 as template. Plasmid pCF8 was constructed by Gibson assembly as described for plasmid pCF7 by assembly of the linearized pBV2mp plasmid (restriction endonuclease Kpnl) and the PCR fragment of the proHJAA region. The reaction mixture was transformed into competent E. coli DH10B cells (Invitrogen) and the correct plasmid recovered. The resulting plasmid pCF8 places the proHJAA region under the control of the promoter of the mdh gene of Bacillus methanolicus MGA3 present on plasmid pBV2mp.

Plasmid pCF9 - PproBA-proBAI

For the construction of a synthetic anabolic proline biosynthetic gene cluster the the proBA operon of Bacillus methanolicus MGA3 with its native 5’ gene regulatory region (SEQ ID NO 11) was PCR-amplified with oligonucleotides SEQ ID NO. 7 and SEQ ID NO. 8 and the prol gene region of B. methanolicus MGA3 with oligonucleotides SEQ ID NO. 9 and SEQ ID NO 10. The PCR products were cloned into the linearized (Smal restriction endonuclease cleavage) low- copy number vector pSC101 -derivative pHSG575 (Cmr) (Takeshita, S., Hashimoto-Gotoh, T., High-copy-number and low-copy-number plasmid vectors for lacZa- complementation and chloramphenicol- or kanamycin-resistance selection, Gene, Volume 61, Issue 1 ,1987, Pages 63-74) by Gibson assembly (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs). The reaction mixture was transformed into E. coli proline auxotrophic strain MG165548 and plated on LB-agar plates supplemented with 30 pg/ml chloramphenicol. After overnight incubation at 37°C, positive clones were selected by transferring of transformants to minimal salt agar plates supplemented with 30 pg/ ml chloramphenicol. Clones which were able to growth on the minimal salt agar plates exhibiting a functional proBAI gene operon, were selected for sequencing. The correct plasmid carrying a complete synthetic proline biosynthetic operon under the control of the native promoter of the proBA genes of Bacillus methanolicus MGA3 was named pCF9.

Plasmid pCF10 - PproBA-(delta)T-box-proBAI

For deletion of the T- box mediated regulatory mechanism of proline transcription, the Q5 site-directed mutagenesis (Q5 Site) kit (New England BioLabs) was used with oligonucleotides SEQ ID NO 12 and SEQ ID NO 13. with plasmid pCF9 serving as the template. The reaction mixture was transformed into E. coli proline auxotrophic strain MG165548 and plated on LB-agar plates supplemented with 30 pg/ml chloramphenicol. After overnight incubation at 37°C, positive clones were selected by transferring of transformants to minimal salt agar plates supplemented with 30 pg/ ml chloramphenicol. Clones which were able to growth on the minimal salt agar plates exhibiting a functional proBAI gene operon, were selected for sequencing. The correct plasmid carrying a complete synthetic proline biosynthetic operon under the control the 5’ gene regulatory region of proBA operon with deleted T-box Box (PproBA-(delta)T-box) was named pCF10.

Plasmid pCF11 - PproBA-(delta)T-box-proB*AI

A point mutation within ProB (Glutamate 5-kinase 1) to change the amino acid E142 to R142 (E142R mutation is indicated with an asterix *)was introduced into the synthetic proline biosynthetic operon of plasmid pCF10 using the Q5 site-directed mutagenesis (Q5 Site) kit (New England BioLabs) with oligonucleotides SEQ ID NO 14 and SEQ ID NO 15. The construction was performed as described for plasmid pCF10. The resulting plasmid was named pCF11 with the mutagenized proBAI (ProB E142R) indicated as proB*AI.

