Screening,Cultivation And Study On Mechanism Of Starch Mutant Of Chlorella Sorokiniana GT-1 With High Lipid Production

Chlorella sorokiniana GT-1 is a mixotrophic microalga that can be used for large-scale cultivation and biofuels production due to its fast-growing capabilites.Like most green algae,C.sorokiniana GT-1 can accumulated other types of macromolecules storing energy and carbon sources in addition to lipids,such as carbohydrates(e.g.starch)under adverse environmental stresses(such as nitrogen deficiency),which limited the efficient accumulation of lipids in this promising production strain.Therefore,this work aims to improve lipid production of C.sorokiniana GT-1 by screening the starchless mutants from a random chemical mutagenesis library.In this work,growth and biochemical compositions of the starchless mutant were characterized.The main results and findings of this work are as follows:1.In this study,a mutant library of C.sorokiniana GT-1 was constructed by using a Ethyl Methane Sulphonate(EMS)-mediated mutagenesis method,and 17 potential starchless were screened from a library containing 35,000 mutants,including 3 mutants(i.e.SLM1,SLM2 and SLM3)with lowered starch and enhanced lipid content,respectively.Among them,SLM2 accumulated the highest content of lipids.2.Under heterotrophic conditions,the cell division of SLM2 was faster than that of wild type(WT),which was beneficial for us to obtain a large number of algae cells as induction oilstrains in a short period of time.Under the nitrogen-limited conditions,the oil content,yield and productivity of SLM2 were higher than that of WT.In addition,the glucose consumption was lower in SLM2 than that of WT,but the efficiency of conversion of glucose into oil was higher in SLM2 than that of WT,thus reducing the cultivation cost.3.By comparing the oil production between heterotrophically or photoautotrophically-grown SLM2 and WT cells subjected to the high-light and nitrogen-limited conditions,this study revealed that lipid production of photoautotrophically-grown SLM2 cells was superior to WT.Combining the advantages of heterotrophic and photoautotrophic culture,a suitable culturing mode was established for SLM2,which involves producing high-cell-density inocula under heterotrophic conditions,followed by adaption under low-light and nitrogen-replete photoautotrophic conditions prior to inducing lipid production under high-light and nitrogen-limited condtions.This culture mode enabled producing a large amounts of inoclula in a short period of time and meanwhile enhanced the lipid production eventually.4.Metabolomic methods were used to study the metabolites and metabolic pathways attributable for enhanced lipid production in the photoautotrophically-grown SLM2 subjected high-light and nitrogen-limited conditions.When the starch synthesis pathway of SLM2 was blocked,the sucrose synthesis pathway was augmented,which can in turn provide sufficient precursors for glycolysis.The photoautotrophically-grown SLM2 can produce more NADPH via the pentose phosphate pathway and more glycerol-3-phosphate from glycolysis than WT.Additionally,the Glutamate-GABA-Succinate shunt was increased in photoautotrophically-grown SLM2 than that of WT,which can facilitate the transfer of more carbon skeletons into the lipid biosynthesis pathway.Phosphocreatine was detected in C.sorokiniana GT-1 cells,of which the content was higher in photoautotrophically-grown SLM2 than that in WT.These "ATP buffers" were beneficial for the synthesis of TAG.SLM2 was featured by lower contents of L-carnitine and acetyl carnitine content than wild type,which may promot the synthesis of TAG and reduce the degradation of fatty acids.For the first time,a metabolic network map related to oil synthesis was constructed.The above findings will provide theoretical guidance for the industrial application of the starchless mutant SLM2.