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  • 1.
    Englund, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Andersen-Ranberg, Johan
    Miao, Rui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Hamberger, Björn
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide2015In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 4, no 12, p. 1270-1278Article in journal (Refereed)
    Abstract [en]

    Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.

  • 2.
    Eungrasamee, Kamonchanock
    et al.
    Chulalongkorn Univ, Fac Sci, Dept Biochem, Lab Cyanobacterial Biotechnol, Bangkok 10330, Thailand.
    Miao, Rui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Incharoensakdi, Aran
    Chulalongkorn Univ, Fac Sci, Dept Biochem, Lab Cyanobacterial Biotechnol, Bangkok 10330, Thailand.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Jantaro, Saowarath
    Chulalongkorn Univ, Fac Sci, Dept Biochem, Lab Cyanobacterial Biotechnol, Bangkok 10330, Thailand.
    Improved lipid production via fatty acid biosynthesis and free fatty acid recycling in engineered Synechocystis sp. PCC 68032019In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 12, p. 1-13, article id 8Article in journal (Refereed)
    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.

  • 3.
    Liu, Xufeng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Miao, Rui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Metabolic engineering of Synechocystis PCC 6803 for photosynthetic 1-butanol productionManuscript (preprint) (Other academic)
  • 4.
    Liu, Xufeng
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Miao, Rui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2 in cyanobacteria2019In: Energy & Environmental Science, ISSN 1754-5692, E-ISSN 1754-5706, Vol. 12, no 9, p. 2765-2777Article in journal (Refereed)
    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.

  • 5.
    Miao, Rui
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Metabolic Engineering of Synechocystis PCC 6803 for Butanol Production2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    There is an urgent demand for renewable alternatives to fossil fuels since the extraction and utilization cause a series of environmental problems in the world. Thus, the utilization of solar energy has attracted much attention in the last decades since there is excess amount of light on Earth. Photosynthetic microorganisms, such as cyanobacteria, can be a good biological chassis to convert solar energy directly to chemical energy. It has been demonstrated that cyanobacteria can produce various compounds which can be used asfourth-generation biofuels. This thesis focuses on the photo-autotrophic production of two biofuel compounds, isobutanol and 1-butanol, in the unicellular cyanobacterial strain Synechocystis PCC 6803. In the studies of isobutanol production, the endogenous alcohol dehydrogenase of Synechocystis encoded by slr1192 showed impressive activity in isobutanol formation. In addition, a-ketoisovalerate decarboxylase (Kivd) was identified as the only heterologous enzyme needed to be introduced for isobutanol production in Synechocystis. Kivd was further recognized as a bottleneck in the isobutanol production pathway. Therefore, Kivd was engineered via rational design to shift the preferential activity towards the production of isobutanol instead of the by-product 3-methyl-1-butanol. The best strain pEEK2-ST expressing KivdS286T showed dramatically increased productivity, and the activity of Kivd was successfully shifted further towards isobutanol production. A cumulative isobutanol titer of 911 mg L-1 was observed from this strain after 46 days growth under 50 μmol photons m−2 s−1 with pH adjusted to between 7 and 8. A maximum production rate of nearly 44 mg L-1d-1was reached between days 4 and 6. Similar metabolic engineering strategies were employed to generate 1-butanol producing Synechocystis strains and then to stepwise enhance the production. By selecting the best enzymes and promotors, 836 mg L-1 in-flask 1-butanol was produced. By optimizing the cultivation condition, an in-flask titer of 2.1 g L-1 and a maximal cumulative titer of 4.7 g L-1 were observed in the long-term cultivation. This thesis demonstrates different metabolic engineering strategies for producing valuable compounds in Synechocystis, exemplified with butanol, and how to enhance production systematically. 

  • 6.
    Miao, Rui
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Liu, Xufeng
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Englund, Elias
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindberg, Pia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases2017In: Metabolic Engineering Communications, ISSN 2214-0301, Vol. 5, p. 45-53Article in journal (Refereed)
    Abstract [en]

    Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. In this study, we examined the capacity of engineered strains of Synechocystis PCC 6803 containing the α-ketoisovalerate decarboxylase from Lactococcus lactis and different heterologous and endogenous alcohol dehydrogenases (ADH) for isobutanol production. A strain expressing an introduced kivdwithout any additional copy of ADH produced 3 mg L−1 OD750−1 isobutanol in 6 days. After the cultures were supplemented with external addition of isobutyraldehyde, the substrate for ADH, 60.8 mg L−1 isobutanol was produced after 24 h when OD750 was 0.8. The in vivo activities of four different ADHs, two heterologous and two putative endogenous in Synechocystis, were examined and the Synechocystis endogenous ADH encoded by slr1192 showed the highest efficiency for isobutanol production. Furthermore, the strain overexpressing the isobutanol pathway on a self-replicating vector with the strong Ptrc promoter showed significantly higher gene expression and isobutanol production compared to the corresponding strains expressing the same operon introduced on the genome. Hence, this study demonstrates that Synechocystis endogenous AHDs have a high capacity for isobutanol production, and identifies kivd encoded α-ketoisovalerate decarboxylase as one of the likely bottlenecks for further isobutanol production.

