# Patent

## Title
Processes and recombinant microorganisms for the production of fine chemicals

## Abstract

Recombinant microorganisms with deregulated succinyl-CoA synthetase activity, as well as the uses for producing lysine, β-lysine, cadaverine or N-acetylcadaverine thereby are provided. Recombinant polypeptides comprising an amino acid sequence being at least 80% identical to SEQ ID NO: 51 or 53 are also provided. The method of producing fine chemicals using said recombinant microorganisms, in particular the method of producing lysine, or derivatives thereof, such as β-lysine, cadaverine or N-acetylcadaverine is further provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is the U.S. national phase of International Application No. PCT/IB2012/057312, filed Dec. 14, 2012, which claims the benefit of European Patent Application No. 11195301.4, filed Dec. 22, 2011, U.S. Provisional Patent Application No. 61/578,929, filed Dec. 22, 2011, incorporated herein by reference in its entirety, European Patent Application No. 12165935.3, filed Apr. 27, 2012, and U.S. Provisional Patent Application No. 61/639,109, filed Apr. 27, 2012, incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates to the use of recombinant microorganisms comprising DNA molecules in a deregulated form which improve the production of fine chemicals, in particular lysine, or derivatives thereof, such as β-lysine, cadaverine or N-Acetylcadaverine, especially cadaverine, as well as to recombinant DNA molecules and polypeptides used to produce the microorganism, said microorganism comprising a deregulated succinyl-CoA synthetase. The present invention also relates to processes for the production of fine chemicals using such recombinant microorganisms, in particular processes for the production of lysine, or derivatives thereof, such as β-lysine, cadaverine or N-Acetylcadaverine, especially cadaverine.
PRIOR ART
U.S. Pat. No. 7,435,584, discloses a method for enhanced production of L-lysine by culturing corynebacteria having a high expression of the lysE (lysine export carrier) gene.
WO 2008092720 discloses a method for producing cadaverine by fermenting high lysine producing microorganisms comprising an intracellular expressed lysine decarboxylase. Whereby, such microorganisms may have a reduced or eliminated expression of a lysine/cadaverine antiporter.
Further background on sucCD of Corynebacterium glutamicum can be found in: Cho H Y, Lee S G, Hyeon J E, Han S O; (2010) Identification and characterization of a transcriptional regulator, SucR, that influences sucCD transcription in Corynebacterium glutamicum. Biochemical and biophysical research communications, 401 (2):300-5, and in Han S O, Inui M, Yukawa H (2008) Effect of carbon source availability and growth phase on expression of Corynebacterium glutamicum genes involved in the tricarboxylic acid cycle and glyoxylate bypass. Microbiology (Reading, England), 154 (Pt 10):3073-83.
WO 2005/058945 discloses different mutations in Corynebacterium glutamicum succinyl-CoA synthetase genes.
Becker, J., C. Klopprogge, H. Schroder and C. Wittmann. (2009). Tricarboxylic acid cycle engineering for improved lysine production in Corynebacterium glutamicum. Appl Environ Microbiol 75:7866-7869 disclosed showed positive effects on the production of lysine due to a reduction of the metabolic flux trough the tricarboxylic acid cycle (TCA) by replacing the start codon of the isocitrate dehydrogenase (ICD) gene with a less preferred start codon.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a microorganism having a deregulated succinyl-CoA synthetase activity, preferably the deregulated succinyl-CoA synthetase activity is at least partially due to deregulation of one or more succinyl-CoA synthetase polypeptides comprising an amino acid sequence being at least 80% identical to SEQ ID NO: 51 or 53 or due to deregulation of one or more succinyl-CoA synthetase polypeptides comprising an amino acid sequence being at least 80% identical to SEQ ID NO: 51 and deregulation of one or more succinyl-CoA synthetase polypeptides comprising an amino acid sequence being at least 80% identical to SEQ ID NO: 53. The provided microorganism may have further a deregulated cadaverine exporter activity, preferably the deregulated cadaverine exporter activity is at least partially due to deregulation of one or more cadaverine exporter polypeptides comprising an amino acid sequence being at least 80% identical to SEQ ID NO: 1. In one embodiment of the invention, microorganism has an enhanced cadaverine exporter activity, while in another embodiment of the invention, the microorganism has a decreased cadaverine exporter activity. Preferably the microorganism has an enhanced lysine decarboxylase activity, even more preferred, the enhanced lysine decarboxylase activity is due to expression of one or more lysine decarboxylase polypeptides comprising an amino acid sequence being at least 80% identical to SEQ ID NO: 3 or SEQ ID NO: 4. In a further embodiment of the invention, the microorganisms described above have also at least one deregulated gene selected from the group consisting of the genes of aspartokinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, succinyl-amino-ketopimelate transaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelate epimerase, diaminopimelate dehydrogenase, arginyl-tRNA synthetase, diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase, homoserine dehydrogenase, phophoenolpyruvate carboxykinase, methylmalonyl-CoA mutase, diamine acteyltransferase. Preferably the microorganisms described above have a deregulated lysine import activity. In an even more preferred embodiment, the microorganism have a deregulated lysine import activity being at least partially due to an enhanced or a decreased lysine exporter activity or an enhanced or a decreased lysine permease activity or an enhanced or a decreased lysine/cadaverin antiporter activity or any combination thereof. The deregulated lysine import activity is preferably due to an enhanced or a decreased activity of at least one lysine exporter polypeptide comprising an amino acid sequence which has at least 80% identity to SEQ ID NO: 5. In another embodiment of the invention, the microorganisms described above have a deregulated lysine import activity, being at least partially due to an enhanced or a decreased lysine permease activity or at least partially due to an enhanced or a decreased lysine/cadaverin antiporter activity or any combination thereof.
The invention comprises microorganism as described above, further having a deregulated N-acetylcadaverine-forming activity. In one embodiment of the invention the microorganism having a deregulated N-acetylcadaverine-forming activity has no or a decreased N-acetylcadaverine-forming activity. In another embodiment of the invention, the microorganism having a deregulated N-acetylcadaverine-forming activity has an enhanced N-acetylcadaverine-forming activity and a decreased cadaverine exporter activity. Preferably the deregulated N-acetylcadaverine-forming activity of the microorganism described above is at least partially due to deregulation of a N-acetylcadaverine-forming polypeptide comprising an amino acid sequence, being at least 80% identical to SEQ ID NO: 13. Preferably any one of the microorganism described above belongs to the clade Eubacteria, even more preferred, to the genus Corynebacterium, most preferred the microorganisms described above belong to the species Corynebacterium glutamicum. 
The invention encompases a process for the production of a fine chemical, preferably a fine chemical selected from the group of lysine, β-lysine, cadaverine or N-Acetylcadaverine, using any one of the microorganism described above. Further, the invention comprises the use of any one of the microorganism described above for the production of a fine chemical, preferably a fine chemical selected from the group of lysine, β-lysine, cadaverine or N-Acetylcadaverine. Other embodiments of the invention are the use of lysine produced by fermenting any one of the microorganism described above for the production of food or feed or the production of food or feed supplements. Further embodiments of the invention are the use of cadaverine produced by fermenting any one of the microorganism described above for the production of polyamines and polyamines produced by using cadaverine produced by fermenting any one of the microorganism described above.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Strategy for metabolic engineering of lysine production in C. glutamicum: Coupling of the TCA cycle to the succinylase branch of lysine biosynthesis via deletion of the TCA cycle enzyme succinyl-CoA synthetase competing for succinyl-CoA.
FIG. 2: Metabolic fluxes of succinyl-CoA metabolism in different lysine producing C. glutamicum strains taken from published 13C flux studies (Becker, J., E. Heinzle, C. Klopprogge, O. Zelder, and C. Wittmann. 2005. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol 71:8587-8596, and Becker, J., C. Klopprogge, A. Herold, O. Zelder, C. J. Bolten, and C. Wittmann. 2007. Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum-over expression and modification of G6P dehydrogenase. J Biotechnol 132:99-109 and Becker, J., C. Klopprogge, and C. Wittmann. 2008. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum. Microb Cell Fact 7:8 and Kiefer, P., E. Heinzle, O. Zelder, and C. Wittmann. 2004; Comparative metabolic flux analysis of lysine-producing Corynebacterium glutamicum cultured on glucose or fructose. Appl Environ Microbiol 70:229-39 and Kim, H. M., E. Heinzle, and C. Wittmann. 2006; Deregulation of aspartokinase by single nucleotide exchange leads to global flux rearrangement in the central metabolism of Corynebacterium glutamicum. J Microbiol Biotechnol 16:1174-1179 and Krömer, J. O., O. Sorgenfrei, K. Klopprogge, E. Heinzle, and C. Wittmann. 2004; In-depth profiling of lysine-producing Corynebacterium glutamicum by combined analysis of the transcriptome, metabolome, and fluxome. J Bacteriol 186:1769-84 and Wittmann, C., and E. Heinzle. 2002; Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing Corynebacteria. Appl Environ Microbiol 68:5843-59. The fluxes comprise succinyl-CoA supply by alpha-ketoglutarate dehydrogenase and maximum succinyl-CoA demand in the lysine pathway, assuming sole contribution of the succinylase branch. In addition, the straight lines indicate the corresponding fluxes for exclusive utilization of succinyl-CoA, supplied by the TCA cycle, for lysine biosynthesis with varied contribution of the succinylase branch. The distance of the real flux distribution to these lines denotes the potential for improvement of the lysine production by pathway coupling.
FIG. 3: Growth and production characteristics of lysine-producing C. glutamicum BS87 (A, B) and the corresponding succinyl-CoA synthetase deletion mutant C. glutamicum BS87 □sucCD (C, D) during batch cultivation on glucose. The linear correlation between biomass and lysine production and consumption of glucose, respectively, indicates metabolic steady state during cultivation. The given data represent values from three biological replicates.
FIG. 4: In vivo fluxes of succinyl-CoA metabolism in lysine producing C. glutamicum BS87 and the corresponding succinyl-CoA synthetase deletion mutant C. glutamicum BS87 □sucCD. The fluxes given comprise succinyl-CoA supply in the TCA cycle, given as flux through alpha-ketoglutarate dehydrogenase, and maximum succinyl-CoA consumption in lysine biosynthesis, assuming sole contribution of the succinylase branch. The errors reflect the 90% confidence interval, obtained by a Monte-Carlo analysis from 100 independent estimations.



