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Durall de la Fuente, C., Lindberg, P., Yu, J. & Lindblad, P. (2020). Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803. Biotechnology for Biofuels, 13, Article ID 16.
Open this publication in new window or tab >>Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803
2020 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 13, article id 16Article in journal (Refereed) Published
Abstract [en]

Background: Cyanobacteria can be metabolically engineered to convert CO2 to fuels and chemicals such as ethylene. A major challenge in such efforts is to optimize carbon fixation and partition towards target molecules.

Results: The efe gene encoding an ethylene-forming enzyme was introduced into a strain of the cyanobacterium Synechocystis PCC 6803 with increased phosphoenolpyruvate carboxylase (PEPc) levels. The resulting engineered strain (CD-P) showed significantly increased ethylene production (10.5 +/- 3.1 mu g mL(-1) OD-1 day(-1)) compared to the control strain (6.4 +/- 1.4 mu g mL(-1) OD-1 day(-1)). Interestingly, extra copies of the native pepc or the heterologous expression of PEPc from the cyanobacterium Synechococcus PCC 7002 (Synechococcus) in the CD-P, increased ethylene production (19.2 +/- 1.3 and 18.3 +/- 3.3 mu g mL(-1) OD-1 day(-1), respectively) when the cells were treated with the acetyl-CoA carboxylase inhibitor, cycloxydim. A heterologous expression of phosphoenolpyruvate synthase (PPSA) from Synechococcus in the CD-P also increased ethylene production (16.77 +/- 4.48 mu g mL(-1) OD-1 day(-1)) showing differences in the regulation of the native and the PPSA from Synechococcus in Synechocystis.

Conclusions: This work demonstrates that genetic rewiring of cyanobacterial central carbon metabolism can enhance carbon supply to the TCA cycle and thereby further increase ethylene production.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-392232 (URN)10.1186/s13068-020-1653-y (DOI)000513591700001 ()32010220 (PubMedID)
Funder
NordForsk, 82845
Available from: 2019-09-01 Created: 2019-09-01 Last updated: 2020-03-25Bibliographically approved
Durall de la Fuente, C., Kanchugal Puttaswamy, S., Selmer, M. & Lindblad, P. (2020). Phosphoenolpyruvate carboxylase in Synechococcus PCC 7002: Oligomerization, structure, and characteristics. Scientific Reports, 10, Article ID 3607.
Open this publication in new window or tab >>Phosphoenolpyruvate carboxylase in Synechococcus PCC 7002: Oligomerization, structure, and characteristics
2020 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 10, article id 3607Article in journal (Refereed) Published
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-392227 (URN)10.1038/s41598-020-60249-2 (DOI)
Available from: 2019-09-01 Created: 2019-09-01 Last updated: 2020-03-10Bibliographically approved
Jin, H., Lindblad, P. & Bhaya, D. (2019). Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803. ACS Synthetic Biology, 8(4), 655-660
Open this publication in new window or tab >>Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803
2019 (English)In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 8, no 4, p. 655-660Article in journal (Refereed) Published
Abstract [en]

To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high, GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc10 and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2019
Keywords
Synechocystis sp. PCC6803, T7 RNA polymerase/T7 promoter circuit, orthogonal promoter, nickel inducible promoter
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-383202 (URN)10.1021/acssynbio.8b00515 (DOI)000465487100006 ()30935196 (PubMedID)
Available from: 2019-07-23 Created: 2019-07-23 Last updated: 2019-07-23Bibliographically approved
Hing, N. Y., Liang, F., Lindblad, P. & Morgan, J. A. (2019). Combining isotopically non-stationary metabolic flux analysis with proteomics to unravel the regulation of the Calvin-Benson-Bassham cycle in Synechocystis sp. PCC 6803. Metabolic engineering, 56, 77-84
Open this publication in new window or tab >>Combining isotopically non-stationary metabolic flux analysis with proteomics to unravel the regulation of the Calvin-Benson-Bassham cycle in Synechocystis sp. PCC 6803
2019 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 56, p. 77-84Article in journal (Refereed) Published
Abstract [en]

