WO2013172928A1 - Production photosynthétique de 3-hydroxybutyrate à partir de dioxyde de carbone - Google Patents

Production photosynthétique de 3-hydroxybutyrate à partir de dioxyde de carbone Download PDF

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WO2013172928A1
WO2013172928A1 PCT/US2013/029997 US2013029997W WO2013172928A1 WO 2013172928 A1 WO2013172928 A1 WO 2013172928A1 US 2013029997 W US2013029997 W US 2013029997W WO 2013172928 A1 WO2013172928 A1 WO 2013172928A1
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synechocystis
pbs
production
hydroxybutyrate
culture
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Bo Wang
Weiwen Zhang
Deirdre Meldrum
David Nielsen
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Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizonia State University
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Publication of WO2013172928A1 publication Critical patent/WO2013172928A1/fr
Priority to US14/479,893 priority Critical patent/US9944955B1/en
Priority to US15/938,742 priority patent/US10246726B2/en

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    • C12N9/10—Transferases (2.)
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    • C12P7/00—Preparation of oxygen-containing organic compounds
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/52—Propionic acid; Butyric acids
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/133—Renewable energy sources, e.g. sunlight
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/59—Biological synthesis; Biological purification

Definitions

  • Lipid-rich cyanobacteria and microalgae have most notably been employed to produce fuels such as biodiesel.
  • Cyanobacteria are also natural producers of the naturally-occurring biodegradable plastic poly- -hydroxybutyrate (PHB).
  • PHB biosynthesis by cyanobacteria was a multistage cultivation process that involved nitrogen starvation followed by supplementation of fructose or acetate, which does not capitalize on the important photosynthetic potential of cyanobacteria.
  • the required processes for their extraction are energy-intensive and remain as one of the major hurdles for commercial applications.
  • 3-hydroxybutyrate is a small molecule that could possibly be secreted out of the cells into extracellular environment, thereby facilitating its collection.
  • 3HB can then be chemo- catalytically polymerized to produce PHB or be co-polymerized with other organic acid compounds to synthesize renewable plastics with a broader range of chemical and material properties (including adjustable molecular weight and improved purity) relative to naturally-synthesized PHB.
  • R - or (5)-3HB can also serve as a precursor for many stereo-specific fine chemicals such as antibiotics, pheromones and amino acids.
  • (R)-3HB has been found to be an advanced nutrition source for tissue cells and can reduce the death rate of the human neuronal cells, improve mice memory and promote growth of osteoblasts.
  • 3HB synthetic pathways in cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) were constructed and demonstrated highly efficient photosynthetic production and secretion of 3HB using solar energy and CO 2 as the sole carbon and energy sources. Thus, multi-cycle or continuous production of 3HB from engineered Synechocystis are possible.
  • Fig. 1 is a schematic representation of (5)-3HB and (R)-3HB biosynthesis from CO2 in engineered Synechocystis.
  • Thil thiolase from C. acetobutilicum ATCC824; PhaA, thiolase from R. eutropha H16; PhaA2 (Slrl993), native thiolase in Synechocystis 6803; Hbd (5)-3-hydroxybutyryl-CoA dehydrogenase from C. acetobutilicum ATCC824; PhaB, (R)-3-hydroxybutyryl-CoA dehydrogenase from R.
  • Fig. 2 is a schematic representation of the modification of Synechocystis chromosome for 3HB production.
  • Site 1 the site on the genome of Synechocystis 6803 between slrl495 and slll397;
  • Site 2 the site between slrl362 and sill 274;
  • Site 3 the site between sir 1828 and sill 736 for phaEC deletion;
  • Site 4 the site between sir 1992 and phaA2 (sir 1993).
  • Fig. 3 depicts the cell density of different Synechocystis strains cultivated in shaking flasks.
  • Fig. 4 depicts extracellular production of 3HB by Synechocystis 6803 and engineered derivatives. 3HB titers were symbolized as grey bars and acetate titers were symbolized as yellow bars.