DETAILED DESCRIPTION OF THE INVENTION
In the description that follows, a number of terms are utilized extensively. Definitions are herein provided to facilitate understanding of the invention.
The term lysine means L-Lysine.
The term R-lysine means L-β-lysine.
The term cadaverine means 1,5-diaminopentane (CAS-Number: 462-94-2).
The term N-acetylcadaverine means N-acetyldiaminopentan (CAS-Number: 102029-76-5).
The methodologies of the present invention feature recombinant microorganisms, preferably including vectors or genes (e.g., wild-type and/or mutated genes) as described herein and/or cultured in a manner which results in the production of a fine chemical, preferably a fine chemical selected from the group of lysine, β-lysine, cadaverine or N-Acetylcadaverine.
The term “recombinant” microorganism includes a microorganism (e.g., bacteria, yeast cell, fungal cell, etc.) which has been genetically altered, modified or engineered (e.g., genetically engineered) such that it exhibits an altered, modified or different genotype and/or phenotype (e.g., when the genetic modification affects coding nucleic acid sequences of the microorganism) as compared to the naturally-occurring microorganism from which it was derived.
Promoter. A DNA sequence which directs the transcription of a structural gene to produce mRNA. Typically, a promoter is located in the 5′ region of a gene, proximal to the start codon of a structural gene. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent, if the promoter is a constitutive promoter.
Enhancer. A promoter element. An enhancer can increase the efficiency with which a particular gene is transcribed into mRNA irrespective of the distance or orientation of the enhancer relative to the start site of transcription.
Cloning vector. A DNA molecule, such as a plasmid, cosmid, phagemid or bacteriophage which has the capability of replicating autonomously in a host cell and which is used to transform cells for gene manipulation. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences may be inserted in a determinable fashion without loss of an essential biological function of the vector, as well as a marker gene which is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
Expression vector. A DNA molecule comprising a cloned structural gene encoding a foreign protein which provides the expression of the foreign protein in a recombinant host. Typically, the expression of the cloned gene is placed under the control of (i.e. operably linked to) certain regulatory sequences such as promoter and enhancer sequences. Promoter sequences may be either constitutive or inducible.
Recombinant host. A recombinant host may be any prokaryotic or eukaryotic cell which contains either a cloning vector or an expression vector. This term is also meant to include those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell. For examples of suitable hosts, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) [“Sambrook”].
The terms “express,” “expressing,” “expressed” and “expression” refer to expression of a gene product (e.g., a biosynthetic enzyme of a gene of a pathway or reaction defined and described in this application) at a level that the resulting enzyme activity of this protein encoded for, or the pathway or reaction that it refers to allows metabolic flux through this pathway or reaction in the organism in which this gene/pathway is expressed in. The expression can be done by genetic alteration of the microorganism that is used as a starting organism. In some embodiments, a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product at an increased level relative to that produced by the starting microorganism or in a comparable microorganism which has not been altered. Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g. by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins).
In some embodiments, a microorganism can be physically or environmentally altered to express a gene product at an increased or lower level relative to level of expression of the gene product by the starting microorganism. For example, a microorganism can be treated with or cultured in the presence of an agent known or suspected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased. Alternatively, a microorganism can be cultured at a temperature selected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased.
The terms “deregulate,” “deregulated” and “deregulation” refer to alteration or modification of at least one gene in a microorganism, wherein the alteration or modification results in increasing efficiency of lysine or β-lysine production in the microorganism relative to of lysine or β-lysine production in absence of the alteration or modification, or in case a lysine decarboxylase activity is present, results in increasing efficiency of cadaverine or N-acetylcadaverine production in the microorganism relative to cadaverine or N-acetylcadaverine production in absence of the alteration or modification. In some embodiments, a gene that is altered or modified encodes an enzyme in a biosynthetic pathway or a transport protein, such that the level or activity of the biosynthetic enzyme in the microorganism is altered or modified or that the transport specificity or efficiency is altered or modified. In some embodiments, at least one gene that encodes an enzyme in a biosynthetic pathway is altered or modified such that the level or activity of the enzyme is enhanced or increased relative to the level in presence of the unaltered or wild type gene. Deregulation also includes altering the coding region of one or more genes to yield, for example, an enzyme that is feedback resistant or has a higher or lower specific activity. Also, deregulation further encompasses genetic alteration of genes encoding transcriptional factors (e.g., activators, repressors) which regulate expression of genes coding for enzymes or transport proteins.
The term “deregulated” includes expression of a gene product (e.g., lysine decarboxylase) at a level lower or higher than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated. In one embodiment, the microorganism can be genetically manipulated (e.g., genetically engineered) to express a level of gene product at a lesser or higher level than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated. Genetic manipulation can include, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by removing strong promoters, inducible promoters or multiple promoters), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, decreasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene routine in the art (including but not limited to use of antisense nucleic acid molecules, or other methods to knock-out or block expression of the target protein).
The term “deregulated gene activity”, e.g. deregulated lysine decarboxylase activity, also means that a gene activity, e.g. a lysine decarboxylase activity, is introduced into a microorganism where the respective gene activity, e.g. the lysine decarboxylase activity, has not been observed before, e.g. by introducing a heterologous gene, e.g. a lysine decarboxylase gene in one or more copies into the microorganism, preferably by means of genetic engineering.
The terms “overexpress”, “overexpressing”, “overexpressed” and “overexpression” refer to expression of a gene product, in particular to enhancing the expression of a gene product (e.g. a lysine biosynthetic enzyme or sulfate reduction pathway enzyme or cysteine biosynthetic enzyme or a gene or a pathway or a reaction defined and described in this application) at a level greater than that present prior to a genetic alteration of the starting microorganism. In some embodiments, a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product at an increased level relative to that produced by the starting microorganism. Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a particular gene using routine in the art (including but not limited to use of antisense nucleic acid molecules, for example, to block expression of repressor proteins). Another way to overexpress a gene product is to enhance the stability of the gene product to increase its life time. Examples for the overexpression of genes in organisms such as C. glutamicum can be found in Eikmanns et al (Gene. (1991) 102, 93-8).
The phrase “deregulated pathway or reaction” refers to a biosynthetic pathway or reaction in which at least one gene that encodes an enzyme in such biosynthetic pathway or reaction is altered or modified such that the level or activity of at least one biosynthetic enzyme is altered or modified. The phrase “deregulated pathway” includes a biosynthetic pathway in which more than one gene has been altered or modified, thereby altering level and/or activity of the corresponding gene products/enzymes. In some cases the ability to “deregulate” a pathway (e.g., to simultaneously deregulate more than one gene in a given biosynthetic pathway) in a microorganism arises from the particular phenomenon of microorganisms in which more than one enzyme (e.g., two or three biosynthetic enzymes) are encoded by genes occurring adjacent to one another on a contiguous piece of genetic material termed an “operon.” In other cases, in order to deregulate a pathway, a number of genes must be deregulated in a series of sequential engineering steps.
To express the deregulated genes according to the invention, the DNA sequence encoding the enzyme must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into either a prokaryotic or eukaryotic host cell. In addition to transcriptional regulatory sequences, such as promoters and enhancers, expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector.
Suitable promoters for expression in a prokaryotic host can be repressible, constitutive, or inducible. Suitable promoters are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, T5, Sp6 and T7 polymerases, the PR and PL promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac, lpp-lac pr, phoA, gal, trc and lacZ promoters of E. coli, the alpha-amylase and the sigma 28-specific promoters of B. subtilis, the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda, the bla promoter of the β-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene as well as PGRO, PSOD, PEFTU, PEFTS from Corynebacterium and combinations of these promoters as described in WO2005059144, WO2007011939, WO2007012078, WO2005059093, WO2008049782, WO2006069711, WO2007020295. Prokaryotic promoters are reviewed by Glick, J. Ind. Microbiol. 1:277 (1987); Watson et al., MOLECULAR BIOLOGY OF THE GENE, 4th Ed., Benjamin Cummins (1987); Ausubel et al., supra, and Sambrook et al., supra.
A preferred promoter for the expression of the E. coli lysine decarboxylase is the PsodA, the PGRO and the PEFTU promoter of C. glutamicum described in WO2005059144, WO2007011939, WO2007012078, WO2005059093, WO2008049782, WO2006069711, WO2007020295.
Methods for expressing proteins in prokaryotic hosts are well-known to those of skill in the art. See, for example, Williams et al., “Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies,” in DNA CLONING 2: EXPRESSION SYSTEMS, 2nd Edition, Glover et al. (eds.), pages 15-58 (Oxford University Press 1995). Also see, Ward et al., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc. 1995); and Georgiou, “Expression of Proteins in Bacteria,” in PROTEIN ENGINEERING: PRINCIPLES AND PRACTICE, Cleland et al. (eds.), pages 101-127 (John Wiley & Sons, Inc. 1996). Further methods to increase or decrease gene expression and protein production can be found in WO2008049782.
An expression vector can be introduced into bacterial host cells using a variety of techniques including calcium chloride transformation, electroporation, and the like. See, for example, Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rd Edition, pages 1-1 to 1-24 (John Wiley & Sons, Inc. 1995).
As used herein, a substantially pure protein means that the desired purified protein is essentially free from contaminating cellular components, as evidenced by a single band following polyacrylamide-sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The term “substantially pure” is further meant to describe a molecule which is homogeneous by one or more purity or homogeneity characteristics used by those of skill in the art. For example, a substantially pure lysine decarboxylase will show constant and reproducible characteristics within standard experimental deviations for parameters such as the following: molecular weight, chromatographic migration, amino acid composition, amino acid sequence, blocked or unblocked N-terminus, HPLC elution profile, biological activity, and other such parameters. The term, however, is not meant to exclude artificial or synthetic mixtures of lysine decarboxylase with other compounds. In addition, the term is not meant to exclude lysine decarboxylase fusion proteins isolated from a recombinant host.
As used herein, the phrase “is at least partially due” means that the deregulated activity to which the phrase refers to can be restored by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% by reversing the described genotype.
For example: A decreased succinyl-CoA synthetase activity of a recombinant microorganism is at least partially due to deregulation of one or more succinyl-CoA synthetase polypeptides comprising an amino acid sequence being at least 80% identical to SEQ ID NO: 51, if the succinyl-CoA synthetase activity can be restored to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% of the succinyl-CoA synthetase activity of the non-recombinant microorganism, which has been used to produce the recombinant microorganism, by reversing the genetic modification of the recombinant microorganism, i.e. the deregulation of one or more succinyl-CoA synthetase polypeptides comprising an amino acid sequence being at least 80% identical to SEQ ID NO: 51.
The microorganism can be any prokaryotic or eukaryotic microorganism, in particular bacteria, archaea, yeasts and fungi. Preferred are microorganisms being selected from the genus of Corynebacterium with a particular focus on Corynebacterium glutamicum, the genus of Escherichia with a particular focus on Escherichia coli, the genus of Bacillus, particularly Bacillus subtilis, and the genus of Streptomyces. 
As set out above, a preferred embodiment of the invention relates to the use of host cells which are selected from coryneform bacteria such as bacteria of the genus Corynebacterium. Particularly preferred are the species Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium callunae, Corynebacterium ammoniagenes, Corynebacterium thermoaminogenes, Corynebacterium melassecola and Corynebacterium effiziens. Other preferred embodiments of the invention relate to the use of Brevibacteria and particularly the species Brevibacterium flavum, Brevibacterium lactofermentum and Brevibacterium divarecatum. 
In other preferred embodiments of the invention the host cells may be selected from the group comprising Corynebacterium glutamicum ATCC13032, C. acetoglutamicum ATCC15806, C. acetoacidophilum ATCC13870, Corynebacterium thermoaminogenes FERMBP-1539, Corynebacterium melassecola ATCC17965, Corynebacterium effiziens DSM 44547, Corynebacterium effiziens DSM 44549, Brevibacterium flavum ATCC14067, Brevibacterium lactoformentum ATCC13869, Brevibacterium divarecatum ATCC 14020, Corynebacterium glutamicum KFCC10065 and Corynebacterium glutamicum ATCC21608 as well as strains that are derived thereof by e.g. classical mutagenesis and selection or by directed mutagenesis.
Other particularly preferred strains of C. glutamicum may be selected from the group comprising ATCC13058, ATCC13059, ATCC13060, ATCC21492, ATCC21513, ATCC21526, ATCC21543, ATCC13287, ATCC21851, ATCC21253, ATCC21514, ATCC21516, ATCC21299, ATCC21300, ATCC39684, ATCC21488, ATCC21649, ATCC21650, ATCC19223, ATCC13869, ATCC21157, ATCC21158, ATCC21159, ATCC21355, ATCC31808, ATCC21674, ATCC21562, ATCC21563, ATCC21564, ATCC21565, ATCC21566, ATCC21567, ATCC21568, ATCC21569, ATCC21570, ATCC21571, ATCC21572, ATCC21573, ATCC21579, ATCC19049, ATCC19050, ATCC19051, ATCC19052, ATCC19053, ATCC19054, ATCC19055, ATCC19056, ATCC19057, ATCC19058, ATCC19059, ATCC19060, ATCC19185, ATCC13286, ATCC21515, ATCC21527, ATCC21544, ATCC21492, NRRL B8183, NRRL W8182, B12NRRLB12416, NRRLB12417, NRRLB12418 and NRRLB11476.
