Preliminary Study On The Structure And Function Of GPDH And Its Relationship With TAG Generation In Dunaliella Salina

Biodiesel from microalgae is the third generation of bioenergy and has received a lot of attention due to its wider availability,carbon neutrality and sustainability.As an important member of the microalgae family,Dunaliella salina is the first microalgae to be commercially produced.It not only contains rich oil,but also can grow strongly in harsh environment,and can effectively obtain a large number of algae through large-scale open culture,which greatly reduces the cost of culture.It is a very suitable candidate algal species for high lipid engineering algae.As the main rate-limiting enzyme in the glycerol synthesis pathway,glycerol-3-phosphate dehydrogenase(GPDH)has a close relationship with triglyceride(TAG),an important reaction material for biodiesel production.Unlike GPDH in traditional oil crops,which catalyzes the reduction of DHAP to glycerol 3-phosphate(G3P),Dunaliella salina’s GPDH2,reported in 2020,has a special dual domain(GPDH-serb)and a unique quaternary structure(tetrameter polymerization state)that can directly and rapidly convert DHAP to glycerol.From the perspective of metabolic pathway,this reaction consumes G3 P,an important precursor of TAG synthesis,and consumes the carbon source of acetyl-Co A,a precursor of fatty acid synthesis,thus inhibiting TAG synthesis.Therefore,this study speculated that the glycerol pathway catalyzed by Dunaliella salina’s GPDH might be competitive with the TAG biosynthesis pathway.On this basis,four GPDH isoenzymes containing special dual domains were selected as the research object for the first time to preliminarily explore the relationship between GPDH and TAG production.In previous studies,we have successfully obtained four GPDH proteins(GPDH1,GPDH2,GPDH3 and GPDH4)from Dunaliella salina,and preliminarily explored their properties and functions.Moreover,it is known that their unique dual domain structure and polymerization state are very important for the rapid synthesis of glycerol.In view of this,this paper mainly used GPDH2 as a reference and template protein,continued to analyze the threedimensional structure of GPDH1,GPDH3 and GPDH4 proteins,and further elucidate the relationship between the structure of GPDH isozyme and its functional characteristics to explore their possible impact on TAG biosynthesis.In addition,we preliminarily attempted to provide stress conditions to down-regulate the expression level of GPDH gene and/or inhibit the activity of classical GPDH domain of GPDH isozyme.At the same time,glycerol and TAG production levels were detected to explore whether the inhibition of GPDH by inhibiting glycerol synthesis could lead to the carbon flow to the TAG production pathway,which makes the TAG pile up.The main results obtained are as follows:1.It was preliminarily inferred that GPDH1 was monomer,and GPDH4 may contain multiple polymerization forms or have been denaturated,while GPDH3 was confirmed to be dimer protein by negative staining and the primary conditions for its crystal growth have been successfully screened.At the same time,the inactivity of the classical GPDH domains of GPDH1 and GPDH4 is most likely due to the mutation of amino acids in their active sites and the inability to effectively bind to NAD+.Therefore,we preliminarily speculated that the different polymerization states and non-bidomain activities of the four GPDH isoenzymes might affect the rapid synthesis of glycerol and then affect the biosynthesis of TAG.2.Under high salt stress,the contents of glycerol and TAG in Dunaliella salina cells increased,which was consistent with the reported results.However,the addition of high phosphate to inhibit GPDH did not significantly inhibit the synthesis of glycerol;Under nitrogen deficiency stress,the increase of TAG production was consistent with the reported results.After mixed treatment with high phosphate,glycerol content increased first and then decreased sharply,while TAG showed an opposite trend.These results indicate that under the condition of GPDH inhibition,some glycerol carbon sources may flow to the direction of TAG production,suggesting that TAG production may be negatively correlated with the down-regulation of GPDH gene expression and/or the inhibition of GPDH isozyme activity of classical GPDH domain,which can be further verified by experiments.In conclusion,this paper has preliminarily completed the exploration of the structure and function of GPDH isozyme proteins in Dunaliella salina and their relationship with TAG generation.The obtained results not only enrich the protein structure database in salina system,but also reveal the close relationship between the structure and function of GPDH protein.At the same time,it also provides a theoretical basis for further analysis of the reaction mechanism of algae in the process of oil production.