  • Fig. 5 depicts 3HB productivity for unit density of Synechocystis TAB1 cell cultures subjected to nitrogen starvation and normal BG11 medium (control) cultures. 3HB production by the normal BG11 medium control dramatically increased by day 3. Culture grown under low light intensity (LL, -45 ⁇ / ⁇ 2 /8), middle light intensity (ML, -60 ⁇ / ⁇ 2 /8) and high light intensity (HL, -90 ⁇ / ⁇ 2 /8) were studied.
  • LL low light intensity
  • ML middle light intensity
  • HL high light intensity
  • Fig. 6 depicts Synechocystis TAB 1 cells subjected to nitrogen starvation and normal BG1 1 medium (control) cultures. Overall, higher 3HB production and cell density of the control versus that of the nitrogen-starved counterparts was observed.
  • Fig. 7 depicts the effect on 3HB production by nutrient supplementation. When the indicated amount of nutrient was supplemented into the culture after day 7, both biomass and 3HB production started to increase again.
  • Fig. 8 depicts biomass and 3HB production curves of Synechocystis strain TABd under different nutrient supplementation conditions.
  • A Biomass curves. Grey squares indicate non-N-supplementation; Open squares indicate 5%N-supplementation; solid squares indicate 10%N-supplementation.
  • B 3HB production curves. Grey triangles indicate non-N-supplementation; Open triangles indicate 5%N- supplementation; solid triangles indicate 10%N-supplementation.
  • Fig. 9 depicts, after 10 days culture, chlorosis in the non-N and 5%-N (shown) cultures but not in the 10%-N cultures.
  • Fig. 10 depicts biomass and 3HB production curves of Synechocystis strain TABd under 5%N-supplementation condition for 3HB production by medium exchange.
  • Fig. 11 depicts continuous production of 3HB directly from atmospheric C0 2 by Synechocystis strain TABd. Solid triangles indicate 3HB titers; solid squares indicate cell densities represented by OD 730 .
  • this disclosure relates to engineered strains of Synechocystis .
  • 3HB synthetic pathways in cyanobacterium Synechocystis 6803 were constructed and demonstrated highly efficient photosynthetic production of 3HB using solar energy as the sole energy source.
  • this disclosure relates to highly efficient photosynthetic production of 3HB using bicarbonate or CO 2 as the sole carbon source by engineered Synechocystis.
  • this disclosure relates to biosynthesis of 3HB in a process coupled with oxygenic photosynthesis in engineered Synechocystis.
  • this disclosure relates to highly efficient secretion of hydrophilic 3HB molecules by engineered Synechocystis without overexpression of specific transporters.
  • this disclosure relates to multi-cycle or continuous photosynthetic production of 3HB from engineered Synechocystis. [0023] In a sixth aspect, this disclosure relates to photosynthetic production of 3HB from engineered cyanobacteria.
  • Synechocystis strains and culture conditions A series of Synechocystis strains were constructed using marker modification and markerless modification methods. Synechocystis 6803 and its derivatives were grown in BGl l medium under a light intensity of 35 ⁇ / ⁇ 2 /8 unless otherwise specified. For BGl l plates for Synechocystis growth, 10 mM TES (pH 8.2), 3 g/1 thiosulfate and 1.5% agar was supplemented before autoclaving. E. coli XLl-Blue MRF' (Stratagene, La Jolla, CA) was used as host to construct and store all recombinant plasmids. All strains of E.
  • Bacillus subtilis strain 168 was obtained from American Type Culture Collection (ATCC) and was cultured in LB medium at 30°C. [0027] Synechocystis 6803 genomic DNA was purified by DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA) and subsequently used as template for PCR amplification of SR12 ⁇ sir 1495) and SL12 ⁇ sill 397) DNA fragments.
  • SR12 and SL12 were recombined together by overlapping PCR and were inserted into the Sacl and Kpnl restriction sites of the plasmid pBluescript II SK(+) (Stratagene, La Jolla, CA) to construct pBS-SRSL.
