FROM CARBON DIOXIDE

Cr oss-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 61/646,807 filed on May 14, 2012.

Background of the Invention

[0002] Although human society has progressed significantly over past centuries through the development and use of petroleum-derived products (e.g. fuels, plastics, solvents, etc.), their over-utilization has caused environmental issues including increasing atmospheric concentration of CO ₂ (a greenhouse gas), pollution from petrochemical production and use, and disposal of non-biodegradable plastic materials. More importantly, petroleum resources are finite and not renewable in nature. For these reasons it is necessary to seek alternative approaches to produce fuels and chemicals using renewable resources. Photosynthetic cyanobacteria have attracted significant attention in recent years as a 'microbial factory' to produce biofuels and chemicals due to their capability to utilize solar energy and C0 ₂ as the sole energy and carbon sources, respectively.

[0003] 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). Despite efforts to enhance PHB biosynthesis through both genetic engineering strategies and the optimization of culture conditions, 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. Most importantly, as neither lipids nor PHB are secreted by the cells, the required processes for their extraction are energy-intensive and remain as one of the major hurdles for commercial applications. As a result, researchers have recently focused on engineering cyanobacteria to instead produce secretable biofuels and chemicals. However, most production titers are below 200 mg/1 and to our knowledge no report demonstrated the potential of employing photosynthetic microorganisms in a continuous production process.

Summary of the Invention

[0004] Different from PHB, which accumulates inside cells, 3-hydroxybutyrate (3HB) 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. Moreover, (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. [0005] 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.

[0006] Various other purposes and advantages of the invention will become clear from its description in the specification that follows. Therefore, to the accomplishment of the objectives described above, this invention includes the features hereinafter fully described in the detailed description of the preferred embodiments, and particularly pointed out in the claims. However, such description discloses only some of the various ways in which the invention may be practiced. Brief Description of the Figures

[0007] 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. eutropha H16; PhaB2 (Slrl994), native (R)-3-hydroxybutyryl-CoA dehydrogenase in Synechocystis 6803; TesB, thioesterase II from E. coli XLl-Blue MRF'; PhaEC, native PHB polymerase in Synechocystis 6803.

[0008] 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).

[0009] Fig. 3 depicts the cell density of different Synechocystis strains cultivated in shaking flasks.

[0010] 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. [0011] 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 μΕ/ιη ² /8), middle light intensity (ML, -60 μΕ/ιη ² /8) and high light intensity (HL, -90 μΕ/ιη ² /8) were studied.

[0012] 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. [0013] 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.

[0014] 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.

[0015] Fig. 9 depicts, after 10 days culture, chlorosis in the non-N and 5%-N (shown) cultures but not in the 10%-N cultures.

[0016] Fig. 10 depicts biomass and 3HB production curves of Synechocystis strain TABd under 5%N-supplementation condition for 3HB production by medium exchange. [0017] 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 .

Detailed Description of the Invention

[0018] In a first aspect, 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. [0019] In a second aspect, this disclosure relates to highly efficient photosynthetic production of 3HB using bicarbonate or CO ₂ as the sole carbon source by engineered Synechocystis. [0020] In a third aspect, this disclosure relates to biosynthesis of 3HB in a process coupled with oxygenic photosynthesis in engineered Synechocystis.

[0021] In a fourth aspect, this disclosure relates to highly efficient secretion of hydrophilic 3HB molecules by engineered Synechocystis without overexpression of specific transporters.

[0022] In a fifth aspect, 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.

[0024] While the embodiments described below utilize Synechocystis, those of ordinary skill will appreciate that other cyanobacterial species may be engineered to produce 3HB following the strategies and genetic engineering guidance provided herein. Therefore, this disclosure is not limited to 3HB production from Synechocystis but rather extends to 3HB production from all cyanobacteria capable of genetic manipulation of 3HB biosynthesis pathways. [0025] Construction and expression of synthetic pathways to produce (S) or (R)-3- hydroxybutyrate (3HB) as enantiomerically-pure products using cyanobacterium Synechocystis sp. PCC 6803 was undertaken as described below. However, this disclosure is not limited to the exact methods and materials described. [0026] 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 μΕ/ιη 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. coli were cultivated in Luria-Bertani (LB) medium at 37°C. Antibiotics were supplemented as appropriate at the following concentrations: 100 ng/μΐ ampicillin, 30 ng/μΐ kanamycin, and 25 ng/μΐ chloramphenicol. 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. From the genomic DNA of Clostridium acetobutylicum ATCC 824, 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. Next, 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. Two fragments of the cat (Cm ^R ) gene on pACYC 184 (New England Biolabs, Ipswich, MA) were amplified using primer pairs Cat3 and Cat4, Cat5 and Cat6, and then were recombined by overlapping PCR using primers Cat3 and Cat6 to remove the Ncol restriction site in the open reading frame. The Ncol-removed cat gene was then inserted between the Pstl and BamHI sites of pBS-SRSL to construct pBS- SCat. 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. The R. 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.