  • 7.
    Miao, Rui
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Wegelius, Adam
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Durall de la Fuente, Claudia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Liang, Feiyan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Khanna, Namita
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Engineering Cyanobacteria for Biofuel Production2017In: Modern Topics in the Phototrophic Prokaryotes: Environmental and Applied Aspects / [ed] Hallenbeck, Patrick, USA: Springer, 2017, p. 351-393Chapter in book (Refereed)
  • 8.
    Miao, Rui
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Xie, Hao
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Ho, Felix M.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Protein engineering of α-ketoisovalerate decarboxylase for improved isobutanol production in Synechocystis PCC 68032018In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 47, p. 42-48Article in journal (Refereed)
    Abstract [en]

    Protein engineering is a powerful tool to modify e.g. protein stability, activity and substrate selectivity. Heterologous expression of the enzyme α-ketoisovalerate decarboxylase (Kivd) in the unicellular cyanobacterium Synechocystis PCC 6803 results in cells producing isobutanol and 3-methyl-1-butanol, with Kivd identified as a potential bottleneck. In the present study, we used protein engineering of Kivd to improve isobutanol production in Synechocystis PCC 6803. Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. Single replacement, either Val461 to isoleucine or Ser286 to threonine, increased the Kivd activity significantly, both in vivo and in vitro resulting in increased overall production while isobutanol production was increased more than 3-methyl-1-butanol production. Moreover, among all the engineered strains examined, the strain with the combined modification V461I/S286T showed the highest (2.4 times) improvement of isobutanol-to-3M1B molar ratio, which was due to a decrease of the activity towards 3M1B production. Protein engineering of Kivd resulted in both enhanced total catalytic activity and preferential shift towards isobutanol production in Synechocystis PCC 6803.

  • 9.
    Miao, Rui
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Xie, Hao
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Lindblad, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Enhancement of photosynthetic isobutanol production in engineered cells of Synechocystis PCC 68032018In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, article id 267Article in journal (Refereed)
    Abstract [en]

    Background: Cyanobacteria, oxygenic photoautotrophic prokaryotes, can be engineered to produce various valuable chemicals from solar energy and CO2 in direct processes. The concept of photosynthetic production of isobutanol, a promising chemical and drop-in biofuel, has so far been demonstrated for Synechocystis PCC 6803 and Synechococcus elongatus PCC 7942. In Synechocystis PCC 6803, a heterologous expression of alpha-ketoisovalerate decarboxylase (Kivd) from Lactococcus lactis resulted in an isobutanol and 3-methyl-1-butanol producing strain. Kivd was identified as a bottleneck in the metabolic pathway and its activity was further improved by reducing the size of its substrate-binding pocket with a single replacement of serine-286 to threonine (Kivd(S286T)). However, isobutanol production still remained low. Results: In the present study, we report on how cultivation conditions significantly affect the isobutanol production in Synechocystis PCC 6803. A HCl-titrated culture grown under medium light (50 mu mol photons m(-2) s(-1)) showed the highest isobutanol production with an in-flask titer of 194 mg l(-1) after 10 days and 435 mg l(-1) at day 40. This corresponds to a cumulative isobutanol production of 911 mg l(-1), with a maximal production rate of 43.6 mg l(-1) day(-1) observed between days 4 and 6. Additional metabolic bottlenecks in the isobutanol biosynthesis pathway were further addressed. The expression level of Kivd(S286T) was significantly affected when co-expressed with another gene downstream in a single operon and in a convergent oriented operon. Moreover, the expression of the ADH encoded by codon-optimized slr1192 and co-expression of IlvC and IlvD were identified as potential approaches to further enhance isobutanol production in Synechocystis PCC 6803. Conclusion: The present study demonstrates the importance of a suitable cultivation condition to enhance isobutanol production in Synechocystis PCC 6803. Chemostat should be used to further increase both the total titer as well as the rate of production. Furthermore, identified bottleneck, Kivd, should be expressed at the highest level to further enhance isobutanol production.

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