Methods for fermentative production of massoia lactone

Abstract

The present invention relates to the field of fermentation biotechnology, more particularly to methods for the fermentative production of massoia lactone by Aureobasidium species.

1 . A pure culture of  Aureobasidium melanogenum  that expresses no functional Aureobasidin A biosynthesis complex gene mRNA when cultured. 
     
     
         2 . The pure culture of  claim 1 , wherein the  Aureobasidium melanogenum  strain is  Aureobasidium melanogenum  strain W5-2. 
     
     
         3 . The pure culture of  claim 2 , wherein  Aureobasidium melanogenum  W5-2 is deposited with Agricultural Research Culture Collection (NRRL) and assigned Accession Number NRRL 67063. 
     
     
         4 . The pure culture of  claim 1 , wherein the  Aureobasidium melanogenum  GDP1 sequence shares at least 97.5% identity over at least 98.5% of SEQ ID NO:2, preferably 99%-100% identity to over at least 98% of SEQ ID NO:2. 
     
     
         5 . The pure culture of  claim 1 , wherein the  Aureobasidium melanogenum  TEF1A sequence shares at least 98% identity over at least 94% of SEQ ID NO:8, preferably 99%-100% identity to over at least 99% of SEQ ID NO:8. 
     
     
         6 . The pure culture of  claim 1 , wherein the  Aureobasidium melanogenum  RBP1 sequence shares at least 91% identity over at least 92% of SEQ ID NO:10, preferably 96%-100% identity to over at least 98% of SEQ ID NO:10. 
     
     
         7 . The pure culture of  claim 1 , wherein the  Aureobasidium melanogenum  GDP1 sequence shares at least 97.5% identity over at least 98.5% of SEQ ID NO:2, preferably 99%-100% identity to over at least 98% of SEQ ID NO:2, wherein the  Aureobasidium melanogenum  TEF1A sequence shares at least 98% identity over at least 94% of SEQ ID NO:8, preferably 99%-100% identity to over at least 99% of SEQ ID NO:8 and wherein the  Aureobasidium melanogenum  RBP1 sequence shares at least 91% identity over at least 92% of SEQ ID NO:10, preferably 96%-100% identity to over at least 98% of SEQ ID NO:10. 
     
     
         8 . Use of the pure culture of  claim 1  for the fermentative production of massoia lactone. 
     
     
         9 . A method of fermentative production of massoia lactone comprising culturing an  Aureobasidium melanogenum  species in a culture medium comprising high levels of phosphate ions, ammonium ions, calcium ions, at least two trace elements selected from the group consisting of Fe 2+ , Cu 2+ , Zn 2+  and MoO 4   2− , urea and a carbon source selected from the group consisting of glucose, mannose, xylose and mixtures thereof for a sufficient period of time to produce massoia lactone in a fermentation product. 
     
     
         10 . The method of  claim 9 , wherein the pH of the culture medium is from about 5.5 to about 6.5, preferably 6.0 
     
     
         11 . The method of  claim 9 , wherein the amount of urea present in the culture medium is from about 1.5 g/l to about 2.5 g/l, preferably from about 1.8 g/l to about 2.2 g/l, more preferably about 2 g/l. 
     
     
         12 . The method of  claim 9 , wherein the carbon source present in the culture medium is from about 4% to about 12%, preferably from about 5% to about 12%, preferably from about 5% to about 11%, more preferably from about 5% to about 10%. 
     
     
         13 . The method of  claim 9 , wherein each trace element in the culture medium is present in an amount from about 0.1 μM to about 1.0 mM, from about 1.0 μM to about 1.0 mM, from about 10.0 μM to about 1.0 mM, or from about 100 μM to about 1.0 mM. 
     
     
         14 . The method of  claim 9 , wherein the culture comprises about 10.0 g/l to about 15 g/l, preferably about 12.5 g/l KH 2 PO 4 , about 0.5 g/l to about 2.0 g/l, preferably about 1.0 g/l Na 2 HPO 4 , about 3.5 g/l to about 6.5 g/l, preferably about 5.0 g/l (NH4) 2 SO 4 , about 1.0 g/l to about 4.0 g/l, preferably about 2.5 g/l MgSO 4 .7H 2 O and about 0.10 g/l to about 0.40 g/l, preferably about 0.25 g/l CaCl 2 .2H 2 O. 
     