  • pBS-SRSL Sacl and Kpnl restriction sites of the plasmid pBluescript II SK(+) (Stratagene, La Jolla, CA) to construct pBS-SRSL.
  • thil gene was PCR amplified using primers Th5 and Th8. The purified product was then again PCR amplified by primers Ptac and Th8 to construct Ptac-thil, wherein thil was under the control of the Ptac promoter.
  • the gel-purified Ptac-thil product was then again PCR amplified using primers TAC5 and Th8, the product of which was purified and restriction digested before being inserted into the BamHI and Sail sites of pBS-SRSL to construct pBS-SPT.
  • hbd of C. acetobutylicum was PCR amplified with primers HBD3 and HBD6.
  • the resultant fragment was purified and restriction digested before being inserted between the Ncol and Sail sites of pBS-SPT to construct pBS-SPTH.
  • the Ptac-thil-hbd fragment of pBS-SPTH was PCR amplified using primers TAC5 and HBD6 and then inserted between the BamHI and Sail sites of pBS-SCat to construct pBS-SCPTH.
  • the Ptac-thil fragment from pBS-SCPTH was PCR amplified using primers TAC5 and primer ThlO and was used to replace the original Ptac-thil fragment of pBS-SCPTH between BamHI and Ncol to construct pBS-SCPTH2.
  • eutropha H16 gene phaB was PCR amplified with primers PHAB1 1 and PHAB 12 using pETphaAphaB (reconstructed based on the methods of Tseng et al. in constructing pET-P-P) as template and was inserted between the M and Hindlll sites of pBS-SCPTH2 to construct pBS-SCPTB.
  • the gene phaA from R. eutropha H16 was PCR amplified using primers PHAA 11 and PHAA 12 with pETphaAphaB as template.
  • the purified product was then amplified using primers Ptac and primer PHAA 12 to construct the Ptac-phaA fragment.
  • Ptac-phaA was further PCR amplified using primers TAC5 and PHAA 12 before being inserted between the BamHI and M sites of pBS-SCPTB to construct pBS-SCPAB.
  • the DNA fragment containing GTP from Synechocystis 6803 was PCR amplified using primers GTP1 and GTP2 and was inserted between the Sacl and Pstl sites of pBS-SCat to construct pBS-SCG.
  • the DNA fragment PHAU from Synechocystis 6803 was PCR amplified using primers PHAUl and PHAU2 before being further PCR amplified using primers Ptac and PHAU2 to construct Ptac-PHA U.
  • Ptac-PHA U was then amplified using primers TAC5 and PHAU2 and the product was inserted between the BamHI and Kpnl sites of pBS-SCG to construct pBS-GCPU.
  • the DNA fragments SR56 and SL56 were PCR amplified using primer pairs SR5 and SR6 and SL5 and SL6 with Synechocystis 6803 genomic DNA as template. Fragments SR56 and SL56 were recombined together by overlapping PCR before being inserted into the Sacl and Xhol restriction sites of the plasmid pBluescript II SK(+) to construct pBS-S2.
  • pBS-S2 was digested with MM and Sail before being ligated with kan (Kan R ) which was amplified from pET-30a(+) (Novagen, Madison, WI) using primers Kanl and Kan2 to construct pBS-S2K.
  • Kan R kan
  • coli gene tesB was amplified with primers TESB 1 and primer TESB2 using the E. coli XL 1 -Blue MRF' genomic DNA as template.
  • Ptac promoter was PCR amplified with primers TAC11 and TACTESB1 using pBS-SPTH as template. The Ptac and tesB containing PCR products were then recombined by overlapping PCR using primers TAC1 1 and TESB2 to construct the fragment Ptac-tesB.
  • Ptac-tesB was digested with Bglll and Hindlll before being inserted between the corresponding sites of pBS-S2K to construct pBS- SPtTeK.
  • the DNA fragment PpasD56 was PCR amplified from the Synechocystis 6803 genomic DNA using primers PpsaD5 and PpsaD6.
  • the thil gene was PCR amplified from C. acetobutylicum ATCC 824 genomic DNA using primers Thl and Th2.