The abbreviation KFCC stands for Korean Federation of Culture Collection, ATCC stands for American-Type Strain Culture Collection and the abbreviation DSM stands for Deutsche Sammlung von Mikroorganismen. The abbreviation NRRL stands for ARS cultures collection Northern Regional Research Laboratory, Peorea, Ill., USA.
In certain embodiments, a microorganism of the invention is a “Campbell in” or “Campbell out” microorganism (or cell or transformant). As used herein, the phrase “Campbell in” transformant shall mean a transformant of an original host cell in which an entire circular double stranded DNA molecule (for example a plasmid) has integrated into a chromosome of the cell by a single homologous recombination event (a cross in event), and which effectively results in the insertion of a linearized version of the circular DNA molecule into a first DNA sequence of the chromosome that is homologous to a first DNA sequence of the circular DNA molecule. The phrase “Campbelled in” refers to the linearized DNA sequence that has been integrated into the chromosome of the “Campbell in” transformant. A “Campbell in” transformant contains a duplication of the first homologous DNA sequence that includes and surrounds the homologous recombination crossover point.
“Campbell out” refers a cell descended from a “Campbell in” transformant, in which a second homologous recombination event (a cross out event) has occurred between a second DNA sequence that is contained on the linearized inserted DNA of the “Campbelled in” DNA, and a second DNA sequence of chromosomal origin, which is homologous to the second DNA sequence of the linearized insert, the second recombination event resulting in the deletion (jettisoning) of a portion of the integrated DNA sequence, but, importantly, also resulting in a portion (this can be as little as a single base) of the integrated DNA sequence remaining in the chromosome, such that compared to the original host cell, the “Campbell out” cell contains one or more intentional changes in the chromosome (for example, a single base substitution, multiple base substitutions, insertion of a heterologous gene or DNA sequence, insertion of an additional copy or copies of a homologous gene or a modified homologous gene, or insertion of a DNA sequence comprising more than one of these aforementioned examples listed above).
A “Campbell out” cell or strain is usually, but not necessarily, obtained by a counter selection against a gene that is contained in a portion (the portion that is desired to be jettisoned) of the “Campbelled in” DNA sequence, for example the Bacillus subtilis sacB gene, which is lethal when expressed in a cell that is grown in the presence of about 5% to 10% sucrose. Either with or without a counter selection, a desired “Campbell out” cell can be obtained or identified by screening for the desired cell, using any screenable phenotype, such as, but not limited to, colony morphology, colony color, presence or absence of antibiotic resistance, presence or absence of a given DNA sequence by polymerase chain reaction, presence or absence of an auxotrophy, presence or absence of an enzyme, colony nucleic acid hybridization, and so on.
The homologous recombination events that leads to a “Campbell in” or “Campbell out” can occur over a range of DNA bases within the homologous DNA sequence, and since the homologous sequences will be identical to each other for at least part of this range it is not usually possible to specify exactly where the crossover event occurred. In other words, it is not possible to specify precisely which sequence was originally from the inserted DNA, and which was originally from the chromosomal DNA. Moreover, the first homologous DNA sequence and second homologous DNA sequence are usually separated by a region of partial non-homology, and it is this region of non-homology that remains deposited in the chromosome of the “Campbell out” cell.
For practicality, in C. glutamicum, typical first and second homologous DNA sequence are at least about 200 base pairs in length, and can be up to several thousand base pairs in length, however, the procedure can be made to work with shorter or longer sequences. A preferred length for the first and second homologous sequences is about 500 to 2000 bases, and the obtaining of a “Campbell out” from a “Campbell in” is facilitated by arranging the first and second homologous sequences to be approximately the same length, preferably with a difference of less than 200 base pairs and most preferably with the shorter of the two being at least 70% of the length of the longer in base pairs.
Succinyl-CoA synthetase activity refers to the conversion of succinyl-CoA to succinate. The Succinyl-CoA synthetase activity of a given microorganism can be determined by using the enzymatic test described in the Examples below.
Lysine decarboxylase activity refers to the decarboxylation of L-lysine into cadaverine, which is catalyzed by a Lysine decarboxylase. The enzyme has been classified as E.C. 4.1.1.18. For example, enzymes isolated from Escherichia coli having lysine decarboxylase activity are the cadA gene product (Kyoto Encyclopedia of Genes and Genomes, Entry b4131, SEQ ID NO: 4) and the IdcC gene product (Kyoto Encyclopedia of Genes and Genomes, Entry JW0181 SEQ ID NO: 3).
Methods to measure and compare enzymatic activities are known in the art and are described for example in “Handbook of Corynebacetrium glutamicum 2005 Eggeling, Borth eds. CRC Press Boca Raton USA and references within) or in Methods in Enzymology Volume 17, Part 1 pp. 3-1098 (1970), Volume 17, Part 2, pp. 3-961 (1971) Volume 41, pp. 3-564 (1975), Volume 42 pp. 3-537 (1975), Volume 63, pp. 3-547 (1979), Volume 64, pp. 3-418 (1980), Volume 89, pp. 3-656 (1982), Volume 90 pp. 3-602 (1982), Volume 142, pp. 3-732 (1987), Volume 143 pp. 3-582 (1987) and references therein.”. A standard strain used for comparison of Corynebacterium glutamicum strains is ATCC13032 (Handbook of Corynebacterium glutamicum 2005 Eggeling, Borth eds. CRC Press Boca Raton USA)
The amino acid sequences of E. coli IdcC is disclosed in accession number SEQ ID NO 3: and of E. coli cadA is disclosed in SEQ ID NO: 4.
DNA molecules encoding the E. coli lysine decarboxylase gene can be obtained by screening cDNA or genomic libraries with polynucleotide probes having nucleotide sequences reverse-translated from the amino acid sequence of SEQ ID NO: 3 or 4.
Alternatively, the E. coli lysine decarboxylase genes or any genes described herein can be obtained by synthesizing DNA molecules using mutually priming long oligonucleotides. See, for example, Ausubel et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to 8.2.13 (1990) [“Ausubel”]. Also, see Wosnick et al., Gene 60:115 (1987); and Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rd Edition, pages 8-8 to 8-9 (John Wiley & Sons, Inc. 1995). Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least 2 kilobases in length. Adang et al., Plant Molec. Biol. 21:1131 (1993); Bambot et al., PCR Methods and Applications 2:266 (1993); Dillon et al., “Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes,” in METHODS IN MOLECULAR BIOLOGY, Vol. 15: PCR PROTOCOLS: CURRENT METHODS AND APPLICATIONS, White (ed.), pages 263-268, (Humana Press, Inc. 1993); Holowachuk et al., PCR Methods Appl. 4:299 (1995).
Variants of polypeptides e.g. cadaverine exporter polypeptides, or variants of any gene described herein can be produced that contain conservative amino acid changes, compared with the parent enzyme. That is, variants can be obtained that contain one or more amino acid substitutions of e.g. SEQ ID NO: 1, in which an alkyl amino acid is substituted for an alkyl amino acid in the polypeptide sequence, an aromatic amino acid is substituted for an aromatic amino acid e.g. in the cadaverine exporter polypeptide amino acid sequence, a sulfur-containing amino acid is substituted for a sulfur-containing amino acid e.g. in the cadaverine exporter polypeptide amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy-containing amino acid e.g. in the cadaverine exporter polypeptide amino acid sequence, an acidic amino acid is substituted for an acidic amino acid e.g. in the cadaverine exporter polypeptide amino acid sequence, a basic amino acid is substituted for a basic amino acid e.g. in the cadaverine exporter polypeptide amino acid sequence.
Among the common amino acids, for example, a “conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) cysteine and methionine, (4) serine and threonine, (5) aspartate and glutamate, (6) glutamine and asparagine, and (7) lysine, arginine and histidine.
Conservative amino acid changes e.g. in the cadaverine exporter polypeptide can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID NO: 1. Such “conservative amino acid” variants can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like. Ausubel et al., supra, at pages 8.0.3-8.5.9; Ausubel et al. (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rd Edition, pages 8-10 to 8-22 (John Wiley & Sons, Inc. 1995). Also see generally, McPherson (ed.), DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press (1991). The ability of cadaverine exporter polypeptide variants to export cadaverine can be determined using an HPLC Assay for extracellular cadaverine.
The microorganisms of the invention comprise at least one deregulated succinyl-CoA synthetase polypeptides. The succinyl-CoA synthetase acitivty is usually due to the expression and activity of two polypetides, sucC and sucD, together referred to as sucCD. Typical examples are sucC (database accession No. cgl2566) and sucD (database accession No. cgl2565). Thus, a decreased succinyl-CoA synthetase activity usually requires the downregulation of at least one of those polypetides, meaning either sucC or sucD, wherein an enhanced succinyl-CoA synthetase activity usually requires the up-regulation of both polypeptides, meaning sucC and sucD.
Preferred sucC polypeptides are polypeptides comprising an amino acid sequence being at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 51.
Preferred sucD polypeptides are polypeptides comprising an amino acid sequence being at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53.
Accordingly, the deregulated succinyl-CoA synthetase acitivty is at least partly, preferably to more than 30% or to more than 50% or to more than 60% or to more than 70% or to more than 75%, 80%, 85%, 90%, 95% 98%, 99% due to one or more polypeptides comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 51 or 59, or due to one or more polypeptides comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 53, or due to one or more polypeptides comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 51 or 59 and due to one or more polypeptides comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 53.
The succinyl-CoA synthetase acitivty can be reduced by reducing the expression of the genomic genes coding for a succinyl-CoA synthetase, for example by introducing a stop codon in a gene coding for a succinyl-CoA synthetase, or by deleting parts or the complete sequence of the promoter of such gene or by deleting parts or the complete sequence of the coding region of the gene.
In the case of Corynebacteria, in particular Corynebacterium glutamicum, by reducing expression of a polynucleotide sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 52 or 60, or by reducing expression of a polynucleotide sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 54, or by reducing expression of a polynucleotide sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 52 or 60 and by reducing expression of a polynucleotide sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 54.
In the case of Escherichia, in particular Escherichia coli, by reducing expression of a polynucleotide sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 62, or by reducing expression of a polynucleotide sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 64, or by reducing expression of a polynucleotide sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 62 and by reducing expression of a polynucleotide sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 64.
The respective amino acid sequences of succinyl-CoA synthetase polypeptides of Escherichia coli are described by SEQ ID NO: 61 and SEQ ID NO: 63.
Preferred cadaverine exporter polypeptides according to the invention are the cadaverine exporter polypeptide from Corynebacterium glutamicum (dapE) and their equivalent genes, which have up to 80%, preferably 90% and most preferred 95% and 98% sequence identity (based on amino acid sequence) with the corresponding “original” gene product and have still the biological activity of a cadaverine exporter polypeptide. These equivalent genes can be easily constructed by introducing nucleotide substitutions, deletions or insertions by methods known in the art or by cloning homolog genes of other organisms, which can be identified and cloned according to methods well known in the art e.g. database searches, library screenings, complementation assays or enzymatic activity tests. Preferably the nucleotide sequence of cloned homolog genes is optimized for expression in the intended host microorganism e.g. by adapted to the codon usage of Corynebacterium glutamicum or Escherichia coli.
Sequences homologous to the sequence of SEQ ID NO: 1 can be found. Examples are given as the following: A4QH10_CORGB protein from Corynebacterium glutamicum strain R TX=40322 SEQ ID NO 25, Q8FMK8_COREF protein from Corynebacterium efficiens SEQ ID NO 26, ZP_03711358 protein from Corynebacterium matruchotii ATCC 33806 SEQ ID NO: 27, ZP_03392764 protein from Corynebacterium amycolatum SK46 SEQ ID NO: 28, YP_003696648 protein from Arcanobacterium haemolyticum DSM 20595 SEQ ID NO 29, ZP_06042915 protein from Corynebacterium aurimucosum ATCC 700975 SEQ ID NO 30, ZP_03936401 protein from Corynebacterium striatum ATCC 6940 SEQ ID NO 31, ZP_06837496 protein from Corynebacterium ammoniagenes DSM 20306 SEQ ID NO 32.
Accordingly, in a preferred embodiment the intracellular or the cadaverine exporter activity is at least partly, preferably to more than 30% or to more than 50% or to more than 60% or to more than 70% or to more than 75%, 80%, 85%, 90%, 95% 98%, 99% due to one or more polypeptides comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 1 and having cadaverine exporter activity.
Other preferred embodiments of the invention use the cadaverine exporter polypeptide of Corynebacterium glutamicum (SEQ ID NO: 1) which may be retranslated into DNA by applying the codon usage of the microorganism intended to express the cadaverine exporter polypeptide e.g E. coli, or to optimize the codon usage for expression in Corynebacterium glutamicum. 
A preferred microorganism of the invention comprises an intracellular lysine decarboxylase activity, preferably a high intracellular decarboxylase activity. An intracellular lysine decarboxylase activity can be created or enhanced by transforming the microorganism with one or more lysine decarboxylase genes to be expressed by the microorganism. Additionally or alternatively lysine decarboxylase genes can be mutated to enhance expression or the enzymatic activity of the encoded lysine decarboxylase.
In another embodiment, the intracellular lysine decarboxylase activity is combined with an extracellular lysine decarboxylase activity. A microorganism lacking either an intracellular or extracellular lysine decarboxylase activity may be transformed to express a lysine decarboxylase either intracellular or extracellular. Extracellular expression can be achieved by mutating a lysine decarboxylase gene in order to comprise signal sequences for extracellular expression, e.g. secretion signals or signals for molecular anchors, which are functional in the microorganism to be transformed. Examples for extracellular expression can be found in Choi and Lee Appl Microbiol Biotechnol 2004 64: 625-635, Current Opinion in Biotechnology 2005, 16:538-545, Trends in Biotechnology 2007 16 73-79.
