Improving hydrocarbon production by engineering cyanobacterial acyl-(acyl carrier protein) reductase

Kudo, Hisashi; Hayashi, Yasumasa; Arai, Munehito

doi:10.1186/s13068-019-1623-4

Cited by 17 publications

(16 citation statements)

References 30 publications

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“…Interestingly, GFC elution profile of AAR does not correspond to a monomeric fraction, instead, a broadly distributed peak-like pattern has been observed indicative of the co-existence of multiple-sized states of the protein in solution (Fig. S2 ), which was also previously reported by Kudo et al group 17 . The presence of other protein contaminants was ruled out as only a single band was observed in SDS-PAGE corresponding to the monomeric molecular weight of the AAR protein (Figs.…”

Section: Discussionsupporting

confidence: 82%

Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase

Sharma

Shakeel

Gupta

et al. 2021

Sci Rep

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Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding. Characterization by dynamic light scattering experiment and intact mass spectrometry revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles due to diverse lipidation. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution suggesting that the protein milieu plays a significant role in dictating its folding. Apparent standard free energy $$\left( {\Delta {\text{G}}_{{{\text{NU}}}}^{{{\text{H}}_{2} {\text{O}}}} } \right)$$ Δ G NU H 2 O of ~ 4.5 kcal/mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show a path for building superior biocatalyst for higher biofuel production.

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Section: Discussionsupporting

confidence: 82%

Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase

Sharma

Shakeel

Gupta

et al. 2021

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Interestingly, GFC elution profile of AAR does not correspond to a monomeric fraction, instead, a broadly distributed peak-like pattern has been observed indicative of the co-existence of multiple-sized states of the protein in solution (Fig. S1), which was also previously reported by Kudo et al group [18]. The presence of other protein contaminants was ruled out as only a single band was observed in SDS-PAGE corresponding to the monomeric molecular weight of the AAR protein ( Fig.…”

Section: Discussionsupporting

confidence: 81%

Biophysical and structural studies reveal marginal stability of Acyl ACP Reductase: a crucial hydrocarbon biosynthetic enzyme

Sharma¹,

Shakeel²,

Gupta³

et al. 2020

Preprint

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Background: Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in E. coli, leading to varying alkane levels. Intriguingly, the structural characterization remains largely elusive as AAR alone failed to form stable crystals, possibly due to a number of intrinsically flexible random regions. The present work attempts to fill a gap in the literature by investigating the crucial structural aspects of AAR protein associated with its stability and folding. Results: The AAR protein was recombinant expressed in E. coli and purified by metal affinity and gel filtration chromatography. Characterization by dynamic light scattering experiment revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles in the range of 36.4 to 51.6 nm. Intact mass spectrometry revealed that recombinant AAR was heterogenous due to diverse lipidation and de-lipidation resulted in a single mass peak of 40296.87 Da as predicted. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution. Lower concentration of GdnHCl appeared to be stabilizing the protein, suggesting that the protein milieu plays a significant role in dictating it’s folding. Standard free energy (∆GH2ONU) of ~4.5 kcal/mol for steady-state unfolding of AAR indicated borderline stability of the protein. Molecular dynamics simulation conducted on AAR structure in presence of KCl, an ionic solvent with similar properties as GdnHCl at lower concentrations, suggested that KCl mediates structural stabilization especially at the concentration of 375 mM, and thus was responsible for enhancing its activity. KCl presence also resulted in regional alteration towards the binding site of its neighbouring pathway enzyme, ADO, thus paving the way for coordinated catalysis.Conclusion: Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show path for building superior biocatalyst for higher biofuel production.

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“…Example for such strategies can be understood by the research of Kudo et al (2019), wherein they had shown that when non-conserved ADO was engineered conversion efficiency of this enzyme was increased for hydrocarbon production. This strategy can be utilized for the enhanced synthesis of biofuels using type I polyketide synthases that has low solubility and high catalytic functioning, through polyketide biosynthetic pathway [ 57 ]. Clostridium acetobutylicum was also engineered to increase the butanol-to-ethanol ratio.…”

Section: Metabolic Engineering For Biofuel Productionmentioning

confidence: 99%

Microbial biotechnological approaches: renewable bioprocessing for the future energy systems

et al. 2021

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The accelerating energy demands of the increasing global population and industrialization has become a matter of great concern all over the globe. In the present scenario, the world is witnessing a considerably huge energy crisis owing to the limited availability of conventional energy resources and rapid depletion of non-renewable fossil fuels. Therefore, there is a dire need to explore the alternative renewable fuels that can fulfil the energy requirements of the growing population and overcome the intimidating environmental issues like greenhouse gas emissions, global warming, air pollution etc. The use of microorganisms such as bacteria has captured significant interest in the recent era for the conversion of the chemical energy reserved in organic compounds into electrical energy. The versatility of the microorganisms to generate renewable energy fuels from multifarious biological and biomass substrates can abate these ominous concerns to a great extent. For instance, most of the microorganisms can easily transform the carbohydrates into alcohol. Establishing the microbial fuel technology as an alternative source for the generation of renewable energy sources can be a state of art technology owing to its reliability, high efficiency, cleanliness and production of minimally toxic or inclusively non-toxic byproducts. This review paper aims to highlight the key points and techniques used for the employment of bacteria to generate, biofuels and bioenergy, and their foremost benefits.

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Improving hydrocarbon production by engineering cyanobacterial acyl-(acyl carrier protein) reductase

Cited by 17 publications

References 30 publications

Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase

Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase

Biophysical and structural studies reveal marginal stability of Acyl ACP Reductase: a crucial hydrocarbon biosynthetic enzyme

Microbial biotechnological approaches: renewable bioprocessing for the future energy systems

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