WO2004113373A1 - Surexpression du transporteur cyddc - Google Patents

Surexpression du transporteur cyddc Download PDF

Info

Publication number
WO2004113373A1
WO2004113373A1 PCT/GB2004/002679 GB2004002679W WO2004113373A1 WO 2004113373 A1 WO2004113373 A1 WO 2004113373A1 GB 2004002679 W GB2004002679 W GB 2004002679W WO 2004113373 A1 WO2004113373 A1 WO 2004113373A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cysteine
fold
nucleic acid
cydd
Prior art date
Application number
PCT/GB2004/002679
Other languages
English (en)
Inventor
Robert Poole
Marc Pittman
Original Assignee
University Of Sheffield
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0314561A external-priority patent/GB0314561D0/en
Application filed by University Of Sheffield filed Critical University Of Sheffield
Publication of WO2004113373A1 publication Critical patent/WO2004113373A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)

Definitions

  • the mvention relates to a cell culture system for the enhanced production of amino acids, peptides and: polypeptides by bacterial cells and including genetically modified cells for use in said system.
  • Bacterial expression systems for the production of molecules, in particular amino acids, peptides and polypeptides, are well known in the art.
  • bacterial host cells are transformed with a vector (e.g. plasmid, phagemid) that is provided with expression signals (e.g. promoter sequences) that are operably linked to a nucleic acid molecule that encodes a polypeptide sequence the expression of which is desired.
  • vectors are also provided with replication origins that facilitate the replication of the vector inside the host bacterium.
  • the large scale production of recombinant proteins requires a high standard of quality control since many of these proteins are used as pharmaceuticals, for example: growth hormone; leptin; erythropoietin; prolactin; TNF, interleukins (IL), IL-2, IL-3, IL-4, IL-5, IL-6, ⁇ L-7, IL-9, IL-IO, ⁇ L-11; the p35 subunit of IL-12, IL-13, IL-15; granulocyte colony stimulating factor (G-CSF); granulocyte macrophage colony stimulating factor (GM-CSF); ciliary neurotrophic factor (CNTF); cardiotrophin-1 (CT-1); leukemia inhibitory factor (LIF); oncostatin M (OSM); interferon, IFN ⁇ , TFN ⁇ , and extracellular receptor domains from any cell surface receptor.
  • growth hormone for example: growth hormone; leptin; erythropoietin; prolactin; TNF, interleukins
  • vaccines particularly subunit vaccines, (vaccines based on a defined antigen, for example gpl20 of HIV)
  • a defined antigen for example gpl20 of HIV
  • Bacterial expression systems are also used in the industrial production of amino acids, for example the sulphur containing amino acids cysteine and cystine and the tri-peptide anti-oxidant glutathione.
  • the use of bacterial expression systems is known for the industrial production of amino acids.
  • WO0127307 describes the deregulation of the cysB gene resulting in elevated levels of cysteine and cysteine derivatives as a consequence of over- expression of CysB.
  • US6, 027, 888 describes the bacterial production of di-sulphide containing polypeptides by co-expression of a eukaryotic foldase (e.g. protein disulphide isomerase) to facilitate recombinant protein production.
  • a eukaryotic foldase e.g. protein disulphide isomerase
  • JPl 1155571 discloses a bacterial strain genetically engineered to suppress the activity of a cysteine desulphydrase, which reduces cysteine degradation in the cell.
  • peptides are also expressed on an industrial scale.
  • WO02061106 describes the transformation of bacteria with a gene encoding serine acetyltransferase and its over-expression. These transformed cells show enhanced production of cysteine, cystine and the tri-peptide glutathione (L glutamate: L cysteine: glycine).
  • Glutathione is an anti-oxidant and is used extensively in the food industry. It is thought to. have beneficial properties with respect to boosting the immune system by neutralising oxygen free radical activity in the body. GSH is usually depleted by oxidative stress which sometimes occurs during trauma, illness or infection. GSH is synthesised in two steps by the enzymes ⁇ -glutamylcysteine synthetase (encoded by gshA) and glutathione synthetase (gshB), and serves as a reductant in many cellular reactions. An important function of GSH is to reduce disulphide bonds, which form within cytoplasmic proteins when exposed to oxidative stress.
  • This phenomenon occurs as a result of exposure to elevated levels of reactive oxygen species such as superoxide (O 2 " ), hydrogen peroxide (H 2 O ), and alkyl hydroperoxides (ROOH) such as cumene hydroperoxides and t-butyl hydroperoxide.
  • reactive oxygen species such as superoxide (O 2 " ), hydrogen peroxide (H 2 O ), and alkyl hydroperoxides (ROOH) such as cumene hydroperoxides and t-butyl hydroperoxide.
  • GSH oxidised glutathione
  • Escherichia coli possesses two major membrane-bound terminal respiratory oxidases, namely cytochromes bo ' (" ⁇ o 3 " encoded by cyoABCDE) and bd, comprising two polypeptide subunits (encoded by cydA and cydB) and hemes bsss, bsps, and d (1-3). Both oxidases catalyse ubiquinol oxidation and oxygen reduction but differ in the efficiency with which electron transfer is coupled to proton translocation (2, 4), and the pattern of expression in response to environment (1, 2, 4). Significantly, cytochrome bd is required for resistance to a number of environmental stresses and its loss attenuates virulence in several bacteria (5, 6).
  • cytochrome bd Assembly of cytochrome bd is dependent not only on the structural genes cydAB, but also on the unlinked cydDC operon (7-9).
  • the latter genes are predicted to encode a heterodimeric ABC 1 -type transporter (traffic ATPase) (9) with a previously unknown export function (10, 11). Therefore, unlike traffic ATPases involved in uptake, CydDC is not thought to interact with a cognate periplasmic-binding protein.
  • CydDC exports thiol-containing compounds, for example and not by way of limitation, cysteine and glutathione, to the periplasm.
  • thiol-containing compounds for example and not by way of limitation, cysteine and glutathione
  • cysteine-transport hypothesis we show that cydD mutant cells have higher cytoplasmic levels of cysteine and are more susceptible to growth inhibition by external cysteine, whereas strains that over-express CydDC exhibit increased resistance to cytotoxic levels of cysteine.
  • GSH like L-cysteine, can restore defects associated with periplasmic stress in a cydD mutant. Moreover, a gshA mutant also displays phenotypes indicative of periplasmic redox stress. Assembly of cytochrome d was not restored in a cydD mutant by the exogenous addition of GSH.