In case the intracellular or extracellular lysine decarboxylase activity is due to expression of more than one lysine decarboxylase genes coding for different polypeptides having lysine decarboxylase activity, the total intracellular or extracellular lysine decarboxylase activity will be the result of those different lysine decarboxylase activities. The contribution of a particular lysine decarboxylase gene to the total lysine decarboxylase activity can be measured by comparing the expression level of different lysine decarboxylase genes in a particular microorganism and comparing the specific enzymatic activities of the lysine decarboxylases expressed from these genes. The expression level of different genes can be measured according to methods well known in the art, preferably the expression level is measured on the protein level, e.g. by Western Blots or ELISA assays. Methods to measure the specific enzymatic activity of a lysine decarboxylase are also well known in the art. A preferred method is disclosed in WO2007113127.
In case a endogenous lysine decarboxylase activity is enhanced by transgenic expression of at least one further polypeptide having lysine decarboxylase activity, the contribution of this or those additional polypeptides can be measured by comparing the total lysine decarboxylase activity of the transformed and untransformed microorganism.
In a preferred embodiment the intracellular or the extracellular decarboxylase activity or both activities are at least partly, preferably to more than 30% or to more than 50% or to more than 60% or to more than 70% or to more than 75%, 80%, 85%, 90%, 95% 98%, 99% due to one or more polypeptides comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 3 and having lysine decarboxylase activity, preferably having a high lysine decarboxylase activity.
In one embodiment the intracellular or the extracellular decarboxylase activity or both activities are to more than 98% or to more than 99% due to one or more polypeptides comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 3 and having lysine decarboxylase activity, preferably having a high lysine decarboxylase activity.
In one embodiment the intracellular or the extracellular decarboxylase activity or both activities are to more than 98% or to more than 99% due to one or more polypeptides comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 4 and having lysine decarboxylase activity, preferably having a high lysine decarboxylase activity.
In one embodiment, the microorganisms comprising a cadaverine exporter activity does also comprise an enhanced lysine import capacity.
In improved lysine import capacity, can be achieved by any measure, which enhances the flow of lysine from the fermentation medium into the microorganism or by reducing the flow of lysine from microorganism to the fermentation medium.
Methods to determine the lysine export and import activity can be found in Bellmann, A, Vrljic, M, Patek, M, et al. MICROBIOLOGY-SGM 147 pp. 1765-1774, 2001, and in Burkovski, A, Kramer, R. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 58 pp. 265-274. 2002 and in references cited within.
For example, the lysine import capacity can be improved by reducing or eliminating lysine exporter activity or an enhancing lysine permease activity or an enhancing lysine/cadaverine antiporter activity or any combination thereof.
A lysine exporter activity can be due to any polypeptide, being able to transport lysine from the medium to the cell, e.g. lysine-exporter, -symporter or -antiporter polypeptides.
In case the microorganism has an extracellular lysine decarboxylase activity and a high lysine production capacity, it may be of advantage to enhance the lysine export capacity by taking measures contrary to the ones described for enhancing the lysine import capacity, e.g. by enhancing a certain gene expression instead of reducing the expression of a certain gene. Examples for microorganisms having a high lysine production capacity and a reduced or eliminated expression of a lysine exporter polypeptide, but lacking an extracellular lysine decarboxylase activity can be found in WO 97/23597 and WO2005073390, which disclose methods for enhanced production of amino acids by culturing microorganism having an enhanced expression or activity of amino acid export proteins and in U.S. Pat. No. 7,435,584, which discloses a method for enhanced production of L-lysine by culturing corynebacteria having a high expression of the lysE (lysine export carrier) gene.
In one embodiment of the invention the activity of at least one lysine exporter polypeptide is decreased, wherein the lysine exporter polypeptide comprises an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 5 and having lysine export activity or wherein the lysine exporter polypeptide comprises an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 5 and having lysine export activity, or wherein the activity of at least two lysine exporter polypeptides are decreased, at least one comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO:5 and having lysine export activity and at least one comprising an amino acid sequence being at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 5 and having lysine export activity.
The lysE gene has been described in the literature, Eggeling, L, Sahm, H JOURNAL OF BIOSCIENCE AND BIOENGINEERING 92, 3 201-213, Eggeling, L, Sahm, H ARCHIVES OF MICROBIOLOGY 180, 3 155-160 2003, see also the German patent application DE 95-01048222.
The YbjE gene product (SEQ ID NO: 6) and mutants thereof have been described, for example in WO2005073390.
The term “decreased activity” includes the expression of a gene product, e.g. of a cadaverine exporter polypeptide, or a lysine exporter polypeptide, at a lower level than expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated, preferably the expression of a gene product is compared to a well known strain of a particular microorganism species, which is grown under the same conditions, e.g. in the case of Corynebacterium, the expression of a gene is preferably compared with the expression level in the strain ATCC13032. In the case of E. coli, the expression of a gene is preferably compared with the expression level in the strain MG1665, deposited in the strain collection ATCC in the case of Saccharomyces cerevisiae, the expression level is compared to the strain W303 deposited in the strain collection ATCC. In one embodiment, the microorganism can be genetically manipulated (e.g., genetically engineered) to express a level of gene product at a lesser than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated. Genetic manipulation can include, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by removing strong promoters, inducible promoters or multiple promoters), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, decreasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of decreasing expression of a particular gene routine in the art (including but not limited to use of antisense nucleic acid molecules, or other methods to knock-out or block expression of the target protein). In particular the gene can be manipulated that 1 or more nucleotides are being deleted from the chromosome of the host organism. The decreased activity of the gene product e.g. of a lysine exporter polypeptide can also be obtained by introducing one or more gene mutations which lead to a decreased activity of the gene product. In addition the activity of a gene product can be also decreased by influencing regulatory proteins that are regulating the expression or activity of said gene product e.g. by influencing the transcription of the said gene. Examples are transcriptional repressors and transcriptional activators. For example lysE gene expression is negatively influenced by the lysG gene product. The sequence of the lysG gene (herein disclosed as SEQ ID NO: 7) and the LysG gene product (herein disclosed as SEQ ID NO: 8 can also be found under the following accession numbers: P94632, X96471
In another embodiment the lysine import capacity is enhanced by an enhanced lysine permease activity or by an enhanced lysine/cadaverine antiporter activity or a combination of both.
The lysine permease activity of a given microorganism can be enhanced for example by increasing the expression of one or more endogenous lysine permease genes for example as disclosed in SEQ ID NO: 9, or increasing the permease activity of lysine permease polypeptides for example as disclosed in SEQ ID NO: 10, e.g. by random mutagenesis of the microorganism or by transforming the microorganism with one or more lysine permease genes or by any combination thereof.
In one embodiment the lysine permease activity of the microorganism is enhanced, by recombinant expression of one or more lysine permease polypeptides comprising an amino acid sequence, which is at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 10 and having lysine permeate activity, e.g. homologues or mutants having lysine permease activity. Methods to determine the lysine permease activity can be found in Bellmann, A, Vrljic, M, Patek, M, et al. MICROBIOLOGY-SGM 147 pp. 1765-1774 2001, and in Burkovski, A, Kramer, R APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 58 pp. 265-274 2002, in Fujii, T, et al. as well as in BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66, pp 1981-1984, 2002 and in references cited therein.)
Recombinant expression can be archived for example by transformation of one or more lysine permease genes, by providing endogenous lysine permease genes with a deregulated promoter having a higher expression activity or by reducing negative regulators of lysine permease gene expression or gene product activity.
In another embodiment the lysine import capacity is enhanced by an enhanced lysine/cadaverine antiporter activity. Lysine/cadaverine antiporter are proteins transporting lysine and cadaverine at the same time in different directions across the membrane of the cell. Enhanced lysine/cadaverine antiporter activity can be archived by increasing the expression of one or more endogenous lysine/cadaverine antiporter genes, by random mutation of the microorganism, transforming the microorganism with one or more lysine/cadaverine antiporter genes or by optimizing the lysine/cadaverine antiporter activity of lysine/cadaverine antiporter polypeptides e.g. for a preference of lysine import and cadaverine export, or by any combination thereof.
In one embodiment the lysine/cadaverine antiporter activity of a microorganism is enhanced by recombinant expression of one or more lysine/cadaverine antiporter polypeptides comprising an amino acid sequence, which is at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 11 having lysine/cadaverine antiporter activity, e.g. homologues or mutants having lysine/cadaverine antiporter activity.
Recombinant expression can be archived for example by transformation of one or more lysine/cadaverine antiporter genes, by providing endogenous lysine/cadaverine antiporter genes with a deregulated promoter having a higher expression activity or by reducing negative regulators of lysine/cadaverine antiporter gene expression or gene product activity.
It is known that the import or export activity of the cadB gene product, herein disclosed as SEQ ID NO: 11, depends on the lysine and cadaverine concentrations in the cell and the fermentation medium. Therefore, the lysine import capacity of a certain microorganism expressing a functional cadB gene product can be improved by enhancing the lysine concentration in the fermentation medium, preventing a low pH-value of the fermentation medium or by inserting mutations, which promote the lysine import at a given lysine concentration or pH-value in the fermentation medium or by a combination thereof (Soksawatmaekhin W. et al.; Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli; Molecular Microbiology; 2004; volume 51,5; pages 1401 to 1412).
Several mutations are described, which promote the lysine import of the Escherichia coli cadB gene product a given lysine concentration or pH-value in the fermentation medium (Soksawatmaekhin W. et al.; Identification of the Cadaverine Recognition Site on the Cadaverine-Lysine Antiporter Cad B; The Journal of Biological Chemistry; 2006; volume 281, 39; pages 29213 to 29220). A person having skill in the art will be able to identify similar mutations in other cadB proteins, e.g. homologues or mutants of cadB.
Accordingly, in one embodiment the lysine/cadaverine antiporter polypeptides comprise an amino acid sequence, which is at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 11 comprises one or more of the following mutations: C12S, W41L, W43L, Y55L, Y57L, Y73L, Y73F, Y73W, Y89L, Y89F, Y89W, Y90L, Y90F, Y90W, Y107L, Y174L, C125S, D185N, E204Q, E204D, Y235L, Y235F, Y235W, W289L, D303N, D303E, Y310L, Y366L, Y368L, D372N, E377Q, E408Q, Y423L, Y423F and Y423W. The mutations mentioned above are named according to the amino acid sequence of the cadB gene product. A person skilled in the art will be able to transfer these mutations to other gene products, e.g. gene products being homologous of the cadB gene product, by using sequence comparison tools e.g. by using sequence alignments.
Preferably the lysine/cadaverine antiporter polypeptides comprise an amino acid sequence, which is at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 11 comprises one or more of the following mutations: W41L, W43L, Y57L, Y89L, Y107L, Y174L, D185N, E204Q, Y235L, W289L, D303N, Y366L, Y368L, D372N and E408Q.
More preferred the lysine/cadaverine antiporter polypeptides comprise an amino acid sequence, which is at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 11 comprises one or more of the following mutations: W41L, W43L, Y57L, Y107L, Y174L, D185N, Y366L, Y368L and E408Q.
In a further embodiment the invention provides for a microorganism comprising an intracellular lysine decarboxylase activity and an enhanced lysine import activity or comprising an intracellular and an extracellular lysine decarboxylase activity or comprising an intracellular and an extracellular lysine decarboxylase activity and an enhanced lysine import activity and having a high lysine production capacity. A microorganism having a high lysine production capacity is able to enrich lysine inside the cell or in the surrounding medium, if the lysine is not further metabolized e.g. to cadaverine.
Preferably the microorganism having a high lysine production capacity has at least one deregulated gene selected from the group (i). The group (i) is a group of genes which play a key role in the biosynthesis of lysine and consists of the genes of aspartokinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, succinyl-amino-ketopimelate transaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelate epimerase, diaminopimelate dehydrogenase, arginyl-tRNA synthetase, diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase, homoserine dehydrogenase, phophoenolpyruvate carboxykinase, methylmalonyl-CoA mutase, diamine-acteyltransferase.
At least one gene of the group (i) has to be deregulated according to the inventive process. Preferably more than one gene of group (i), e.g. two, three, four, five, six, seven, eight, nine, ten genes are deregulated in the microorganism according to the invention.
Preferred genes of the group (i) to be deregulate are: aspartokinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, diaminopimelate dehydrogenase, diaminopimelate decarboxylase, pyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase, homoserine dehydrogenase, diamine-actyltransferase.
The genes and gene products of group (i) are known in the art. EP 1108790 discloses mutations in the genes of homoserinedehydrogenase and pyruvate carboxylase which have a beneficial effect on the productivity of recombinant corynebacteria in the production of lysine. WO 00/63388 discloses mutations in the gene of aspartokinase which have a beneficial effect on the productivity of recombinant corynebacteria in the production of lysine. EP 1108790 and WO 00/63388 are incorporated by reference with respect to the mutations in these genes described above.
In the table below for every gene/gene product possible ways of deregulation of the respective gene are mentioned. The literature and documents cited in the row “Deregulation” of the table are herewith incorporated by reference with respect to gene deregulation. The ways mentioned in the table are preferred embodiments of a deregulation of the respective gene.









