WO2011083059A1 - Bactéries mutantes et leurs emplois dans la production de protéines - Google Patents

Bactéries mutantes et leurs emplois dans la production de protéines Download PDF

Info

Publication number
WO2011083059A1
WO2011083059A1 PCT/EP2010/070683 EP2010070683W WO2011083059A1 WO 2011083059 A1 WO2011083059 A1 WO 2011083059A1 EP 2010070683 W EP2010070683 W EP 2010070683W WO 2011083059 A1 WO2011083059 A1 WO 2011083059A1
Authority
WO
WIPO (PCT)
Prior art keywords
escherichia coli
coli
mutated
arca
iclr
Prior art date
Application number
PCT/EP2010/070683
Other languages
English (en)
Inventor
Joeri Beauprez
Original Assignee
Universiteit Gent
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
Application filed by Universiteit Gent filed Critical Universiteit Gent
Publication of WO2011083059A1 publication Critical patent/WO2011083059A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for obtaining increased product yields and/or for eliminating by-product formation in micro-organisms, and to mutants and/or transformants for use in said methods. More particularly, it relates to bacterial mutants and/or transformants that are affected in the primary metabolism for enhanced product formation, for enhanced product yield and/or for eliminating production of by-products, especially mutants and/or transformants that are affected in the repression of the glyoxylate bypass and in the chromosomal gene expression in response to deprivation of oxygen or in response to absence/presence of carbon compounds such as glycerol, fatty acids, glyoxylate, acetate and the like.
  • biotechnology uses living cells and enzymes to synthesize a wide range of products such as bulk and fine chemicals, food ingredients, pharmaceutical ingredients, bio-fuels, bio-plastics, etc. from renewable resources, using a wide range of production hosts varying from bacteria (e.g. Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas sp ), yeasts (e.g. Saccharomyces cerevisiae, Pichia pastoris) to fungi (e.g Penicillium chrysogenus).
  • bacteria e.g. Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas sp
  • yeasts e.g. Saccharomyces cerevisiae, Pichia pastoris
  • fungi e.g Penicillium chrysogenus
  • Cultivated Escherichia coli strains e.g. E. coli K12
  • E. coli K-12 are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine.
  • Well- known examples of the E. coli K-12 strains are K-12 Wild type, W3110, MG1655, M182, MC 1000, MC 1060, MC 1061 , MC4100, JM 101 , NZN 1 11 and AA200.
  • Table 1 gives an overview of microbially derived peptides and proteins for therapeutic use [3]. 29.8% of protein-based recombinant pharmaceuticals licensed up to January 2009 by the FDA and EMEA are obtained in Escherichia coli [4].
  • the state of the art strategies to reduce acetate formation anticipate on these two mechanisms by modifying the bioprocess conditions or the genotype of the production host.
  • the former strategies comprise the limitation of the glucose uptake rate by applying specific glucose feeding patterns, the application of alternative substrates, the addition of supplements to the medium, the control of a range of fermentation parameters and the application of systems to remove acetate from the fermentation broth [7-15].
  • the latter strategies are based on the alteration of the central metabolism of E. coli. These strategies mainly target the glucose uptake mechanism, the blocking the acetate pathway, the pyruvate branch point (starting point to direct the carbon flow to acetate) [7, 12, 13, 16-26].
  • Known regulators of the central metabolism of E. coli are: IclR, ArcA, CRP, Cra, IHF.
  • different regulators will affect transcription e.g. ArcA regulates the aerobic/anaerobic metabolism, and CRP and Cra regulate carbohydrate metabolism.
  • ArcA regulates the aerobic/anaerobic metabolism
  • CRP and Cra regulate carbohydrate metabolism.
  • the aerobic respiration control protein ArcA is considered to be active under anaerobic conditions although its activity varies with the fermentation type (batch vs. chemostat). These differences occur in a similar way for crp knock out mutants. In batch cultures this knock out seems to have minor effects on the fluxes and transcription; in chemostat conditions CRP is described as the most dominant regulator [32, 33].
  • the third regulator isocitrate lyase regulator IclR, shows more activity in glucose abundant environments in comparison with glucose deprived environments because it has to bind either to pyruvate or PEP to be active and intracellular metabolite concentrations in chemostat cultures are very low [34, 35].
  • Cra is a dual transcriptional regulator that plays an important role in the regulation of E. colVs carbohydrate metabolism [12]. It is considered to be the major transcription inducer of the aceBAK operon [13, 14].
  • the activity of this transcriptional regulator is modulated by fructose- 1- phosphate and fructose- 1 ,6-bisphosphate in micromolar and millimolar concentrations, respectively [36, 37].
  • the decreased activity of Cra in a batch culture may be the effect of an increased intracellular fructose- 1 -phosphate and fructose- 1 ,6-bisphosphate concentration, which are generally low in a steady state culture.
  • An interesting observation in this regulatory network is the antagonistic effect of CRP and Cra on the glyoxylate route in glucose limited cultures. While Cra activates the route, CRP represses it. Furthermore Cra affects also isocitrate dehydrogenase expression.
  • IHF or the Integration Host Factor has two different modes of operation. It induces gene expression when acetate is the primary carbon source, gene repression occurs under glucose abundant conditions. For gene induction, IHF interferes with IclR binding, which seems to be still active in the presence of acetate. An IHF knock out reduces glyoxylate route activity, an effect that disappears when iclR is knocked out as well. Under glucose abundant conditions (the gene repression mode of IHF) IHF does not show any effect on the operon. However, an iclR knock out still increases glyoxylate pathway activity [16].
  • IHF is probably absent in repressing conditions or lacks activity.
  • the intracellular IHF concentration in exponentially growing cells with glucose as carbon sources is quite low, while the concentration increases dramatically when the cells reach a stationary phase metabolism [17].
  • this phase the cell prepares for growth on alternative substrates, which might be formed during the initial exponential phase.
  • this is mainly acetate.
  • the increase in IHF might thus play a role in the diauxic growth pattern of E. coli by activating the glyoxylate shunt.
