Design of a recombinant Escherichia coli for producing L-phenylalanine from glycerol.

A recombinant Escherichia coli was engineered to produce the commercially important amino acid L-phenylalanine (L-Phe) using glycerol as the carbon source. Compared to the conventionally used glucose and sucrose, glycerol is a less expensive carbon source. As phenylalanine dehydrogenase (PheDH) activity is involved in the last step of L-Phe synthesis in E. coli, a phenylalanine dehydrogenase gene (phedh) from the thermotolerant Bacillus lentus was cloned into pRSFDuet-1 (pPheDH) and expressed in E. coli BL21(DE3). The resulting clone had a limited ability to produce L-Phe from glycerol, possibly because of a poor glycerol uptake by the cell, or an inability to excrete L-Phe, or both. Therefore, yddG gene encoding an aromatic amino acid exporter and glpF gene encoding a glycerol transport facilitator were coexpressed with the phedh in a reengineered E. coli. In a glycerol medium, the maximum L-Phe production rates of the clones pPY (phedh and yddG genes) and pPYF (phedh, yddG and glpF genes) were 1.4- and 1.8-fold higher than the maximum production rate of the pPheDH clone. The better producing pPYF clone was further evaluated in a 5 l stirred-tank fermenter (37 °C, an aeration rate of 1 vvm, an agitation speed of 400 rpm). In the fermenter, the maximum concentration of L-Phe (366 mg/l) was achieved in a much shorter period compared to in the shake flasks. In the latter, the highest titer of L-Phe was only 76 % of the maximum value attained in the fermenter.

Evidence of the involvement of asparagine deamidation in the formation of cyclodextrin glycosyltransferase isoforms in Paenibacillus sp. RB01.

Cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 and its recombinant enzyme exhibit three isoforms (I, II, and III) with the same apparent size but different charge. Here, we demonstrate for the first time that the deamidation of labile Asns causes the change in molecular forms of CGTase. The faster increase in number of isoforms was observed upon incubation in deamidation buffer at the more alkaline pH. The increase in levels of isoform II and III over time correlated with the increase in isoaspartate, a unique deamidation product. The predicted labile Asns were individually mutated to Asp, then the selected mutant and wild type isoforms were tryptic digested and labile Asns were investigated by MALDI-TOF. From the results, Asn427 was the most susceptible residue for deamidation, followed by Asn336, Asn415, and Asn567. In addition, Gln389 might also share a role. The advantage of using appropriate CGTase isoform in cyclodextrin production is reported.

Production of L-phenylalanine from glycerol by a recombinant Escherichia coli.

The production of L-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing L-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37 degrees C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300-500 rpm corresponding to 0.97-1.62 m s(-1) impeller tip speed) and aeration rates (2-8 L min(-1), or 1-4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed approximately 1.62 m s(-1)) reduced the biomass and L-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of L-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s(-1) impeller tip speed, 4 vvm aeration rate), the yield of L-phenylalanine on glycerol was 0.58 g g(-1), or more than twice the best yield attainable on sucrose (0.25 g g(-1)). In the best case, the peak concentration of L-phenylalanine was 5.6 g L(-1), or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of L-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.

Molecular characterization of lysine 6-dehydrogenase from Achromobacter denitrificans.

An inducible lysine 6-dehydrogenase (Lys 6-DH), which catalyzes the oxidative deamination of the 6-amino group of L-lysine in the presence of NAD(+), was purified to homogeneity from Achromobacter denitrificans, yielding a homodimeric protein of 80 kDa. The enzyme was specific for the substrate L-lysine and NAD(+) served as a cofactor. The dimeric enzyme associated into a hexamer in the presence of 10 mM L-lysine. The K(m) values for L-lysine and NAD(+) were 5.0 and 0.09 mM, respectively. The lys 6-dh gene was cloned and overexpressed in E. coli. The open reading frame was 1,107 nucleotides long and encoded a peptide containing 368 amino acids with 39,355 Da. The recombinant enzyme was purified to homogeneity and characterized. Enzyme activities and kinetic properties of the recombinant enzyme were almost the same as those of the endogenous enzyme obtained from A. denitrificans. Crystals of the enzyme were obtained using the hanging drop method.