L-tyrosine production by recombinant Escherichia coli: fermentation optimization and recovery.
L-tyrosine (L-tyr) overproducing Escherichia coli strain derived from an L-phenylalanine (L-phe) overproducing strain is characterized in 10 L and 200 L scale fermentations. Deletion of the chromosomal region encoding for the pheA gene, chorismate mutase/prephenate dehydratase, its leader peptide (pheL) and its associated promoter resulted in significant increase in L-tyr production (Olson et al., 2007. Appl Microbiol Biotechnol 74(5):1031-1040). Further increase in titer was achieved by overexpressing tyrA, encoding chorismate mutase/prephenate dehydrogenase, from a strong non-native trc promoter (Olson et al., 2007. Appl Microbiol Biotechnol 74(5):1031-1040). Fermentation optimization studies include media component selection; glucose feed optimization, antifoam agent selection, and understanding fermentation parameters affecting foaming. Generational stability of the strain was evaluated along with rate, titer, and yield of tyrosine formation from glucose. L-tyr titer of 55 g/L in 48 h was demonstrated in 200 L batches, is the highest titer published till date. We have also evaluated two primary separations schemes to isolate and purify L-tyr from the fermentation broth. Physical separation of L-tyr crystals from biomass using a decanter type centrifuge, based on the density difference between the solids, is compared and contrasted with a strategy where L-tyr is first dissolved at pH 11.5 and then acid precipitated from clarified supernatants following removal of biomass using membrane filtration. L-tyr product purity of 98% with yields ranging from 90% to 95% is demonstrated.
Patnaik
Ranjan
R
DuPont Central Research & Development, Glasgow 300, PO Box 6300, Newark, Delaware 19714, USA. ranjan.patnaik@usa.dupont.com
Zolandz
Raymond R
RR
Green
Daniel A
DA
Kraynie
David F
DF
D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D005591:Chemical Fractionation / Q000379:methods*; D004926:Escherichia coli / Q000502:physiology*; D005285:Fermentation; D024861:Genetic Enhancement / Q000379:methods*; D014443:Tyrosine / Q000302:isolation & purification* / Q000378:metabolism*; D014462:Ultrafiltration / Q000379:methods*
D014443:Tyrosine