High-Density Cultivation of Terrestrial Nostoc Strains Leads to Reprogramming of Secondary Metabolome

Guljamow, Arthur; Kreische, Marco; Ishida, Keishi; Liaimer, Anton; Altermark, Bjørn; Bähr, Lars; Hertweck, Christian; Ehwald, R.; Dittmann, Elke

doi:10.1128/aem.01510-17

Cited by 36 publications

(67 citation statements)

References 31 publications

(42 reference statements)

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“…Indeed, final bisabolene titers were even higher in the 6-well-plate experiment than in the MC system, which can be due to better light penetrance, higher mixing efficiency that favours cell-dodecane contacts, and higher evaporation-derived final cell densities. A recently developed HDC system allows for sustainably fast photoautotrophic growth, with a high potential for efficient biomass accumulation and high yields of natural products 28,29 . In this study, we tested HDC for its value regarding heterologous terpenoid production.…”

Section: Sesquiterpene Production Under Small-scale High Density Cultmentioning

confidence: 99%

“…High density cultivation (HDC) is a recently developed cultivation system that tackles these problems by implementing a membrane-mediated CO 2 supply technology, combined with optimised nutrient supply and high light intensities 27 . This system facilitates rapid, sustainable biomass accumulation and was demonstrated to further raise the potential for the recovery of natural products from cyanobacteria [28][29][30] .…”

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confidence: 99%

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High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803

Dienst

Wichmann

Mantovani

et al. 2020

Sci Rep

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cyanobacteria and microalgae are attractive photoautotrophic host systems for climate-friendly production of fuels and other value-added biochemicals. However, for economic applications further development and implementation of efficient and sustainable cultivation strategies are essential. Here, we present a comparative study on cyanobacterial sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803 using a commercial lab-scale High Density Cultivation (HDC) platform in the presence of dodecane as in-situ extractant. operating in a two-step semi-batch mode over a period of eight days, volumetric yields of (E)-α-bisabolene were more than two orders of magnitude higher than previously reported for cyanobacteria, with final titers of 179.4 ± 20.7 mg * L −1. Likewise, yields of the sesquiterpene alcohols (−)-patchoulol and (−)-α-bisabolol were many times higher than under reference conditions, with final titers of 17.3 ± 1.85 mg * L −1 and 96.3 ± 2.2 mg * L −1 , respectively. While specific productivity was compromised particularly for (E)-α-bisabolene in the HDc system during phases of high biomass accumulation rates, volumetric productivity enhancements during linear growth at high densities were more pronounced for (E)-α-bisabolene than for the hydroxylated terpenoids. together, this study provides additional insights into cell density-related process characteristics, introducing HDC as highly efficient strategy for phototrophic terpenoid production in cyanobacteria. Cyanobacteria are oxygenic phototrophs with a versatile metabolism and therefore constitute an economically appealing platform for the sustainable production of a diversity of industrially relevant chemicals. In particular, the natural occurrence of the methylerythritol-4-phosphate (MEP) pathway 1 allows the engineered biosynthesis of a diversity of high-value terpenoid compounds such as pharmaceuticals, cosmetics, and biofuels (reviewed in 2,3). While numerous pioneering proof-of-concept studies already demonstrated the heterologous biosynthesis of isoprene (C 5), mono-(C 10), sesqui-(C 15), di-(C 20) and tri-(C 30) terpenes in cyanobacterial host strains 4-9 , the reported titers of these compounds are typically two to three orders of magnitudes lower than those of e.g. alcoholic fuels (cf. overviews in 3,10). The major limitation of these processes arise from the inherent carbon partitioning characteristics, which favour primary metabolism and biomass accumulation, resulting in weak carbon fluxes through the native MEP pathway 11. In accordance, a strategy to bypass these inherent flux limitations by heterologous expression of the complete bacterial mevalonate (MVA) pathway in Synechocystis sp. PCC 6803 (from here: Synechocystis) resulted in 2.5-fold higher isoprene yields 12. Likewise, numerous rational design studies targeting selected bottleneck steps within the MEP pathway as well as selected steps of upstream carbon fluxes lent support to this concept 8,9,13-19. Further strategies sought to challenge the problem of inherently weak terpene s...

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Section: Sesquiterpene Production Under Small-scale High Density Cultmentioning

confidence: 99%

mentioning

confidence: 99%

High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803

Dienst

Wichmann

Mantovani

et al. 2020

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Indeed, final bisabolene titers were even higher in the 6-well-plate experiment than in the MC system, which can be due to better light penetrance, higher mixing efficiency that favors cell-dodecane contacts, and higher evaporation-derived final cell densities. A recently developed HDC system allows for sustainably fast photoautotrophic growth, with a high potential for efficient biomass accumulation and high yields of natural products [28,29]. In this study, we tested HDC for its value regarding heterologous terpenoid production.…”

Section: Sesquiterpene Production Under Small-scale High Density Cultmentioning

confidence: 99%

“…High density cultivation (HDC) is a recently developed cultivation system that tackles these problems by implementing a membrane-mediated CO2 supply technology, combined with optimized nutrient supply and high light intensities [27]. This system facilitates rapid, sustainable biomass accumulation and was demonstrated to further raise the potential for the recovery of natural products from cyanobacteria [28][29][30].…”

Section: Introductionmentioning

confidence: 99%

High density cultivation for efficient sesquiterpenoid biosynthesis inSynechocystissp. PCC 6803