  • An overexpression of ihfA and ihfB, encoding for the two subunits of IHF, in exponentially grown cells slightly affects synthesis of rpoH dependent heat shock proteins (Nystrom, 1995).
  • EP1382686 and US2007249018 disclose an arcA mutant to obtain increased biomass and/or product yields.
  • the present invention relates to the surprising finding that modification of the expression of both the genes encoding for the regulators ArcA and IclR in Escherichia coli leads to nearly theoretical biomass yield and hence elimination of co-product formation, and, more importantly, leads in a synergistic manner to an unexpectedly high and efficient production of proteins by fermentation.
  • FIG. 1 Construction of promoter delivery system for gene overexpression [21]
  • FIG. 3 Batch (A) and chemostat (B) product yields of the E. coli MG1655 (wild type), E. coli MG1655 AarcA (AarcA), E. coli MG1655 AiclR (AiclR), E. coli MG1655 AarcA-AiclR (AarcA- AiclR) and E. coli MG1655 AackA-pta, ApoxB, Apppc ppc-p37 (3KO ppc) mutant strains in c- mole per c-mole glucose.
  • the oxygen yield is represented as a positive number although it is actually consumed during the fermentation.
  • the values represented in this graph are the average of at least two separate fermentations, the error bars are the standard deviations calculated on the yields.
  • FIG. 4 Pedigree of GFP.
  • Figure 5 Strategy used to construct pCX-prom22-6His-GFPmut3b.
  • Figure 6 Sensitivity and reliability of fluorescence measurement for 5 different fixation solutions (GA (Glutaraldehyde), MeOH, acetone, formaline and PBS (phosphate buffer)) at 8 different excitation wavelengths.
  • GA Glutaraldehyde
  • MeOH MeOH
  • acetone acetone
  • formaline phosphate buffer
  • Figure7 Sensitivity of fluorescence measurement in function of time for 2 different fixation solutions (formaline and PBS) at 8 different excitation wavelengths.
  • Figure 8 Fluorescence in function of time (top) and fluorescence/OD 6 oo in function of OD 6 oo
  • Figure 9 Fluorescence per gram cell dry weight (g/1) of mutant strains compared to the wild type strain and the industrial strain BL21.
  • WT E. coli MG1655;
  • arcA-iclR E. coli MG1655 AarcA AiclR;
  • BL21 E. coli BL21(DE3);
  • PPC E. coli MG1655 AackA-pta, ApoxB, Apppc ppc-p37;
  • arcA E. coli MG1655 AarcA;
  • iclR E. coli MG1655 AiclR.
  • Arc A is an anaerobic respiration regulating protein
  • IclR is a glyoxylate shunt regulating protein
  • Ppc indicates the overexpression of PEP carboxylase with the acetate pathway knocked out
  • Figure 10 Fluorescence per cell of mutant strains compared to the wild type strain and the industrial strain BL21.
  • WT E. coli MG1655;
  • arcA-iclR E. coli MG1655 AarcA AiclR;
  • BL21 E. coli BL21(DE3);
  • PPC E. coli MG1655 AackA-pta, ApoxB, Apppc ppc-p37;
  • arcA E. coli MG1655 AarcA;
  • iclR E. coli MG1655 AiclR.
  • ArcA is an anaerobic respiration regulating protein
  • IclR is a glyoxylate shunt regulating protein
  • Ppc indicates the overexpression of PEP carboxylase with the acetate pathway knocked out. Description of invention
  • the present invention relates to the finding that modification of the expression of both the genes encoding for the regulators ArcA and IclR in Escherichia coli leads to nearly theoretical biomass yield and hence elimination of co-product formation. Moreover, modifying the expression of both of the latter genes results -in a synergistic manner- in an unexpectedly high and efficient production of (recombinant) proteins.
  • the gene products are not directly involved in the production of co-products.
  • Escherichia coli MG1655 wild-type, WT was chosen as reference to the constructed strains.
  • Escherichia coli MG1655 AackA-pta, ApoxB, Apppc ppc-p37 was chosen as reference of the state-of-the-art strategy to reduce by-product formation.
  • the single knock outs E. coli MG1655 AarcA and E. coli MG16 AiclR were chosen to confirm that a combined modification of the universally regulators ArcA and IclR are obligatory to eliminate co-product formation and to increase product formation and product yield.
  • Escherichia coli BL21(DE3) is chosen as industrial reference.
  • an expression plasmid carrying a reporter protein has been constructed: pCX-prom22-6His- GFPmut3b. Two different measurements were used, flow cytometry and SpectramaxTM fluorescence measurement to compare green fluorescent protein (GFP) production in the different strains.
  • the production of the recombinant protein GFP was significantly higher in the E. coli MG1655 AarcA AiclR mutant compared to the WT MG1655. It was also demonstrated that both genes (arcA and iclR) have to be knocked out to obtain a higher biomass yield and, more important, an unexpectedly high protein production.
  • the strains E. coli MG16 AarcA AiclR and E. coli BL21 perform significantly -and in a synergistic manner compared to the single knock-outs- better than the other 4 strains.
  • the E. coli MG16 AarcA AiclR and E. coli BL21 perform significantly -and in a synergistic manner compared to the single knock-outs- better than the other 4 strains.
  • the E. coli MG16 AarcA AiclR and E. coli BL21 perform significantly -and in a synergistic manner compared to the single knock-outs- better than the other 4 strains
  • E. coli MG1655 AarcA AiclR mutant strain gave a higher fluorescence in comparison to the E. coli BL21 strain. The same effect could be seen when the data were normalized per cell in a flow cytometer. E. coli BL21 and E. coli MG1655 AarcA AiclR gave -in a synergistic manner compared to the single knock-outs- better results than the other strains, and, E. coli MG1655 AarcA AiclR performed better than E. coli BL21.
  • the present invention relates to a mutated and/or transformed Escherichia coli (E. coli) strain comprising a genetic change leading to a modified expression of the transcriptional regulators: the aerobic respiration control protein ArcA and the isocitrate lyase regulator IclR.
  • E. coli Escherichia coli
  • a mutated and/or transformed E. coli as used here can be obtained by any method known to the person skilled in the art, including but not limited to UV mutagenesis and chemical mutagenesis.
  • a preferred manner to obtain the latter E. coli is by disrupting (knocking-out) the genes (arc A and iclR) encoding for the proteins ArcA and IclR, or, by replacing the endogenous promoters of said genes by artificial promoters.
  • the present invention specifically relates to a mutated and/or transformed Escherichia coli strain as indicated above wherein said genetic change is disrupting the genes encoding for ArcA and IclR, or, is replacing the endogenous promoters of the genes encoding for ArcA and IclR by artificial promoters.