TABLE 1





Enzyme (gene product)
Gene
Deregulation







Aspartokinase
ask
Releasing feedback inhibition by point




mutation (Eggeling et al., (eds.), Hand-




book of Corynebacterium glutamicum,




pages 20.2.2 (CRC press, 2005)) and




amplification)


Aspartatesemialdehyde dehydrogenase
asd
Amplification


Dihydrodipicolinate synthase
dapA
Amplification


Dihydrodipicolinate reductase
dapB
Amplification


Tetrahydrodipicolinate succinylase
dapD
Amplification


Succinyl-amino-ketopimelate transaminase
dapC
Amplification


Succinyl-diamino-pimelate desuccinylase
dapE
Amplification


Diaminopimelate dehydrogenase
ddh
Amplification


Diaminopimelate epimerase
dapF
Amplification


Arginyl-tRNA synthetase
argS
Amplification


Diaminopimelate decarboxylase
lysA
Amplification


Pyruvate carboxylase
pycA
Releasing feedback inhibition by point




mutation (EP1108790) and amplification


Phosphoenolpyruvate carboxylase
ppc
Amplification


Glucose-6-phosphate dehydrogenase
G6PDH
Releasing feedback inhibition by point



zwf
mutation (US2003/0175911) and amplifi-




cation


Transketolase
tkt
Amplification


Transaldolase
tal
Amplification


6-Phosphogluconolactonase
pgl
Amplification


6-Phosphogluconate dehydrogenase

point mutation and amplification


Fructose 1,6-biphosphatase
fbp
Amplification


Homoserine dehydrogenase
hom
Attenuating by point mutation




(EP1108790) decrease of gene activity,




Knock-out or silencing by mutation


Phophoenolpyruvate carboxykinase
pck
Knock-out or silencing by mutation, de-




crease of gene activity or others


Methylmalonyl-CoA mutase
MMCM
Attenuating by point mutation (WO




05/58945) decrease of gene activity,




Knock-out or silencing by mutation


Diamine Acetyltransferase
RXA2240
Weakening by decrease of gene activity,




Knock-out or silencing by mutation, dele-




tion









A preferred way of deregulation of the genes of aspartokinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, succinyl-amino-ketopimelate transaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelate epimerase, diaminopimelate dehydrogenase, arginyl-tRNA synthetase, diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase is an “up”-mutation which increases the gene activity e.g. by gene amplification using strong expression signals and/or point mutations which enhance the enzymatic activity.
A preferred way of deregulation of the genes of homoserine dehydrogenase, phophoenolpyruvate carboxykinase, methylmalonyl-CoA mutase Acetyltransferase is a “down”-mutation which decreases the gene activity e.g. by gene deletion or disruption, using weak expression signals and/or point mutations which destroy or decrease the enzymatic activity.
If aspartokinase is deregulated as a member of gene (i) group at least a second gene (i) member—other than aspartokinase—has to be deregulated also.
It has been observed that a significant portion of the cadaverine produced in the microorganism according to the inventive process may become acetylated later on (WO 2007/113127). In order to block the acetylation reaction which is attributed to an N-acetylcadaverine-forming polypeptide, which is defined as an enzymatic active polypeptide being able to produce N-acetylcadaverine. In order to increase the yield of cadaverine it is a preferred embodiment of the invention to deregulate the diamine acetyltransferase of the producing microorganism, especially to decrease its activity, e.g. by deletion or disruption of the gene. One example for an N-acetylcadaverine-forming polypeptide is the acetyl-CoA dependent diamine acetyltransferase of Corynebacterium glutamicum (NP_600742 protein) for example as disclosed in SEQ ID NO: 12 and SEQ ID NO: 13
In one embodiment of the invention, the N-acetylcadaverine-forming activity is decreased by decreasing the activity of at least one N-acetylcadaverine-forming polypeptide comprising an amino acid sequence, being at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to SEQ ID NO: 13 and has N-acetylcadaverine-forming activity. N-actylcadaverine-forming activity can be tested as described in WO 2007/113127.
It has been observed that a significant portion of the cadaverine produced in the microorganism according to the inventive process may be converted to aminopropylcadaverine by an aminopropylcadaverine-forming polypeptide. In order to block this reaction and in order to increase the yield of cadaverine it is a preferred embodiment of the invention to deregulate the aminopropylcadaverine-forming polypeptide of the producing microorganism, especially to decrease its activity, e.g. by deletion or disruption of the gene.
One example for an aminopropylcadaverine-forming polypeptide is the spermidine synthase of Escherichia coli, as described in Soksawatmaekhin W. et al; Molecular Microbiology; (2004) Vol. 51,5; pages 1401 to 1412)), SEQ ID NO: 14
Accordingly, in one embodiment of the invention, the aminopropylcadaverine-forming activity is decreased by decreasing the activity of at least one aminopropylcadaverine-forming polypeptide comprising an amino acid sequence, being at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to SEQ ID NO: 14.
It has been observed that the production capacity of a microorganism for lysine can be improved by deregulating the activity of a homoserine dehydrogenase polypeptide of the cadaverine producing microorganism as described in JP2004222569, preferably by decreasing its activity via deletion or disruption of the gene coding for the homoserine dehydrogenase polypeptide.
Examples for a homoserine dehydrogenase polypeptide is the homoserine dehydrogenase of Corynebacterium glutamicum, herein disclosed as SEQ ID NO: 15, or homoserine dehydrogenases of Escherichia coli, herein disclosed as SEQ ID NO: 16 and SEQ ID NO: 17.
Accordingly, in one embodiment of the invention, the homoserine dehydrogenase activity is decreased by decreasing the activity of a homoserine dehydrogenase polypeptide comprising an amino acid sequence, being at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to SEQ ID NO: 15, 16 or 17.
Methods for the determination of homoserine dehydrogenase activity can be found in. M. B. Jenkins, V. W. Woodward, Biochimica et Biophysica Acta, 1970, 212, 21-32.
In another embodiment of the invention the microorganism has a reduced capacity to degrade lysine other than by decarboxylation, e.g. by having a decreased lysine hydroxylase activity.
A lysine hydroxylase polypeptide is a polypeptide having lysine hydroxylase activity also described as lysine N6-hydroxylase [EC:1.14.13.59]. Tests for having lysine hydroxylase activity can be found in Meneely K M, and Lamb A L BIOCHEMISTRY 46 Pages: 11930-11937 2007 and in I J, Hsueh L C, Baldwin J E, et al. EUROPEAN JOURNAL OF BIOCHEMISTRY 268 Pages: 6625-6636.
One example is the lysine hydroxylase iucD of Escherichia coli CFT073 (SEQ ID NO: 18).
In a further embodiment of the invention, the lysine degradation activity is decreased by decreasing the activity of at least one polypeptide comprising an amino acid sequence being at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to SEQ ID NO: 18 lysine hydroxylase, having lysine hydroxylase activity.
In a further embodiment of the invention, the microorganism has a deregulated spermidine forming or uptake activity or putrescine forming or uptake activity or a combination thereof.
A spermidine forming activity is brought about by a polypeptide being able to synthesize spermidine. One example for a spermidine forming polypeptide is the spermidine synthase of SEQ ID NO: 14.
A putrescine forming activity is brought about by a polypeptide being able to synthesize putrescine. One example for a putrescine forming polypeptide are the putrescine synthase of E. coli speE (e.g. as disclosed in SEQ ID NO: 19) or the ornithin decarboxylases of E. coli, speF (e.g as disclosed in SEQ ID NO: 20)
A polypeptide having spermidine or putrescine uptake activity is a polypeptide being able to transport spermidine or putrescine or both into the cell. One example for a spermidine and or an putrescine uptake polypeptide is potE of E. coli (e.g as disclosed in SEQ ID NO: 21), which functions as a putrescine/ornithine antiporter.
In one embodiment of the invention, the microorganism has a decreased spermidine forming or uptake activity or putrescine forming or uptake activity, wherein

    a) the spermidine forming activity is deregulated by decreasing the activity of a spermidine forming polypeptide comprising an amino acid sequence being at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to SEQ ID NO: 14 or,
    b) the putrescine forming activity is deregulated by decreasing the activity of a putrescine forming polypeptide comprising an amino acid sequence being at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to SEQ ID NO: 19, or
    c) the putrescine forming activity is deregulated by decreasing the activity of a ornithine decarboxylase polypeptide comprising an amino acid sequence being at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to SEQ ID NO: 20, or
    d) the spermidine or putrescine or spermidine and putrescine uptake activity is deregulated by decreasing the activity of a spermidine or putrescine or spermidine and putrescine uptake polypeptide comprising an amino acid sequence being at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% identical to SEQ ID NO: 21 or
    e) wherein the spermidine forming or uptake activity or the putrescine forming or uptake activity or a combination thereof is decreased by decreasing the activity of a combination of a), b), c) or d).