  • the PCR product was recombined with PpsaD56 by overlapping PCR using primers PpsaD5 and Th2 and the resultant PpsaD-thil product was inserted between the BamHI and Mlul sites of pBS-S2K to construct pBS-SPTK.
  • Ptac was amplified from pBS-SPTH using primers TAC5 and TAC-PTB3 and then inserted between the BamHI and Ndel sites of pBS-SPTK to construct pBS-SPtK.
  • the sacB gene was PCR amplified using primers SACB8 and SACB9 using B. subtillus genomic DNA as template.
  • the product was restriction digested and inserted between the Ndel and Mlul sites of the pBS-SPtK plasmid to construct pBS-SPSK2.
  • DNA fragments PHA1 and PHA2 were each PCR amplified from Synechocystis 6803 genomic DNA using primer pairs PHA11 and PHA12 and PHA21 and PHA22.
  • PHA1 and PHA2 were then recombined together by overlapping PCR using primers PHA11 and PHA22 to construct the DNA fragment PHA.
  • PHA was then inserted between the Xhol and Sacl sites of pBS-S2 to construct pBS-PHA.
  • the Ptac-sacB-kan fragment was removed from pBS-SPSK2 by digestion with BamHI and Sail and then inserted between the corresponding sites of pBS-PHA to construct pBS-SPSK3.
  • Synechocystis strains were grown to an OD7 30 of 0.2-0.4, at which time point 0.5 ml culture was pelleted by centrifugation at 2700 xg for 10 min at room temperature.
  • the cell pellet was re- suspended in 50 ⁇ fresh BG11 medium to which approximately 2 ⁇ g of the chromosome-targeting plasmid was added and mixed.
  • the mixture was incubated at 30°C under light (-25 ⁇ / ⁇ 2 /8) for 5 h before being plated on BG11 solid agar plates with appropriate antibiotics supplements, 10 ng/ ⁇ kanamycin or 5 ng/ ⁇ chloramphenicol. The plates were placed at 30 °C under light and colonies could be seen within two weeks.
  • the strain were grown in BG1 1 medium to an OD730 of 0.2-0.4, when cells were centrifuged at 2700xg for 10 min at room temperature and was resuspended to OD 730 of 4.0 by 50 ⁇ BG1 1.
  • About 2 ⁇ g of chromosome-targeting plasmid pBS-PHA was added and mixed well with the cells. The mixture was incubated at 30°C under light (25 ⁇ / ⁇ 2 /8) for 5 h before being transferred into 25 ml BG11 medium in a 50 ml flask.
  • RT-qPCR Approximately 1.67x l0 8 Synechocystis cells (assuming OD 730 of 0.6 equals to 10 8 cells/ml) were collected by centrifugation at 17,000 xg, 4°C for 1 min. The supernatant was discarded and the cell pellet was kept under -80°C until RNA extraction. Total RNA extraction, cDNA synthesis and RT-qPCR were conducted using methods described previously.
  • Enzyme activity assay 3.3> ⁇ 10 9 cells were collected by centrifugation at 5000 xg at 4°C for 10 min. The supernatant was discarded and the cell pellet was used either immediately or frozen at -80°C for assaying at a later date.
  • the cell pellet was first re-suspended in 1.0 ml 100-mM Tris-HCl (pH7.5) and then subjected to sonication in ice bath using a Branson Digital Sonifier Model 102C CE (Branson Ultrasonics, Danbury, CT) and Sonic Dismembrator Model 500 (Fisher Scientific, Waltham, MA) to lyse cells.
  • the sonication program consisted of: 3-sec-on / 3-sec-off for 100 cycles. Cellular debris was removed by centrifugation at 17,000 xg at 4°C for 10 min. The resultant supernatant was used for enzyme assays. [0036] The thiolase (encoded by phaA2, phaA or thil) activity was determined using acetoacetyl-CoA and CoA as substrates. The decrease in absorbance at 303 nm was monitored as function of time and specific enzyme activity was calculated by using a molar extinction coefficient of 14,000 M ⁇ cm "1 .
  • the decrease in absorbance at 340 nm was monitored over time and specific enzyme activity was calculated by using a molar extinction coefficient of 6,220 M _1 cm ⁇ ⁇
  • the thioesterase activity was determined using butyryl-CoA, decanoyl-CoA or acetyl-CoA as substrate and the release of CoA was monitored at 412 nm by using 5,5'- dithiobis(2-nitrobenzoic acid) (DTNB; Sigma-Aldrich, St. Louis, MO).
  • the molar extinction coefficient was taken as 13,600.
  • Thioesterase (TesB) activity specificity assay The thioesterase activities were examined using different acyl-CoA substrates including decanoyl-CoA (10 carbon acyl group), butyryl-CoA (4 carbon acyl group) and acetyl-CoA (2 carbon acyl group). The release of CoA was monitored at 412 nm by using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; Sigma-Aldrich, St. Louis, MO). The molar extinction coefficient was taken as 13,600.
  • DTNB 5,5'-dithiobis(2-nitrobenzoic acid)
  • Synechocystis strain TESB cells was collected after 5 days cultivation. The cell pellet was first re-suspended in 1.0 ml 100-mM Tris-HCl (pH7.5) and then subjected to sonication in ice bath using a Branson Digital Sonifier Model 102C CE (Branson Ultrasonics, Danbury, CT) and Sonic Dismembrator Model 500 (Fisher Scientific, Waltham, MA) to lyse cells. The sonication program consisted of: 3-sec-on / 3-sec-off for 100 cycles. Cellular debris was removed by centrifugation at 17,000 xg at 4°C for 10 min. The resultant supernatant was used for enzyme assays using same molar concentration of different acyl-CoA substrates.
  • strain XLl-Blue/pBS-SPtTeK cells was collected after ⁇ 10 hours cultivation in a 50 ml tube containing 10 ml LB medium at 37 °C, 200 rpm. After 10 h cultivation, the OD 600 of is. coli XLl-Blue/pBS-SPtTeK and E. coli XLl-Blue/pBS-S2K was 5.3 and 4.6, respectively, and thus 3.8 ml and 4.4 ml culture was pelletted for each before sonication. The cells were lysed using same method as described above.
  • the resultant supernatant was used for enzyme assays using same molar concentration of butyryl-CoA and acetyl-CoA as substrates.
  • E. coli XL1- Blue/pBS-S2K was used as a control in these enzyme assays.
  • Cell pellets were re-suspended in 10 ml BG11 containing 10 mM TES-NaOH (pH8.0) and 50 mM aHC0 3 in 50 ml-flasks with an initial cell density of OD7 30 of 2.0.
  • the initial pH of culture medium was adjusted to 7.5 by adding 10 N HCl.
  • 1 ml of the culture was sampled for analysis and replaced with 1 ml fresh BG1 1 containing 500 mM NaHC0 3 and 1.5 g/1, 0.75 g/1 and none of NaN0 3 , as appropriate.
  • Synechocystis was inoculated into a 125 ml flask containing 75 ml autoclaved BG11 (10 mM TES-NaOH) medium to an initial OD7 30 of 0.2.
  • the culture was placed at 30 °C with continuous illumination of 120 ⁇ / ⁇ 2 /8 and was bubbled with ambient air.
  • the aeration rate was initially set as 75 ml/min. When the culture OD730 surpassed about 0.6, the aeration rate was then increased to 250 ml/min. Daily, 1 ml of culture was sampled and 1 ml 5-fold concentrated sterilized BG1 1 medium was added back into the culture until day 18.
  • the mobile phase consisted of 5 mM H2SO4 at an initial flow rate of 0.55 ml/min before immediately and linearly increasing to a final flow rate of 0.8 ml/min over 12 min, followed by an 8 min hold.
  • the column temperature was maintained at 35°C throughout.
  • 3HB is the precursor for synthesizing the biodegradable plastics poly- ⁇ - hydroxybutyrate (PHB), as well as many chiral fine chemicals.
  • PHB poly- ⁇ - hydroxybutyrate
  • gene pairs from three different bacterial sources were comparatively examined: native Synechocystis slrl993 (phaA2) and slrl994 (phaB2) for (R)-3HB, phaA and phaB from Ralstonia eutropha H16 for (R)-3HB, and thil and hbd from Clostridium acetobutylicum ATCC824 for (5)-3HB.
  • coli thioesterase II encoded by tesB gene has been utilized in each scheme to directly hydrolyze (5)- or (R)-3-hydroxybutyryl-CoA to generate (5)- or (R)-3HB, respectively.
  • the resultant strains included HB5 and TAB 1, which respectively harbored thil and hbd from C. acetobutylicum ATCC 824 or phaA and phaB from Ralstonia eutropha HI 6.
  • a third strain TPU3 was constructed by placing a Ptac promoter just upstream of the native sir 1993 (phaA2)-slrl994 (phaB2) operon to enhance its expression (Fig. 1).
  • the Ptac promoter has been reported as a strong promoter in Synechococcus and Synechocystis and was used to initiate high-level expression of isobutanol biosynthetic genes in Synechococcus.
  • the Ptac promoter was used to express all of the 3HB pathway genes (Fig. 2). All foreign genes were integrated into the neutral sites of the Synechocystis genome where no effect was expected (Fig. 2). Additionally, because native PHB synthesis would compete with 3HB biosynthesis for the intermediate (R)-3-hydroxybutyryl-CoA (Fig.
  • strain TPU3 in which tesB as well as the native phaA2 and phaB2 were over-expressed with the use of the Ptac promoter, achieved no detectable increase of 3HB production compared to that of strain TESB.
  • Co-expression of tesB with thil and hbd of C. acetobutylicum (strain HB5) resulted in 33.2 mg/1 3HB production.
  • Production of 3HB was boosted to 45.1 mg/1 in the culture medium of strain TAB1 which co-expressed tesB with phaA and phaB of R. eutropha.
  • strain TABd achieved similar cell densities in about 5 days.
  • the cell density of the non-N and 5%-N supplementation cultures apparently started to decrease after day 7, which could be partially attributed to the daily sampling rather than severe collapse of the cell culture; in contrast, the 10%-N supplementation cultures were apparently able to maintain a relatively stable cell density after day 5.
  • Synechocystis would alkalize the culture medium and thus also cause stress to cells.
  • Synechocystis TABd exhibited durable and repeatable activity in continuous production of 3HB under our experimental condition.
  • the titers of 3HB in the culture medium could achieve repeatable linear increase after a 2-3 days lag phase at the beginning of each cycle and could finally reach 3HB titers of 191.0 ⁇ 10.3 mg/1 (for Cycle I) and 203.3 ⁇ 10.1 mg/1 (for Cycle II), respectively (Fig. 10B). Carbonate also is believed to be usable for the carbon source.
  • PCC6803 Wild-type ATCC TESB Ptac-fes_S- an R integrated at S2 site in Synechocystis 6803
  • TPU3 Ptac-fes_S- an R integrated at S2 site and Cm R -Ptac integrated at S4 site
  • HB5 Ptac-fes_S- an R integrated at S2 site and Cm R -Ptac-f zz7- z& ⁇ i integrated This study at SI site
  • pBS- -SCPTB phaB inserted between the MM and Hindlll sites of pBS-SCPTH2
  • pBS- -SCPAB Ftac-phaA inserted between the BamHI and MM sites of pBS-SCPTB
  • pBS- -SCG GTP fragment inserted between the Sacl and Pstl sites of pBS-SCat
  • pBS- ⁇ GCPU Ptac-PHAU inserted between the BamHI and Kpnl sites of pBS-SCG
  • pBS- -S2 SR56-SL56 inserted into the Sacl and Xhol sites of pBluescript II

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Abstract

Cette invention concerne l'élaboration et l'expression de voies de synthèse pour la production de (S) ou de (R)-3-hydroxybutyrate (3HB) à titre de produits énantiomères purs par modification génétique de la cyanobactérie Synechocystis sp. PCC 6803 (Fig. 2). Dans des conditions de croissance optimisées, la voie phaA et phaB de R. eutropha s'est avérée la plus efficace, produisant jusqu'à 533,4 ± 5,5 mg/l de (R)-3HB après 21 jours de culture photosynthétique. Pour la première fois, la faisabilité et l'efficacité élevée de la production de 3HB à l'aide de l'énergie solaire et de CO2 à titre de seule énergie et de sources de carbone par des cyanobactéries génétiquement modifiées est démontrée.
