ABSTRACT
Erucic acid (cis-docosa-13-enoic acid, C22:1∆13) and nervonic acid (cis-tetracosa-15-enoic acid, C24:1 ∆15) are important renewable feedstocks in plastic, cosmetic, nylon, and lubricant industries. Furthermore, nervonic acid is also applied to the treatment of some neurological diseases. However, the production of these two very long-chain fatty acids (VLCFA) is very limited as both are not present in the main vegetable oils (e.g., soybean, rapeseed, sunflower, and palm). Ectopic integration and heterologous expression of fatty acid elongases (3-ketoacyl-CoA synthases, KCS) from different plants in Rhodosporidium toruloides resulted in the de novo synthesis of erucic acid and nervonic acid in this oleaginous yeast. Increasing KCS gene copy number or the use of a push/pull strategy based on the expression of elongases with complementary substrate preferences increased significantly the amount of these two fatty acids in the microbial oils. Oil titers in 7-L bioreactors were above 50 g/L, and these two VLCFA represented 20-30% of the total fatty acids. This is the first time that microbial production of these types of oils is reported.
Subject(s)
Fatty Acids, Monounsaturated/metabolism , Microorganisms, Genetically-Modified , Rhodotorula/metabolism , Acetyltransferases/genetics , Acetyltransferases/metabolism , Bioreactors , Erucic Acids/chemistry , Fatty Acid Elongases , Fatty Acids, Monounsaturated/chemistry , Gene Dosage , Palm Oil/chemistry , Plant Oils/chemistry , Rapeseed Oil/chemistry , Rhodotorula/genetics , Soybean Oil/chemistry , Sunflower Oil/chemistryABSTRACT
We have engineered Rhodosporidium toruloides to produce fatty alcohols by expressing a fatty acyl-CoA reductase from Marinobacter aquaeolei VT8. Production of fatty alcohols in flasks was achieved in different fermentation media at titers ranging from 0.2 to 2 g/L. In many of the conditions tested, more than 80 % of fatty alcohols were secreted into the cultivation broth. Through fed-batch fermentation in 7 L bioreactors, over 8 g/L of C(16)-C(18) fatty alcohols were produced using sucrose as the substrate. This is the highest titer ever reported on microbial production of fatty alcohols to date.
Subject(s)
Aldehyde Oxidoreductases/metabolism , Basidiomycota/metabolism , Bioreactors , Fatty Alcohols/metabolism , Aldehyde Oxidoreductases/genetics , Basidiomycota/genetics , Batch Cell Culture Techniques , Culture Media/chemistry , Culture Media/metabolism , Fatty Alcohols/analysis , Fermentation , Gammaproteobacteria/enzymology , Gammaproteobacteria/genetics , Sucrose/metabolismABSTRACT
Fructo-oligosaccharides (FOS) represent the most abundantly supplied and utilized group of nondigestible oligosaccharides as food ingredients. These prebiotics can be produced from sucrose using the transglycosylating activity of beta-fructofuranosidases (EC 3.2.1.26) at high concentrations of the starting material. The main problem during FOS synthesis is that the activity of the enzyme is inhibited by the glucose generated during the reaction, and therefore the maximum FOS content in commercial products reaches up to 60% on a dry substance basis. The glucose oxidase (gox) gene from Aspergillus niger BT18 was cloned and integrated, as part of an expression cassette, into the ribosomal DNA of a Saccharomyces cerevisiae host strain. One of the recombinant strains with a high copy number of the gox gene and showing a high GOX specific activity was used to produce the enzyme. Addition of the extracellular glucose oxidase to the FOS synthesis reaction helped to remove the glucose generated, avoiding the inhibition of the fungal beta-fructofuranosidase. As a result, a final syrup containing up to 90% of FOS was obtained.