  • a bacterial cell wherein said cell is genetically modified which modification is the transformation of said cell with a nucleic acid molecule wherein said nucleic acid molecule encodes a polypeptide with the specific enzyme activity associated with the CydDC transporter and further wherein said enzyme activity is overexpressed when compared to a non-transformed reference cell ofthe same species.
  • said nucleic acid molecule is selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented by Figure 13a and 13b; ii) a nucleic acid molecule which hybridises to the nucleic acid molecule in
  • nucleic acid molecule consisting of a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequence defined in (i) and (ii) above.
  • nucleic acid molecule hybridises under stringent hybridisation conditions to the sequence presented in Figure 13a and 13b.
  • nucleic acid hybrids are stable after washing in O.lx SSC, 0.1% SDS at 60°C. It is well known in the art that optimal hybridisation conditions can be calculated if the sequence of the nucleic acid is known.
  • hybridisation conditions can be determined by the GC content of the nucleic acid subject to hybridisation. .
  • a common formula for calculating the stringency conditions required to achieve hybridisation between nucleic acid molecules of a specified homology is:
  • hybridisation conditions uses 4 - 6 x SSPE (20x SSPE contains 175.3g NaCl, 88.2g NaH 2 PO 4 H 2 O and 7.4g EDTA dissolved to 1 litre and the pH adjusted to 7.4); 5- lOx Denhardt's solution (50x Denhardt's solution contains 5g Ficoll (type 400, Pharmacia), 5g polyvinylpyrrolidone and 5g bovine serum albumen; lOO ⁇ g-l.Omg/ml sonicated salmon/herring DNA; 0.1-1.0% sodium dodecyl sulphate; optionally 40-60% deionised formamide.
  • Hybridisation temperature will vary depending on the GC content ofthe nucleic acid target sequence but will typically be between 42°- 65° C.
  • nucleic acid molecule consists of the nucleic acid sequence presented in Figure 13a and 13b.
  • said cell over-expresses said enzyme activity by at least two-fold when compared to a non-transformed reference cell of the same species.
  • said enzyme activity is over-expressed at least 3-fold; 4-fold; 5-fold; 6-fold; 7-fold; 8-fold; 9-fold; or at least 10-fold.
  • said enzyme activity is over-expressed at least 20-fold; 30-fold; 40-fold; or at least 50-fold.
  • said enzyme activity is over-expressed by at least 100-fold.
  • the over-expression of enzyme activity can be achieved by means known to those skilled in the art. For example, placing the gene encoding an enzyme with the activity ofthe CydDC transporter on a high copy number plasmid. Alternatively, or in addition, said gene can be operably linked to a promoter sequence which provides for high level expression of said gene, said promoter can be constitutively active or inducible. Adaptations also include the provision of selectable markers, which select for cells containing high copy plasmids. These adaptations are well known in the art. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general.
  • said enzyme over-expression is provided by a variant gene which has the activity ofthe CydDC transporter wherein said activity is enhanced when compared to an unmodified reference gene as represented by the amino acid sequence in Figure 14a or 14b.
  • said bacterium is transformed with a gene which encodes a variant polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue and wherein said variant polypeptide has the activity associated with the CydDC transporter; preferably said activity is enhanced.
  • a variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination.
  • substitutions are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics.
  • amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or enhance the same biological function and activity as the reference polypeptide from which it varies.
  • a functionally equivalent polypeptide is a variant wherein one in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues, or one in which one or more amino acid residues includes a substituent group.
  • Conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Tfrr; exchange of the acidic residues Asp and Glu; substitution between amide residues Asn and Gin; exchange ofthe basic residues Lys and Arg; and replacements among aromatic residues Phe and Tyr.
  • the invention features polypeptide sequences having at least 75% identity with the polypeptide sequences as herein disclosed, or fragments and functionally equivalent polypeptides thereof.
  • the polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the amino acid sequences illustrated herein.
  • said bacterial cell is a Gram negative bacterial cell, for example Escherichia coli.
  • said bacterial cell is a Gram positive bacterial cell, for example, a bacterium of the genus Bacillus spp. (e.g. B. subtilis; B. licheniformis; B. amyloliquefaciens).
  • a Gram positive bacterial cell for example, a bacterium of the genus Bacillus spp. (e.g. B. subtilis; B. licheniformis; B. amyloliquefaciens).
  • Gram positive and Gram negative bacteria differ in many respects from one another. A difference exists in the nature of their respective cell walls.
  • the biochemical composition of the B. subtilis cell wall is quite different from that of E. coli.
  • the cell walls of E. coli and B. subtilis contain a framework that is composed of peptidoglycan, a complex of polysaccharide chains covalently cross-linked by peptide chains. This forms a semi-rigid structure that confers physical protection to the cell since the bacteria have a high internal .osmotic pressure and can be exposed to variations in external osmolarity.
  • the peptidoglycan framework may represent as little as 50% ofthe cell wall complex and these bacteria are characterised by having a cell wall that is rich in accessory polymers such as teichoic acids.
  • Gram negative bacteria do not readily secrete polypeptides into the surrounding growth medium although Gram positive bacteria, in particular those of the genus Bacillus spp, do have cell transport mechanisms to secrete polypeptides, these secreted polypeptides can be endogenous polypeptides, (e.g. amylases) or recombinant heterologous polypeptides. However, both are known to have transport mechanisms that transport amino acids and small peptides into the surrounding growth medium.
  • Methods to transform bacteria are well known in the art and have been> established for many years. These include chemical methods (e.g. calcium permeabilisation) or physical permeabilisation (e.g. electro oration).
  • a cell culture vessel comprising a cell according to the invention and medium sufficient to support the growth of said cell.
  • said vessel is a fermentor.
  • a method for the manufacture of at least one molecule comprising the steps: i) providing a vessel comprising a cell according to the invention; ii) providing cell culture conditions which facilitate the growth of a cell culture contained in said vessel; and optionally iii) isolating said molecule from said cell or said cells surrounding growth medium.
  • said molecule is a protein.