Plasmid pCF21 - Pmdh-proB*AI

The synthetic proline biosynthetic operon proB*AI with the E142 point mutation within ProB (Glutamate 5-kinase 1) was placed under the control of the Bacillus methanolicus MGA3 promoter. The proB*AI gene cluster of plasmid pCF11 was PCR- amplified with oligonucleotides SEQ ID NO 16 and SEQ ID NO 17. The vector pBV2mp was linearized with restriction endonuclease Kpnl. Both linear fragments were assembled by Gibson assembly (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs) following transformation into E. coli DH10B cells (Invitrogen). The correct assembled plasmid was recovered and verified by sequencing. The plasmid was named pCF21.

Plasmid pCF22 - PproBA-(delta)T-box-proB*AI

The proline biosynthetic genes proB*AI with the E142 point mutation within ProB (Glutamate 5-kinase 1) under the control of the native proBA promoter with deleted T- Box (PproBA- (delta)T-box) was PCR- amplified with oligonucleotides SEQ ID NO 18 and SEQ ID NO 19 with plasmid pCF11 serving as template and subsequently cloned into the linearized pCF6 vector using Gibson assembly as described above. The resulting plasmid pCF22 carries the proB*AI gene cluster controlled by the native proBAI ((delta)TBox) promoter. Example 1 :

The proHJAA osmoadaptive proline biosynthetic system of Bacillus licheniformis provided on plasmid pCF7 under the control of the native promoter PproH JAA and provided on plasmid pCF8 in addition under the control of the promoter of the mdh gene of Bacillus methanolicus MGA3 were transformed into Bacillus subtilis JH642, Bacillus subtilis JSB8 (Bacillus subtilis JH642 (delta)( proHJ::tet)) Bacillus methanolicus MGA3 strains (see Table 1).

Table 1: Bacillus strains

Single colonies of Bacillus subtilis strains carrying either plasmid pCF7, pCF8 or empty vector control pBV2mp from fresh LB-agar plates (supplemented with 20 pg/ml kanamycin) were used to inocculate 5 ml of LB-Miller medium (supplemented with 20 pg/ml kanamycin) following cultivation for 5 h at 37°C and 220 rpm. 20 pl of these cultures were used to inoculate 20ml SMM media (20 pg/ml kanamycin) provided in 250 ml shake flasks, following cultivation overnight at 37°C with 220 rpm until cell cultures reached an optical density OD578 of 1.0. Main cultures containing 20ml prewarmed SMM medium or SMM medium with 0.5 M NaCI, both supplemented with 20 pg/ml kanamycin, in 250ml shake flasks were subsequently incocculated with the respective overnight cultures with a start optical density OD578 of 0.05. Main cultures were again cultivated at 37°C at 220 rpm until cell cultures reached an optical density OD578 of 1.0. 8 ml of each culture was harvested by centrifugation and the intracellular proline content quantified as described in the ‘proline quantification section’ above.

Bacillus methanolicus MGA3 strains carrying either plasmid pCF7, plasmid pCF8 or emptyl plasmid control pBV2mp, were grown on SOB agar plates (supplemented with 20 pg/ml kanamycin) at 50°C overnight and 5 ml of prewarmed SOB medium supplemented with 20 pg/ml kanamycin were inoculated each with a single colony following cultivation for 6h at 50°C and 220 rpm until cell cultures reached an optical density OD578 of 1.0. 30 pl of these cultures were used to inoculate 20 ml MVcM medium supplemented with methanol (200mM), d-Biotin (0.1 mg/L), vitamin B12 (0.01 mg/L) and 20 pg/ml kanamycin provided in 250 ml shake flasks, following cultivation overnight at 50°C with 220 rpm until cell cultures reached an optical density OD578 of 1.0. Main cultures containing 20 ml prewarmed MVcM medium or MVcM medium with 0.5 M NaCI, both supplemented with methanol (200mM), d-Biotin (0.1 mg/L), vitamin B12 (0.01 mg/L) and 20 pg/ml kanamycin, in 250ml shake flasks were subsequently incocculated with the respective overnight cultures with a start optical density OD578 of 0.05. Main cultures were again cultivated at 50°C at 220 rpm until cell cultures reached an optical density OD578 of 1.0. 8 ml of each culture was harvested by centrifugation and the intracellular proline content quantified as described in the ‘proline quantification section’ above.