  • the mutant and/or transformant according to the present invention may further comprise an additional genetic change in one or more other genes within its genome.
  • Escherichia coli relates to any strain belonging to this bacterial species. More specifically, the latter term relates to cultivated Escherichia coli strains -the so-called E.
  • the present invention specifically relates to a mutated and/or transformed Escherichia coli strain as indicated above wherein said E. coli strain is a K12 strain. More specifically, the present invention relates to a mutated and/or transformed Escherichia coli strain as indicated above wherein said 12 strain is E. coli MG1655.
  • the present invention relates to a mutated and/or transformed Escherichia coli strain as indicated above wherein said modified expression is a decreased expression, and, to a mutated and/or transformed Escherichia coli strain as indicated above wherein said decreased expression is an abolished expression.
  • the present invention further relates to the usage of a mutated and/or transformed Escherichia coli strain as indicated above to produce proteins and peptides.
  • the present invention relates to a usage as indicated above wherein said protein is green fluorescent protein (GFP), an antibody, a beta-galactosidase, an oxidoreductase, a lyase, a ligase, a hydrolase, a transferase, an isomerase, a protease, an amylase, a pullulinase, an oxidase, a glucose isomerase, a beta-glucanase, an asparaginase, a hormone, a colony stimulating factor, an interferon, a interleukin, an interferon receptor, an interleukin receptor, an interleukin receptor agonist or antagonist, a plasminogen activator, glucagon, a tumor necrosis factor, a growth factor, a toxin, a viral protein or an engineered variant of a said proteins.
  • GFP green fluorescent protein
  • an antibody an antibody
  • a beta-galactosidase
  • Escherichia coli MG1655 [ ⁇ ⁇ , F " , rph-l] was obtained from the Netherlands Culture Collection of Bacteria (NCCB).
  • Escherichia coli BL21(DE3) was obtained from Novagen.
  • Escherichia coli MG1655 AackA-pta, poxB, Apppc ppc-p37 [19] the single knock-outs E. coli MG1655 AarcA and E. coli MG1655 AiclR and the double knock-out E. coli MG1655 AarcA, AiclR were constructed using the method of Datsenko & Wanner [20]. 1.2. Media
  • the Luria Broth (LB) medium consisted of 1 % tryptone peptone (Difco, Erembodegem, Belgium), 0.5 % yeast extract (Difco) and 0.5 % sodium chloride (VWR, Leuven, Belgium).
  • Shake flask medium contained 2 g/1 NH 4 C1, 5 g/1 (NH 4 ) 2 S0 4 , 2.993 g/1 KH 2 P0 4 , 7.315 g/1 K 2 HP0 4 , 8.372 g/1 MOPS, 0.5 g/1 NaCl, 0.5 g/1 MgS0 4 7H 2 0, 16.5 g/1 glucose H 2 0, 1 ml/1 vitamin solution, 100 ⁇ / ⁇ molybdate solution, and 1 ml/1 selenium solution.
  • the medium was set to a pH of 7 with 1M KOH.
  • Vitamin solution consisted of 3.6 g/1 FeCl 2 ⁇ 4H 2 0, 5 g/1 CaCl 2 ⁇ 2H 2 0, 1.3 g/1 MnCl 2 ⁇ 2H 2 0, 0.38 g/1 CuCl 2 ⁇ 2H 2 0, 0.5 g/1 CoCl 2 ⁇ 6H 2 0, 0.94 g/1 ZnCl 2 , 0.0311 g/1 H 3 B0 4 , 0.4 g/1 Na 2 EDTA- 2H 2 0 and 1.01 g/1 thiamine ⁇ HC1.
  • the molybdate solution contained 0.967 g/1 Na 2 Mo0 4 ⁇ 2H 2 0.
  • the selenium solution contained 42 g/1 Se0 2 .
  • the minimal medium for fermentations contained 6.75 g/1 NH 4 C1, 1.25 g/1 (NH 4 ) 2 S04, 1.15 g/1 KH 2 P0 4 , 0.5 g/l NaCl, 0.5 g/1 MgS0 4 -7H 2 0, 16.5 g/1 glucose H 2 0, 1 ml/1 vitamin solution, 100 ⁇ / ⁇ molybdate solution, and 1 ml/1 selenium solution with the same composition as described above.
  • a preculture, from a single colony on a LB-plate, in 5 ml LB medium was incubated during 8 hours at 37 °C on an orbital shaker at 200 rpm. From this culture, 2 ml was transferred to 100 ml minimal medium in a 500 ml shake flask and incubated for 16 hours at 37 °C on an orbital shaker at 200 rpm. 4 % inoculum was used in a 2 1 Biostat B Plus culture vessel with 1.5 1 working volume (Sartorius Stedim Biotech, Melsoder, Germany). The culture conditions were: 37 °C, stirring at 800 rpm, and a gas flow rate of 1.5 1/min.
  • Aerobic conditions were maintained by sparging with air, anaerobic conditions were obtained by flushing the culture with a mixture of 3 % C0 2 and 97 % of N 2 .
  • the pH was maintained at 7 with 0.5 M H 2 S04 and 4 M KOH.
  • the exhaust gas was cooled down to 4 °C by an exhaust cooler (Frigomix 1000, Sartorius Stedim Biotech, Melsungen, Germany).
  • 10 % solution of silicone antifoaming agent (BDH 331512K, VWR Int Ltd., Poole, England) was added when foaming raised during the fermentation (approximately 10 ⁇ ).
  • the off-gas was measured with an EL3020 off-gas analyser (ABB Automation GmbH, 60488 Frankfurt am Main, Germany).
  • the bioreactor contains in its interior a harvest pipe (BD Spinal Needle, 1.2x152 mm (BDMedical Systems, Franklin Lakes, NJ - USA) connected to a reactor port, linked outside to a Masterflex-14 tubing (Cole-Parmer, Antwerpen, Belgium) followed by a harvest port with a septum for sampling. The other side of this harvest port is connected back to the reactor vessel with a Masterflex-16 tubing.
  • This system is referred to as rapid sampling loop.
  • reactor broth is pumped around in the sampling loop. It has been estimated that, at a flow rate of 150 ml/min, the reactor broth needs 0.04 s to reach the harvest port and 3.2 s to re-enter the reactor.
  • reactor broth was sucked through the harvest port in a syringe filled with 62 g stainless steel beads precooled at -20 °C, to cool down 5 ml broth immediately to 4 °C.
  • Sampling was immediately followed by cold centrifugation (15000 g, 5 min, 4 °C).
  • a sample for OD 6 oonm and extracellular measurements was taken each hour using the rapid sampling loop and the cold stainless bead sampling method. When exponential growth was reached, the sampling frequency was increased to every 20 to 30 minutes. 1.5. Analytical methods
  • Cell density of the culture was frequently monitored by measuring optical density at 600 nm (Uvikom 922 spectrophotometer, BRS, Brussel, Belgium). Cell dry weight was obtained by centrifugation (15 min, 5000 g, GSA rotor, Sorvall RC-5B, Goffm Meyvis, Kapellen, Belgium) of 20 g reactor broth in pre-dried and weighted falcons. The pellets were subsequently washed once with 20 ml physiological solution (9 g/1 NaCl) and dried at 70 °C to a constant weight. To be able to convert OD 6 oonm measurements to biomass concentrations, a correlation curve of the OD6oonm to the biomass concentration was made.
