Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803

Choi, Yun‐Nam; Park, Jong

doi:10.1016/j.biortech.2016.02.056

Cited by 58 publications

(28 citation statements)

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“…PCC 6803 to enhance the reduction of NADP into NADPH. As a result of improved NADPH level intracellularly, the biomass and ethanol concentrations were increased (6). In the present study, the increased NADPH regeneration mediated by metal oxides has been achieved without engineering other natural pathways of Synechocystis and the production of ethanol is also higher than that reported by Choi and Park (6).…”

Section: Resultsmentioning

confidence: 99%

“…Apart from thepathway engineering strategies, there are certain factors such as cellular transport system, antioxidant enzymes, and cofactors influencing ethanol production and tolerance (5). Among them, NADPH plays an important role in photosynthesis, carbon metabolism, and more importantly NADPH plays an essential role in the conversion of acetaldehyde to ethanol (6). The engineering of various enzymes involved in NADPH regeneration to improve various functions of cyanobacteria has been demonstrated (6,7).…”

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confidence: 99%

“…Among them, NADPH plays an important role in photosynthesis, carbon metabolism, and more importantly NADPH plays an essential role in the conversion of acetaldehyde to ethanol (6). The engineering of various enzymes involved in NADPH regeneration to improve various functions of cyanobacteria has been demonstrated (6,7). On the other hand, the regeneration of NADPH is also reported as an effective process; however, it needs further study about activation of regenerating factor especially under photoautotrophic condition (8,9).…”

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Nanoparticle Mediated NADPH Regeneration for Enhanced Ethanol Production by EngineeredSynechocystissp. PCC 6803

Velmurugan

Incharoensakdi

2019

Preprint

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10The ethanol synthesis pathway engineered Synechocystis sp. PCC 6803 was used to investigate 11 the influence of metal oxide mediated NADPH regeneration on ethanol synthesis. Among the 12 metal oxides, Fe 2 O 3 and MgO showed considerable improvement in growth, chlorophyll a 13 content and ethanol synthesis. The in-vitro studies proved that the selected metal oxides have the 14 potential to regenerate the NADPH under light illumination. The results clearly indicate that the 15 light energy is the key factor for activation of metal oxides and to a less extent light itself has the 16 possibility for direct regeneration of NADPH. Under optimized light intensity and NADP 17 addition, the maximum MgO mediated ethanol production of 5100mg/L, about a 2-fold increase 18 compared to the control, was obtained after 20 days cultivation at 5L level. This study indicates 19 that the efficient NADPH regeneration aided by metal oxide is crucial to improve ethanol 20 productivity in Synechocystis sp. PCC 6803. 21 IMPORTANCE 22 23 Cyanobacteria are efficient ethanol producing organisms from atmospheric CO 2 upon 24 engineering of pathway. In cyanobacterial ethanol synthesis pathway, NADPH plays an 25 important role acetaldehyde to ethanol conversion. Here we elucidated the NADPH regeneration 26 through extracellular addition of metal oxides. The metal oxide mediated NADPH regeneration 27 study allows us to dissect the importance of metal oxides in enhancing ethanol production 28 through NADPH regeneration while also providing insight into the regulatory functions of metal 29 oxides in growth, photosynthetic apparatus and various carbon metabolisms.30 31 KEYWORDS 32 ethanol, Synechocystis, NADPH, metal oxides, light intensity 33 34To replace the fossil fuels, extensive studies have been focused on developing efficient 35 techniquefor the production of ethanol from cyanobacteria (1,2). The incorporation of ethanol 36 synthesis pathwayfrom various microorganisms into cyanobacteriahas already been 37 demonstrated as a route of direct carbon fixation for ethanol production (3). However, the 38 longercultivation time and lower yield at large scale makes the process ineffective (4). Apart 39 from thepathway engineering strategies, there are certain factors such as cellular transport 40 system, antioxidant enzymes, and cofactors influencing ethanol production and tolerance (5). 41Among them, NADPH plays an important role in photosynthesis, carbon metabolism, and more 42 importantly NADPH plays an essential role in the conversion of acetaldehyde to ethanol (6). The 43 engineering of various enzymes involved in NADPH regeneration to improve various functions 44 of cyanobacteria has been demonstrated (6,7). On the other hand, the regeneration of NADPH is 45 3 also reported as an effective process; however, it needs further study about activation of 46 regenerating factor especially under photoautotrophic condition (8,9). Generally, the 47 regeneration of cofactor (NADPH/NADH) has been performed in the presence of 48 oxidoreductases (...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Nanoparticle Mediated NADPH Regeneration for Enhanced Ethanol Production by EngineeredSynechocystissp. PCC 6803

Velmurugan

Incharoensakdi

2019

Preprint

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“…PCC 6803 ('Synechocystis' hereafter) and Synechococcus sp. PCC 7002 have been successfully engineered to produce 2,3-butanediol 1,2 , lactate 3 , isobutanol 4 , plant terpenoids 5 and ethanol [6][7][8][9] , and to allow the utilisation of xylose 10 . Cyanobacteria, particularly Synechocystis, are also used as model organisms for fundamental studies of important processes such as photosynthesis [11][12][13][14][15][16] , circadian rhythms [17][18][19] and carbon-concentrating mechanisms [20][21][22][23] .…”

Section: Introductionmentioning

confidence: 99%

A rhamnose-inducible system for precise and temporal control of gene expression in cyanobacteria

Kelly

Hitchcock

Torres-Méndez

et al. 2017

Preprint

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Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

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“…G6PDH is the enzyme responsible for the first committing step in OPPP and generates NADPH upon oxidation of glucose-6-phosphate (G6P). It has been shown that overexpression of G6PDH in an ethanol-producing strain of Synechocystis 6803 led to an enhanced yield of ethanol, as a result of an increased NADPH pool [10]. …”

Section: Introductionmentioning

confidence: 99%

An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria

et al. 2016

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BackgroundCyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls, lysis of cyanobacterial cells is inefficient and often laborious. In some cases radioisotope-labeled substrates can be fed directly to intact cells; however, label-free assays are often favored due to safety and practical reasons.ResultsHere we show an easy and highly efficient method for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism in cyanobacteria. Incubation of the cyanobacterial cells in the commercially available B-PER reagent for 10 min permeabilized the cells, as confirmed by the SYTOX Green staining. There was no significant change in the cell shape and no major loss of intracellular proteins was observed during the treatment. When used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at −20 °C without losing the enzyme activities. The permeabilization process and subsequent activity assays were successfully adapted to the 96-well plate system.ConclusionsAn easy, efficient and scalable permeabilization protocol was established for cyanobacteria. The permeabilized cells can be directly applied for measurement of G6PDH and Rubisco activities without using radioisotopes and the protocol may be readily adapted to studies of other cyanobacterial species and other intracellular enzymes. The permeabilization and enzyme assays can be performed in 96-well plates in a high-throughput manner.

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Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803

Cited by 58 publications

References 18 publications

Nanoparticle Mediated NADPH Regeneration for Enhanced Ethanol Production by EngineeredSynechocystissp. PCC 6803

Nanoparticle Mediated NADPH Regeneration for Enhanced Ethanol Production by EngineeredSynechocystissp. PCC 6803

A rhamnose-inducible system for precise and temporal control of gene expression in cyanobacteria

An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria

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