Lipases And Their Role In Regulating Lipid Metabolism In The Oleaginous Fungus Mucor Circinelloides

Oleaginous microorganisms can produce a substantial amount of triacylglycerol of20-80%of their cell dry weight.Among them,the filamentous fungus and microalgae can accumulate the long-chain polyunsaturated fatty acids?PUFA?which have many biological activities and have been identified as important nutritional and food resources.Fatty acids produced by oleaginous yeast species can be converted into biodiesel,which is an important alternative to fossil energy.The oleaginous filamentous fungus Mucor circinelloides has drawn many attentions because it produces high levels of?-linolenic acid.A lot of work has been done to analyze the molecular mechanism of lipid accumulation in M.circinelloides by genomics,proteomics,metabolic flux and key enzyme activity analysis.The cytosolically-located,NADP⁺-dependent malic enzyme and the pentose phosphate pathway have proved to be the main provider of NADPH in M.circinelloides.However,the lipid catabolic metabolism in M.circinelloides has not been studied,especially the lipases which can regulate lipid degradation.This study systematically analyzed the characteristics of lipase genes in a high lipid-producing strain M.circinelloides WJ11 and a low lipid-producing strain M.circinelloides CBS 277.49 based on gene annotations and extensive bioinformatics analysis.This study also found four key lipase genes involved in the lipid metabolism through transcription analysis,whose functions were preliminarily studied by gene over-expression.Dual roles of lipase Lip6 and Lip10 in the lipid metabolism were proved by site-directed mutagenesis and enzyme activity analysis.In addition,the effect of different glucose-oil mixed media on cell growth and lipid accumulation was studied.The main research contents and results include the following sections:The gene annotations and extensive bioinformatics analysis found 47 potential lipase genes in WJ11 and 30 potential lipase genes in CBS 277.49.All of these lipases were divided into four types including?/?-hydrolase₁,?/?-hydrolase₃,class₃ and GDSL lipase.25lipases in WJ11 not only contain the typical lipase motif?G/A?XSXG,but also the consensus sequence motif H-?X?₄-D?a typical of acyltransferase motif?,while only 16 similar lipases are observed in CBS 277.49.Analysis of sub-cellular location,signal peptide and transcriptional level of lipase genes showed that lipases?WJ₁6,29,39,40 and 43?in WJ11and lipases?116027,106404 and 170034?in CBS 277.49 contained signal peptide sequences and their transcriptional levels were inhibited during the lipid accumulation,indicating that these lipases belong to the family of secretary lipases.Transcriptional analysis revealed that lipase genes?161426 and 115761,No.6 and 10?expression increased significantly during lipid accumulation while lipase genes?143450 and 107413,No.19 and 24?were down-regulated along with the increase in lipid accumulation.These four lipase genes maybe take part in lipid accumulation or degradation in CBS 277.49.The cell growth,lipid accumulation,and lipase activity of CBS 277.49 and WJ11 grown on different glucose-oil mixed media were analyzed.The highest biomass of 21.2 g/L was obtained for CBS 277.49 grown on soybean oil,while the highest biomass of 18.9 g/L was obtained for WJ11 grown on soybean oil soybean phospholipids.The highest lipid content of50%was obtained when CBS 277.49 was grown on phytosterol esters,while the highest lipid content of 65%was obtained when WJ11 was grown on soybean phospholipids.M.circinelloides preferred to assimilate exogenous palmitic,stearic,and?-linolenic acid to synthesize intracellular triacylglycerols.Soybean phospholipids and phytosterol esters stimulated the production of intracellular lipases in both strains more efficiently than soybean oils.The maximal lipase activity of 2200 U/g protein was observed for CBS 277.49 grown on phytosterol esters,while the maximal lipase activity of 2000 U/g protein was observed for WJ11 grown on soybean phospholipids.Although the transcriptional levels of the lipase genes were sharply up-regulated when strains were grown on glucose-oil mixed media,the extracellular lipase activity in the medium was still low at 4-210 U/g protein.The result of transcription and bioinformatics analysis suggested that lipases?Lip6,Lip10,Lip19,and Lip24?may be involved in lipid accumulation in M.circinelloides.Over-expression of either lipase Lip6 or Lip10 led to a slight increase in cell dry weight,but no significant effect on lipid accumulation.Over-expression of Lip6 resulted in a 17%increase in C18:3 content of the lipids.Transcriptional levels of Lip6 or Lip10 were enhanced notably during lipid accumulation,but no significant lipid degradation was observed in strains Mc-Lip6 and Mc-Lip10.However,over-expression of lipase Lip19 or Lip24 decreased the cell dry weight by 9-19%and decreased the lipid content by 38-41%,respectively.The fatty acid profiles of strains with Lip19 and Lip24 over-expression were also significantly changed as compared to control.The sequences of Lip6 and Lip10 contained not only the typical lipase motif?G/A?XSXG,but also the consensus sequence motif HXXXXD?a typical of acyltransferase motif?.However,the lower homology between Lip6/Lip10,LPAT,and S.cerevisiae lipases indicated that Lip6 and Lip10 in M.circinelloides are functionally different from Tgl3p,Tgl4p and Tgl5p in S.cerevisiae.These results suggest that Lip6 or Lip10 may control acyltransferase activity in M.circienlloides and play a role in fatty acid reconstruction of the TAG.Lip19 or Lip24 works as a TAG lipase involved in TAG metabolism/degradation.To demonstrate that Lip6 or Lip10 might play dual roles as a lipase and an acyltransferase,we performed site-directed mutagenesis in the lipase motif,GXSXG,and the acyltransferase motif,H-?X?₄-D,of both Lip6 and Lip10.Over-expression of the acyltransferase motif of Lip6 and Lip10 increased cell growth by 12%-18%and promoted lipid accumulation by 9%-24%,whilst over-expression of the lipase motif induced lipid degradation in M.circinelloides.Enzyme activity assays in vitro proved that purified Lip6 and Lip10 have slight lipase activity but have stronger phospholipid:DAG acyltransferase activity.Lip6 exhibited a preference for phosphatidyl ethanolamine?PE?and phosphatidyl serine?PS?over phosphatidylcholine?PC?,while Lip10 can form more TAG when PS was used as acyl donors.Enzyme activity assays in vivo and phospholipid synthesis pathway analysis suggested that PS and PE can be the supplier of a fatty acyl moiety for the acyltransferase to form TAG in M.circinelloides.