  • the concentrations of glucose and organic acids were determined on a Varian Prostar HPLC system (Varian, Sint-Katelijne- Waver, Belgium), using an Aminex HPX-87H column (Bio-Rad, Eke, Belgium) heated at 65 °C, equipped with a 1 cm precolumn, using 5 mM H2S04 (0.6 ml/min) as mobile phase.
  • a dual- wave UV-VIS (210 nm and 265 nm) detector Varian Prostar 325) and a differential refractive index detector (Merck LaChrom L-7490, Merck, Leuven, Belgium) was used for peak detection. By dividing the absorptions of the peaks in both 265 and 210 nm, the peaks could be identified. The division results in a constant value, typical for a certain compound (formula of Beer-Lambert).
  • Plasmids were maintained in the host E. coli DH5cc (F ⁇ , cp80d/acZAM15, A(lacZYA-argF)XJ 169, deoR, recA , endA l , hsdR ⁇ l ⁇ x ⁇ , mk + ), phoA, supE44, X, thi-l, gyrA96, relA X).
  • Plasmids. pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT-flanked kanamycin resistance (kan) gene), and pCP20 (expresses FLP recombinase activity) plasmids were obtained from Prof. Dr. J-P Hernalsteens (Vrije Universiteit Brussel, Belgium).
  • the plasmid pBluescript Fermentas, St. Leon-Rot, Germany
  • Plasmid pPS1880 (of pUT miniTn5-kan GFPmut3b) was kindly provided by the laboratory of Microbial Ecology and Technology (Ghent University). Plasmid pCX-prom22-6His (a pUC derived plasmid baring a constitutive promoter 22, 6x His-tag, Enterokinase cleavage site, MCS, Transcription terminators rrnB Tl and T2, ⁇ -lactamase gene and pMBl origin of replication) was constructed by the Centre of Expertise - Industrial Biotechnology and Biocatalysis at Ghent University and used to construct plasmid pCX-prom22-6His-GFPmut3b.
  • This expression plasmid contains the model recombinant protein GFP coded by the gene GFPmut3b. Mutations. The mutations consisted in gene disruption (knock-out, KO), replacement of an endogenous promoter by an artificial promoter (knock- in, KI) [21], Figure 1 (left) and Figure 1 (right), respectively. They were introduced using the concept of Datsenko and Wanner [20]. The primers for the mutation strategies are described in Table 2.
  • Transformants carrying a Red helper plasmid were grown in 10 ml LB media with ampicillin (100 mg/1) and L-arabinose (10 mM) at 30 °C to an OD 6 oonm of 0.6.
  • the cells were made electrocompetent by washing them with 50 ml of ice-cold water, a first time, and with 1 ml ice- cold water, a second time. Then, the cells were resuspended in 50 ⁇ of ice-cold water. Electroporation was done with 50 ⁇ l of cells and 10-100 ng of linear double-stranded-DNA product by using a Gene PulserTM (BioRad) (600 ⁇ , 25 ⁇ , and 250 volts).
  • BioRad Gene PulserTM
  • cells were added to 1 ml LB media incubated 1 h at 37 °C, and finally spread onto LB-agar containing 25 mg/1 of chloramphenicol or 50 mg/1 of kanamycin to select antibiotic resistant transformants.
  • the selected mutants were verified by PCR with primers upstream and downstream of the modified region and were grown in LB-agar at 42 °C for the loss of the helper plasmid. The mutants were tested for ampicillin sensitivity
  • Linear double-stranded-DNA The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivates as template.
  • the primers used had a part of the sequence complementary to the template and another part complementary to the side on the chromosomal DNA where the recombination has to take place (see Table 4.1).
  • the region of homology was designed 50-nt upstream and 50-nt downstream of the start and stop codon of the gene of interest.
  • the transcriptional starting point (+1) had to be respected.
  • PCR products were PCR-purified, digested with Dpn ⁇ , repurified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0).
  • the selected mutants (chloramphenicol or kanamycin resistant) were transformed with pCP20 plasmid, which is an ampicillin and chloramphenicol resistant plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis.
  • the ampicillin-resistant transformants were selected at 30 °C, after which a few were colony purified in LB at 42 °C and then tested for loss of all antibiotic resistance and of the FLP helper plasmid.
  • the gene knock outs and knock ins are checked with control primers (Fw/Rv-gene-out). These primers are given in Table 2.
  • Plasmids were transformed in CaCl 2 competent cells using the simplified procedure of Hanahan [22] or via electroporation as described above.
  • the yields were calculated on at least ten data points within the exponential phase of the batch fermentation. Each sample resulted in a substrate concentration S and a product concentration P. By plotting P in function of S, a regression line can be fitted. The slope of this regression is the yield of product P on substrate S. The dilution effect due to base and antifoam addition was assumed negligible for these calculations because the total amount of fluid added to the reactor was less than 1% of the total volume of the reactor. On each of the slopes a standard deviation was calculated which was used in the error propagation to obtain an error on the averaged yields of the duplicate fermentations.
  • the maximal growth rate was obtained by plotting the Neperian logarithm of the optical density in function of time. A linear model was fitted on this plot. The slope of this regression is the maximal growth rate ⁇ . Maximal specific production rates were calculated by first determining the bio mass specific yields of all products P and substrates S. These calculations are similar to the yield calculations described above (with the same assumptions concerning dilution effects). A regression curve was fitted on the substrate and product concentrations in function of the bio mass concentration plots. The obtained slope was multiplied by the maximal growth rate to obtain the maximal specific production rates and substrate uptake rates. On each of the slopes and on the ⁇ ⁇ a standard deviation was calculated which was used in the error propagation to obtain an error on the averaged specific production rates and substrate uptake rates of the duplicate fermentations.
  • LB supplemented with ampicillin were inoculated with a single colony carrying the plasmid pCX-prom22-6His-GFPmut3b from an agar-plate and grown overnight at 37 °C on an orbital shaker (200 rpm).
  • This seed culture was used to inoculate 100 ml minimal shake-flask medium to obtain a start optical density at 600 nm (OD 6 oo) of 0.05-0.1.
  • the culture were incubated at 37 °C on an orbital shaker (200 rpm) until the required OD 6 oo was obtained.
  • Escherichia coli MG1655 is chosen as reference to the constructed strains.
  • Escherichia coli MG1655 AackA-pta, ApoxB, Apppc ppc-p37 is chosen as reference of the state-of-the-art strategy to reduce by-product formation.