In Figure 2b the intracellular L-proline content per biomass is plotted against the respective Bacillus subtilis strains. B. subtilis strain defective in osmoadaptive proline synthesis (Bacillus subtilis JH642 (delta)( proHJ::tet)) with empty control plasmid pBV2mp is not able to synthesize L-proline under high salt stress conditions (media supplemented with 0.5 M NaCI), whereas the B. subtilis JH642 WT strain with empty control plasmid pBV2mp shows strong induction of L-proline synthesis upon salt stress. Introduction of the proHJAA osmoadaptive proline biosynthetic system of Bacillus licheniformis provided on plasmid pCF7 and pCF8 respectively into B. subtilis strain defective in osmoadaptive proline synthesis (Bacillus subtilis JH642 (delta)( proHJ::tet)) results in functional osmostress-dependent L- proline synthesis (0.5 M NaCI cultivation conditions.) and hence shows functionality of the Bacillus licheniformis derived proline building block at mesophilic temperatures.

In Figure 2c the intracellular L-proline content per biomass of Bacillus methanolicus carrying the plasmids pBV2mp, pCF7 and pCF8 is shown for cultivations in MVcM medium and MVcM medium with 0.5 M NaCI. Surprisingly, no L-proline is synthesized under 50°C cultivation condition in Bacillus methanolicus, neither in the presence or absence of salt stress.

Example 2:

Construction of a synthetic anabolic proline synthesis building block in Bacillus methanolicus.

The anabolic proline synthesis genes proBA and prol were fused together to form a synthetic proBAI operon. Moreover, the allosteric feedback inhibition of ProB Glutamate 5-kinase by L- proline was removed by the exchange of amino acid E142 into aminoacid R142 (referred to as *). Transcriptional regulation of the synthetic proB*AI operon is brought about either by placing the genes under control of the promoter of the mdh gene of Bacillus methanolicus (see construction of plasmid pCF21) or by removing the T-Box riboswitch (Brill J, Bremer E. T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis; Microbiology; 2011 Apr;157(Pt 4):977-987) mediated transcriptional regulation within the natural promoter of proBA of Bacillus methanolicus (see construction of plasmid pCF22).

Bacillus methanolicus MGA3 strains carrying either plasmid pCF21, plasmid pCF22 or empty plasmid control pBV2mp, were grown on SOB agar plates (supplemented with 20 pg/ml kanamycin) at 50°C overnight and 5 ml of prewarmed SOB medium supplemented with 20 pg/ml kanamycin were inoculated each with a single colony following cultivation for 6h at 50°C and 220 rpm until cell cultures reached an optical density OD578 of 1.0. 30 pl of these cultures were used to inoculate 20 ml MVcM medium supplemented with methanol (200mM), d-Biotin (0.1 mg/L), vitamin B12 (0.01 mg/L) and 20 pg/ml kanamycin provided in 250 ml shake flasks, following cultivation overnight at 50°C with 220 rpm until cell cultures reached an optical density OD578 of 1.5. Cell pellets and culture supernatants were recovered by centrifugation and the cell dry weight (CDW) was determined and the intracellular and extracellular amount of L-proline content was quantified as described above in the ‘quantification of proline using HPLC section’. In Figure 4 the intracellular amount of proline (Figure 4a) and the extracellular amount of proline (Figure 4b) are plotted for Bacillus methanolicus strains carrying either empty vector control pBV2mp, plasmid pCF22, or plasmid pCF21.

L-proline is synthesized in the presence of the thermotolerant proB*AI building block.