  • a protein that contains a sulphur-containing amino acid is preferable.
  • Recombinant protein production relates to the synthesis of protein in expression systems.
  • Heterologous polypeptides include commercially important polypeptides, for example enzymes used in biocatalysis (e.g. restriction enzymes, enzymes used in industrial processing; e.g. amylases, proteases, nucleases, lipases) and therapeutic polypeptides.
  • enzymes used in biocatalysis e.g. restriction enzymes, enzymes used in industrial processing; e.g. amylases, proteases, nucleases, lipases
  • therapeutic polypeptides e.g. restriction enzymes, enzymes used in industrial processing; e.g. amylases, proteases, nucleases, lipases
  • the large scale production of recombinant proteins requires a high standard of quality control since many of these proteins are used as pharmaceuticals, for . example, interleukins, growth hormone, erythropoietin, and interferon.
  • vaccines particularly subunit vaccines, (vaccines based on a defined antigen, " for example gpl20 of HIV)
  • a defined antigen for example gpl20 of HIV
  • the ability to secrete polypeptides into growth medium offers an opportunity- to purify polypeptides without the need for extraction from a host cell expressing said polypeptide.
  • said polypeptide is a peptide, preferably a peptide that includes a sulphur-containing amino acid.
  • said peptide is glutathione, or a structural variant of glutathione.
  • said molecule is an amino acid, preferably a sulphur-containing amino acid.
  • amino acid is cysteine or cystine.
  • said cell is a Gram positive bacterium.
  • said cell is a Gram negative bacterium.
  • Figure 1 illustrates a two-dimensional polyacrylamide gel electrophoresis of polypeptides from periplasmic fractions of wild-type strain AN2342 (A) and cydD mutant AN2343 (B). Arrows identify polypeptides whose levels were found either to decrease (1, 2, 3, 4, 7, 8, 9, 10 in panel A) or to increase (5 and 6 in panel B) in the cydD mutant;
  • Figure 2 illustrates that exogenous cysteine partially restores swarming behaviour of E. coli cells.
  • Strains AN2342 (cydD + ) (white bars) and AN2343 (cydD) (black bars) were grown in liquid broth to stationary phase at 30 °C, then inoculated with sterile toothpicks onto LB (0.3% Difco agar) with or without cysteine and incubated at 30 °C for 8 h. Bars show mean values and standard deviations in three experiments;
  • Figure 3 illustrates that exogenous cysteine confers resistance to benzylpenicillin differentially in wild-type and cydD mutant strains.
  • Figure 4 illustrates that exogenous cysteine modulates cytochrome c levels.
  • Strains AN2342 (cydD + ) (white bars) and AN2343 (cydD) (black bars) were grown anaerobically in the presence of 0, 0.2 and 2 mM cysteine.
  • Periplasmic fractions were isolated and reduced minus oxidised spectra were recorded.
  • Total amounts of c-type cytochromes were calculated; values shown are means of three determinations with standard deviations;
  • Figure 5 illustrates that mutants, affected in cydD and orf299 are each hypersensitive to exogenous cysteine. Effects of cysteine on the growth of strains AN2342 (cydD + , orf299 + ) (A), AN2343 (cydD, orf299 + ) (B), RKP4611 (cydD + , orf299) (C) and R P4612 (cydD, orf299) (D). Cells were grown in LB broth at 37 °C in the presence of 0 mM (gray rhombus), 2 mM (black squares), and 5 mM (open triangles) cysteine. The experiment is typical of three sets of growth curves;
  • FIG. 6 illustrates that CydDC is an ATP-driven cysteine transporter in everted membrane vesicles.
  • the cells used were strain R P4611 (cydD + , or/299; white circles in A, B, and C) and RKP4612 (cydD, orf299; black circles in A, B, and C).
  • Panels A and B both show ATP-driven uptake for the wild-type strain only in the absence of inhibitors (A) or in the presence of 2 ⁇ M CCCP (B).
  • Panel C shows data for strain RKP4611 (white circles, no vanadate; white triangles, + vanadate) and RKP4612 (black circles) in the presence of 50 mM sodium orthovanadate.
  • the arrow represents the time point at which 10 mM ATP was added.
  • A the bars show standard deviations of three experiments.
  • B and C a result typical of three replicates is illustrated;
  • Figure 7 illustrates that strains AN2342 (wild type, black bars) and AN2343 (cydD, white bars) were grown to stationary phase in MOPS minimal media (pH 7.0) supplemented with 40 mM glucose at 30 °C. A 5 ⁇ l aliquot of each strain was plated onto LB (0.3% Difco agar) and incubated at 30 °C for up to 2 d. The diameter of the swarm was measured, and the average of three experiments was taken;
  • Figure 8 illustrates that strains AN2342 (wild type, black bars) and AN2343 (cydD, white bars) were grown to stationary phase in LB at 37 °C. The OD 600 of each strain was measured and equalised. Serial dilutions (1 x 10 " ) were made of each strain and 5 ⁇ l aliquots of each of the diluted cultures were drop-plated onto LB containing benzylpenicillin (20 ⁇ g ml "1 ) and/or GSH (2 mM). Bars show the mean of 10 replicates with standard deviations. Total colony counts are shown; note that in the presence of both penicillin and GSH, AN2343 (cydD) produced colonies of normal size in 16-18 h whilst the AN2342 (wild-type) produced 'pin-prick' colonies;
  • Figure 9 illustrates that strains Fragl (wild type) and MJF355 (gshA) were grown to stationary phase in MOPS minimal media (pH 7.0) supplemented with 40 mM glucose at 30 °C. A 5 ⁇ l aliquot of each strain was plated onto LB (0.3% Difco agar) and incubated at 30 °C for up to 2 d. The data represent the average of three individual experiments;
  • Figure 10 illustrates that strains Fragl (wild type, black bars) and MJF355 (gshA, white bars) were grown to stationary phase in LB at 37 °C. The OD 600 of each strain was measured and equalised. Serial dilutions (1 x 10 " ) were made of each strain and 5 ⁇ l aliquots of each of the diluted cultures were drop-plated onto LB containing benzylpenicillin (20 ⁇ g ml "1 ) and/or GSH (2 mM). Bars show the mean of 10 replicates with standard deviations. Total colony counts are shown; note that in the presence of both penicillin and GSH, MJF355 (gshA) produced colonies of normal size in 16-18 h whilst Fragl (wild-type) produced 'pin-prick' colonies;
  • Figure 11 illustrates that the everted membrane vesicles used were from strain AN2342 (wild type, black circles in A, B, and C) and AN2343 (cydD, black circles in A, B, and C).
  • Panels A and B both show ATP-driven uptake for the wild-type strain only in the absence of inhibitors (A) or in the presence of 2 ⁇ M CCCP (B).
  • Panel C shows data for strain AN2342 (black circles, no vanadate; white triangles, + vanadate) and AN2343 (white circles) in the presence of 50 mM sodium orthovanadate.
  • the arrow represents the time point at which 10 mM ATP was added; and