An important aspect of the present invention involves cultivating or culturing the recombinant microorganisms described herein, such that the desired compound cadaverine is produced. Accordingly one embodiment of the invention is a cadaverine production system comprising a microorganism, comprising an intracellular lysine decarboxylase activity and an enhanced lysine import activity or comprising an intracellular and an extracellular lysine decarboxylase activity or comprising an intracellular and an extracellular lysine decarboxylase activity and an enhanced lysine import activity and a fermentation medium suitable to cultivate this microorganism, preferably the fermentation medium comprises lysine.
A cadaverine production system is a technical system for the production of cadaverine, e.g. a culture medium comprising a cadaverine producing microorganism or a lysine comprising solution or culture medium and a lysine decarboxylase producing microorganism. Usually the cadaverine production system comprises technical systems to support the production of cadaverine, e.g. a fermenter.
The term “cultivating” includes maintaining and/or growing a living microorganism of the present invention (e.g., maintaining and/or growing a culture or strain). In one embodiment, a microorganism of the invention is cultured in liquid media. In another embodiment, a microorganism of the invention is cultured on solid media or semi-solid media. In a preferred embodiment, a microorganism of the invention is cultured in media (e.g., a sterile, liquid media) comprising nutrients essential or beneficial to the maintenance and/or growth of the microorganism.
Carbon sources which may be used include sugars and carbohydrates, such as for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as for example soy oil, sunflower oil, peanut oil and coconut oil, fatty acids, such as for example palmitic acid, stearic acid and linoleic acid, alcohols, such as for example glycerol and ethanol, and organic acids, such as for example acetic acid. In one preferred embodiment, glucose, fructose or sucrose are used as carbon sources. These substances may be used individually or as a mixture.
In another preferred embodiment, the microorganisms described herein are cultivated in or on liquid, solid or semi-solid media comprising xylose, arabinose, cellobiose or mixtures thereof, such media may or may not comprise other carbon sources like the ones described above. Media for cultivating microorganisms of the invention can comprise only a limited number of different carbon sources e.g. 1, 2, 3, 4, 5 or more carbon sources, or might comprise very complex mixtures of carbon sources, hydrolysates of lignocellulose substrates or agricultural residues, e.g. hydrolysates of starches from sources such as but not limited to corn, wheat, rye, barley, rice, cassava, or hydrolysates of straw, wood, paper, or other material of plant origin. Preferred combinations of carbon sources are media comprising a high content of glucose and xylose, fructose and xylose, sucrose and xylose, or sucrose and glucose, or sucrose and fructose, or sucrose, glucose and fructose and sucrose, glucose, fructose and xylose, or glucose, fructose and xylose or glucose, fructose, xylose and arabinose or sucrose, xylose and arabinose, or glucose, xylose and arabinose and of other combination of sugars mentioned.
In case the media comprises xylose it is of advantage to use a microorganism of the invention expressing or overexpressing genes of the xylose metabolism.
Genes of the xylose metabolism are for example the genes of the xyIABFGHR locus of E. coli, comprising genes for a xylose transport systems (xylE, xylT and the xylFGH gene), genes for xylose utilization (xylA and xylB gene) and genes for xylose transcriptional activator (xylR gene). Microorganisms overexpressing genes of the xyIABFGHR locus are described in EP1577396 and EP1577369. Corynebacteria overexpressing genes of the xylA alone or with the xylB gene of E. coli, encoding a xylose isomerase and xylB encoding a xylulokinase have been described e.g. (Kawaguchi, et al. Engineering of a xylose metabolic pathway in Corynebacterium gutamicum, Applied and Environmental Microbiology, 2006, Vol. 72, 5, pages 3418 to 3428).
In a preferred embodiment, the microorganism of the invention expresses or over expresses at least the xylA or the xylB gene or even more preferred the xylA and the xylB gene.
In case the media comprises arabinose, it is of advantage to use a microorganism of the invention expressing or overexpressing genes of the arabinose metabolism. Genes of the arabinose metabolism are for example genes of the araBAD operons, e.g. the araA, araB, araD and areE genes of E. coli, coding for L-arabinose isomerase (araA), L-ribolokinase (araB) and L-ribulose-5-phosphate-4-epimerase (araD) or the genes of the araBDA operon of Corynebacterium gluctamicum, comprising homologs of the araA, araB and araD genes and the araE coding for a L-arabinose isomerase, the araR gene coding for a transcriptional regulator and the galM gene coding for a putative aldose 1-epimerase. Preferably the microorganism of the invention expresses or overexpresses at least the araA, araB and araD gene, more preferably at least the araA, araB, araD and the araE gene. Kawaguchi et al. Identification and Functional Analysis of the Gene Cluster for L-Arabinose Utilization in Corynebacterium glutamicum, Applied and Environmental Microbiology, 2009, 75, Vol. 11, pages 3419-3429).
The E. coli homologs of the genes of the arabinose metabolism can also be used in heterologous microorganism such as Corynebacterium glutamicum (Kawaguchi et al. Engineering of an L-arabinose metabolic pathway in Corynebacterium glutamicum, Applied Microbiology and Biotechnology, 2008, 77, Vol, 5, pages 1053 to 1062).
In case the media comprises cellobiose, it is of advantage to use a microorganism of the invention expressing or overexpressing genes of the cellobiose metabolism. Genes of the cellobiose metabolism are for example the bglA genes of Corynebacterium glutamicum, encoding phosphenolpyruvate:carbohydratephosphotransferase system (PTS) beta-glucoside-specific enzyme IIBCA component and phosphor-beta-glucosidase examples of these genes and the respective proteins from the Corynebacterium R strain can be found under the accession number AF508972.
In case the media comprises combinations of xylose, arabinose, cellobiose or other carbon sources, it is of advantage to use microorganisms of the invention expressing or overexpressing genes of the xylose metabolism, arabinose metabolism or cellobiose metabolism. For example, in case the media has a high content of xylose- and arabinose it is of advantage to use microorganisms expressing or overexpressing genes of the xylose- and arabinose metabolism for example expressing the xylA and xylB genes and the araA, araB, araD genes, preferably the xylA and xylB araA, araB, araD and araE gene.
In case the media has a high content xylose and cellubiose it is of advantage to use microorganisms expressing or overexpressing genes of the xylose- and cellobiose metabolism for example expressing the xylA and xylB and the bglA genes (Sasaki et al. Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions, Applied Microbiology and Biotechnology, 2008, Vol. 81, 4, pages 691 to 699)
Nitrogen sources which may be used comprise organic compounds containing nitrogen, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture. Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding salts containing sodium. The culture medium must furthermore contain metal salts, such as for example magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins may also be used in addition to the above-stated substances. Suitable precursors may furthermore be added to the culture medium. The stated feed substances may be added to the culture as a single batch or be fed appropriately during cultivation.
Preferably, microorganisms of the present invention are cultured under controlled pH. The term “controlled pH” includes any pH which results in production of the desired fine chemical, e.g. cadaverine. In one embodiment, microorganisms are cultured at a pH of about 7. In another embodiment, microorganisms are cultured at a pH of between 6.0 and 8.5. The desired pH may be maintained by any number of methods known to those skilled in the art. For example, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or ammonia water, or acidic compounds, such as phosphoric acid or sulfuric acid, are used to appropriately control the pH of the culture.
Also preferably, microorganisms of the present invention are cultured under controlled aeration. The term “controlled aeration” includes sufficient aeration (e.g., oxygen) to result in production of the desired fine chemical, e.g., cadaverine. In one embodiment, aeration is controlled by regulating oxygen levels in the culture, for example, by regulating the amount of oxygen dissolved in culture media. Preferably, aeration of the culture is controlled by agitating the culture. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the growth vessel (e.g., fermenter) or by various pumping equipment. Aeration may be further controlled by the passage of sterile air or oxygen through the medium (e.g., through the fermentation mixture). Also preferably, microorganisms of the present invention are cultured without excess foaming (e.g., via addition of antifoaming agents such as fatty acid polyglycol esters).
Moreover, microorganisms of the present invention can be cultured under controlled temperatures. The term “controlled temperature” includes any temperature which results in production of the desired fine chemical, e.g., cadaverine. In one embodiment, controlled temperatures include temperatures between 15° C. and 95° C. In another embodiment, controlled temperatures include temperatures between 15° C. and 70° C. Preferred temperatures are between 20° C. and 55° C., more preferably between 30° C. and 45° C. or between 30° C. and 50° C.
Microorganisms can be cultured (e.g., maintained and/or grown) in liquid media and preferably are cultured, either continuously or intermittently, by conventional culturing methods such as standing culture, test tube culture, shaking culture (e.g., rotary shaking culture, shake flask culture, etc.), aeration spinner culture, or fermentation. In a preferred embodiment, the microorganisms are cultured in shake flasks. In a more preferred embodiment, the microorganisms are cultured in a fermentor (e.g., a fermentation process). Fermentation processes of the present invention include, but are not limited to, batch, fed-batch and continuous methods of fermentation. The phrase “batch process” or “batch fermentation” refers to a closed system in which the composition of media, nutrients, supplemental additives and the like is set at the beginning of the fermentation and not subject to alteration during the fermentation, however, attempts may be made to control such factors as pH and oxygen concentration to prevent excess media acidification and/or microorganism death. The phrase “fed-batch process” or “fed-batch” fermentation refers to a batch fermentation with the exception that one or more substrates or supplements are added (e.g., added in increments or continuously) as the fermentation progresses. The phrase “continuous process” or “continuous fermentation” refers to a system in which a defined fermentation medium is added continuously to a fermenter and an equal amount of used or “conditioned” medium is simultaneously removed, preferably for recovery of the desired cadaverine. A variety of such processes have been developed and are well-known in the art.
The methodology or process of the present invention can further include a step of recovering the produced fine chemical e.g. lysine, β-lysine, cadaverine or N-Acetylcadaverine. The term “recovering”, for example as used in “recovering cadaverine” includes extracting, harvesting, isolating or purifying the compound from culture media. In case of “recovering cadaverine”, the recovered compound is cadaverine. Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like. For example cadaverine can be recovered from culture media by first removing the microorganisms. The broth removed biomass is then passed through or over a cation exchange resin to remove unwanted cations and then through or over an anion exchange resin to remove unwanted inorganic anions and organic acids having stronger acidities than cadaverine. In addition the broth can be treated with caustic agents and the cadaverine be extracted with organic solvents such as alkohols by phase separation. The cadaverine can be retrieved from the extracted phase by distillation to purity sufficient for diverse applications. Possible applications include the production of polyamides by polycondensation with dicarboxylic organic acids.
Accordingly, in another aspect, the present invention provides a process for the production of polyamides (e.g. NylonTM) comprising a step as mentioned above for the production of cadaverine. The cadaverine is reacted in a known manner with di-, tri- or polycarboxylic acids to get polyamides. Preferably the cadaverine is reacted with dicarboxylic acids containing 4 to 10 carbons such as succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, and so forth. The dicarboxylic acid is preferably a free acid.
The microorganism of the invention are useful for producing a fine chemical, e.g. cadaverine by fermenting these microorganism, which is growing the microorganism in culture, preferably growing the microorganism under culture conditions as described above.
Accordingly the invention includes a process for the production of cadaverine, comprising fermenting a microorganism, comprising an intracellular lysine decarboxylase activity and an enhanced lysine import activity or comprising an intracellular and an extracellular lysine decarboxylase activity or comprising an intracellular and an extracellular lysine decarboxylase activity and an enhanced lysine import activity. Preferably the process includes recovering of cadaverine from the culture medium.
In one embodiment the microorganism comprises an enhanced lysine/cadaverine antiporter activity, e.g. a microorganism overexpressing a polypeptide which is at least 80% or at least 85% or at least 90% or at least 95% or at least 98% identical to SEQ ID NO: 11 and having lysine/cadaverine antiporter activity, e.g. homologues or mutants having lysine/cadaverine antiporter activity and the process includes fermenting the microorganism in a medium comprising lysine. Preferably the culture medium comprises more than 0.1 mM, or more than 0.5 mM, or more than 1 mM, or more than 3 mM, or more than 5 mM, or more than 7 mM, or more than 8 mM, or more than 9 mM, or more than 10 mM lysine. More preferred the culture medium comprises more than 15 mM lysine. Even more preferred, the culture medium comprises more than 20 mM lysine. Most preferred the culture medium comprises more than 30 mM lysine
In a further embodiment the microorganism comprises an extracellular lysine decarboxylase activity or comprises an extracellular lysine decarboxylase activity and an enhanced lysine/cadaverine antiporter activity.
In another embodiment of the invention is a process to produce cadaverine, wherein the concentration (mol/l) of cadaverine in the culture medium is at least 1.2 times higher, or more than 1.3 times higher, or more than 1.4 times, or more than 1.5 times, or more than 1.6 times, or more than 1.7 times, or more than 1.8 times, or more than 1.9 times or more than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times higher than the concentration (mol/l) of N-acetylcadaverine or aminopropylcadaverine or of both. Another embodiment of the invention is the culture medium produced in the process.
The cadaverine produced by the processes described above can be recovered or purified by a work up of the cadaverine (DAP) comprising fermentation broth, i.e. the culture medium in its state after the process to produce cadaverine is finished or has been terminated.
The process to recover or purify the cadaverine from the fermentation broth as described in the following medium is solely for exemplary reasons. The person skilled in the art will know alternative methods or variants of the process described below, which can also successfully be applied.
In order to recover the produced cadaverine, it is of advantage to thicken or to concentrate the fermentation broth. The fermentation broth can be thickened or concentrated by known methods, such as, for example, with the aid of a rotary evaporator, thin-film evaporator, falling film evaporator, by reverse osmosis or by nanofiltration. If necessary, salts which may have precipitated due to the concentration procedure may be removed, for example by filtration or centrifugation. This concentrated fermentation broth can then be worked up in the manner of the invention to obtain cadaverine. For the work up in accordance with the present invention, such a concentration procedure is feasible, but not absolutely necessary.
According to the invention, cadaverine is extracted from the fermentation broth with the aid of an organic extractant. More specifically, use is made of here of an organic solvent having a miscibility gap with water that is as polar as possible and stable at alkaline pH, such as in particular a polar, dipolar protic, organic solvent. Suitable solvents are in particular cyclic or open-chain, optionally branched alkanols having from 3 to 8 carbon atoms, in particular n- and isopropanol, n-, sec- and iso-butanol, or cyclohexanol, and also n-pentanol, n-hexanol-n-heptanol, n-octanol, 2-octanol and the mono- or polybranched isomeric forms thereof. Particular mention is to be made here of n-butanol.
In a preferred embodiment, the extraction and/or subsequent phase separation are carried out batchwise at an elevated temperature which is limited by the boiling points of water and of the extractant or of possibly forming azeotropes. Using the extractant n-butanol, extraction and phase separation could be carried out, for example, at about 25-90° C. or, preferably, at 40-70° C. For extraction, the two phases are stirred until the partition equilibrium has been established, for example over a period of from 10 seconds to 2 hours, preferably 5 to 15 min. The phases are then left to settle until they have separated completely; this takes preferably from 10 seconds to 5 hours, for example 15 to 120 or 30 to 90 minutes, in particular also at a temperature in the range from about 25-90° C. or 40-70° C. in the case of n-butanol.
In further preferred embodiments, cadaverine is extracted from the fermentation broth continuously in a multi-stage process (for example in mixer-settler combinations) or continuously in an extraction column.
The skilled worker may establish the configuration of the extraction columns which can be employed according to the invention for the phases to be separated in each case as part of optimization routines. Suitable extraction columns are in principle those without power input or those with power input, for example pulsed columns or columns with rotating internals. The skilled worker may also, as part of routine work, select in a suitable manner types and materials of internals, such as sieve trays, and column trays, to optimize phase separation. The basic theories of liquid-liquid extraction of small molecules are well known (cf. e.g. H.-J. Rehm and G. Reed, Eds., (1993), Biotechology, Volume 3 Bioprocessing, Chapter 21, VCH, Weinheim). The configuration of industrially applicable extraction columns is described, for example, in Lo et al., Eds., (1983) Handbook of Solvent Extraction, John Wiley & Sons, New York. Explicit reference is made to the disclosure of the textbooks above.
After phase separation, cadaverine is isolated and purified from the cadaverine-comprising extract phase in a manner known per se. Possible measures of recovering cadaverine are in particular, without being limited thereto, distillation, precipitation as salt with suitable organic or inorganic acids, or combinations of such suitable measures.
Distillation may be carried out continuously or batchwise. A single distillation column or a plurality of distillation columns coupled to one another may be used. Configuring the distillation column apparatus and establishing the operational parameters are the responsibilities of the skilled worker. The distillation columns used in each case may be designed in a manner known per se (see e.g. Sattler, Thermische Trennverfahren [Thermal separation methods], 2nd Edition 1995, Weinheim, p. 135ff; Perry's Chemical Engineers Handbook, 7th Edition 1997, New York, Section 13). Thus, the distillation columns used may comprise separation-effective internals, such as separation trays, e.g. perforated trays, bubble-cap trays or valve trays, arranged packings, e.g. sheet-metal or fabric packings, or random beds of packings. The number of plates required in the column(s) used and the reflux ratio are essentially governed by the purity requirements and the relative boiling position of the liquids to be separated, with the skilled worker being able to ascertain the specific design and operating data by known methods.
Precipitation as salt may be achieved by adding suitable organic or inorganic acids, for example sulfuric acid, hydrochloric acid, phosphoric acid, acetic acid, formic acid, carbonic acid, oxalic acid, etc. In another preferred embodiment, an organic dicarboxylic acid is used, forming a salt which can be used, either directly or after purification, for example by recrystallization, in a subsequent polycondensation to give the polyamide. More specifically, such dicarboxylic acids are C4-C12-dicarboxylic acids.
The organic cadaverine phase produced in the extraction procedure may also be worked up chromatographically. For chromatography, the cadaverine phase is applied to a suitable resin, for example a strongly or weakly acidic ion exchanger (such as Lewatit 1468 S, Dowex Marathon C, Amberlyst 119 Wet or others), with the desired product or the contaminants being partially or fully retained on the chromatographic resin. These chromatographic steps may be repeated, if necessary, using the same or other chromatographic resins. The skilled worker is familiar with selecting the appropriate chromatographic resins and their most effective application. The purified product may be concentrated by filtration or ultrafiltration and stored at an appropriate temperature.
The identity and purity of the compound(s) isolated may be determined by prior art technologies. These comprise high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzyme assay or microbiological assays. These analytical methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.
As the cadaverine produced and recovered or purified by the processes described above can be used to produce polyamides by known techniques, those polyamides represent another embodiment of the invention.
The skilled worker will realize that analogous processes can be used to produce and/or recover lysine, β-lysine or N-Acetylcadaverine. Methods to recover lysine, β-lysine or N-Acetylcadaverine are available in the prior art.
Processes to produce and or recover lysine or β-lysine, will employ microorganisms of the invention having a high capacity to produce and or export lysine, but a low capacity to degrade Lysine and/or a low capacity to convert Lysine into cadaverine, N-Acetylcadaverine, or other derivatives of cadaverine described herein.
For example, a microorganism employed to produce lysine or β-lysine, will have a decreased succinyl-CoA synthetase activity and preferably a low lysine decarboxylase activity. Such microorganism will preferably also comprise at least one deregulated gene selected from the group consisting of the genes of aspartokinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, succinyl-amino-ketopimelate transaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelate epimerase, diaminopimelate dehydrogenase, arginyl-tRNA synthetase, diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase, homoserine dehydrogenase, phophoenolpyruvate carboxykinase, methylmalonyl-CoA mutase, diamine acteyltransferase.
For example, a microorganism employed to produce cadaverine or N-Acetylcadaverine, will have a decreased succinyl-CoA synthetase activity but preferably a high lysine decarboxylase activity. Such microorganism will preferably also comprise at least one deregulated gene selected from the group consisting of the genes of aspartokinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, succinyl-amino-ketopimelate transaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelate epimerase, diaminopimelate dehydrogenase, arginyl-tRNA synthetase, diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase, homoserine dehydrogenase, phophoenolpyruvate carboxykinase, methylmalonyl-CoA mutase, diamine acteyltransferase, and may also comprise an enhanced lysine import activity, in case the lysine decarboxylase is expressed intracellularly. The microorganism employed to produce cadaverine preferably comprise a low N-acetylcadaverine-forming activity and may also comprise a enhanced cadaverine exporter activity, while a microorganism employed to produce N-Acetylcadaverine will preferably comprise a high N-acetylcadaverine-forming activity and may also comprise a decreased cadaverine exporter activity.
The invention will now be described in more detail on the basis of the following non-limiting examples and with reference to the accompanying figures
EXAMPLES
Microorganisms and strain construction: The rational lysine-producing strain, C. glutamicum BS87 (Becker, J., C. Klopprogge, H. Schroder, and C. Wittmann. 2009. Metabolic engineering of the tricarboxylic acid cycle for improved lysine production by Corynebacterium glutamicum. Appl Environ Microbiol 75:7866-9.), derived from the wild-type ATCC 13032 was used as parent strain. The deletion of sucCD, cg12565 and cg12566, encoding the two subunits of succinyl-CoA synthetase, was realized by replacement of the two genes by a shortened DNA fragment as previously applied to other deletions in C. glutamicum (Becker, J., E. Heinzle, C. Klopprogge, 0. Zelder, and C. Wittmann. 2005. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol 71:8587-8596.). Transformation was performed with an integrative vector, using kanamycin resistance and sucrose tolerance as positive selection markers (Becker, J., E. Heinzle, C. Klopprogge, O. Zelder, and C. Wittmann. 2005. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol 71:8587-8596 and Jäger, W., A. Schäfer, A. Pühler, G. Labes, and W. Wohlleben. 1992. Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans. J Bacteriol 174:5462-5). Vector construction was carried out with standard cloning strategies involving PCR, enzymatic digestion and DNA ligation. Strain validation was performed by PCR and determination of enzyme activity.
Cultivation and growth conditions: Cultivation was performed as described previously in two pre-cultures and a main culture (Becker, J., C. Klopprogge, A. Herold, O. Zelder, C. J. Bolten, and C. Wittmann. 2007. Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum-over expression and modification of G6P dehydrogenase. J Biotechnol 132:99-109.). First pre-culture was carried out in complex medium containing 10 g L−1 peptone, 5 g L−1 beef extract, 5 g L−1 yeast extract, 2.5 g L−1 NaCl, 10 g L−1 glucose and 2 g L−1 urea. Second pre-culture and main culture were performed in minimal medium as described (Kind, S., W. K. Jeong, H. Schröder, and C. Wittmann. 2010. Systems-wide metabolic pathway engineering in Corynebacterium glutamicum for bio-based production of diaminopentane. Metab Eng 12:341 351). Main cultivation was performed in triplicate using 500 mL baffled shake flasks with 50 mL minimal medium. During the cultivation the pH remained constant within a range of 7.0±0.2 and sufficient oxygen supply was ensured.
Substrate and product analysis: Glucose was quantified with a biochemical analyzer (YSI 2700 Select, Kreienbaum, Langenfeld, Germany). Concentration of organic acids and trehalose was determined by HPLC (Aminex HPX-87H, 300×7.8, Bio-Rad, Hercules, Calif.) at 45° C., with 12.5 mM H2SO4 as mobile phase and a flow rate of 0.5 mL min−1 and detection via refraction index (trehalose) or UV absorbance (organic acids) at 210 nm. Amino acids were quantified as described previously (Kromer, J. O., M. Fritz, E. Heinzle, and C. Wittmann. 2005. In vivo quantification of intracellular amino acids and intermediates of the methionine pathway in Corynebacterium glutamicum. Anal Biochem 340:171-3). Cell concentration was determined as optical density (OD660) (Libra S11, Biochrome, Cambridge, UK) (Kiefer, P., E. Heinzle, O. Zelder, and C. Wittmann. 2004. Comparative metabolic flux analysis of lysine-producing Corynebacterium glutamicum cultured on glucose or fructose. Appl Environ Microbiol 70:229-39). The correlation factor between OD660 and cell dry mass (CDM) was CDM [g L−1]=0.255×OD660 (Becker, J., C. Klopprogge, H. Schroder, and C. Wittmann. 2009. Tricarboxylic acid cycle engineering for improved lysine production in Corynebacterium glutamicum. Appl Environ Microbiol 75:7866-7869.).
Determination of succinyl-CoA synthetase activity: Cell preparation was performed as previously described, whereby 20 mM potassium phosphate buffer (pH 7.2) containing 20 mM MgCl2 was used as washing and disruption buffer (Becker, J., E. Heinzle, C. Klopprogge, O. Zelder, and C. Wittmann. 2005. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol 71:8587-8596.). Cell disruption was carried out by a ribolyzer (MM301, Retsch, Haan, Germany) at 30 Hz (2×5 min; 5 minutes break in between). Crude cell extract was obtained as supernatant after centrifugation (10 min, 13000×g, 4° C.). Protein content was quantified by the method of Bradford (Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-54.). The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.2), 10 mM MgCl2, 100 mM KCl, 10 mM sodium succinate, 0.1 mM CoA, and 0.4 mM ATP (Noda, S., Y. Takezawa, T. Mizutani, T. Asakura, E. Nishiumi, K. Onoe, M. Wada, F. Tomita, K. Matsushita, and A. Yokota. 2006. Alterations of cellular physiology in Escherichia coli in response to oxidative phosphorylation impaired by defective F1-ATPase. J Bacteriol 188:6869-76.). The reaction was initiated at 30° C. by the addition of ATP with thorough mixing. The formation of succinyl-CoA was then monitored via the absorbance increase at 230 nm. The molar extinction coefficient for succinyl-CoA of 4900 M−1 cm−1 at this wavelength was used to calculate the enzyme activity.
Estimation of in vivo fluxes: The in vivo flux through alpha-ketoglutarate dehydrogenase (□AKD) was estimated compiling a correlation between biomass yield (g/mol), lysine yield (%) and alpha-ketoglutarate dehydrogenase flux (%), as described recently for other reactions of the TCA cycle (Becker, J., C. Klopprogge, H. Schroder, and C. Wittmann. 2009. Tricarboxylic acid cycle engineering for improved lysine production in Corynebacterium glutamicum. Appl Environ Microbiol 75:7866-7869.) using the following equation:

uAKD=Y0+aYLys/S+bYX/S+cY2Lys/S+dY2X/S  [1]

Stoichiometric parameters from the paraboloid fitting were determined to Y0=120.5, a=−1.70, b=−0.47, c=6.06×10−3 and d=−4.49×10−3. Flux mean value and confidence intervals were calculated from 100 statistically varied values for biomass yield and lysine yield using a Monte-Carlo-approach.
Strain construction and validation. Mutants from the second recombination were validated by site specific PCR. Using the primer set in Table 1, successful deletion of sucCD was visualized by a resulting DNA fragment, which was 1050 bp shorter than that of the wild type. Positive clones, subsequently analyzed, lacked specific activity of succinyl-CoA synthetase (<0.1 mU mg−1), whereas the enzyme was active in the parent strain C. glutamicum BS87 (86.2±12.9 mU mg−1).







TABLE 1





Forward (F) and Reverse (R) Primer Sequences
















scs-1: CCGCTCGAGGAAATGGAATTGGCAGTGGA
(F)


 


scs-2: AGTAATAATCACGCACAGTGTGTCCTCATCAATACCAGTG 
(R)


 


scs-3: CACTGGTATTGAGGACACACTGTGCGTGATTATTACT
(F)


 


scs-4: CTAGTCTAGAGTTACAAAGCTGCAACTACC
(R)





Primer pairs used for construction and verification of the deletion of sucCD (cgl2565, cgl2566) encoding subunits A and B of succinyl-CoA synthetase from the genome of C. glutamicum (scs-1 (SEQ ID No. 55), scs-2 (SEQ ID No. 56), scs-3 (SEQ ID No. 57), scs.4 (SEQ ID No. 58))






Growth and production characteristic: To investigate the impact of the sucCD deletion on production characteristics, growth, lysine production and biomass formation of the different mutants was compared (Table 2). As response to deletion of succinyl-CoA synthetase, the lysine yield on glucose was increased by 60%. The deletion mutant (μ=0.27 h−1) grew almost as efficient as the parent strain (0.34 h−1). Moreover, it did not exhibit significantly changed secretion of byproducts, indicating respiratory metabolism and obviously sustained cyclic operation of the TCA cycle. In general by-product formation of both strains was rather small. Only low levels of trehalose, lactate, alpha-ketoglutarate and glutamate, each below 5 mmol mol−1, were observed in the culture supernatant. The yield for lysine and biomass as well as the specific growth rate was constant throughout the whole cultivation showing that the strains were in metabolic steady state (FIG. 3). The observed differences are therefore clearly attributed to the genetic modification.







TABLE 2







Production characteristics of lysine-producing C. glutamicum


BS87 and C. glutamicum BS87 delta-sucCD on glucose. The data given


are lysine yield (YLys/S) and biomass yield (YX/S) and represent


mean values from three parallel cultivation experiments and


corresponding deviations. The yields were determined as slope


of the linear best fit when plotting product formation against


substrate consumption (FIG. 2).











