Comparative Dose–Response Analysis of Inducible Promoters in Cyanobacteria

Behle, Anna; Saake, Pia; Germann, Anna T.; Dienst, Dennis; Axmann, Ilka M.

doi:10.1021/acssynbio.9b00505

Cited by 49 publications

(72 citation statements)

References 49 publications

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“…The expression module was further linked to a codon optimised version of the fluorescence reporter mVenus 40 , which was deployed as isogenic negative control for sesquiterpene accumulation and quantitative expression proxy during all experiments. All expression cassettes were located on the RSF1010-derived, autonomously replicating plasmid pSHDY*in 41 (Fig. S1).…”

Section: Resultsmentioning

confidence: 99%

“…plasmid construction and transformation. The common vector was the conjugative plasmid RSF1010based pSHDY*in, which is a derivative of pSHDY (version w/o mobAY25F point mutation) 41 with optimised insulation of the default expression cassette. The latter consists of the copper-inducible promoter P petE (from Synechocystis), the 5′UTR BCD2 55 , a codon optimised coding sequence (CDS) for the mVenus fluorophore 40 including a second stop codon and the rrnB T1/T7Te double terminator (BBa_B0015).…”

Section: Methodsmentioning

confidence: 99%

“…In conclusion, under these conditions the P petE promoter enables sensitive and strong induction of gene expression, however, exhibits a rather poor dynamic range, in accordance with previous observations 42 . 41 ). Gene expression is mediated by synthetic expression modules, consisting of the native copper-inducible promoter P petE 61,62 linked to the bicistronic design BCD2 for reliable translation initiation 55 .…”

Section: Assessment Of Sesquiterpene Biosynthesis In 6 Well Platesmentioning

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: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Assessment Of Sesquiterpene Biosynthesis In 6 Well Platesmentioning

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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“…This appears to be the case in cyanobacteria cultures grown at constant light. In a study on the induction kinetics of YFP from various promoters in Synechocystis , the protein accumulated for five days after induction with rhamnose before reaching a steady state ( Behle et al, 2020 ). In day/night cultivations, the change in the target’s protein abundance will be slower still, as total transcription and/or translation is globally downregulated at night, by inactivation of RNA polymerases and/or ribosomes ( Hood et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

Slow Protein Turnover Explains Limited Protein-Level Response to Diurnal Transcriptional Oscillations in Cyanobacteria

et al. 2021

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Metabolically engineered cyanobacteria have the potential to mitigate anthropogenic CO2 emissions by converting CO2 into renewable fuels and chemicals. Yet, better understanding of metabolic regulation in cyanobacteria is required to develop more productive strains that can make industrial scale-up economically feasible. The aim of this study was to find the cause for the previously reported inconsistency between oscillating transcription and constant protein levels under day-night growth conditions. To determine whether translational regulation counteracts transcriptional changes, Synechocystis sp. PCC 6803 was cultivated in an artificial day-night setting and the level of transcription, translation and protein was measured across the genome at different time points using mRNA sequencing, ribosome profiling and quantitative proteomics. Furthermore, the effect of protein turnover on the amplitude of protein oscillations was investigated through in silico simulations using a protein mass balance model. Our experimental analysis revealed that protein oscillations were not dampened by translational regulation, as evidenced by high correlation between translational and transcriptional oscillations (r = 0.88) and unchanged protein levels. Instead, model simulations showed that these observations can be attributed to a slow protein turnover, which reduces the effect of protein synthesis oscillations on the protein level. In conclusion, these results suggest that cyanobacteria have evolved to govern diurnal metabolic shifts through allosteric regulatory mechanisms in order to avoid the energy burden of replacing the proteome on a daily basis. Identification and manipulation of such mechanisms could be part of a metabolic engineering strategy for overproduction of chemicals.

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“…Another interesting approach is to use on/off switches or even titratable systems, using defined external cues, to tightly control levels of natural competence ( Figure 3 B). Several inducible promoter systems have been characterised in cyanobacteria [ 110 , 111 , 112 ] and multiple types of genome editing tools have been developed (recently reviewed in [ 113 ]). These tools could also be deployed to engineer natural competence.…”

Section: Targeted Manipulation Of Natural Competencementioning

confidence: 99%

Function and Benefits of Natural Competence in Cyanobacteria: From Ecology to Targeted Manipulation

Schirmacher

Hanamghar

Zedler

2020

Life

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Natural competence is the ability of a cell to actively take up and incorporate foreign DNA in its own genome. This trait is widespread and ecologically significant within the prokaryotic kingdom. Here we look at natural competence in cyanobacteria, a group of globally distributed oxygenic photosynthetic bacteria. Many cyanobacterial species appear to have the genetic potential to be naturally competent, however, this ability has only been demonstrated in a few species. Reasons for this might be due to a high variety of largely uncharacterised competence inducers and a lack of understanding the ecological context of natural competence in cyanobacteria. To shed light on these questions, we describe what is known about the molecular mechanisms of natural competence in cyanobacteria and analyse how widespread this trait might be based on available genomic datasets. Potential regulators of natural competence and what benefits or drawbacks may derive from taking up foreign DNA are discussed. Overall, many unknowns about natural competence in cyanobacteria remain to be unravelled. A better understanding of underlying mechanisms and how to manipulate these, can aid the implementation of cyanobacteria as sustainable production chassis.

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Comparative Dose–Response Analysis of Inducible Promoters in Cyanobacteria

Cited by 49 publications

References 49 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

Slow Protein Turnover Explains Limited Protein-Level Response to Diurnal Transcriptional Oscillations in Cyanobacteria

Function and Benefits of Natural Competence in Cyanobacteria: From Ecology to Targeted Manipulation

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