  • Figure 12 illustrates that transport of GSH (black circles) and GSSG (white circles) were each measured in everted membrane vesicles derived from strain AN2342 (wild type).
  • GSSG was obtained by extraction of DTT from [ 35 S]GSH followed by oxidation in air as described in Materials and Methods. Equal concentrations of both [ 35 S]GSH/GSSG and non-radio-labelled GSG/GSSG were used in each assay.
  • the arrow represents the addition of 10 mM ATP to initiate transport;
  • Figure 13a is the nucleotide sequence of cydD
  • Figure 13b is the nucleotide sequence of cydC
  • Figure 14a is the amino acid sequence of cydD;
  • Figure 14b is the amino acid sequence of cyd C.
  • E. coli strain AN2343 carrying the mutant cydDl allele and its isogenic wild-type parent strain AN2342 have been described before (8).
  • Strains RKP4611 and RKP4612 were constructed by PI transduction (16) of the or ⁇ 99: ⁇ a.n allele from strain MC4100 ⁇ 299 (24) into strains AN2342 and AN2343, respectively.
  • Strains RKP2634 and RKP2005 were obtained by transformation of the wild-type and cydD mutant strain, respectively, with plasmid pRP33 (9) that has the cydDC* operon cloned into vector pBR328.
  • L-Cysteine was added as a filter-sterilised 100 mM stock solution to media, giving the final concentrations in the text.
  • Aerated cultures were grown in Erlenmeyer flasks containing one fifth their volume by shaking (200 rpm) at 30 °C or 37 °C.
  • a ⁇ aerobically-grown cultures were obtained by filling growth vessels to the brim with LB (supplemented with 20 mM KNO 3 ) and incubating without shaking at 37 °C for 14 h. Where reduced glutathione was added to the media, the concentrations are described in the text.
  • Agar and other dehydrated media were from Difco and Oxoid. Other chemicals were from Sigma.
  • Aerated cultures were grown with shaking (200 rpm) at 30 °C or 37 °C.
  • Cells were grown to stationary phase in LB broth or MOPS minimal media (pH 7.0) at 30 °C, and 5 ⁇ l drops were spotted onto semi-solid LB medium (0.3% Difco agar). The cells were incubated at 30 °C for up to 3 d, and the diameter of the resultant swarm of growth was measured.
  • Periplasmic fractions were isolated using a modified procedure of Willis et al (20). In brief, 200 ml culture was conditioned for osmotic shock by the addition of 6 ml 1 M NaCl and 6 ml 1 M Tris-HCl buffer (pH 7.3). An equal volume of a 40% (w/v) sucrose solution containing 33 mM Tris-HCl (pH 7.3) and 2 mM EDTA was added, and incubated at room temperature for 20 min. Cells were harvested and to each pellet 6 ml ice cold water was added. After 45 s on ice, MgCl 2 was added to 1 mM and the cells kept on ice for 10 min.
  • the periplasmic fraction was obtained by centrifugation (10,000 x g for 5 min) at 4 °C to remove cell debris and stored at 4 °C until ready for use.
  • the cytoplasmic fraction for enzyme assays was produced from the pellet (spheroplasts), which was resuspended in a buffer (6 ml) that contained (final concentration) 20 % (w/v) sucrose, 200 mM Tris-HCl (pH 7.5), and 1 mM Na EDTA.
  • Assays of ⁇ -galactosidase and alkaline phosphatase activities were used to determine the purity of periplasmic and cytoplasmic fractions, ⁇ -galactosidase (18) and alkaline phosphatase (21-22) activities were measured at room temperature by monitoring at 420 nm the hydrolysis of o-nitrophenyl- ⁇ -D-galactopyranoside or 4-nitrophenyl phosphate, respectively.
  • Periplasmic samples were concentrated approximately 2-fold with a Centricon YM-3 centrifugal filter device (Amicon Bioseparations - Millipore Corporation) with a maximum volume of 2 ml and a molecular mass cut-off of 3,000 Da. A portion (2 ml) of each sample was spun (5,000 x g for 120 min) without the retentate vial. A further 2 ml of sample was centrifuged exactly as above, and samples were pooled.
  • a Centricon YM-3 centrifugal filter device Ananton Bioseparations - Millipore Corporation
  • Concentrated periplasm (approximately 0.2 mg protein) was included in 125 ⁇ l (total volume) of rehydration solution (8 M urea, 2 % (w/v) CHAPS, 0.5 % (v/v) JPG buffer pH 3-10 (non-linear) (Amersham Pharmacia Biotech), 0.28 % dithiothreitol, and a few grains of bromophenol blue) and applied to a 7 cm PG strip.
  • rehydration solution 8 M urea, 2 % (w/v) CHAPS, 0.5 % (v/v) JPG buffer pH 3-10 (non-linear) (Amersham Pharmacia Biotech), 0.28 % dithiothreitol, and a few grains of bromophenol blue
  • 2D gel electrophoresis was carried out using a Multiphor II horizontal unit with immobilised pH gradients (pre-cast JPG strip, pH 3-10, non- linear) in the first dimension and a sodium dodecyl sulfate (SDS)-polyacrylamide gel (8-18 %> polyacrylamide) in the second dimension, according to the manufacturer's instructions (Amersham Pharmacia Biotech). Gels were stained with Coomassie Blue.
  • SDS sodium dodecyl sulfate
  • Proteins were electroblotted onto ProBlott (Applied Biosystems) membranes at 400-500 mA for 1.5-2 h before staining with Coomassie Blue.
  • the N-terminal sequences ofthe protein spots were determined by sequential Edman degradation (23). Sequence identity was computed using the Colibri website (http://genohst.pasteiir.fr/colibri7) FASTA function (24). Further information on sequenced proteins was found on the SWISS- PROT website (http Awww.expasy.cbT) .
  • Cytochrome d was quantified in cells grown aerobically to stationary phase in 50 ml LB and harvested at 6000 x g for 15 min. Cells were washed with 100 mM K-phosphate buffer (pH 7.2) and used to record reduced minus oxidised difference spectra and CO + reduced minus reduced difference spectra at room temperature as before (8) except that a SDB4 dual wavelength scanning spectrophotometer (26) was used. An absorption coefficient ⁇ (622 minus 644 nm) of 12.6 mM "1 cm “1 (27) was used. For c-type cytochromes, periplasmic fractions were isolated as described above to minimise interference by other cytochromes with overlapping spectral features.
  • Everted vesicles were thawed slowly on ice and diluted to 1.0 mg protein ml "1 in 10 mM Tris-HCl (pH 8.0) containing 140 M choline chloride and 5 mM MgCl 2 . Vesicles were added to glass tubes containing buffer (pre-equilibrated at 30, °C) to a final volume of 200 ⁇ l, and were incubated at 30 °C for 15 min without shaking. To initiate [ 14 C]lactose transport, vesicles were energised for 15 min prior to lactose addition with 20 mM D-lactate.
  • Periplasmic fractions of wild-type and cydD mutant strains have different levels of periplasmic transport proteins
  • cydC mutant The periplasm of a cydC mutant is more oxidised than that of a wild-type strain (12).
  • the cydDC operon is adjacent to the trxB (thioredoxin reductase) gene (34) on the E. coli chromosome and Goldman et al. (12) suggested that TrxB might be a substrate for CydDC.
  • TrxB (and trxA) mutants do synthesise cytochromes c and bd (10, 15), ruling out TrxB as a substrate of CydDC.
