Study On Fermentation Regulation And Proteomics Of Odd Carbon Fatty Acids Produced By Schizochytrium

Odd-carbon fatty acids(OCFA)refer to a class of fatty acids with an odd number of carbon atoms.They have an active role in anti-inflammatory,treatment of cardiovascular diseases and cancer,and are widely used in food,pharmaceuticals,and chemical industry fields.Schizochytrium sp.is a kind of heterotrophic marine microalgae,which has the characteristics of simple culture conditions,fast growth,high biological yield,high oil content and good safety performance.It is an approved algal oil production strain in China,and has a wide range of industrial application prospects.Under the existing fermentation technology conditions,the yield of OCFA in Schizochytrium sp.is low,and the content in lipids is generally not higher than 10%,which is difficult to meet the industrial demand.This research systematically investigated the influence of culture medium composition and environmental factors on OCFA production by Schizochytrium sp.during fermentation,established a high-density fermentation process for OCFA production,and clarified the molecular mechanism regulating the synthesis of OCFA in Schizochytrium sp.by proteomics analysis technology.The specific research contents and results are as follows:1.The most suitable medium combination was determined by investigating the influence of medium composition and concentration on the growth of Schizochytrium sp.and OCFA content.The optimized culture medium composition was as follows:total carbon ratio 10:1,ratio of organic nitrogen to inorganic nitrogen 8:1,glucose concentration 80 g/L,sodium glutamate 30 g/L,and ammonium sulfate concentration1.2 g/L.Isopropanol of 5%(v/v)could effectively increase the proportion and yield of OCFA in total fatty acids from 9.5% to 18.8%,with an increase of 97.89%.2.The key factors of OCFA production by Schizochytrium sp.were explored,and a high-density fermentation process was established with its stability verification in small-scale production.The 5-L bioreactor was used for scale-up culture,and the influences of dissolved oxygen,pH,temperature and other environmental factors on the production of OCFA were investigated.Compared with 33 ? and 28 ?,the highest OCFA yield at 23 ? increased by 64.51% and 110.8%,respectively.Under optimal pH,dissolved oxygen conditions,and batch feeding,the highest OCFA content and yield reached 32.19% and 7.48 g/L,respectively.Six batches of stability verification were performed using 5-L bioreactors.The lipid yields and OCFA proportions in total fatty acids were 28.0%-32.0% and 27.0%-32.0%,respectively.3.Tandem Mass Tags(TMT)proteomics was used to analyze the differential protein function and metabolic pathway,and to explore the molecular mechanism of temperature regulating the high production of OCFA in Schizochytrium sp..Compared with the control group(33?),the glycolysis(EMP)pathway and tricarboxylic acid(TCA)cycle in the bacterial cells cultured at 23? were significantly strengthened,providing a large amount of succinyl-CoA and propionyl-CoA.At the same time,propionyl-COA produced by branched chain amino acid metabolism was also significantly increased.And the expression of FAS synthetase,acetyl-CoA carboxylase,fatty acyl-CoA elongase,etc.was significantly increased,prompting propionyl-CoA to enter the OCFA synthesis pathway,thereby greatly increasing the yield of OCFA.Our study investigated the composition of the culture medium and high-density fermentation conditions of the Schizochytrium sp..The lipid yield increased from 8.64g/L to 28.95 g/L,and the proportion of OCFA in total fatty acids increased from 9.5%to over 32%,and the possible metabolic pathway of OCFA in the Schizochytrium was explained by proteomic analysis.This study will help to clarify the regulatory mechanism of fatty acid biosynthesis pathway of Schizochytrium sp.,provide the theoretical basis for genetic engineering of Schizochytrium sp.,expand the application field of Schizochytrium sp.,and promote the industrialization of OCFA from microalgae.