Photosynthetic microorganisms are increasingly being investigated as a sustainable alternative to existing bio-industrial processes, converting CO2 into desirable end products without the use of carbohydrate feedstock. The Calvin-Benson-Bassham (CBB) cycle is the main pathway of carbon fixation metabolism in photosynthetic organisms. In this study, we analyzed the metabolic fluxes in two strains of the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) that overexpressed fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) and transketolase (TK), respectively. These two potential carbon flux control enzymes in the CBB cycle had previously been shown to improve biomass accumulation when overexpressed under air and low light (15 mu mol m(-2) s(-1)) conditions (Liang and Lindblad, 2016). We measured the growth rates of Synechocystis under atmospheric and high (3% v/v) CO2 conditions at 80 mu mol m(-2) s(-1). Surprisingly, the cells overexpressing transketolase (tktA) demonstrated no significant increase in growth rates when CO2 was increased, suggesting an altered carbon flux distribution and a potential metabolic bottleneck in carbon fixation. Moreover, the tktA strain had an increased susceptibility to oxidative stress under high light as revealed by its chlorotic phenotype under high light conditions. In contrast, the fructose-1,6/sedoheptulose-1,7-bisphosphatase (70glpX) and wildtype cells demonstrated increases in growth rates as expected. To investigate the disparate phenotypical responses of these different Synechocystis strains, isotopically non-stationary metabolic flux analysis (INST-MFA) was used to estimate the carbon flux distribution of tktA, 70glpX, and a kanamycin-resistant control (Km), under atmospheric conditions. In addition, untargeted label-free proteomics, which can detect changes in relative enzymatic abundance, was employed to study the possible effects caused by overexpressing each enzyme. Fluxomic and proteomic results indicated a decrease in oxidative pentose phosphate pathway activity when either FBP/SBPase or TK were overexpressed, resulting in increased carbon fixation efficiency. These results are an example of the integration of multiple omic-level experimental techniques and can be used to guide future metabolic engineering efforts to improve performances and efficiencies.

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2019
Keywords
INST-MFA, CBB cycle, Cyanobacteria, FBP/SBPase, Transketolase, Proteomics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-399084 (URN)10.1016/j.ymben.2019.08.014 (DOI)000491860400008 ()31470115 (PubMedID)
Funder
Swedish Energy Agency, P46607-1
Available from: 2019-12-16 Created: 2019-12-16 Last updated: 2019-12-16Bibliographically approved
Lindblad, P., Fuente, D., Borbe, F., Cicchi, B., Conejero, J. A., Couto, N., . . . Wuenschiers, R. (2019). CyanoFactory, a European consortium to develop technologies needed to advance cyanobacteria as chassis for production of chemicals and fuels. Algal Research, 41, Article ID 101510.
Open this publication in new window or tab >>CyanoFactory, a European consortium to develop technologies needed to advance cyanobacteria as chassis for production of chemicals and fuels
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2019 (English)In: Algal Research, ISSN 2211-9264, Vol. 41, article id 101510Article, review/survey (Refereed) Published
Abstract [en]