  • the proteins, represented by spots I and 9 were identified as OppA (36) and AnsB (37), respectively, and were expressed at significantly higher levels in the periplasm of the wild-type than that ofthe mutant (Fig. 1).
  • a minor spot (number 8) was also OppA and may result from post-translational alteration or modification of lysine residues during electrophoresis (38).
  • Proteins OsmY (39) and HisJ (40) spots 5 and 6 respectively) were expressed at slightly more elevated levels in the cydD mutant periplasm compared to that of the wild-type (Fig 1).
  • the remaining five sequenced proteins (MalE, GlnH, ProX, HisJ, and DppA) were expressed at slightly higher levels in the wild-type compared to the cydD mutant periplasm and are the periplasmic binding-proteins of secondary type transport systems in E. coli (41 and references therein). Transport mechanisms for all of these proteins are already established, so it seems unlikely that they are substrates ofthe CydDC transporter.
  • a cydD mutant displays a cysteine-reversible defect in motility
  • Thiol compounds have been shown to correct lesions caused by mutations in dsbA and dsbB (46). These genes " encode thio oxidoreductases (47, 48) that, with DsbD, control the redox balance in the periplasm by maintaining the cysteine residues of periplasmic C-X-X-C motifs as disulfides. We therefore postulated that cysteine might reverse the lack of motility.
  • the cydD mutant was inoculated onto LB (0.3% agar) containing 2 mM L-cysteine and incubated at 30 °C for 8 h.
  • cysteine suppressed the antibacterial effect more markedly for the cydD mutant than for the wild-type.
  • the colony morphologies of the two strains when plated on cysteine with penicillin were markedly different: cysteine allowed growth of the cydD mutant to give normal colonies (1-2 mm diam) after overnight incubation of the plates, whereas colonies of the wild-type strain were extremely small ( ⁇ 0.5 mm) even after prolonged incubation.
  • the suppressing effect of cysteine on the inhibition by penicillin of growth of the cydD mutant was dose-dependent in the range 0.5 to 2 mM cysteine (not shown).
  • Exogenous cysteine modulates levels of c-type cytochromes
  • DsbD is an integral membrane protein and translocates electrons from the cytoplasm to the periplasm (54), thereby providing the reducing power to enable apo- cytochromes to ligate heme (55). Loss of DsbD results in a loss of c-type cytochromes
  • Wild-type and cydD strains were grown anaerobically in LB plus 20 mM KNO to elevate cytochrome c levels, without or with cysteine (0.2 and 2 mM) supplements.
  • Reduced minus oxidised difference spectra of the periplasm from the wild-type strain showed a Soret band at 423 nm, a ⁇ -band at 525 nm, and a - band at 552.5 nm (data not shown).
  • These signals are attributable to the NrfA/NrfB cytochrome c nitrite reductase, maxima for reduced NrfA being, for example, 420.5,
  • cysteine increased cytochrome c levels in a cydD mutant, we hypothesised that cysteine might also restore cytochrome bd.
  • CO difference spectra ofthe wild-type strain showed a band at 644 nm, corresponding to the carbonmonoxy form of cytochrome d, as described before (8, 9).
  • Cytochrome bd levels (approx. 0.05 nmol mg protein "1 ) were unaffected by the addition of exogenous cysteine (0.2 and 2 mM).
  • Spectra of the cydD mutant revealed no cytochrome d signal at 644 nm and cells grown in the presence of 0.2 and 2 mM cysteine also lacked cytochrome d.
  • This gene product is a putative member ofthe major facilitator superfamily (MFS) of transport proteins and its expression promotes cysteine and OAS excretion (17).
  • MFS major facilitator superfamily
  • the growth phenotypes of the orf299 and cydD orf299 strains were compared to those of wild-type and cydD mutant strains in the presence of 0, 2, and 5 mM cysteine (Fig. 5). Growth of the wild-type was not substantially affected by cysteine. At 5 mM cysteine, there was a slight lag in reaching the stationary phase but optical density was not significantly different after 8 h (Fig. 5A). Growth of the cydD mutant was slightly inhibited at 2 mM cysteine, as characterised by a slower growth rate and a reduction in optical density after 8 h. However, 5 mM cysteine was severely inhibitory and growth was arrested after 4 h (Fig. 5B).
  • the or/299 mutant displayed an extended lag phase and did not reach mid-exponential growth until 3-4 h after inoculation compared to 2 h for the wild-type and cydD strains.
  • Cysteine (5 mM) extended the lag phase by about 1 h.
  • the double mutant (or ⁇ 99 cydD) displayed growth that was highly sensitive to cysteine. At 2 mM, growth was arrested after 4 h, and at 5 mM, OD 600 was further reduced (Fig. 5D). Therefore a cydD mutant strain displays greater cysteine sensitivity than an orf299 mutant, but defects in both genes result in extreme sensitivity to cysteine. The data suggest that both gene products are involved in cysteine resistance.
  • Cysteine is transported by CydDC in an ATP-dependent manner
  • cysteine In view of the ability of cysteine to reverse some of the pleiotropic defects of a cydD mutant, particularly those associated with periplasm physiology, it was considered a candidate substrate of CydDC. To test this, we measured uptake of [ 35 S]cysteine by everted membrane vesicles ofthe orf299 and cydD or ⁇ 99 strains. Extensive studies (32, 58-60) have already demonstrated the efficacy of French pressure cell treatment or ultrasonication of Gram-negative bacteria in producing predominantly everted (inside- out) vesicles that actively take up solutes, particularly toxic metal or metalloid ions, that would be exported in vivo.
  • the lactose transporter LacY
  • LacY can support lactose transport in either right-sided or everted vesicles, provided a protonmotive force ( ⁇ p) of the appropriate polarity is applied.
  • ⁇ p protonmotive force
  • Everted vesicles prepared from cells grown in MOPS medium supplemented with glucose have a low rate of lactose transport (15). • Therefore, everted vesicles were prepared from the or ⁇ 99 strain grown with lactose.
  • CydDC transporter derives energy for transport directly from ATP was tested with transport inhibitors.
  • CCCP (2 ⁇ M) had no discernible effect on the uptake of [ 35 S] cysteine by vesicles of the or ⁇ 99 mutant (Fig. 6B), and the rate of [ 35 S]cysteine uptake was approximately 0.21 nmol (mg protein) "1 .
  • Glutathione complements defects associated with redox stress in the periplasm
  • AN2342 wild type
  • AN2343 cydD
  • MOPS minimal media supplemented with 40 mM glucose as described in Materials and Methods.
  • a 5 ⁇ l aliquot of each strain was plated onto semi-solid agar containing one of the following reduced GSH concentrations 0, 1, and 2 mM, and incubated at 30 °C for a period of 2-3 days.
  • the zone of swarming in AN2342 was approximately 58 mm after 2 days (Fig. 7).
  • the zone of swarming for AN2343 at 0 mM GSH was severely reduced compared to AN2342 and was only ' 5 mm from the site of inoculation (Fig. 7).