  • the single knock outs E. coli MG1655 AarcA and E. coli MG1655 AiclR are chosen to confirm that a combined modification of the universally regulators ArcA and IclR are obligatory to eliminate co-product formation and increases product formation and product yield.
  • Escherichia coli BL21(DE3) is chosen as industrial reference.
  • the effect of arcA and iclR was both tested in batch cultures, in which the strain grows at maximal growth rate and in a chemostat culture with a growth rate around 0.1 h "1 .
  • the measured dilution rates for the chemostat cultures and the maximal growth rates are listed in Table 3.
  • the maximal growth rate is dependent on the mutations that have been introduced in the strain.
  • the differences in dilution rate are due to technical issues of the used setup, in which the pump rate is manually adjusted to obtain a certain dilution rate. We assumed that the small deviations in dilution rate of 0.02 h "1 from the average are negligible for this study
  • the arcA and iclR single knock outs do not have a very large effect on the maximal growth rate in comparison with the wild type strain. However the maximal growth rate of the double mutant seems to be affected more.
  • the combined effect oiarcA and iclR reduces the maximal growth rate about 38 % in comparison with the wild type strain. This might be caused by an increase in glyoxylate shunt activity and a decreased activity of isocitrate dehydrogenase, which is phosphorylated by isocitrate dehydrogenase kinase.
  • the outer branch of the TCA cycle yields 2 NAD(P)H, with the loss of two carbon dioxide molecules. There should thus be a difference in carbon dioxide excretion rate between the different strains.
  • Green Fluorescent Protein (GFP) of the jellyfish Aequorea victoria absorbs light with an excitation maximum of 395 nm and fluorescences with an emission maximum of 510 nm. Since this can occur in absence of co factors, GFP is a good candidate as a marker for gene expression and as a tag in studying protein localization in a variety of organisms.
  • GFPmut3b is a good candidate and this GFPmut3 has some advantages:
  • spectra can be measured with flow cytometer.
  • GFPmut3b was chosen as reporter protein.
  • the strategy followed to construct pCX-prom22-6His GFPmut3b is depicted in Figure 5.
  • the backbone of the expression plasmid was obtained by restriction digest of pCX-prom22-6His with Nhel and Pstl.
  • the reporter protein GFPmut3b was amplified from the plasmid pPS1880 (or pUT miniTn5-kan GFPmut3b) via PCR using the primers Memo045 GFP fw (5'-
  • the obtained plasmid pCX-prom22-6His-GFPmut3b was transformed via electroporation in following strains:
  • Figure 6 depicts the sensitivity and reliability of the florescence. This figure shows that the sensitivity is the highest for GA, formaline and PBS. For the former, the netto fluorescence of cultures treated with GA (glutaraldehyde) increases with increasing excitation wavelength, while for the latter two the netto fluorescence stays stable. The reliability is the highest for cells treated with formaline and PBS. From these results, phosphate buffer solution (PBS) and formaline seems the best candidates as fixation solution. Hence, it was decided to investigate the stability of fluorescence over time for cells treated with PBS or formaline (Figure 7).
  • PBS phosphate buffer solution
  • Figure 8 shows that the fluorescence of the cells for both fixation solutions is stable during several days at excitation wavelengths higher than 490 nm. Because PBS gave better results for fluorescence determination using flow cytometry (data not shown), it was decided to use PBS as fixation solution and 491 nm as excitation wavelength.
  • the strains arcA- iclR and BL21 performs significantly better than the other 4 strains.
  • the arcA- iclR mutant strain gave a slightly higher absorbance in comparison to the BL21 strain. The same effect can be seen when the data is normalized per cell in a flow cyto meter.
  • BL21 and arcA-iclR gave better results than the other strains, but arcA-iclR performed better than BL21 ( Figure 10).
  • the results above showed that by altering the gene expression of two universally existing regulator proteins ArcA and IclR eliminates co-product formation and increases product formation and product yield.
  • prokaryotes are being used or have potential to be used for the production of a wide variety of products, ranging from (recombinant) proteins such as but not limited to GFP, ⁇ -galactosidase, antibodies.... This alleviates the problem of co-product formation, e.g., acetate, which might inhibit biomass growth and product formation, reduction of carbon dioxide formation and results in an increased biomass and product yields.
  • Applications of this invention are the production of all classes of proteins and peptides (oxidoreductases, lyases, ligases, hydrolases, transferases, isomerases) and engineered variants thereof with a prokaryote micro-organism as the iclR and arcA genes are global transcriptional regulators in prokaryotes. Examples hereof are the production of peptides and proteins that are currently produced in microbial hosts, a non limitative list is given in Table 1. This invention may also be used for the production of proteins and peptides such as, but not limited to, proteases, amylases, pullulanase, glucose isomerase, mannanase, beta-glucanase, and engineered variants thereof. References
  • Microbial factories for recombinant pharmaceuticals (2009) Microbial factories for recombinant pharmaceuticals.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode d'augmentation des rendements en produits et/ou d'élimination de la formation de sous-produits chez des micro-organismes, ainsi que des mutants et/ou des transformants pour emploi dans de telles méthodes. Plus particulièrement, la présente invention concerne des bactéries mutantes et/ou transformantes affectées au niveau du métabolisme primaire pour une augmentation de la formation de produits, pour un rendement supérieur en produits et/ou pour l'élimination de la production de sous-produits, spécialement des mutants et/ou des transformants affectés au niveau de l'inhibition du cycle du glyoxylate et de l'expression de gènes chromosomiques en réponse à la privation d'oxygène ou en réponse à l'absence ou à la présence de composés carbonés comme le glycérol, les acides gras, le glyoxylate, l'acétate, etc.
PCT/EP2010/070683 2010-01-06 2010-12-23 Bactéries mutantes et leurs emplois dans la production de protéines WO2011083059A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US29251610P 2010-01-06 2010-01-06
US61/292,516 2010-01-06