CyanoFactory, Design, construction and demonstration of solar biofuel production using novel (photo) synthetic cell factories, was an R&D project developed in response to the European Commission FP7-ENERGY-2012-1 call "Future Emerging Technologies" and the need for significant advances in both new science and technologies to convert solar energy into a fuel. CyanoFactory was an example of "purpose driven" research and development with identified scientific goals and creation of new technologies. The present overview highlights significant outcomes of the project, three years after its successful completion. The scientific progress of CyanoFactory involved: (i) development of a ToolBox for cyanobacterial synthetic biology; (ii) construction of DataWarehouse/Bioinformatics web-based capacities and functions; (iii) improvement of chassis growth, functionality and robustness; (iv) introduction of custom designed genetic constructs into cyanobacteria, (v) improvement of photosynthetic efficiency towards hydrogen production; (vi) biosafety mechanisms; (vii) analyses of the designed cyanobacterial cells to identify bottlenecks with suggestions on further improvements; (viii) metabolic modelling of engineered cells; (ix) development of an efficient laboratory scale photobioreactor unit; and (x) the assembly and experimental performance assessment of a larger (1350 L) outdoor flat panel photobioreactor system during two seasons. CyanoFactory - Custom design and purpose construction of microbial cells for the production of desired products using synthetic biology - aimed to go beyond conventional paths to pursue innovative and high impact goals. CyanoFactory brought together ten leading European partners (universities, research organizations and enterprises) with a common goal - to develop the future technologies in Synthetic biology and Advanced photobioreactors.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Cyanobacterial synthetic biology toolbox, DataWarehouse, Chassis robustness, Biosafety, Improved electron chain, Large-scale photobioreactor cultivation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-390421 (URN)10.1016/j.algal.2019.101510 (DOI)000472593800014 ()
Funder
EU, FP7, Seventh Framework Programme, 308518
Available from: 2019-08-12 Created: 2019-08-12 Last updated: 2019-08-12Bibliographically approved
Eungrasamee, K., Miao, R., Incharoensakdi, A., Lindblad, P. & Jantaro, S. (2019). Improved lipid production via fatty acid biosynthesis and free fatty acid recycling in engineered Synechocystis sp. PCC 6803. Biotechnology for Biofuels, 12, 1-13, Article ID 8.
Open this publication in new window or tab >>Improved lipid production via fatty acid biosynthesis and free fatty acid recycling in engineered Synechocystis sp. PCC 6803
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2019 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 12, p. 1-13, article id 8Article in journal (Refereed) Published
Abstract [en]

Background

Cyanobacteria are potential sources for third generation biofuels. Their capacity for biofuel production has been widely improved using metabolically engineered strains. In this study, we employed metabolic engineering design with target genes involved in selected processes including the fatty acid synthesis (a cassette of accD, accA, accC and accB encoding acetyl-CoA carboxylase, ACC), phospholipid hydrolysis (lipA encoding lipase A), alkane synthesis (aar encoding acyl-ACP reductase, AAR), and recycling of free fatty acid (FFA) (aas encoding acyl-acyl carrier protein synthetase, AAS) in the unicellular cyanobacterium Synechocystis sp. PCC 6803.

Results

To enhance lipid production, engineered strains were successfully obtained including an aas-overexpressing strain (OXAas), an aas-overexpressing strain with aar knockout (OXAas/KOAar), and an accDACB-overexpressing strain with lipA knockout (OXAccDACB/KOLipA). All engineered strains grew slightly slower than wild-type (WT), as well as with reduced levels of intracellular pigment levels of chlorophyll a and carotenoids. A higher lipid content was noted in all the engineered strains compared to WT cells, especially in OXAas, with maximal content and production rate of 34.5% w/DCW and 41.4mg/L/day, respectively, during growth phase at day 4. The OXAccDACB/KOLipA strain, with an impediment of phospholipid hydrolysis to FFA, also showed a similarly high content of total lipid of about 32.5% w/DCW but a lower production rate of 31.5mg/L/day due to a reduced cell growth. The knockout interruptions generated, upon a downstream flow from intermediate fatty acyl-ACP, an induced unsaturated lipid production as observed in OXAas/KOAar and OXAccDACB/KOLipA strains with 5.4% and 3.1% w/DCW, respectively.

Conclusions

Among the three metabolically engineered Synechocystis strains, the OXAas with enhanced free fatty acid recycling had the highest efficiency to increase lipid production.

Keywords
Total lipid, Unsaturated lipid, Synechocystis sp, PCC 6803, Acyl-acyl carrier protein synthetase, Lipase A, Acyl-ACP reductase, Acetyl-CoA carboxylase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-374428 (URN)10.1186/s13068-018-1349-8 (DOI)000454948200008 ()30622650 (PubMedID)
Available from: 2019-01-28 Created: 2019-01-28 Last updated: 2019-01-28Bibliographically approved
Wutthithien, P., Lindblad, P. & Incharoensakdi, A. (2019). Improvement of photobiological hydrogen production by suspended and immobilized cells of the N-2-fixing cyanobacterium Fischerella muscicola TISTR 8215. Journal of Applied Phycology, 31(6), 3527-3536
Open this publication in new window or tab >>Improvement of photobiological hydrogen production by suspended and immobilized cells of the N-2-fixing cyanobacterium Fischerella muscicola TISTR 8215
2019 (English)In: Journal of Applied Phycology, ISSN 0921-8971, E-ISSN 1573-5176, Vol. 31, no 6, p. 3527-3536Article in journal (Refereed) Published
Abstract [en]

