Genetically modified fungi and their use in lipid production

Abstract

The invention refers to fungal cells, and especially to oleaginous fungal cells that have been genetically modified to produce enzymes of the pyruvate dehydrogenase bypass route to enhance their lipid production. Especially the cells are modified to overexpress genes encoding pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD) and/or acetyl-CoA synthetase (ACS), optionally together with a gene encoding diacylglycerol acyltransferase (DAT), or to express genes encoding PDC together with ALD and/or ACS. Methods of producing lipids, biofuels and lubricants using the modified fungi are also disclosed as well as expression cassettes useful therein. A new enzyme having phosholipid:diacylglycerol acyltransferase (PDAT) activity and a polynucleotide encoding it are also disclosed, which are useful in the lipid production. A recombinant Cryptococcus cell and its construction is described.

1 . A genetically modified oleaginous fungal cell comprising:
 a) a nucleic acid with enhanced expression encoding an acetaldehyde dehydrogenase (ALD), and   b) a nucleic acid with enhanced expression encoding a diacylglycerol acyltransferase (DAT),   wherein said genetically modified oleaginous fungal cell has enhanced lipid production in an aerobic bioreactor compared to a genetically unmodified oleaginous fungal cell.   
     
     
         2 . The genetically modified oleaginous fungal cell of  claim 1 , wherein the encoded ALD is a cytosolic ALD. 
     
     
         3 . The genetically modified oleaginous fungal cell of  claim 2 , wherein the encoded ALD is a fungal ALD, preferably  Saccharomyces cerevisiae  ALD6. 
     
     
         4 . The genetically modified oleaginous fungal cell of  claim 1 , wherein the DAT encoding nucleic acid encodes a phosholipid:diacylglycerol acyltransferase (PDAT). 
     
     
         5 . The genetically modified oleaginous fungal cell of  claim 4 , wherein the encoded DAT is a fungal DAT, preferably of  Rhizopus oryzae , and most preferably it encodes a phosholipid:diacylglycerol acyltransferase (PDAT) having at least 40% sequence identity to SEQ ID NO:52, or an enzymatically active fragment or variant thereof. 
     
     
         6 . The genetically modified oleaginous fungal cell of  claim 1 , further comprising a nucleic acid with enhanced expression encoding acetyl-CoA synthetase (ACS). 
     
     
         7 . The genetically modified oleaginous fungal cell of  claim 6 , wherein the nucleic acid encoding ACS is a gene that is not under glucose repression or its gene product is not subject to post-translational regulation. 
     
     
         8 . The genetically modified oleaginous fungal cell of  claim 7 , wherein the encoded ACS is a fungal ACS, preferably  Saccharomyces cerevisiae  ACS2. 
     
     
         9 . The genetically modified oleaginous fungal cell of  claim 1 , which is a yeast cell selected from the genera  Cryptococcus, Candida, Galactomyces, Hansenula, Lipomyces, Rhodosporidium, Rhodotorula, Trichosporon  and  Yarrowia , preferably from the group consisting of  Candida  sp.,  Cryptococcus  curvatus,  Cryptococcus albidus, Galactomyces geotrichum, Hansenula ciferri, Lipomyces lipofer, Lipomyces  ssp.,  Lipomyces starkeyi, Lipomyces tetrasporus, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica , or
 a filamentous fungal cell selected from the genera  Aspergillus, Cunninghamella, Fusarium, Glomus, Humicola, Mortierella, Mucor, Penicillium, Pythium  and  Rhizopus , preferably from the group consisting of  Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Aspergillus niger, Cuninghamella japonica, Fusarium oxysporum, Glomus caledonius, Humicola lanuginose, Mortierella isabellina, Mortierella pusilla, Mortierella vinacea, Mucor circinelloides, Mucor plumbeus, Mucor ramanniana, Penicillium lilacinum, Penicillium spinulosum, Pythium ultimum  and  Rhizopus oryzae.      
     
     
         10 . The genetically modified oleaginous fungal cell of  claim 9 , which is from the genera  Cryptococcus  or  Mucor , preferably it is a cell of  Cryptococcus curvatus  or  Mucor  circinelloides. 
     
     
         11 . A method of producing lipids, comprising cultivating a genetically modified oleaginous fungal cell according to  claim 1  in a medium containing carbon and nitrogen sources, and recovering the lipids produced. 
     
     
         12 . The method of  claim 11 , wherein the lipids are recovered from the culture medium. 
     
     
         13 . The method of  claim 11 , wherein lipids comprising acylglycerols, preferably triacylglycerols (TAG) are produced. 
     
     
         14 . The method of  claim 11 , wherein the carbon source is a hexose or pentose sugars containing material. 
     
     
         15 . The method of  claim 11 , comprising producing precursors for functional fatty acids. 
     
     
         16 . A method of producing biofuel, or lubricant, said method comprising cultivating a genetically modified oleaginous fungal cell according to  claim 1  in a medium containing carbon and nitrogen sources, and recovering the lipids produced, and optionally esterifying said lipids to obtain biodiesel or lubricant, or hydrogenizing the lipids to obtain renewable diesel or lubricant. 
     
     
         17 . A method of preparing an oleaginous fungal cell of  claim 1 , said method comprising transforming a fungal cell with
 a) a nucleic acid with enhanced expression encoding an acetaldehyde dehydrogenase (ALD) enzyme, and   b) a nucleic acid with enhanced expression encoding a diacylglycerol acyltransferase (DAT).   
     
     
         18 . Use of a genetically modified fungal cell of  claim 1  for producing lipids, precursors of functional fatty acids, functional fatty acids, biofuels, biodiesel, renewable diesel or lubricants.