Publications (1)

Publication Number Publication Date
WO2011083059A1 true WO2011083059A1 (fr) 2011-07-14

Family

ID=43795051

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2010/070683 WO2011083059A1 (fr) 2010-01-06 2010-12-23 Bactéries mutantes et leurs emplois dans la production de protéines

Country Status (1)

Country Link
WO (1) WO2011083059A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013087884A1 (fr) * 2011-12-16 2013-06-20 Universiteit Gent Microorganismes mutants pour la synthèse de l'acide colanique, d'oligosaccharides mannosylés et/ou fucosylés
CN110195070A (zh) * 2019-05-31 2019-09-03 天津大学 大肠杆菌全局调控因子的突变基因crp及应用
CN110564659A (zh) * 2019-09-17 2019-12-13 天津大学 耐乙酸钠、氯化钠和异丁醇的大肠杆菌及其构建方法
WO2020218876A1 (fr) * 2019-04-26 2020-10-29 고려대학교 산학협력단 Production d'acide colanique à l'aide d'e. coli mutant
CN113430155A (zh) * 2021-05-31 2021-09-24 天津大学 高产游离脂肪酸重组大肠杆菌菌株及其构建方法和用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1382686A1 (fr) 2002-07-12 2004-01-21 Ajinomoto Co., Inc. Procédé pour la production fermentative d'une substance déesirée en utilisant une souche bactérienne qui manque le gen ArcA
WO2006031424A2 (fr) * 2004-08-27 2006-03-23 Rice University Souche e. coli mutante avec production accrue d'acide succinique
WO2006034156A2 (fr) * 2004-09-17 2006-03-30 Rice University Bacteries produisant un taux eleve de succinates
US20070249018A1 (en) 2006-02-23 2007-10-25 Goutham Vemuri Reduced overflow metabolism and methods of use
EP2233562A1 (fr) * 2009-03-24 2010-09-29 Metabolic Explorer Procédé de fabrication d'une grande quantité d'acide glycolique par fermentation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1382686A1 (fr) 2002-07-12 2004-01-21 Ajinomoto Co., Inc. Procédé pour la production fermentative d'une substance déesirée en utilisant une souche bactérienne qui manque le gen ArcA
WO2006031424A2 (fr) * 2004-08-27 2006-03-23 Rice University Souche e. coli mutante avec production accrue d'acide succinique
WO2006034156A2 (fr) * 2004-09-17 2006-03-30 Rice University Bacteries produisant un taux eleve de succinates
US20070249018A1 (en) 2006-02-23 2007-10-25 Goutham Vemuri Reduced overflow metabolism and methods of use
EP2233562A1 (fr) * 2009-03-24 2010-09-29 Metabolic Explorer Procédé de fabrication d'une grande quantité d'acide glycolique par fermentation