[0028] 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.

[0029] 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. The E. 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. [0030] 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. Fragments 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. [0031] Modification of Synechocystis genome. 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 μΐ fresh BG11 medium to which approximately 2 μg of the chromosome-targeting plasmid was added and mixed. The mixture was incubated at 30°C under light (-25 μΕ/ιη ² /8) for 5 h before being plated on BG11 solid agar plates with appropriate antibiotics supplements, 10 ng/μΐ kanamycin or 5 ng/μΐ chloramphenicol. The plates were placed at 30 °C under light and colonies could be seen within two weeks. Individual colonies were then isolated and re-streaked on BG1 1 solid agar plates with appropriate antibiotics for additional one to two weeks to achieve full chromosome segregation, as was verified by colony PCR. Alternatively, markerless modification of the Synechocystis genome was conducted using the method described previously with minor modifications. [0032] Briefly, fragment Ptac-sacB-kan was inserted into the neutral site of Synechocystis 6803 using a marker modification method as described in the text. After confirming that the resultant strain was genotypically pure as verified using colony PCR, 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 μΐ BG1 1. About 2 μg of chromosome-targeting plasmid pBS-PHA was added and mixed well with the cells. The mixture was incubated at 30°C under light (25 μΕ/ιη ² /8) for 5 h before being transferred into 25 ml BG11 medium in a 50 ml flask. Cells were then further cultivated for 4-5 days after which about 1.3 ^χ 10 ⁸ cells (assuming OD ₇₃₀ of 0.6 equals to 10 ⁸ cells/ml (3)) were spread onto a BG1 1 plate containing 4.5% (w/v) sucrose for counter-selection. The plates were incubated at 30°C under light for one or two weeks before colonies appeared. Individual colonies were then re-streaked on fresh BG11 plates with 4.5% sucrose for additional one to two weeks until full chromosome segregation was achieved, as verified by colony PCR.

[0033] Gene expression analysis: Synechocystis strains were inoculated in 50 ml flasks, each containing 10 ml BG1 1 (10 mM TES-NaOH), to an initial OD7 ₃₀ of 1.5. Then cells were incubated in a shaking bed (150 rpm) at 30 °C with light intensity of 35 μΕ/ιη ² /8 for 5 days. Every 24 h, 0.5 ml 1.0M aHC03 was added to each culture and the pH of the culture medium was adjusted to 7.5 by addition of 10 N HC1.

[0034] 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.

[0035] 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. For all enzyme assays, 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 activity of (R)-3-hydroxybutyryl- CoA dehydrogenase (encoded by phaB2 or phaB) was determined using acetoacetyl- CoA and NADPH as substrates. The activity of (5)-3-hydroxybutyryl-CoA dehydrogenase (encoded by hbd) was determined using acetoacetyl-CoA and NADH as substrates. 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.

[0037] 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.

[0038] For TesB assay in Synechocystis , 20 OD ₇₃ o-mL 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.

[0039] For TesB assay in E. coli, 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.