     
         15 . The method of  claim 14 , wherein the culture medium further comprises about 2 g/l urea, about 5% to about 10% carbon source and at least two of the trace elements. 
     
     
         16 . The method of  claim 9 , wherein the period of time is for about 4 days to about 12 days, preferably for about 5 days to about 12 days, preferably for about 5 days to about 11 days, preferably for about 6 days to about 11 days, more preferably for about 7 days to about 10 days. 
     
     
         17 . The method of  claim 9 , wherein the  Aureobasidium melanogenum  is a strain that expresses no functional Aureobasidin A synthase gene mRNA in culture. 
     
     
         18 . The method of  claim 17 , wherein the  Aureobasidium melanogenum  strain is  Aureobasidium melanogenum  W5-2. 
     
     
         19 . The method of  claim 18 , wherein  Aureobasidium melanogenum  W5-2 is deposited with Agricultural Research Culture Collection (NRRL) and assigned Accession Number NRRL 67063. 
     
     
         20 . The method of  claim 9 , which further comprises hydrolyzing the fermentation product by a strong inorganic acid and purifying the massoia lactone from the fermentation product. 
     
     
         21 . The method of  claim 9 , wherein the fermentation product is substantially free of contaminant 3-hydroxyl delta-decalactone (3-hydroxydecan-5-olide). 
     
     
         22 . A culture medium for the fermentative production of massoia lactone by  Aureobasidium melanogenum  species, the culture medium comprising high levels of phosphate ions, ammonium ions, calcium ions, at least two trace elements selected from the group consisting of Fe 2+ , Cu 2+ , Zn 2+  and MoO 4   2− , urea and a carbon source selected from the group consisting of glucose, mannose, xylose and mixtures thereof. 
     
     
         23 . The culture medium of  claim 22 , wherein the pH of the culture medium is from about 5.5 to about 6.5, preferably 6.0 
     
     
         24 . The culture medium of  claim 22 , wherein the amount of urea present in the culture medium is from about 1.5 g/l to about 2.5 g/l, preferably from about 1.8 g/l to about 2.2 g/l, more preferably about 2 g/l. 
     
     
         25 . The culture medium of  claim 22 , wherein the carbon source present in the culture medium is from about 4% to about 12%, preferably from about 5% to about 12%, preferably from about 5% to about 11%, more preferably from about 5% to about 10%. 
     
     
         26 . The culture medium of  claim 22 , wherein each trace element present in the culture medium is present in an amount from about 0.1 μM to about 1.0 mM, from about 1.0 μM to about 1.0 mM, from about 10.0 μM to about 1.0 mM, or from about 100 μM to about 1.0 mM. 
     
     
         27 . The culture medium of  claim 22 , wherein the culture medium comprises about 10.0 g/l to about 15 g/l, preferably about 12.5 g/l KH 2 PO 4 , about 0.5 g/l to about 2.0 g/l, preferably about 1.0 g/l Na 2 HPO 4 , about 3.5 g/l to about 6.5 g/l, preferably about 5.0 g/l (NH4) 2 SO 4 , about 1.0 g/l to about 4.0 g/l, preferably about 2.5 g/l MgSO 4 .7H 2 O and about 0.10 g/l to about 0.40 g/l, preferably about 0.25 g/l CaCl 2 .2H 2 O. 
     
     
         28 . The culture medium of  claim 27 , wherein the culture medium further comprises about 2 g/l urea, about 5% to about 10% carbon source and at least two of the trace elements. 
     
     
         29 . The culture medium of  claim 22 , wherein the culture medium further comprises an  Aureobasidium melanogenum  species. 
     
     
         30 . The culture medium of  claim 29 , wherein the  Aureobasidium melanogenum  is a strain that expresses no functional Aureobasidin A synthase gene mRNA in culture. 
     
     
         31 . The culture medium of  claim 30 , wherein the  Aureobasidium melanogenum  strain is  Aureobasidium melanogenum  W5-2. 
     
     
         32 . The culture medium of  claim 31 , wherein  Aureobasidium melanogenum  W5-2 is deposited with Agricultural Research Culture Collection (NRRL) and assigned Accession Number NRRL 67063. 
     
     
         33 . The culture medium of  claim 22  further comprising more than 10 g/l of massoia lactone.