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
CLONTECH: "Living colors user manual", 1999, CLONTECH LABORATORIES, INC. 51
CORMACK, B.P.; VALDIVIA, R.H.; FALKOW, S.: "FACS-optimized mutants of the green fluorescent protein (GFP)", GENE, vol. 173, 1996, pages 33 - 38
DATSENKO, K.A.; WANNER, B.L.: "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 97, no. 12, 2000, pages 6640 - 6645
DAYANIDHI SARKAR ET AL: "Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli", ARCHIVES OF MICROBIOLOGY, vol. 190, no. 5, 22 July 2008 (2008-07-22), pages 559 - 571, XP019652533, ISSN: 1432-072X, DOI: DOI:10.1007/S00203-008-0406-2 *
DE MEY, M.; LEQUEUX, G.J.; BEAUPREZ, J.J.; MAERTENS, J.; VAN HOREN, E.; SOETAERT, W.K.; VANROLLEGHEM, P.A.; VANDAMME, E.J.: "Comparison of different strategies to reduce acetate formation in Escherichia coli", BIOTECHNOL PROG, 2007
DE MEY, M.; MAERTENS, J.; LEQUEUX, G.J.; SOETAERT, W.K.; VANDAMME, E.J.: "Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering", BMC BIOTECHNOLOGY, vol. 7, 2007, pages 34 - 48
DITTO, M.D.; ROBERTS, D.; WEISBERG, R.A.: "Growth phase variation of integration host factor level in Escherichia coli", J BACTERIOL, vol. 176, no. 12, 1994, pages 3738 - 3748
FERRER-MIRALLES, N.; DOMINGO-ESPIN, J.; CORCHERO, J.L.; VAZQUEZ, E.; VILLAVERDE, A.: "Microbial factories for recombinant pharmaceuticals", MICROBIAL CELL FACTORIES 8: 17, 2009
FRAZZETTO, G., WHITE BIOTECHNOLOGY. EMBO REPORTS, vol. 4, no. 9, 2003, pages 835 - 837
GONZALES, D.: "A TD-700 laboratory fluorometer method for the assaying of Green-Fluorescent Protein in whole bacteria", IN: TURNER BIOSYSTEMS APPLICATION NOTE, 2005
GRAUMANN, K.; PREMSTALLER, A.: "Manufacturing of recombinant therapeutic proteins in microbial systems", BIOTECHNOLOGY JOURNAL, vol. 1, no. 2, 2006, pages 164 - 186
HANAHAN, D.; JESSEE, J.; BLOOM, F.R.: "Plasmid transformation of Escherichia coli and other bacteria", METHODS ENZYMOL, vol. 204, 1991, pages 63 - 113
JENSEN, E.B.; CARLSEN, S.: "Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts", BIOTECHNOL BIOENG, vol. 36, no. 1, 1990, pages 1 - 11
LULI, G.W.; STROHL, W.R.: "Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations", APPL ENVIRON MICROBIOL, vol. 56, no. 4, 1990, pages 1004 - 1011
MARCH, J.C.; EITEMAN, M.A.; ALTMAN, E.: "Expression of an Anaplerotic Enzyme, Pyruvate Carboxylase, Improves Recombinant Protein Production in Escherichia coli", APPL ENVIRON MICROBIOL, vol. 68, no. 11, 2002, pages 5620 - 5624
PERRENOUD, A.; SAUER, U.: "Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli", J BACTERIOL, vol. 187, no. 9, 2005, pages 3171 - 3179
RAMSEIER, T.M.: "Cra and the control of carbon flux via metabolic pathways", RES MICROBIOL, vol. 147, no. 6-7, 1996, pages 489 - 493
RAMSEIER, T.M.; NEGRE, D.; CORTAY, J.-C.; SCARABEL, M.; COZZONE, A.J.; SAIER, M.H., JR.: "In vitro binding of the pleiotropic transcriptional regulatory protein, FruR, to the fru, pps, ace, pts and icd operons of Escherichia coli and Salmonella typhimurium", J MOL BIOL, vol. 234, no. 1, 1993, pages 28 - 44
RESNIK, E.; PAN, B.; RAMANI, N.; FREUNDLICH, M.; LAPORTE, D.C.: "Integration host factor amplifies the induction of the aceBAK operon of Escherichia coli by relieving IclR repression", J BACTERIOL, vol. 178, no. 9, 1996, pages 2715 - 2717
RYU, S.; RAMSEIER, T.M.; MICHOTEY, V.; SAIER, M.H., JR.; GARGES, S.: "Effect of the FruR Regulator on Transcription of the pts Operon in Escherichia coli", J BIOL CHEM, vol. 270, no. 6, 1995, pages 2489 - 2496
SANCHEZ A M ET AL: "Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity", METABOLIC ENGINEERING, vol. 7, no. 3, 1 May 2005 (2005-05-01), pages 229 - 239, XP004879518, ISSN: 1096-7176, DOI: DOI:10.1016/J.YMBEN.2005.03.001 *
SARKAR ET AL., ARCH. MICROBIOL., vol. 190, pages 559
SARKAR, D.; SIDDIQUEE, K.A.Z.; ARAUZO-BRAVO, M.J.; OBA, T.; SHIMIZU, K.: "Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli", ARCH MICROBIOL, vol. 190, no. 5, 2008, pages 559 - 571
SCHALLMEY, M.; SINGH, A.; WARD, O.P.: "Developments in the use of Bacillus species for industrial production", CANADIAN JOURNAL OF MICROBIOLOGY, vol. 50, no. 1, 2004, pages 1 - 17
SHALEL-LEVANON, S.; SAN, K.Y.; BENNETT, G.N.: "Effect of ArcA and FNR on the expression of genes related to the oxygen regulation and glycolysis pathway in Escherichia coli under growth conditions", BIOTECHNOL BIOENG, vol. 92, no. 2, 2005, pages 147 - 159
SHALEL-LEVANON, S.; SAN, K.Y.; BENNETT, G.N.: "Effect of oxygen on the Escherichia coli ArcA and FNR regulation systems and metabolic responses", BIOTECHNOL BIOENG, vol. 89, no. 5, 2005, pages 556 - 564
SHALEL-LEVANON, S.; SAN, K.Y.; BENNETT, G.N.: "Effect of oxygen, and ArcA and FNR regulators on the expression of genes related to the electron transfer chain and the TCA cycle in Escherichia coli", METABOLIC ENGINEERING, vol. 7, no. 5-6, 2005, pages 364 - 374