[0040] 3HB production. Each strain was inoculated into 20 ml BG11 medium in a 50 ml flask at an initial OD7 ₃₀ of 0.1, and then grown photosynthetically to an OD7 ₃₀ of 0.5-1.0 before aHC03 was then added to a final concentration of 50 mM. When the cell density reached an OD ₇₃₀ of 1.0-2.0, cells were collected by centrifuging at 5000 xg for 10 min at 20 °C. Cell pellets were re-suspended in 10 ml of fresh BG11 containing 50 mM aHC0 ₃ in 50 ml flasks to a cell density of an OD7 ₃₀ of 1.5. The pH of the medium was adjusted to 7.5. The re-suspended cells were then incubated in a shaking bed (150 rpm) at 30 °C with light intensity of 35 ιΕΙντι Ί$ for 5 days. Every 24 h, 0.5 ml l .OM NaHCC^ was added to each culture and the pH of the culture medium was adjusted to 7.5 by addition of 10 N HCl. All culture experiments were conducted in triplicate for each strain. [0041] Nitrogen limitation test. Synechocystis TABd was grown to an OD ₇₃₀ of 1.0-2.0 as described above before the cells were pelleted and collected. 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. Once daily, 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. Thus, 50 mM fresh NaHC0 ₃ and either 10%, 5% or 0% of fresh NaN0 ₃ were added to the culture medium each day (corresponding to 10%-N, 5%-N or non-N culture, respectively). [0042] Production of 3HB by intermittent medium exchange. After 10 days cultivation (Cycle I) using the 5%-N supplementation strategy as stated above, Synechocystis TABd cells were collected and re-suspended in 10 ml fresh BGl l-lON (10 mM TES-NaOH, pH8.0) medium that contained only 10% of the NaN0 ₃ content in typical BG1 1 medium to re-initiate cultivation in 50 ml flasks for a second 10 days (days 11 through 20; called Cycle II). During this period, 250 μΐ of cell culture was sampled for analysis each day. After sampling, 250 μΐ fresh BG11 medium, 10 μΐ 37.5 g/1 NaN0 ₃ (equivalent to the nitrate content of 250 μΐ BG11) and 250 μΐ 2.0 M aHC0 ₃ were added back into the culture. Note that since evaporative water losses from the culture were calculated to be about 260 μΐ per day, the above supplementation protocol was used to maintain the total culture volume. This protocol resulted in the daily addition of 5% of the NaNC^ to the culture medium. [0043] Photosynthetic 3HB production from CO ₂ . 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 μΕ/Γη 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. After day 18, 1 ml of culture was sampled but no BG1 1 medium was added back into the culture. The experiments were conducted in duplicates. [0044] Product quantification. Standard solutions of 3HB were prepared in water using (±)-3-Hydroxybutyric acid sodium salt. Samples of the culture medium were centrifuged at 17,000 xg for 2 min at room temperature and the supernatant was collected for analysis of products on an 1 100 series HPLC equipped with a refractive index detector (Agilent, Santa Clara, CA). Separation of metabolites was achieved using an Aminex HPX-87H anion-exchange column (Bio Rad Laboratories, Hercules, CA). 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.

[0045] 3HB is the precursor for synthesizing the biodegradable plastics poly-β- hydroxybutyrate (PHB), as well as many chiral fine chemicals. For the first two steps in the constructed pathways, namely thiolase and 3-hydroxybutyryl-CoA dehydrogenase, 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. The final step of all pathways consisted of thioesterase II (encoded by tesB) from Escherichia coli. To facilitate carbon flux towards 3HB, slrl829 and slrl830 (encoding PHB polymerase) were deleted from Synechocystis to eliminate PHB production. [0046] Construction of 3HB- reducing strains. A series of strains were constructed to systematically explore the photosynthetic production of (5)- and (R)- 3HB by engineered Synechocystis (Table 1) using standard molecular biology protocols. The general strategy is presented in Fig. 1. The E. 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. We first introduced the tesB gene from E. coli into the genome of Synechocystis 6803 to construct strain TESB which was expected to realize the conversion of (R)- or («S)-3- hydroxybutyryl-CoA to corresponding 3-HB. To complete the 3HB biosynthesis pathways, we next introduced three different sets of operons into Synechocystis TESB to express both thiolase and 3-hydroxybutyryl-CoA dehydrogenase. 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).

[0047] 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. Here, 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. 1), the native operon harboring phaE (sir 1829) and phaC (sir 1830) which encodes for the PHB polymerase (35, 36) was deleted from the Synechocystis genome (through an intermediate strain Synechocystis SPA; Table 1) to obtain Synechocystis SPA:AphaEC strain. Next, three sets of 3HB pathway genes were each introduced into this PHB polymerase-deletion strain in the same way as described above, resulting in Synechocystis TESBd, HBd, TABd and TPUd. The genotypic purity of each strain was confirmed by colony PCR in all cases.

[0048] Expression of 3HB biosynthetic genes in Synechocystis. The 16S rRNA of both wild-type and engineered Synechocystis strains was used as the reference to calculate the ACT values for individual genes when performing the RT-qPCR analysis. RT-qPCR analysis showed that all target genes were successfully transcribed in all engineered strains, and that no detectible phaE or phaC expression was observed in any of the AphaEC strains (i.e., TESBd, TPUd, HBd, TABd) (Table 2). In addition, by introducing a Ptac promoter upstream of the native phaA2-phaB2 operon, expression of phaA2 and phaB2 was enhanced in TPU3 by nearly 6- and 120-fold, respectively. Similarly, expression of phaA2 and phaB2 was enhanced by about 4- and 90-fold, respectively, in strain TPUd through the addition of Ptac.

[0049] Thus, it was found that the Ptac promoter could be used to effectively transcribe all 3HB pathway genes in Synechocystis 6803 and its derivatives. Enzyme activity assay results (Table 3) revealed that thiolase activities of the enhanced- expressed native PhaA2 and R. eutropha PhaA were 1.14=1=0.14 U ^mol/min/ml cell extract) and 13.12=1=3.36 U, respectively. It is notable that the thiolase activity of PhaA was about 12-fold higher than that of PhaA2. In contrast, no thiolase activity was detected for the C. acetobutylicum Thil. (R)-3-hydroxybutyryl-CoA dehydrogenase activity was detected for PhaB with a value of 0.23±0.15 U, but not for enhanced- expressed PhaB2. (5)-3-hydroxybutyryl-CoA dehydrogenase activity was negative in cell extract of strain HB5 (data not shown). The thioesterase activity reached a value of 0.484±0.044 U using decanoyl-CoA as substrate but 6-fold lower, 0.084±0.021 U, when using butyryl-CoA as substrate (Table 4), which was consistent with the report that TesB biases medium- and long-chain fatty acyl-CoA substrates.

[0050] Production of 3HB by the engineered cyanobacteria. After wild-type Synechocystis and eight engineered strains were grown photosynthetically for five days, they all reached similar OD ₇₃₀ of 8.5-9.0 (Fig. 3). The culture broth was then sampled for HPLC analysis. Under the examined growth conditions, wild-type Synechocystis generated and secreted into the culture medium up to 15.5 mg/1 3HB. The introduction and expression of tesB alone in Synechocystis (strain TESB) led to a final 3HB titer of about 20.6 mg/1. Interestingly, 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. Deletion of phaE and phaC further improved 3HB production, as indicated by the ability of strain TABd to reach final titers of about 100 mg/1, nearly 6.5-fold higher than that of the wild-type (Fig. 4). [0051] Notably, expression of E. coli tesB also resulted in a dramatic increase of acetate production by our engineered Synechocystis relative to the wild-type (Fig. 4), consistent with the previous report about the tes5-over-expressed recombinant E. coli. Further experimental results indicated that TesB can catalyze the hydrolysis of acetyl- CoA to acetate with a 33 -fold lower activity than that in hydrolysis of butyryl-CoA (Table 5]). Nevertheless, with expression of the R. eutropha phaA and phaB in strains TAB1 and TABd (Table 1), the acetate production was significantly reduced (Fig. 4). This is probably due to relatively high activities of PhaA and PhaB which drove an increasing portion of acetyl-CoA to form (R)-3-hydroxybutyryl-CoA (Fig. 1); the latter in turn outcompeted acetyl-CoA as substrate for hydrolysis (by TesB), resulting in increase of 3HB production and decrease of acetate biosynthesis.

[0052] Improving 3HB production by nutrient supplementation. Studies found that nutrient starvation, specifically nitrogen or phosphate depletion, favors PHB accumulation in cyanobacteria. Thus, under these conditions, metabolic flux towards the common pathway intermediate (R)-3-hydroxybutyryl-CoA (3HB pathway & PHB pathway; Fig. 1) should also be increased which might be leveraged to increase 3HB production. We subjected Synechocystis TABl cells to nitrogen starvation and normal BG1 1 medium cultures. The results showed that nitrogen-starved cells were able to produce 3HB at higher titers than that of the control (TABl grown in BGl 1) during the first two days. However, 3HB production by the control then dramatically increased from day 3, continuing on through day 6 (Fig. 5), resulting in overall higher 3HB production than that of the nitrogen-starved counterparts (Fig. 6). Meanwhile, the cell density of nitrogen-starved culture started a gradual decline after day 2, whereas the cell density of the control kept increasing until day 5 (Fig. 6). Once a little amount of nutrient was supplemented into the culture after day 7, both biomass and 3HB production started to increase again (Fig. 7). [0053] From the above, it seemed low level of nutrient (herein most importantly nitrate) might positively support both 3HB production and cell viability, which would lead to increased 3HB production. As shown in Fig. 8A, under all nutrient supplementation conditions, 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.