Dienst

Wichmann

Mantovani

et al. 2019

Preprint

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Cyanobacteria and microalgae are attractive phototrophic host systems for climate-friendly production of fuels and other high-value chemicals. The biosynthesis of an increasing diversity of industrially relevant compounds such as terpenoids has been demonstrated in recent years. To develop economically feasible and sustainable process designs, major challenges still remain regarding intracellular carbon partitioning, specific metabolic pathway activities and efficient cultivation strategies. Here, we present a technical study on comparative characteristics of sesquiterpene and sesquiterpene alcohol accumulation in engineered strains of Synechocystis sp. PCC 6803 (substrain GT-U) under different growth conditions and cell densities. This study particularly focuses on the basic applicability of a commercial High Density Cultivation platform in the presence of a dodecane overlay, which serves as a standard in-situ extractant and sink for various hydrophobic biochemicals. Significantly, the presented data demonstrate high volumetric productivities of (E)-α-bisabolene under high-density conditions that are more than two orders of magnitude higher than previously reported for cyanobacteria. Operating in a two-step semi-batch mode over a period of eight days, average final volumetric titers of 179.4 ± 20.7 mg * L -1 were detected. Likewise, the sesquiterpene alcohols (-)patchoulol and (-)-α-bisabolol accumulated to many times higher levels in high density cultivation than under standard batch conditions, with final titers of 17.3 ± 1.85 mg * L -1 and 96.3 ± 2.2 mg * L -1 , respectively. In contrast, specific product accumulation (mg * L -1 * OD750 -1 ) was compromised particularly for bisabolene in the high density system during phases of high biomass accumulation rates. Volumetric productivities were high during linear growth at high densities, distinctly outperforming standard batch systems. While the presented data highlight the benefits of high-density strategies for highly efficient phototrophic terpenoid production, they further point at the presence of major metabolic bottlenecks for engineered terpenoid biosynthesis and the requirement for systematic and/or targeted strategies to sustainably redirect inherent carbon fluxes in cyanobacteria. Together, our data provide additional insights into growth-and density-related effects on the efficiency of product accumulation, introducing low-scale High Density Cultivation as a rapid and efficient platform for screening of heterologous terpenoid production in cyanobacteria.

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“…Indeed, various cyanobacteria genera are able to synthetize a wild range of secondary metabolites (Welker et al, 2012;Shih et al, 2013), with noticeable bioactivity, comprising high toxicity, and which are potentially harmful to Human health and aquatic organisms (Codd et al, 2005;Pearson et al, 2010). These metabolites are also believed to be noticeably involved in the capability of these organisms to proliferate in various environments (Guljamow et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Global metabolomic characterizations of Microcystis spp. highlights clonal diversity in natural bloom-forming populations and expands metabolite structural diversity

Marie

Manach

Duval

et al. 2018

Preprint

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Cyanobacteria are photosynthetic prokaryotes that are able to synthetize a wild rang of secondary metabolites exhibiting noticeable bioactivity, comprising toxicity. Microcystis represents one of the most common cyanobacteria taxa constituting the intensive blooms that arise nowadays in freshwater ecosystems worldwide. They produce numerous cyanotoxins (toxic metabolites), which are potentially harmful to Human health and aquatic organisms. In order to better understand the variations in cyanotoxins production between clones of the same blooms, we investigate the diversity of several Microcystis strains isolated from different freshwater bloom-forming populations from various geographical area. Twenty-four clonal strains were compared by genotyping with 16S-ITS fragment sequencing and metabolites chemotyping using LC ESI-qTOF mass spectrometry. While, genotyping can only discriminate between the different species, the global metabolomes reveal clear discriminant molecular profiles between strains, which can be clustered primarily according to their global metabolite content, then to their genotype, and finally to their sampling localities. A global molecular network generated from MS/MS fractionation patterns of the various metabolite detected in all strains performed with GNPS tool highlight the production of a wide set of chemically diverse metabolites, comprising only few microcystins, but many aeruginosins, cyanopeptolins and microginins, along with a large set of unknown molecules that still remain to be investigated and characterized at their structure as well as at their potential bioactivity or toxicity levels. Figure 1: Microcystis spp. General view of a representative intense Microcystis sp. bloom in a recreational pound (Champs-sur-Marne) (A). Macrograph of Microcystis colonies at surface water (B). Example of 15-mL vessels containing the monoclonal strains of Microcystis spp. maintained in the Paris' Museum Collection (PMC) of cyanobacteria (MNHN, Paris) (C). Example of micrograph of the isolated monoclonal culture of the Microcystis aeruginosa, where scale bare represents 10 µm (D). Representative picture of Microcystis aeruginosa cell from PMC 156.02 strain (here in division) under transmission electron microscope, where scale bare represents 0.5 µm (E). General structures of various cyanobacterial metabolites belonging to the microcystin, anabaenopeptin, microginin microviridin, aeruginosin and oscillatorin families (F).Microcystis wesenbergii PMC 672.10 84 Microcystis wesenbergii PMC 671.10 Microcystis wesenbergii PMC 673.10 Microcystis wesenbergii PMC 674.10 Microcystis wesenbergii NIES111 (3551457) (AB015388) Microcystis wesenbergii TAC52 (3551459) (AB015390) Microcystis sp. PMC 807.12 Microcystis viridis PMC 566.08 88 Microcystis wesenbergii NIES44 (3551456) (AB015387) Microcystis viridis TAC17 (3551467) (AB015398) Microcystis viridis W191 (295983850) (HM017091) Microcystis viridis W18 (295983846) (HM017087) Microcystis aeruginosa PMC 265.05 Microcystis sp. PMC 816.12 Microcystis aeruginosa PMC...

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High-Density Cultivation of Terrestrial Nostoc Strains Leads to Reprogramming of Secondary Metabolome

Cited by 36 publications

References 31 publications

High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803

High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803

High density cultivation for efficient sesquiterpenoid biosynthesis inSynechocystissp. PCC 6803

Global metabolomic characterizations of Microcystis spp. highlights clonal diversity in natural bloom-forming populations and expands metabolite structural diversity

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