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013087884A1 (fr) * 2011-12-16 2013-06-20 Universiteit Gent Microorganismes mutants pour la synthèse de l'acide colanique, d'oligosaccharides mannosylés et/ou fucosylés
US9719119B2 (en) 2011-12-16 2017-08-01 Universiteit Gent Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides
US9951362B2 (en) 2011-12-16 2018-04-24 Inbiose N.V. Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides
EP3517631A1 (fr) * 2011-12-16 2019-07-31 Inbiose N.V. Micro-organismes mutants pour synthétiser l'acide colanique, des oligosaccharides mannosylatés et/ou fucosylatés
US10738336B2 (en) 2011-12-16 2020-08-11 Inbiose N.V. Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides
WO2020218876A1 (fr) * 2019-04-26 2020-10-29 고려대학교 산학협력단 Production d'acide colanique à l'aide d'e. coli mutant
CN110195070A (zh) * 2019-05-31 2019-09-03 天津大学 大肠杆菌全局调控因子的突变基因crp及应用
CN110564659A (zh) * 2019-09-17 2019-12-13 天津大学 耐乙酸钠、氯化钠和异丁醇的大肠杆菌及其构建方法
CN110564659B (zh) * 2019-09-17 2022-03-11 天津大学 耐乙酸钠、氯化钠和异丁醇的大肠杆菌及其构建方法
CN113430155A (zh) * 2021-05-31 2021-09-24 天津大学 高产游离脂肪酸重组大肠杆菌菌株及其构建方法和用途
CN113430155B (zh) * 2021-05-31 2023-01-31 天津大学 高产游离脂肪酸重组大肠杆菌菌株及其构建方法和用途

Similar Documents

Publication Publication Date Title
Wang et al. Improving the expression of recombinant proteins in E. coli BL21 (DE3) under acetate stress: an alkaline pH shift approach
JP6341936B2 (ja) ５−アミノレブリン酸の高生産株及びその製造方法と使用
Zahoor et al. Metabolic engineering of Corynebacterium glutamicum for glycolate production
Li et al. Engineering Escherichia coli for fumaric acid production from glycerol
US10889842B2 (en) Microorganisms for the enhanced production of amino acids and related methods
Rao et al. Characterization and regulation of the 2, 3-butanediol pathway in Serratia marcescens
US20150072399A1 (en) Methods, Systems And Compositions Related To Reduction Of Conversions Of Microbially Produced 3-Hydroxypropionic Acid (3-HP) To Aldehyde Metabolites
Pérez-García et al. Function of L-pipecolic acid as compatible solute in Corynebacterium glutamicum as basis for its production under hyperosmolar conditions
WO2011083059A1 (fr) Bactéries mutantes et leurs emplois dans la production de protéines
EP2411502A1 (fr) Procédé permettant la production d'une grande quantité d'acide glycolique par fermentation
US20140065697A1 (en) Cells and methods for producing isobutyric acid
Waegeman et al. Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering
Li et al. Directed pathway evolution of the glyoxylate shunt in Escherichia coli for improved aerobic succinate production from glycerol
JP6828038B2 (ja) 低分子量物質およびタンパク質の抗生物質を含まない発酵製造のための微生物株および方法
Halmschlag et al. Poly-γ-glutamic acid production by Bacillus subtilis 168 using glucose as the sole carbon source: a metabolomic analysis
Zhang et al. Improving phloroglucinol tolerance and production in Escherichia coli by GroESL overexpression
US20170166936A1 (en) Processes for the production of hydroxycinnamic acids using polypeptides having tyrosine ammonia lyase activity
Wang et al. Engineering Escherichia coli for high-yield production of ectoine
Da et al. Coutilization of glucose and acetate for the production of pyruvate by engineered Escherichia coli
Hobmeier et al. Metabolic engineering of Halomonas elongata: Ectoine secretion is increased by demand and supply driven approaches
Khandelwal et al. Expression of Escherichia coli malic enzyme gene in Zymomonas mobilis for production of malic acid
WO2017055360A1 (fr) Système de titres de production améliorés dans des fermentations
Liu et al. Lysine acetylation of Escherichia coli lactate dehydrogenase regulates enzyme activity and lactate synthesis
Li et al. Performance and mechanism analysis of succinate production under different transporters in Escherichia coli
Chen et al. Metabolic engineering of Escherichia coli for the efficient production of 5-hydroxyvaleric acid

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10798565

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10798565

Country of ref document: EP

Kind code of ref document: A1