To develop H-2 photoproduction by using the N-2-fixing cyanobacterium Fischerella muscicola TISTR 8215, a novel strain isolated from soil in Thailand, the factors affecting H-2 production were investigated in this study. Enhanced H-2 production in suspension culture was obtained when adapting the cells under N-2-fixing condition (modified AA medium) with continuous illumination of 250 mu mol photons m(-2) s(-1) under aerobic condition for 24 h, followed by further incubation under anaerobic condition for 9 h for production phase. The maximum H-2 production rate was 38.5 mu mol mg(-1) chl a h(-1). Low concentration of Fe2+ and Mo6+, essential elements for nitrogenase, enhanced H-2 production. The enhanced H-2 production was accompanied by the upregulation of nifD. On the other hand, an increased hupL transcript level was observed when there was a decrease of H-2 production. In cells immobilization, 1.5% (w/v) agar-immobilized cells had a 23-fold increase in maximum H-2 yield compared with that using free cell suspension at the same cell concentration, i.e., 7.48 mmol H-2 L-1 by immobilized cells and 0.32 mmol H-2 L-1 by suspended cells. Moreover, cell immobilization in agar could prolong H-2 production up to 108 h. This study underlines the strategies toward enhanced and sustained H-2 production from cyanobacteria. Furthermore, it will pave the way for large-scale production of biohydrogen to be used as an eco-friendly energy resource.

Place, publisher, year, edition, pages
SPRINGER, 2019
Keywords
Cyanobacteria, H-2 production, Cell immobilization, Fischerella
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-406198 (URN)10.1007/s10811-019-01881-y (DOI)000507642200016 ()
Available from: 2020-03-06 Created: 2020-03-06 Last updated: 2020-03-06Bibliographically approved
Douchi, D., Liang, F., Cano, M., Xiong, W., Wang, B., Maness, P.-C., . . . Yu, J. (2019). Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria. Frontiers in Microbiology, 10, Article ID 1356.
Open this publication in new window or tab >>Membrane-Inlet Mass Spectrometry Enables a Quantitative Understanding of Inorganic Carbon Uptake Flux and Carbon Concentrating Mechanisms in Metabolically Engineered Cyanobacteria
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2019 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1356Article in journal (Refereed) Published
Abstract [en]

Photosynthesis uses solar energy to drive inorganic carbon (Ci) uptake, fixation, and biomass formation. In cyanobacteria, Ci uptake is assisted by carbon concentrating mechanisms (CCM), and CO2 fixation is catalyzed by RubisCO in the Calvin-Benson-Bassham (CBB) cycle. Understanding the regulation that governs CCM and CBB cycle activities in natural and engineered strains requires methods and parameters that quantify these activities. Here, we used membrane-inlet mass spectrometry (MIMS) to simultaneously quantify Ci concentrating and fixation processes in the cyanobacterium Synechocystis 6803. By comparing cultures acclimated to ambient air conditions to cultures transitioning to high Ci conditions, we show that acclimation to high Ci involves a concurrent decline of Ci uptake and fixation parameters. By varying light input, we show that both CCM and CBB reactions become energy limited under low light conditions. A strain over-expressing the gene for the CBB cycle enzyme fructose-bisphosphate aldolase showed higher CCM and carbon fixation capabilities, suggesting a regulatory link between CBB metabolites and CCM capacity. While the engineering of an ethanol production pathway had no effect on CCM or carbon fixation parameters, additional fructose-bisphosphate aldolase gene over-expression enhanced both activities while simultaneously increasing ethanol productivity. These observations show that MIMS can be a useful tool to study the extracellular Ci flux and how CBB metabolites regulate Ci uptake and fixation.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2019
Keywords
MIMS, carbon uptake rate, cyanobacteria, FbaA, carbon fixation
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-390822 (URN)10.3389/fmicb.2019.01356 (DOI)000473067000001 ()31293533 (PubMedID)
Funder
Swedish Energy Agency, P46607-1
Available from: 2019-08-15 Created: 2019-08-15 Last updated: 2019-08-15Bibliographically approved
Liu, X., Miao, R., Lindberg, P. & Lindblad, P. (2019). Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2 in cyanobacteria. Energy & Environmental Science, 12(9), 2765-2777
Open this publication in new window or tab >>Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2 in cyanobacteria
2019 (English)In: Energy & Environmental Science, ISSN 1754-5692, E-ISSN 1754-5706, Vol. 12, no 9, p. 2765-2777Article in journal (Refereed) Published
Abstract [en]