Strain
YLys/S [mmol/mol]
YX/S [g/mol]







BS87
124 ± 3
72.5 ± 0.8



BS87 delta-sucCD
195 ± 8
68.3 ± 1.5










In vivo fluxes: Obviously the deletion of succinyl-CoA synthetase strongly enhanced lysine formation. To study the impact of the modified TCA cycle on the metabolic pathways in more detail, we estimated the fluxes of succinyl-CoA metabolism in the two strains. Hereby, alpha-ketoglutarate dehydrogenase was considered as reaction supplying succinyl-CoA. The flux through the lysine pathway was taken as upper boundary for the succinyl-CoA demand. In the parent strain the supply of succinyl-CoA was substantially higher than the maximum demand for lysine biosynthesis, so that probably a high flux was channeled through succinyl-CoA synthetase for energy generation (FIG. 4). In the deletion strain, the flux difference strongly decreased, indicating a certain coupling between the two pathways. The flux coupling between TCA cycle (54%) and lysine pathway (19%) in the deletion mutant was, however, incomplete, even when assuming that only the succinylase branch contributed to lysine production. This indicates further, so far unknown, by-passing or back-up mechanisms in C. glutamicum, which remain as additional targets for strain improvement. The performed t-test clearly revealed a significant difference between the strains considering the succinyl-CoA production flux (t=−24.3) and the succinyl-CoA consumption flux (t=85.3), respectively.
Overall, rational strain optimization was achieved by TCA cycle engineering. By deletion of sucCD, encoding the two subunits of succinyl-CoA synthetase, succinyl-CoA could be directed towards synthesis of lysine via its succinylase branch, which resulted in a 60% increased lysine yield. A complete shut-off of the TCA cycle, suggested from metabolic simulations, can however, hardly be realized, since this does not support biomass formation (Becker, J., C. Klopprogge, H. Schroder, and C. Wittmann. 2009. Tricarboxylic acid cycle engineering for improved lysine production in Corynebacterium glutamicum. Appl Environ Microbiol 75:7866-7869). In this regard the present study displays an important contribution since it maintained the TCA cycle flux at a well balanced level. The high viability and insignificant by-product formation of the deletion strain are clear indications that the cyclic operation of the TCA cycle was sustained, which is partly due to the increased lysine flux. TCA cycle engineering nicely complements with other successful targets in C. glutamicum focusing on the biosynthetic pathway of lysine (Eggeling, L., S. Oberle, and H. Sahm. 1998. Improved L-lysine yield with Corynebacterium glutamicum: use of dapA resulting in increased flux combined with growth limitation. Appl Microbiol Biotechnol 49:24-30 and Kalinowski, J., J. Cremer, B. Bachmann, L. Eggeling, H. Sahm, and A. Pithier. 1991. Genetic and biochemical analysis of the aspartokinase from Corynebacterium glutamicum. Mol Microbiol 5:1197-204.), the complex network around the pyruvate node (Peters-Wendisch, P. G., B. Schiel, V. F. Wendisch, E. Katsoulidis, B. Möckel, H. Sahm, and B. J. Eikmanns. 2001. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mol Microbiol Biotechnol 3:295-300 and Petersen, S., C. Mack, A. A. de Graaf, C. Riedel, B. J. Eikmanns, and H. Sahm. 2001. Metabolic consequences of altered phosphoenolpyruvate carboxykinase activity in Corynebacterium glutamicum reveal anaplerotic regulation mechanisms in vivo. Metab Eng 3:344-61.) or the PPP, supplying reducing power in form of NADPH (Becker, J., E. Heinzle, C. Klopprogge, O. Zelder, and C. Wittmann. 2005. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol 71:8587-8596 and Becker, J., C. Klopprogge, A. Herold, O. Zelder, C. J. Bolten, and C. Wittmann. 2007. Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum-over expression and modification of G6P dehydrogenase. J Biotechnol 132:99-109 and Ohnishi, J., R. Katahira, S. Mitsuhashi, S. Kakita, and M. Ikeda. 2005. A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum. FEMS Microbiol Lett 242:265-74.).
The gene sequence and the polypeptide sequence of sucC of Corynebacterium glutamicum ATCC 13032 is disclosed below:









sucC gene sequence:



GTGGATCTTTTTGAATACCAAGCACGGGACCTCTTTGAAAC



 



CCATGGTGTGCCAGTGTTGAAGGGAATTGTGGCATCAACAC



 



CAGAGGCGGCGAGGAAAGCGGCTGAGGAAATCGGCGGACTG



 



ACCGTCGTCAAGGCTCAGGTCAAGGTGGGCGGACGTGGCTG



 



CGGATGCGATTCTCGGCATGGATATCAAAGGACACACTGTT



 



AATCAGGTGATGGTGGCGCAGGGCGCTGACATTGCTGAGGA



 



ATACTATTTCTCCATTTTGTTGGATCGCGCGAATCGTTCGT



 



ATCTGGCTATGTGCTCTGTTGAAGGTGGCATGGAGATCGAG



 



ATCCTGGCGAAGGAAAAGCCTGAAGCTTTGGCAAAGGTGGA



 



AGTGGATCCCCTCACTGGTATTGATGAGGACAAAGCGCGGG



 



AGATTGTCACTGCTGCTGGCTTTGAAACTGAGGTGGCAGAG



 



AAAGTCATTCCGGTGCTGATCAAGATCTGGCAGGTGTATTA



 



CGAAGAGGAAGCAACACTCGTTGAGGTGAACCCGTTGGTGC



 



TCACGGATGACGGCGATGTGATTGCGCTTGATGGCAAGATC



 



ACGCTGGATGATAACGCTGATTTCCGCCATGATAACCGTGG



 



TGCGTTGGCTGAATCTGCCGGTGGCTTGGACATTTTGGAAC



 



TGAAGGCCAAGAAGAATGATCTGAACTACGTGAAACTTGAT



 



GGCTCTGTGGGCATCATTGGCAATGGTGCAGGTTTGGTGAT



 



GTCCACGTTGGATATCGTGGCTGCAGCTGGTGAACGCCATG



 



GTGGGCAGCGCCCCGCGAACTTCCTAGACATTGGTGGCGGA



 



GCATCAGCTGAATCGATGGCTGCTGGTCTCGATGTGATCCT



 



TGGGGATAGCCAGGTACGCAGTGTGTTTGTGAATGTGTTTG



 



CGTGGCATCACGCGTGTGATGTGGTGGCAAAGGGAATCGTT



 



GGAGCTTTGGATGTGCTCGGCGATCAAGCAACGAAGCCTCT



 



TGTGGTGCGCCTTGATGGCAACAACGTGGTGGAAGGCAGAC



 



GAATCCTCGCGGAATATAACCACCCTTTGGTCACCGTTGTG



 



GAGGGTATGGATGCAGCGGCTGATCACGCTGCCCATTTGGC



 



CAATCTTGCCCAGCACGGCCAGTTCGCAACCGCTAATTAG



 



sucC protein sequence:



MDLFEYQARDLFETHGVPVLKGIVASTPEAARKAAEEIGGL



 



TVVKAQVKVGGRGKAGGVRVAPTSAQAFDAADAILGMDIKG



 



HTVNQVMVAQGADIAEEYYFSILLDRANRSYLAMCSVEGGM



 



EIEILAKEKPEALAKVEVDPLTGIDEDKAREIVTAAGFETE



 



VAEKVIPVLIKIWQVYYEEEATLVEVNPLVLTDDGDVIALD



 



GKITLDDNADFRHDNRGALAESAGGLDILELKAKKNDLNYV



 



KLDGSVGIIGNGAGLVMSTLDIVAAAGERHGGQRPANFLDI



 



GGGASAESMAAGLDVILGDSQVRSVFVNVFGGITACDVVAK



 



GIVGALDVLGDQATKPLVVRLDGNNVVEGRRILAEYNHPLV



 



TVVEGMDAAADHAAHLANLAQHGQFATAN






The gene sequence and the polypeptide sequence of sucD of Corynebacterium glutamicum ATCC 13032 is disclosed below:









sucD gene sequence:



ATGTCTATTTTTCTCAATTCAGATTCCCGCATCATCATTCAG



 



GGCATTACCGGTTCGGAAGGTTCAGAGCATGCGCGTCGAAT



 



TTTAGCCTCTGGTGCGAAGCTCGTGGGTGGCACCAACCCCCG



 



CAAAGCTGGGCAAACCATTTTGATCAATGACACTGAGTTGCC



 



TGTATTTGGCACTGTTAAGGAAGCAATGGAGGAAACGGGTGC



 



GGATGTCACCGTAATTTTCGTTCCTCCAGCCTTTGCCAAAGC



 



TGCGATCATTGAAGCTATCGACGCTCACATCCCACTGTGCGT



 



GATTATTACTGAGGGCATCCCAGTGCGTGACGCTTCTGAGG



 



CGTGGGCTTATGCCAAGAAGGTGGGACACACCCGCATCATTG



 



GCCCTAACTGCCCAGGCATTATTACTCCCGGCGAATCTCTTG



 



CGGGAATTACGCCGGCAAACATTGCAGGTTCCGGCCCGATCG



 



GGTTGATCTCAAAGTCGGGAACACTGACTTATCAGATGATGT



 



ACGAACTTTCAGATATTGGCATTTCTACGGCGATTGGTATTG



 



GCGGTGACCCAATCATCGGTACAACCCATATCGACGCTCTGG



 



AGGCCTTTGAAGCTGATCCTGAGACCAAGGCAATCGTCATGA



 



TCGGTGAGATCGGTGGAGATGCAGAGGAACGCGCTGCTGACT



 



TCATTTCTAAGCACGTGACAAAACCAGTTGTGGGTTACGTGG



 



CAGGCTTTACCGCCCCTGAAGGAAAGACCATGGGGCATGCTG



 



GCGCCATCGTGACAGGTTCAGAAGGCACTGCGCGAGCAAAGA



 



AGCATGCATTGGAGGCCGTGGGTGTTCGCGTGGGAACAACTC



 



CGAGTGAAACCGCGAAGCTTATGCGTGAGGTAGTTGCAGCTT 



 



TGTAA 



 



sucD protein sequence:



MSIFLNSDSRIIIQGITGSEGSEHARRILASGAKLVGGTNPR 



 



KAGQTILINDTELPVFGTVKEAMEETGADVTVIFVPPAFAKA



 



AIIEAIDAHIPLCVIITEGIPVRDASEAWAYAKKVGHTRII



 



GPNCPGIITPGESLAGITPANIAGSGPIGLISKSGTLTYQMM



 



YELSDIGISTAIGIGGDPIIGTTHIDALEAFEADPETKAIVM



 



IGEIGGDAEERAADFISKHVTKPVVGYVAGFTAPEGKTMGHA 



 



GAIVTGSEGTARAKKHALEAVGVRVGTTPSETAKLMREVVAAL

## Claims

1. A microorganism comprising:
a) a reduced expression of a genomic gene coding for a succinyl-CoA synthetase, due to a reduced expression of one or more succinyl-CoA synthetase polypeptides comprising an amino acid sequence being at least 95% identical to SEQ ID NO: 51 and a reduced expression of one or more succinyl-CoA synthetase polypeptides comprising an amino acid sequence being at least 95% identical to SEQ ID NO: 53;
b) an enhanced cadaverine exporter activity being at least partially due to deregulation of one or more cadaverine exporter polypeptides comprising an amino acid sequence being at least 95% identical to SEQ ID NO: 1; and
c) an enhanced lysine decarboxylase activity due to expression of one or more lysine decarboxylase polypeptides comprising an amino acid sequence being at least 95% identical to SEQ ID NO: 3 or 4.

2. A microorganism as claimed in claim 1, having at least one deregulated gene selected from the group consisting of the genes of aspartokinase, aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, succinyl-amino-ketopimelate transaminase, succinyl-diamino-pimelate desuccinylase, diaminopimelate epimerase, diaminopimelate dehydrogenase, arginyl-tRNA synthetase, diaminopimelate decarboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase, homoserine dehydrogenase, phosphoenolpyruvate carboxykinase, methylmalonyl-CoA mutase, and diamine acteyltransferase.

3. A microorganism as claimed in claim 1 having a deregulated lysine import activity.

4. A microorganism as claimed in claim 3 wherein the deregulated lysine import activity is due to an enhanced or a decreased lysine exporter activity or an enhanced or a decreased lysine permease activity or an enhanced or a decreased lysine/cadaverin antiporter activity or any combination thereof.

5. A microorganism as claimed in claim 3 wherein the deregulated lysine import activity is due to an enhanced or a decreased activity of at least one lysine exporter polypeptide comprising an amino acid sequence which has at least 95% identity to SEQ ID NO: 5.

6. A microorganism as claimed in claim 3 wherein the deregulated lysine import activity is due to an enhanced or a decreased lysine permease activity or due to an enhanced or a decreased lysine/cadaverine antiporter activity or any combination thereof.

7. A microorganism as claimed in claim 1, wherein the microorganism has a deregulated N-acetylcadaverine-forming activity.

8. A microorganism as claimed in claim 7, wherein the microorganism has no or a decreased N-acetylcadaverine-forming activity.

9. A microorganism as claimed in claim 7, wherein the microorganism has an enhanced N-acetylcadaverine-forming activity and a decreased cadaverine exporter activity.

10. A microorganism as claimed in claim 7, wherein the N-acetylcadaverine-forming activity is deregulated by deregulating the activity of an N-acetylcadaverine-forming polypeptide comprising an amino acid sequence, being at least 95% identical to SEQ ID NO: 13.

11. A microorganism as claimed in claim 1, wherein the microorganism belongs to the Glade Eubacteria.

12. A microorganism as claimed in claim 1, wherein the microorganism is Corynebacterium glutamicum.

13. A process for the production of cadaverine or N-Acetylcadaverine, comprising fermenting a microorganism as claimed in claim 1.

14. A process as claimed in claim 13, comprising recovering cadaverine or N-Acetylcadaverine from the culture medium.

15. A microorganism as claimed in claim 3 wherein the deregulated lysine import activity is due to an enhanced or a decreased activity of at least one lysine exporter polypeptide comprising an amino acid sequence which has at least 98% identity to SEQ ID NO: 5.