PCT/US2013/029997 2012-05-14 2013-03-08 Production photosynthétique de 3-hydroxybutyrate à partir de dioxyde de carbone WO2013172928A1 (fr)

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US14/479,893 US9944955B1 (en) 2012-05-14 2014-09-08 Photosynthetic production of 3-hydroxybutyrate from carbon dioxide
US15/938,742 US10246726B2 (en) 2012-05-14 2018-03-28 Photosynthetic production of 3-hydroxybutyrate from carbon dioxide

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US61/646,807 2012-05-14

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WO2017016902A1 (fr) * 2015-07-29 2017-02-02 Evonik Degussa Gmbh Production de 3-hydroxybutyrate
WO2017153734A1 (fr) * 2016-03-07 2017-09-14 Chain Biotechnology Limited Procédé et microbes pour la production de composés chiraux
CN111157520A (zh) * 2020-01-19 2020-05-15 江西理工大学 酸铜电镀液中聚二硫二丙烷磺酸钠的定量检测试剂及检测方法
US10844405B2 (en) 2013-09-18 2020-11-24 Arizona Board Of Regents On Behalf Of Arizona State University Enzymes and microorganisms for the production of 1,3-butadiene and other dienes
CN114196607A (zh) * 2021-12-29 2022-03-18 湖北大学 一种生产phb的运动发酵单胞菌的构建方法及其应用
US11613768B2 (en) 2017-07-25 2023-03-28 Arizona Board Of Regents On Behalf Of Arizona State University Microbial production of 2-phenylethanol from renewable substrates
US11708590B2 (en) 2017-07-18 2023-07-25 Arizona Board Of Regents On Behalf Of Arizona State University Synthetic metabolic funneling for biochemical production
US11898157B2 (en) 2019-06-05 2024-02-13 Arizona Board Of Regents On Behalf Of Arizona State University Systems and methods for genetic engineering of a polyploid organism

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10844405B2 (en) 2013-09-18 2020-11-24 Arizona Board Of Regents On Behalf Of Arizona State University Enzymes and microorganisms for the production of 1,3-butadiene and other dienes
WO2017016902A1 (fr) * 2015-07-29 2017-02-02 Evonik Degussa Gmbh Production de 3-hydroxybutyrate
WO2017153734A1 (fr) * 2016-03-07 2017-09-14 Chain Biotechnology Limited Procédé et microbes pour la production de composés chiraux
JP2019509043A (ja) * 2016-03-07 2019-04-04 チェイン バイオテクノロジー リミテッド 光学活性物質の生産のための方法および微生物
US11708590B2 (en) 2017-07-18 2023-07-25 Arizona Board Of Regents On Behalf Of Arizona State University Synthetic metabolic funneling for biochemical production
US11613768B2 (en) 2017-07-25 2023-03-28 Arizona Board Of Regents On Behalf Of Arizona State University Microbial production of 2-phenylethanol from renewable substrates
US11898157B2 (en) 2019-06-05 2024-02-13 Arizona Board Of Regents On Behalf Of Arizona State University Systems and methods for genetic engineering of a polyploid organism
CN111157520A (zh) * 2020-01-19 2020-05-15 江西理工大学 酸铜电镀液中聚二硫二丙烷磺酸钠的定量检测试剂及检测方法
CN114196607A (zh) * 2021-12-29 2022-03-18 湖北大学 一种生产phb的运动发酵单胞菌的构建方法及其应用

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