Claims

1. Method of high temperature fermentative production of any of L-glutamyl-5-phosphate, gamma-glutamate-semialdehyde, 1-pyrroline-5-carboxylate or L-proline, comprising the steps

2) fermentative production of L-glutamyl-5-phosphate, gamma-glutamate- semialdehyde, 1-pyrroline-5-carboxylate and/or proline at a temperature of 40°C to 70°C, wherein the microorganism expresses a proB* gene comprising at least one mutation to remove or reduce inhibition by proline.

2. Method according to claim 1, wherein the microorganism a) expresses a proAA and/or, preferably, a proA gene for conversion of L-glutamyl- 5-phosphate to gamma-glutamate-semialdehyde and/or 1-pyrroline-5- carboxylate, and b) optionally, expresses a proH and/or, preferably, a prol gene for conversion of 1- pyrroline-5-carboxylate to proline.

3. Method according to any of the preceding claims, wherein the at least one mutation of the proB* gene comprises, in the numbering of SEQ ID NO. 26, a mutation selected from L68E, N133D, E142R, E142K and T144A.

4. Method according to any of the preceding claims, wherein the microorganism is selected from the taxonomic family Bacillaceae, preferably from genus Bacillus, Falsibacillus or Mesobacillus, more preferably from any of the genera Bacillus aeolius, Bacillus boroniphilus, Bacillus ciccensis, Bacillus circulans, Bacillus dafuensis, Bacillus foraminis, Bacillus marisflavi, Bacillus novalis, Falsibacillus sp., Mesobacillus jeotgali, Bacillus jeotgali, Bacillus niacini, Bacillus pichinotyi, Bacillus oceanisediminis, Bacillus methanolicus, Bacillus firmus, Bacillus subterraneus.

5. Method according to claim 4, wherein the microorganism is capable of fermenting methanol for producing glutamate.

6. Method according to any of the preceding claims, wherein a) the proA gene codes for a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO. 24, and/or b) the prol gene codes for a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO. 25.

7. Method according to any of the preceding claims, wherein the proB* gene is functionally linked to a promoter which allows expression of the proB* gene without proline-directed repression.

8. Method according to any of the preceding claims, wherein the proB*, proA and preferably, if present, also the prol gene are comprised in a single operon.

9. Method according to claim 8, wherein the proA and prol gene sequences are linked by a linker comprising a ribosome binding site. Expression cassette, comprising a promoter and, operably linked thereto, a proB*, a proA and a prol gene, wherein the proB* gene comprises at least one mutation to remove or reduce inhibition by proline, and preferably the proA and prol gene sequences are linked by a linker comprising a ribosome binding site. Expression cassette according to claim 10, wherein the proB* gene is operably linked to a heterologous promoter. Vector comprising an expression cassette according to any of claims 10 or 11. Microorganism for high temperature fermentative production of any of L-glutamyl-5- phosphate, gamma-glutamate-semialdehyde, 1-pyrroline-5-carboxylate or L-proline, wherein the microorganism expresses a proB* gene comprising at least one mutation to remove or reduce inhibition by proline and preferably comprises an expression cassette according to any of claims 10 to 11 or a vector according to claim 12, wherein the microorganism is capable of glutamate synthesis at a temperature of 40°C to 70°C and preferably is selected from the taxonomic family Bacillaceae, preferably from genus Bacillus, Falsibacillus or Mesobacillus, more preferably from any of the genera Bacillus aeolius, Bacillus boroniphilus, Bacillus ciccensis, Bacillus circulans, Bacillus dafuensis, Bacillus foraminis, Bacillus marisflavi, Bacillus novalis, Falsibacillus sp., Mesobacillus jeotgali, Bacillus jeotgali, Bacillus niacini, Bacillus pichinotyi, Bacillus oceanisediminis, Bacillus methanolicus, Bacillus firmus, Bacillus subterraneus. Use of an expression cassette according to claim 10 or 11, a vector according to claim 12 or a microorganism according to claim 13 for the production of any of L-glutamyl-5- phosphate, gamma-glutamate-semialdehyde, 1-pyrroline-5-carboxylate or L-proline at a temperature of 40°C to 70°C.