  • the zone of swarming of AN2342 is 50 mm and is therefore slightly reduced compared to growth when no GSH is present.
  • the zone of swarming in AN2343 in the presence of 1 mM GSH was approximately 27 mm.
  • the zone of swarming of AN2342 is further reduced and is approximately 42 mm. Under these growth conditions, the zone of swarming of AN2343 was approximately 34 mm.
  • the ability of AN2342 to swarm on semi-solid agar is inhibited by the concentrations of GSH tested, while that of AN2343 is greatly increased as the concentration of GSH increased.
  • Hypersensitivity to benzylpenicillin results when there are defects in disulfide bond formation in the penicillin-binding-protein-4 (PBP4).
  • PBP4 penicillin-binding-protein-4
  • 5 ⁇ l aliquots of 10 "6 serial dilutions of strains AN2342 (wild-type) and AN2343 (cydD) were challenged with benzylpenicillin (20 ⁇ g ml "1 ) in the presence or absence of 2 mM GSH as described in Materials and Methods. All plates were incubated at 37 °C for 14 h.
  • a gshA mutant displays phenotypes associated with redox stress
  • the gshA mutant was challenged with benzylpenicillin in the presence of glutathione to determine if the patterns of hyper-resistance in the presence of glutathione observed for cydD are also evident in a gshA mutant.
  • An identical set of conditions as those used to test sensitivity of AN2342 and AN2343 to benzylpenicillin were employed for Fragl (wild type) and MJF355 (gshA) as described in Materials and Methods. The plates were incubated at 37 °C for 14 h.
  • the gshA mutant showed a decrease in viability compared to that of the isogenic wild type strain (1.1 x 10 9 and 1.9 x 10 9 (ml culture) "1 respectively) (figure 4).
  • the viability ofthe gshA strain increased to almost the level ofthe wild type grown on LB alone (1.7 x 10 9 (ml culture) "1 (figure 4).
  • Fragl showed a slight decrease in viability when grown in the presence of 2 mM glutathione (1.75 x 10 9 (ml culture) "1 ) (figure 4).
  • Glutathione is transported by CydDC in an ATP-dependent manner
  • L-cysteine is a substrate of the CydDC transporter.
  • Uptake into everted membrane vesicles of AN2342 (wild type) was shown to be ATP- dependent and protonophore insensitive.
  • the ATP analogue sodium orthovanadate inhibited transport, as expected from an ATP-dependent transporter.
  • the ability of GSH to rescue phenotypes associated with periplasmic redox stress provides some evidence that it is also a substrate of CydDC.
  • uptake of [ 35 S]glutathione into everted membrane vesicles of AN2342 (wild type) and AN2343 (cydD) was measured.
  • Oxidised glutathione is not a substrate of CydDC
  • GSH periplasmic redox stress
  • periplasmic redox stress such as motility and resistance to benzylpenicillin
  • Dailey and Berg (62) demonstrated that motility defects in a dsbB mutant could be corrected by the exogenous addition of cystine. Therefore, it may be the oxidised form of glutathione that is transported by CydDC, and the [ 35 S]GSH shown to be taken up into everted membrane vesicles (Fig. 10) may in fact be GSSG that was formed as a result of GSH oxidation during the transport assay. Therefore transport assays to measure GSSG uptake by CydDC were performed.
  • the [ 35 S]GSH obtained from Amersham Pharmacia is dissolved in an aqueous solution containing 10 mM DTT, which maintains the GSH in a reduced state. Therefore, to obtain GSSG from GSH, the DTT was first removed by solvent extraction with ethyl acetate as described. Upon removal of the DTT, the GSH was oxidised by exposing to air for 24 h. To determine if oxidation was complete, a DTNB assay was performed as described in Materials and Methods. DTNB undergoes oxidation in the presence of sulfhydryl groups and is therefore useful in monitoring the redox state of glutathione.
  • a cold GSSG solution identical in concentration to that of the [ 35 S]GSH solution was used in the assay as a control to determine the extent of [ 35 S] glutathione oxidation.
  • the transport assay conditions to determine [ 35 S]GSSG uptake into everted membrane vesicles were identical to those employed for [ 35 S]GSH. No uptake of GSSG into everted membrane vesicles of AN2342 was observed, even after the addition of 10 mM ATP (Fig. 11). Conversely, uptake of [ 35 S]GSH was rapid after the addition of 10 mM ATP (Fig. 11). Therefore, CydDC can support uptake of [ 35 S]GSH but not [ 35 S]GSSG.
  • a gshA mutant assembles cytochrome d and exogenous GSH cannot restore a functional bd oxidase in a cydD mutant
  • a clear phenotype of a cydD mutant is the inability to produce a functional cytochrome bd.
  • GSH is a substrate of the CydDC transporter, and therefore it may be required in cytochrome bd assembly.
  • cytochrome bd assays were performed on strain MJF355 (gshA). It would be expected that this strain is deficient in cytochrome bd if indeed GSH is required for cytochrome d assembly.
  • Strains Fragl (wild-type) and MJF355 (gshA) were grown aerobically and difference absorbance spectra (CO + reduced minus reduced) were recorded on whole cell samples.
  • Spectra of the wild-type strain showed a band at 644 nm, corresponding to the carbonmonoxy form of cytochrome d.
  • the levels of cytochrome bd were recorded at approximately 0.05 nmol mg protein "1 .
  • Identical conditions were used to generate CO + reduced minus reduced spectra of the gshA.
  • Identical spectral signals to those obtained for Fragl were recorded, indicating the presence of cytochrome d.
  • the amount of cytochrome d was identical to that produced by Fragl (0.05 nmol mg protein " l ).
  • ⁇ " i culture were required to gain enough material to perform the cytochrome d assays.
  • the (CO + reduced) minus reduced difference spectra were carried out as described in Materials and Methods.
  • the spectra recorded for AN2342 (wild type) revealed a band at 644 nm, corresponding to the carbon onoxy form of cytochrome d, as described before.
  • the conditions under which the cells were grown ensured the production of cytochrome d.
  • Spectra of AN2343 (cydD) grown without the addition of GSH produced no band at 644 nm, indicative that cytochrome d is absent.
  • the spectra of AN2343 to which GSH had been added were identical to this. Therefore no cytochrome d was observed in any ofthe samples to which GSH had been added.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un système de culture cellulaire servant à augmenter la production d'acides aminés, de peptides et de polypeptides par des cellules bactériennes et mettant en application des cellules modifiées génétiquement.
PCT/GB2004/002679 2003-06-21 2004-06-21 Surexpression du transporteur cyddc WO2004113373A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0314561A GB0314561D0 (en) 2003-06-21 2003-06-21 Cell culture
GB0314561.2 2003-06-21
US48202003P 2003-06-25 2003-06-25
US60/482,020 2003-06-25