[0054] Notably, after 10 days culture, chlorosis occurred in the non-N and 5%-N cultures but not in the 10%-N culture (Fig. 9), suggesting pigmented proteins had been degraded in the former cultures (40). Nevertheless, cells from the yellow non-N and 5%-N cultures still maintained viability as they could turn back to green the next day after being re-suspended in fresh BGl 1 or BGl 1-10%N (containing merely 10% of the nitrate relative to BG1 1) medium, respectively (Fig. 9). Under all three culture conditions, 3HB production firstly underwent a 2-3 days lag phase and then started to increase dramatically. The non-N and 5%-N cultures continued this increase of 3HB titers until day 8 after which 5%-N cultures exhibited a slight increase of 3HB titers while non-N cultures showed a slight decrease of 3HB titers. In contrast, the 10%-N cultures exhibited a much lower 3HB production rate compare to that of 5%-N after day 6. Eventually, The 3HB production achieved titers of 152.7±9.9 mg/1 in the 10%-N cultures, 155.9±2.2 mg/1 in the non-N cultures and 191.0±10.3 mg/1 in the 5%-N cultures (Fig. 8B), indicating that compared to non-N and 10%-N supplementation strategy, 5%-N supplementation is more preferable for 3HB production under the examined culture conditions.

[0055] 3HB production from bicarbonate by intermittent medium exchange.

Since 3HB can be secreted out of cells and our engineered Synechocystis cells still maintained viability after 10 days cultivation, we next examined the feasibility of applying engineered Synechocystis in a continuous production mode where cells from the former 3HB production cycle (Cycle I) can still be used for 3HB production in a latter cycle (Cycle II). We replaced the culture broth with fresh culture medium at the end of each 10-day cultivation cycle for two reasons. First, daily supplementation of NaHC0 ₃ would result in increasing Na ⁺ ion in culture medium which would cause salt stress and therefore impair the cellular activity of cyanobacterial cells. Second, a _Ssimi ,a,io» of HCO, ^" (4H ₂ 0 ₊ 4HCO, ^" ÷ C ₄ H _s O, ₊ 40H ^" ₊ £ <¾, and NO, (NO, ₊

3H ₂ 0 - NH ₄ ⁺ + 20H ^" + 20 ₂ ) by Synechocystis would alkalize the culture medium and thus also cause stress to cells. As a result, 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.

[0056] 3HB production from atmospheric CO2. The ability of Synechocystis strain TABd to photosynthetically produce 3HB using CO ₂ as sole carbon source was then investigated by continuous aeration of cultures with ambient air. Upon overcoming a lag phase of nearly one week (during which significant biomass growth was observed), 3HB production by Synechocystis TABd then quickly accelerated, achieving a titer of 446.5±31.0 mg/1 after 18 days of continuous cultivation. At this point, daily BG11 addition into the culture was arrested from days 19 through 21 to probe its effect on continued 3HB production. As can be seen in Fig. 1 1, 3HB titers continued to increase regardless, and reached a final titer of 533.4±5.5 mg/1 by the end of day 21. [0057] It should be noted that at this point, there was no indication that 3HB production would stop; however, we merely elected to stop the experiment. From Fig. 1 1, it was also observed that starting from day 7, as cell growth declined 3HB production rates were found to dramatically increase (Fig. 7). The relationship here between biomass growth and 3HB production rate is consistent with the results of the former experiments in which aHC0 ₃ was used as the sole carbon source (Figs. 8, 6 and 7).

[0058] The above observations pointed to the possibility of using engineered Synechocystis for continuous 3HB production. By following a medium exchange protocol, stable and continuous 3HB production was maintained for a total of 20 days (i.e., two 10-day cycles), resulting in final 3HB titers of -200 mg/1 at the end of each cycle (Fig. 10B). The experiments were stopped after 10 days cultivation due to increasing stresses such as increasing salt concentration and increasing pH in the culture broth. A longer period 3HB production process was developed by using atmospheric CO ₂ rather than bicarbonate as carbon source, and the results showed that a titer of 533.4±5.5 mg/1 3HB in the culture broth was achieved after 21 days cultivation when cells still kept viability (Fig. 11).

