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Dual-function oleaginous biocatalysts for non-sterile cultivation and solvent-free biolipid bioextraction to reduce biolipid-based biofuel production costs.

ABSTRACT: Triacylglycerols (TAGs) are starting materials for the production of biolipid-based fuels such as biodiesel and biojet fuel. While various microorganisms can produce TAGs from renewable resources, the cultivation of TAG-producing microorganisms under sterilization conditions to avoid microbial contamination and application of solvent to extract TAGs from the TAG-filled microorganisms are costly. To overcome these challenges, this study reports the feasibility of a non-sterile cultivation of an oleaginous bacterium Rhodococcus opacus PD631SpAHB under saline conditions, followed by the use of a solvent-free, phage-lysis-protein-based bioextraction approach for TAGs release. The engineered strain PD631SpAHB was developed by introducing a recombinant plasmid carrying a phage lytic gene cassette (pAHB) into Rhodococcus opacus PD631 via transformation, followed by adaptive evolution under saline conditions. This newly developed strain is a salt-tolerant strain with the inducible plasmid pAHB to enable TAGs release into the supernatant upon induction. Cell lysis of PD631SpAHB was confirmed by the decrease of the optical density of cell suspension, by the loss of cell membrane integrity, and by the detection of TAGs in the culture medium. Up to 38% of the total TAGs accumulated in PD631SpAHB was released into supernatant after the expression of the lytic genes. PD631SpAHB strain is a promising candidate to produce TAGs from non-sterile growth medium and release of its TAGs without solvent extraction - a new approach to reduce the overall cost of biolipid-based biofuel production.

SUBMITTER: Hwangbo M

PROVIDER: S-EPMC8061307 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDue to scarcity of fossil fuel, the importance of alternative energy sources is ever increasing. The oleaginous microalgae have demonstrated their potential as an alternative source of energy, but have not achieved commercialization owing to some biological and technical inefficiency. Modern methods of recombinant strain development for improved efficacy are suffering due to inadequate knowledge of genome and limited molecular tools available for their manipulation.ResultsIn the present study, microalga Scenedesmus quadricauda LWG002611 was selected as the preferred organism for lipid production as it contained high biomass (0.37 g L-1 day-1) and lipid (102 mg L-1 day-1), compared to other oleaginous algae examined in the present study as well as earlier reports. It possessed suitable biodiesel properties as per the range defined by the European biodiesel standard EN14214 and petro-diesel standard EN590:2013. To investigate the potential of S. quadricauda LWG002611 in details, the genome of the organism was assembled and annotated. This was the first genome sequencing and assembly of S. quadricauda, which predicted a genome size of 65.35 Mb with 13,514 genes identified by de novo and 16,739 genes identified by reference guided annotation. Comparative genomics revealed that it belongs to class Chlorophyceae and order Sphaeropleales. Further, small subunit ribosomal RNA gene (18S rRNA) sequencing was carried out to confirm its molecular identification. S. quadricauda LWG002611 exhibited higher number of genes related to major activities compared to other potential algae reported earlier with a total of 283 genes identified in lipid metabolism. Metabolic pathways were reconstructed and multiple gene homologs responsible for carbon fixation and triacylglycerol (TAG) biosynthesis pathway were identified to further improve this potential algal strain for biofuel production by metabolic engineering approaches.ConclusionHere we present the first draft genome sequence, genetic characterization and comparative evaluation of S. quadricauda LWG002611 which exhibit high biomass as well as high lipid productivity. The knowledge of genome sequence, reconstructed metabolic pathways and identification of rate-limiting steps in TAG biosynthesis pathway will strengthen the development of molecular tools towards further improving this potentially one of the major algal strains for biofuel production.

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Dual-function oleaginous biocatalysts for non-sterile cultivation and solvent-free biolipid bioextraction to reduce biolipid-based biofuel production costs.

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