Publications (1)

Publication Number Publication Date
WO2004113373A1 true WO2004113373A1 (fr) 2004-12-29

Family

ID=33542689

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2004/002679 WO2004113373A1 (fr) 2003-06-21 2004-06-21 Surexpression du transporteur cyddc

Country Status (1)

Country Link
WO (1) WO2004113373A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008126784A1 (fr) * 2007-04-06 2008-10-23 Kyowa Hakko Bio Co., Ltd. Procédé de production de glutathion ou de ϝ-glutamylcystéine
CN102181500A (zh) * 2010-01-15 2011-09-14 味之素株式会社 使用肠杆菌科细菌生产l-氨基酸的方法
DE102011075656A1 (de) 2011-05-11 2012-03-29 Wacker Chemie Ag Verfahren zur fermentativen Produktion von L-Cystin
WO2013000864A1 (fr) 2011-06-30 2013-01-03 Wacker Chemie Ag Procédé de production par fermentation de l-cystéine naturelle
WO2013171098A2 (fr) 2012-05-18 2013-11-21 Wacker Chemie Ag Procédé de production de l-cystéine et de dérivés de cet acide aminé par fermentation
DE102012216527A1 (de) 2012-09-17 2014-03-20 Wacker Chemie Ag Verfahren zur fermentativen Produktion von L-Cystein und Derivaten dieser Aminosäure
WO2021259491A1 (fr) 2020-06-26 2021-12-30 Wacker Chemie Ag Souches améliorées produisant de la cystéine
WO2023280382A1 (fr) 2021-07-05 2023-01-12 Wacker Chemie Ag Procédé d'oxydation enzymatique d'acides sulfiniques en acides sulfoniques
WO2023165684A1 (fr) 2022-03-01 2023-09-07 Wacker Chemie Ag Souches améliorées produisant de la cystéine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027307A1 (fr) * 1999-10-14 2001-04-19 Consortium für elektrochemische Industrie GmbH Procedes pour produire de la l-cysteine ou des derives de cette derniere par fermentation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027307A1 (fr) * 1999-10-14 2001-04-19 Consortium für elektrochemische Industrie GmbH Procedes pour produire de la l-cysteine ou des derives de cette derniere par fermentation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COOK GREGORY M ET AL: "A novel haem compound accumulated in Escherichia coli overexpressing the cydDC operon, encoding an ABC-type transporter required for cytochrome assembly", ARCHIVES OF MICROBIOLOGY, vol. 178, no. 5, November 2002 (2002-11-01), pages 358 - 369, XP002300370, ISSN: 0302-8933 *
PITTMAN MARC S ET AL: "Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter required for cytochrome assembly.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 51, 20 December 2002 (2002-12-20), pages 49841 - 49849, XP002300371, ISSN: 0021-9258 *
POOLE ROBERT K ET AL: "Cytochrome bd biosynthesis in Escherichia coli: The sequence of the cydC and cydD genes suggest that they encode the components of an ABC membrane transporter", MOLECULAR MICROBIOLOGY, vol. 10, no. 2, 1993, pages 421 - 430, XP009037964, ISSN: 0950-382X *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6018357B2 (ja) * 2007-04-06 2016-11-02 協和発酵バイオ株式会社 グルタチオンおよびγ−グルタミルシステインの製造法
EP2489741A1 (fr) * 2007-04-06 2012-08-22 Kyowa Hakko Bio Co., Ltd. Procédé de production de glutathion ou de la gamma-glutamylcystéine
EP2143804A4 (fr) * 2007-04-06 2010-05-12 Kyowa Hakko Bio Co Ltd Procédé de production de glutathion ou de gamma-glutamylcystéine
CN101715490A (zh) * 2007-04-06 2010-05-26 协和发酵生化株式会社 谷胱甘肽及γ-谷氨酰半胱氨酸的制造方法
WO2008126784A1 (fr) * 2007-04-06 2008-10-23 Kyowa Hakko Bio Co., Ltd. Procédé de production de glutathion ou de ϝ-glutamylcystéine
US8647839B2 (en) 2007-04-06 2014-02-11 Kyowa Hakko Bio Co., Ltd. Method for production of glutathione or gamma-glutamylcysteine
EP2143804A1 (fr) * 2007-04-06 2010-01-13 Kyowa Hakko Bio Co., Ltd. Procédé de production de glutathion ou de gamma-glutamylcystéine
EP2345667A3 (fr) * 2010-01-15 2011-09-21 Ajinomoto Co., Inc. Procédé de production d'un acide L-aminé utilisant une bactérie de la famille des entérobactéries
CN102181500A (zh) * 2010-01-15 2011-09-14 味之素株式会社 使用肠杆菌科细菌生产l-氨基酸的方法
CN104046663B (zh) * 2010-01-15 2016-08-03 味之素株式会社 使用肠杆菌科细菌生产l-氨基酸的方法
EP2484690A1 (fr) * 2010-01-15 2012-08-08 Ajinomoto Co., Inc. Procédé de production d'un acide L-aminé utilisant une bactérie de la famille des entérobactéries
US8852897B2 (en) 2010-01-15 2014-10-07 Ajinomoto Co., Inc. Method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family
US8460903B2 (en) 2010-01-15 2013-06-11 Ajinomoto Co., Inc. Method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family
CN104046663A (zh) * 2010-01-15 2014-09-17 味之素株式会社 使用肠杆菌科细菌生产l-氨基酸的方法
US9074230B2 (en) 2011-05-11 2015-07-07 Wacker Chemie Ag Method for producing L-cystine by fermentation under controlled oxygen saturation
WO2012152664A1 (fr) 2011-05-11 2012-11-15 Wacker Chemie Ag Procédé pour la préparation fermentative de l-cystine à une saturation en oxygène contrôlée
DE102011075656A1 (de) 2011-05-11 2012-03-29 Wacker Chemie Ag Verfahren zur fermentativen Produktion von L-Cystin
US8802399B2 (en) 2011-06-30 2014-08-12 Wacker Chemie Ag Method for production of natural L-cysteine by fermentation
DE102011078481A1 (de) 2011-06-30 2013-01-03 Wacker Chemie Ag Verfahren zur fermentativen Produktion von natürlichem L-Cystein
WO2013000864A1 (fr) 2011-06-30 2013-01-03 Wacker Chemie Ag Procédé de production par fermentation de l-cystéine naturelle
DE102012208359A1 (de) 2012-05-18 2013-11-21 Wacker Chemie Ag Verfahren zur fermentativen Produktion von L-Cystein und Derivaten dieser Aminosäure
WO2013171098A2 (fr) 2012-05-18 2013-11-21 Wacker Chemie Ag Procédé de production de l-cystéine et de dérivés de cet acide aminé par fermentation
US9347078B2 (en) 2012-09-17 2016-05-24 Wacker Chemie Ag Method for the fermentative production of L-cysteine and derivatives of said amino acid
WO2014040955A1 (fr) 2012-09-17 2014-03-20 Wacker Chemie Ag Procédé de production fermentative de l-cystéine et de dérivés de cet acide aminé
DE102012216527A1 (de) 2012-09-17 2014-03-20 Wacker Chemie Ag Verfahren zur fermentativen Produktion von L-Cystein und Derivaten dieser Aminosäure
WO2021259491A1 (fr) 2020-06-26 2021-12-30 Wacker Chemie Ag Souches améliorées produisant de la cystéine
WO2023280382A1 (fr) 2021-07-05 2023-01-12 Wacker Chemie Ag Procédé d'oxydation enzymatique d'acides sulfiniques en acides sulfoniques
WO2023165684A1 (fr) 2022-03-01 2023-09-07 Wacker Chemie Ag Souches améliorées produisant de la cystéine

