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

Abstract

BackgroundFor 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.ResultsL-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 CPhe/CGlucose 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 CPhe/CGlycerol), 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, glpKG232D) 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 CPhe/CGlycerol; 0.25 g l−1 h−1 and 0.21 mM CPhe/CGlycerol; 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 CPhe/CGlycerol.ConclusionsEnhancing 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.

Construction of a novel phenol synthetic pathway in Escherichia coli through 4-hydroxybenzoate decarboxylation

“…Besides eliminating feedback inhibition of DAHP synthase, there are several other strategies to increase the supply of chorismate, such as increasing the carbon flux towards chorismate by activating shikimate kinase (Juminaga et al 2012), increasing the supply of erythrose-4-phosphate (E4P) by activating transketolase (Gosset et al 1996;Gottlieb et al 2014) and increasing the supply of phosphoenolpyruvate (PEP) by activating PEP synthase (Kim et al 2014;Na et al 2013) or replacing the native PTS system with an alternative glucose transport system (Gosset 2009). These strategies can be used in the further study to increase chorismate supply and phenol production.…”

Fed‐batch production of l‐tryptophan from glycerol using recombinant Escherichia coli

“…Glycerol is available in high amount and comparably cheap as it is an inevitable byproduct of biodiesel production (Bozell & Petersen, ) that needs to find reasonable utilization (da Silva, Mack, & Contiero, ; Sprenger, ). For microbial production processes with E. coli , crude glycerol and purified glycerol are applicable (Gottlieb et al, ). While the prices for glucose and purified glycerol are similar around 40 cents lb −1 , crude glycerol is much cheaper (3.5 cents lb −1 ) as it is rather a waste‐ than a value stream (Tan, Abdul Aziz, & Aroua, ).…”

“…But the metabolization of glucose to PEP also contains an ATP‐consuming step from fructose‐6‐phosphate to fructose‐bisphosphate and the subsequent metabolic reaction in glycerol metabolism from glycerol‐3‐phosphate to dihydroxyacetone‐phosphate allows a compensating ubiquinol generation (Weiner, Albermann, Gottlieb, Sprenger, & Weuster‐Botz, ). The applicability of glycerol as carbon source for the production of aromatic amino acids with E. coli was already demonstrated for the fed‐batch production of l ‐phenylalanine (Gottlieb et al, ; Weiner, Albermann et al, ). This study deals with the reaction engineering analysis of a recombinant E. coli strain for the production of l ‐tryptophan from glycerol applying fed‐batch processes in stirred‐tank bioreactors.…”

Metabolic engineering of Escherichia coli to enhance acetol production from glycerol

“…Therefore, it is attractive to consider using fermentative production of biochemicals based on glycerol with metabolically engineered E. coli cells Mazumdar et al 2013;Gottlieb et al 2014). However, the consumption rate of glycerol by E. coli is lower compared to glucose, leading to the reduced cell growth and productivity (Wang and Yang 2013).…”