Cyanobacteria are photoautotrophic microorganisms which can be engineered to directly convert CO2 and water into biofuels and chemicals via photosynthesis using sunlight as energy. However, the product titers and rates are the main challenges that need to be overcome for industrial applications. Here we present systematic modular engineering of the cyanobacterium Synechocystis PCC 6803, enabling efficient biosynthesis of 1-butanol, an attractive commodity chemical and gasoline substitute. Through introducing and re-casting the 1-butanol biosynthetic pathway at the gene and enzyme levels, optimizing the 5 '-regions of expression units for tuning transcription and translation, rewiring the carbon flux and rewriting the photosynthetic central carbon metabolism to enhance the precursor supply, and performing process development, we were able to reach a cumulative 1-butanol titer of 4.8 g L-1 with a maximal rate of 302 mg L-1 day(-1) from the engineered Synechocystis. This represents the highest 1-butanol production from CO2 reported so far. Our multi-level modular strategy for high-level production of chemicals and advanced biofuels represents a blue-print for future systematic engineering in photosynthetic microorganisms.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-395323 (URN)10.1039/c9ee01214a (DOI)000486019600010 ()
Funder
Swedish Energy Agency, P46607-1
Available from: 2019-10-17 Created: 2019-10-17 Last updated: 2019-10-17Bibliographically approved
Junaid, S., Khanna, N., Lindblad, P. & Ahmed, M. (2019). Multifaceted biofuel production by microalgal isolates from Pakistan. Biofuels, Bioproducts and Biorefining, 13(5), 1187-1201
Open this publication in new window or tab >>Multifaceted biofuel production by microalgal isolates from Pakistan
2019 (English)In: Biofuels, Bioproducts and Biorefining, ISSN 1932-104X, E-ISSN 1932-1031, Vol. 13, no 5, p. 1187-1201Article in journal (Refereed) Published
Abstract [en]

Third-generation biofuels are currently considered to be the most resourceful medium for generating bioenergy. In the present study, microalgal strains were isolated from soil samples collected in Pakistan and characterized by 18S rRNA sequencing. The strains were identified as green algae Gloeocystis sp. MFUM-4, Sphaerocystis sp. MFUM-34, and Dictyochloropsis sp. MFUM-35. They were further studied for their potential to produce popular biofuels such as biodiesel, bioethanol, and biohydrogen. Under the test conditions, Gloeocystis sp. MFUM-4 emerged as the most suitable candidate, amongst the three new isolates, for biofuel production with a biodiesel production potential of 33.3% (w/v). Eight different environmental conditions were also tested to identify the most suitable condition for biohydrogen and bioethanol production using the newly isolated strains. Under light but in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), Gloeocystis recorded the highest capacity to produce both biohydrogen and bioethanol compared with the other strains that were examined.

Place, publisher, year, edition, pages
WILEY, 2019
Keywords
microalgae, biohydrogen, biodiesel, bioethanol
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-395852 (URN)10.1002/bbb.2009 (DOI)000485982700006 ()
Available from: 2019-10-25 Created: 2019-10-25 Last updated: 2019-10-25Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-7256-0275

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