Similar Documents

Publication Publication Date Title
US7410788B2 (en) Compositions and methods for production of disulfide bond containing proteins in host cells
Pittman et al. Cysteine Is Exported from the Escherichia coliCytoplasm by CydDC, an ATP-binding Cassette-type Transporter Required for Cytochrome Assembly
US5508192A (en) Bacterial host strains for producing proteolytically sensitive polypeptides
Santini et al. A novel Sec‐independent periplasmic protein translocation pathway in Escherichia coli
US5264365A (en) Protease-deficient bacterial strains for production of proteolytically sensitive polypeptides
Kurokawa et al. Overproduction of bacterial protein disulfide isomerase (DsbC) and its modulator (DsbD) markedly enhances periplasmic production of human nerve growth factor in Escherichia coli
Prinz et al. Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.
Chen et al. Construction of leaky strains and extracellular production of exogenous proteins in recombinant E scherichia coli
Ray et al. Expression and purification of soluble bio-active rice plant catalase-A from recombinant Escherichia coli
WO2004113373A1 (fr) Surexpression du transporteur cyddc
EP2271759B1 (fr) Cellules hôtes et procédés de production de protéines contenant des liaisons disulfure
US20230002722A1 (en) E. coli strains having an oxidative cytoplasm
US7816117B2 (en) Prokaryotic host cells for expressing proteins rich in disulfide bonds
Roszczenko et al. Helicobacter pylori HP0377, a member of the Dsb family, is an untypical multifunctional CcmG that cooperates with dimeric thioldisulfide oxidase HP0231
Scott et al. Characterization of the Lactococcus lactis transcription factor FlpA and demonstration of an in vitro switch
Yang et al. Pdl1 is a putative lipase that enhances Photorhabdus toxin complex secretion
Parrilli et al. Heterologous protein expression in Pseudoalteromonas haloplanktis TAC125
Schlarb et al. Expression of plastocyanin and cytochrome f of the cyanobacterium Phormidium laminosum in Escherichia coli and Paracoccus denitrificans and the role of leader peptides
Richard-Fogal et al. Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III
JP2006174833A (ja) 細胞の成長を促進する方法及びターゲット遺伝子の発現による遺伝子産物の生産量を増加する方法
EP1240338B1 (fr) Production de proteines heterologues
Shields et al. Peptides encoded in the Streptococcus mutans RcrRPQ operon are essential for thermotolerance
US7829687B2 (en) Artificial disulfide isomerases and uses thereof
US20220064227A1 (en) Recombinant strains and medium formulation for enhancing secretion titer using a type iii secretion system
Joos et al. Synthesis of a novel papain-like cysteine peptidase for the proteolytic cleavage of plantaricin 423 from a GFP fusion-based expression system

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase