Radical-based photoinactivation of fatty acid photodecarboxylases

Lakavath, Balaji; Hedison, Tobias M.; Heyes, Derren J.; Shanmugam, Muralidharan; Sakuma, Michiyo; Hoeven, Robin; Tilakaratna, Viranga; Scrutton, Nigel S.

doi:10.1016/j.ab.2020.113749

Cited by 54 publications

(82 citation statements)

References 13 publications

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“…5c). This suggests a gradual decrease in biocatalytic enzyme production, likely due to degradation/inactivation of IPTG and/or CvFAP photoinactivation [23] during the extended fermentation time [36]. Significant propane production in this strain was extended to nearly 5 days, however elimination of the need for IPTG could potentially extend propane production even further.…”

Section: Fermentative Amino Acid To Bio-alkane Production In Halomonasmentioning

confidence: 92%

“…The requirement for blue light in CvFAP-dependent pathways necessitates adaptations to traditional in vivo bacterial cultivation strategies, as prolonged exposure to blue light (380-500 nm) is known to decrease the viability of E. coli [22] and cause the photoinactivation of CvFAP [4,23]. Therefore, dark cycles (light absence) might be required periodically during fermentation to ensure the maintenance of sufficient cell density and the regeneration of biocatalysts.…”

Section: Coa-dependent Pathway Bio-alkane Gas Productionmentioning

confidence: 99%

“…S8-S10). This is attributed to plasmid instability and/or loss, or possibly CvFAP light inactivation [4,23].…”

Section: Fermentative Amino Acid To Bio-alkane Production In Halomonasmentioning

confidence: 99%

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Renewable and tuneable bio-LPG blends derived from amino acids

Amer

Hoeven

Kelly

et al. 2020

Biotechnol Biofuels

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Background: Microbial biorefinery approaches are beginning to define renewable and sustainable routes to cleanburning and non-fossil fuel-derived gaseous alkanes (known as 'bio-LPG'). The most promising strategies have used a terminal fatty acid photodecarboxylase, enabling light-driven propane production from externally fed waste butyric acid. Use of Halomonas (a robust extremophile microbial chassis) with these pathways has enabled bio-LPG production under non-sterile conditions and using waste biomass as the carbon source. Here, we describe new engineering approaches to produce next-generation pathways that use amino acids as fuel precursors for bio-LPG production (propane, butane and isobutane blends). Results: Multiple pathways from the amino acids valine, leucine and isoleucine were designed in E. coli for the production of propane, isobutane and butane, respectively. A branched-chain keto acid decarboxylase-dependent pathway utilising fatty acid photodecarboxylase was the most effective route, generating higher alkane gas titres over alternative routes requiring coenzyme A and/or aldehyde deformylating oxygenase. Isobutane was the major gas produced in standard (mixed amino acid) medium, however valine supplementation led to primarily propane production. Transitioning pathways into Halomonas strain TQ10 enabled fermentative production of mixed alkane gases under non-sterile conditions on simple carbon sources. Chromosomal integration of inducible (~ 180 mg/g cells/day) and constitutive (~ 30 mg/g cells/day) pathways into Halomonas generated production strains shown to be stable for up to 7 days. Conclusions: This study highlights new microbial pathways for the production of clean-burning bio-LPG fuels from amino acids. The use of stable Halomonas production strains could lead to gas production in the field under nonsterile conditions following process optimisation.

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Section: Fermentative Amino Acid To Bio-alkane Production In Halomonasmentioning

confidence: 92%

Section: Coa-dependent Pathway Bio-alkane Gas Productionmentioning

confidence: 99%

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Renewable and tuneable bio-LPG blends derived from amino acids

Amer

Hoeven

Kelly

et al. 2020

Biotechnol Biofuels

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“…Lower than expected correlations could occur when one or both of the ‘activities’ monitored are sub-optimal or absent. For example, photoinactivation or lack of flavin incorporation of CvFAP leads to inactive protein ( 32 , 33 ), while the C-terminal RFP domain may be fully active. Conversely, the RFP maturation rate may not keep pace with high protein expression rates in E. coli ( 34 ).…”

Section: Resultsmentioning

confidence: 99%

Promoter engineering for microbial bio-alkane gas production

et al. 2020

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Successful industrial biotechnological solutions to biofuels and other chemicals production relies on effective competition with existing lower cost natural sources and synthetic chemistry approaches enabled by adopting low-cost bioreactors and processes. This is achievable by mobilising Halomonas as a next generation industrial chassis, which can be cultivated under non-sterile conditions. To increase the cost effectiveness of an existing sustainable low carbon bio-propane production strategy, we designed and screened a constitutive promoter library based on the known strong porin promoter from Halomonas. Comparative studies were performed between E. coli and Halomonas using the reporter gene red fluorescent protein. Later studies with a fatty acid photodecarboxylase-RFP fusion protein demonstrated tuneable propane production in Halomonas and E. coli, with an ~8-fold improvement in yield over comparable IPTG-inducible systems. This novel set of promoters are a useful addition to the synthetic biology toolbox for future engineering of Halomonas to make chemicals and fuels.

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“…The simpler reaction requirements and the higher turnover number of CvFAP compared to ADO suggest the former is a better candidate for scaled hydrocarbon gas production. However, a major limitation of CvFAP is its inherent instability, as seen by a loss of flavin content during protein purification [47]. Studies revealed that blue light-exposure, which is necessary for CvFAP activity, also irreversibly inactivates the enzyme through the formation of protein based organic radicals [47].…”

Section: Decarboxylation: Fatty Acid Photodecarboxylasementioning

confidence: 99%

Engineering nature for gaseous hydrocarbon production

2020

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The development of sustainable routes to the bio-manufacture of gaseous hydrocarbons will contribute widely to future energy needs. Their realisation would contribute towards minimising over-reliance on fossil fuels, improving air quality, reducing carbon footprints and enhancing overall energy security. Alkane gases (propane, butane and isobutane) are efficient and clean-burning fuels. They are established globally within the transportation industry and are used for domestic heating and cooking, non-greenhouse gas refrigerants and as aerosol propellants. As no natural biosynthetic routes to short chain alkanes have been discovered, de novo pathways have been engineered. These pathways incorporate one of two enzymes, either aldehyde deformylating oxygenase or fatty acid photodecarboxylase, to catalyse the final step that leads to gas formation. These new pathways are derived from established routes of fatty acid biosynthesis, reverse β-oxidation for butanol production, valine biosynthesis and amino acid degradation. Single-step production of alkane gases in vivo is also possible, where one recombinant biocatalyst can catalyse gas formation from exogenously supplied short-chain fatty acid precursors. This review explores current progress in bio-alkane gas production, and highlights the potential for implementation of scalable and sustainable commercial bioproduction hubs.

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Radical-based photoinactivation of fatty acid photodecarboxylases

Cited by 54 publications

References 13 publications

Renewable and tuneable bio-LPG blends derived from amino acids

Renewable and tuneable bio-LPG blends derived from amino acids

Promoter engineering for microbial bio-alkane gas production

Engineering nature for gaseous hydrocarbon production

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