Fermentative production of L-glycerol 3-phosphate utilizing a Saccharomyces cerevisiae strain with an engineered glycerol biosynthetic pathway.
Interest in L-glycerol 3-phosphate (L-G3P) production via microbial fermentation is due to the compound's potential to replace the unstable substrate dihydroxyacetone phosphate (DHAP) in one-pot enzymatic carbohydrate syntheses. A Saccharomyces cerevisiae strain with deletions in both genes encoding specific L-G3Pases (GPP1 and GPP2) and multicopy overexpression of L-glycerol 3-phosphate dehydrogenase (GPD1) was studied via small-scale (100 mL) batch fermentations under quasi-anaerobic conditions. Intracellular accumulation of L-G3P reached extremely high levels (roughly 200 mM) but thereafter declined. Extracellular L-G3P was also detected and its concentration continuously increased throughout the fermentation, such that most of the total L-G3P was found outside the cells as fermentation concluded. Moreover, in spite of the complete elimination of specific L-G3Pase activity, the strain showed considerable glycerol formation suggesting unspecific dephosphorylation as a mechanism to relieve cells of intracellular L-G3P accumulation. Up-scaling the process employed fed-batch fermentation with repeated glucose feeding, plus an aerobic growth phase followed by an anaerobic product accumulation phase. This produced a final product titer of about 325 mg total L-G3P per liter of fermentation broth.
Popp
A
A
Department of Microbiology and Genetics, Berlin University of Technology, Seestr. 13, D-13353 Berlin, Germany.
Nguyen
H T T
HT
Boulahya
K
K
Bideaux
C
C
Alfenore
S
S
Guillouet
S E
SE
Nevoigt
E
E
D005285:Fermentation; D005818:Genetic Engineering; D005990:Glycerol / Q000378:metabolism*; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+) / Q000235:genetics; D005994:Glycerophosphates / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D007218:Industrial Microbiology; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D005994:Glycerophosphates; C029620:alpha-glycerophosphoric acid; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+); D010744:Phosphoric Monoester Hydrolases; D005990:Glycerol