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Background  For the production of L-phenylalanine (L-Phe), two molecules of phosphoenolpyruvate (PEP) and one molecule erythrose-4-phosphate (E4P) are necessary. PEP stems from glycolysis whereas E4P is formed in the pentose phosphate pathway (PPP). Glucose, commonly used for L-Phe production with recombinant  E. coli,  is taken up via the PEP-dependent phosphotransferase system which delivers glucose-6-phosphate (G6P). G6P enters either glycolysis or the PPP. In contrast, glycerol is phosphorylated by an ATP-dependent glycerol kinase (GlpK) thus saving one PEP. However, two gluconeogenic reactions (fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, FBPase) are necessary for growth and provision of E4P. Glycerol has become an important carbon source for biotechnology and reports on production of L-Phe from glycerol are available. However, the influence of FBPase and transketolase reactions on L-Phe production has not been reported.    Results  L-Phe productivity of parent strain FUS4/pF81 (plasmid-encoded genes for  aroF, aroB, aroL, pheA ) was compared on glucose and glycerol as C sources. On glucose, a maximal carbon recovery of 0.19 mM C Phe /C Glucose  and a maximal space-time-yield (STY) of 0.13 g l −1  h −1  was found. With glycerol, the maximal carbon recovery was nearly the same (0.18 mM C Phe /C Glycerol ), but the maximal STY was higher (0.21 g l −1  h −1 ). We raised the chromosomal gene copy number of the genes  glpK  (encoding glycerol kinase),  tktA  (encoding transketolase), and  glpX  (encoding fructose-1,6-bisphosphatase) individually. Overexpression of  glpK  (or its feedback-resistant variant,  glpK  G232D ) had little effect on growth rate; L-Phe production was about 30% lower than in FUS4/pF81. Whereas the overexpression of either  glpX  or  tktA  had minor effects on productivity (0.20 mM C Phe /C Glycerol ; 0.25 g l −1  h −1  and 0.21 mM C Phe /C Glycerol ; 0.23 g l −1  h −1 , respectively), the combination of extra genes of  glpX  and  tktA  together led to an increase in maximal STY of about 80% (0.37 g l −1  h −1 ) and a carbon recovery of 0.26 mM C Phe /C Glycerol .    Conclusions  Enhancing the gene copy numbers for  glpX  and  tktA  increased L-Phe productivity from glycerol without affecting growth rate. Engineering of glycerol metabolism towards L-Phe production in  E. coli  has to balance the pathways of gluconeogenesis, glycolysis, and PPP to improve the supply of the precursors, PEP and E4P.

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Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: The role of extra copies of glpK, glpX, and tktA genes