[0059] Both of the experiments above demonstrated that by being cultivated and immobilized in a properly controlled photo-bioreactor system, our engineered Synechocystis strains could be employed into a continuous process for 3HB production using only C0 ₂ , water and low-cost inorganic compounds as feed stocks and sun light as the energy source. We are expecting that such a carbon-neutral and sustainable process would significantly decrease the manufacture cost in production of 3HB as well as other useful chemicals that can be expanded to. Table 1. Strains and plasmids used in this disclosure

Genotype* Reference

Strains

E. coli XL1- A(mcrA)183 A(mcrCB-hsdSMR-mrr)173 endAl supE44 thi-l recA l Stratagene Blue MRF' gyrA96 relAl lac [F'proAB lac ZAM15 ΊηΙΟ (Tef)]

B. subtilis 168 ATCC Synechocystis

PCC6803 Wild-type ATCC TESB Ptac-fes_S- an ^R integrated at S2 site in Synechocystis 6803 This study TPU3 Ptac-fes_S- an ^R integrated at S2 site and Cm ^R -Ptac integrated at S4 site This study HB5 Ptac-fes_S- an ^R integrated at S2 site and Cm ^R -Ptac-f zz7- z&<i integrated This study at SI site

TAB1 Ptac-fes_S- an ^R integrated at S2 site and Cvc -Ptac-phaA-phaB This study integrated at S 1 site

SPA Ftac-adhe2 integrated at S2 site Stored in lab

SPA:SPS 3 Ptac-adhe2 integrated at S2 site and Ptac-sac5- an ^R integrated at S3 This study site

SPA:AphaEC Ftac-adhe2 integrated at S2 site, phaE and phaC deleted at S3 site This study

TESBd phaE and phaC deleted at S3 site, Ptac-fes_S- an ^R integrated at S2 site This study

TPUd phaE and phaC deleted at S3 site, Ptac-fes_S- an ^R integrated at S2 site, This study

Cm ^R -Ptac integrated at S4 site

HBd phaE and phaC deleted at S3 site, Ptac-fes_S- an ^R integrated at S2 site, This study

Cvc -Ftac-thil-hbd integrated at S4 site

TABd phaE and phaC deleted at S3 site, Ptac-fes_S- an ^R integrated at S2 site, This study

Cn^-FXac-phaA-phaB integrated at S4 site

Plasmids

pBlue script II Amp ^R , pUC ori, fl(+) ori Stratagene

S (+)

pACYC184 Cm ^R , Tet ^R , pl5A ori New

England BioLabs pET-30a(+) an ^R , lad, pBR322 ori, fl ori Novagen pETphaAphaB phaA,phaB integrated between the Ncol and Avrll sites of pETDuet-1 Nielsen's

(Amp ^R , pBR322 ori) Lab pBS- ■SRSL SR12-SL12 inserted between the Sacl and pnl sites of pBluescript II This study

S (+)

pBS- ■SPT Ftac-thil integrated between the BamHI and Sail sites of pBS-SRSL This study pBS- ■SPTH hbd integrated between the Ncol and Sail sites of pBS-SPT This study pBS- -SCat Ncol-removed cat (Cm ^R ) integrated into the Pstl and BamHI sites of

pBS-SRSL This study pBS- -SCPTH Ftac-thil-hbd integrated between the Ncol and Sail sites of pBS-SPT This study pBS- ■SCPTH2 M site added between Ml and hbd of pBS-SCPTH

pBS- -SCPTB phaB inserted between the MM and Hindlll sites of pBS-SCPTH2 This study pBS- -SCPAB Ftac-phaA inserted between the BamHI and MM sites of pBS-SCPTB This study pBS- -SCG GTP fragment inserted between the Sacl and Pstl sites of pBS-SCat This study pBS- ■GCPU Ptac-PHAU inserted between the BamHI and Kpnl sites of pBS-SCG This study pBS- -S2 SR56-SL56 inserted into the Sacl and Xhol sites of pBluescript II

SK(+) This study pBS- ■S2K kan (Kan ^R ) inserted between MM and Sail sites of pBS-S2 This study pBS- ■SPtTeK Ptac-fasi? integrated between Bglll and Hindlll site of pBS-S2K This study pBS- ■SPTK PpsaD-f integrated between the BamHI and MM sites of pBS-S2K This study pBS- ■SPtK Ptac inserted between the BamHI and Ndel sites of pBS-SPTK This study pBS- ■SPSI 2 sacB inserted between the Ndel and M sites of the pBS-SPtK This study pBS- ■PHA PHA inserted between the Xhol and Sacl sites of pBS-S2 This study pBS- -SPSK3 Ftac-sacB-kan of pBS-SPSK2 inserted between BamHI and Sail of This study

pBS-PHA

*S1, the site on the genome of Synechocystis 6803 between slrl495 and slll397; S2, the site between slrl362 and sill 274; S3, the site between slrl828 and sill 736; S4, the site between slrl992 and phaA2. Table 2. Expression analysis of 3HB pathway genes by RT-qPCR*

Gene AC _T

WT TPU3 HB5 TAB1 TESBd TPUd HBd TABd tesB ^~ 10.87±0. 10.37±0. 1 1.19±0. 10.44±0. 12.05±0. 10.83±0. 10.94±0. n ^{' '} 07 22 1 1 12 24 15 12 phaA 20.85±0. 18.17±0. 18.75±0.

2 06 16 ^{" " "} 26

phaA 12.24±0. 12.04±0.

n. _d . _ _ _{o i} _ _ _

thil 15.28±0. 15.30±0.

n ^{d "} 09 ^{" " "} 46

phaB 19.30±1. 12.42±0. 12.84±0.

2 07 28 ^{" " "} 07

phaB 12.25±0. 12.50±0.

n. _d . _ _ _ _ _ _o5 hbd 17.14±0. 17.62±0.

n. _d . _ _ _ _ _oi

phaE 15.52±0. 15.76±0

n.d. n.d. n.d. n.d.

28 26

17.17±0. 17.20±0.

n.d. n.d. n.d. n.d. 48 30

*"n.d.", "not detectable". "— ", experimental data was not available. The relative abundance of different mR A molecules could be estimated using 2 ^"AC _T ; the bigger the AC _T value, the lower abundance of the corresponding mRNA is.

Table 3. Enzyme activities for engineered strains.

Examined Strains with same expression cassette

Enzyme (gene) Activities*

strain

Thiolase (phaA2) 1.14±0.14 TPU3 TPUd

Thiolase (thil) n.d. HB5 HBd

Thiolase (phaA) 13.12±3.36 TAB 1 TABd

(R)-3 -Hydro xybutyryl-

CoA dehydrogenase n.d. TPU3 TPUd

(phaB2)

(iS)-3-Hydroxybutyryl-

CoA dehydrogenase n.d. HB5 HBd

(hbd)

(R)-3 -Hydro xybutyryl-

CoA dehydrogenase 0.23±0.15 TAB1 TABd

(phaB)

TPU3, HB5, TAB1, TESBd, TPUd, HBd,

Thioesterase (tesB) 0.084±0.021 TESB

TABd

*Enzyme activities were given in μmol/min/mL cell extract; "n.d." stands for "not detectable". "— " means experimental data was not available. Table 4.

Substrate Thioesterase (TesB) activities* Examined strain

Decanoyl-CoA 0.484±0.044 Synechocystis strain TESB

Butyryl-CoA 0.084±0.021 Synechocystis strain TESB

Acetyl-CoA n.d. Synechocystis strain TESB

Table 5.

Substrate Thioesterase (TesB) activities* Examined strain

E. co/z XLl-Blue/pBS-

Butyryl-CoA 10.451±1.924

SPtTe

E. co/z XLl-Blue/pBS-

Acetyl-CoA 0.319±0.022

SPtTe

Butyryl-CoA n.d. E. coli XLl-Blue/pBS-S2

Acetyl-CoA n.d. E. coli XLl-Blue/pBS-S2

* Enzyme activities were \ given in μηιοΙ/ηιίη/ηιΕ cell extract; "n.d." stands for "not detectable".

[0060] All embodiments of any aspect of the invention can be combined with other embodiments of any aspect of the invention unless the context clearly dictates otherwise.

[0061] Various changes in the details and components that have been described may be made by those skilled in the art within the principles and scope of the invention herein described in the specification and defined in the appended claims. Therefore, while the present invention has been shown and described herein in what is believed to be the most practical and preferred embodiments, it is recognized that departures can be made therefrom within the scope of the invention, which is not to be limited to the details disclosed herein but is to be accorded the full scope of the claims so as to embrace any and all equivalent processes and products.