Effective enhancement of Pseudomonas stutzeri D-phenylglycine aminotransferase functional expression in Pichia pastoris by co-expressing Escherichia coli GroEL-GroES

Abstract

BackgroundD-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT.ResultsIntracellular expression of the codon-optimized synthetic dpgA gene under the PAOX1 promoter in P. pastoris resulted in inactive D-PhgAT associated with insoluble cellular fraction and very low level of D-PhgAT activity in the soluble fraction. Manipulation of culture conditions such as addition of sorbitol to induce intracellular accumulation of osmolytes, addition of benzyl alcohol to induce chaperone expression, or lowering incubation temperature to slow down protein expression and folding rates all failed to increase the active D-PhgAT yield. Co-expression of E. coli chaperonins GroEL-GroES with the D-PhgAT dramatically improved the soluble active enzyme production. Increasing gene dosage of both the dpgA and those of the chaperones further increased functional D-PhgAT yield up to 14400-fold higher than when the dpgA was expressed alone. Optimization of cultivation condition further increased D-PhgAT activity yield from the best co-expressing strain by 1.2-fold.ConclusionsThis is the first report on the use of bacterial chaperones co-expressions to enhance functional intracellular expression of bacterial enzyme in P. pastoris. Only two bacterial chaperone genes groEL and groES were sufficient for dramatic enhancement of functionally active D-PhgAT expression in this yeast. With the optimized gene dosage and chaperone combinations, P. pastoris can be attractive for intracellular expression of bacterial proteins since it can grow to a very high cell density which is translated into the higher volumetric product yield than the E. coli or other bacterial systems.

Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast

“…Co-expression of CbbO2/CbbQ2 did not result in a further increase of Rubisco activity (Figure 2). Co-expression of bacterial chaperones has previously been shown to improve heterologous protein expression in Pichia pastoris and insect cells [21,22]. The positive effect of GroEL/GroES on Rubisco expression in S. cerevisiae demonstrates the potential value of co-expression of heterologous chaperones for metabolic pathway engineering that requires expression of prokaryotic enzymes in the cytosol of eukaryotes.…”

Effects of Halophilic Peptide Fusion on Solubility, Stability, and Catalytic Performance of D-Phenylglycine Aminotransferase

“…Another approach is to employ D-amino-acid aminotransferases for these purposes, but again owing to the low activity and high specificity of the enzymes toward only D-amino acids which are costly, their use was quite limited. Some researchers solved the problem of requiring only D-amino acids by incorporating an amino acid racemase and Lamino acids in the reaction to provide the required D-amino acids, but this may baffle the aminotransferase activity [9]. Therefore, up to now, there was no effective enzymatic reaction for the production of these side-chain compounds.…”

Co-expressing GroEL–GroES, Ssa1–Sis1 and Bip–PDI chaperones for enhanced intracellular production and partial-wall breaking improved stability of porcine growth hormone

“…It is well-known that Pichia pastoris protein expression system offers various advantages [19] including the success in high level production of a variety of heterologous proteins both intra-and extracellularly [20][21][22]. However, Pichia pastoris is a eukaryotic expression system, of which gene expression regulation system and material metabolic system were much more complex than prokaryotes expression systems, and the expression of some heterogeneous proteins in Pichia pastoris is unsatisfactory [11,22].…”

“…It is well-known that Pichia pastoris protein expression system offers various advantages [19] including the success in high level production of a variety of heterologous proteins both intra-and extracellularly [20][21][22]. However, Pichia pastoris is a eukaryotic expression system, of which gene expression regulation system and material metabolic system were much more complex than prokaryotes expression systems, and the expression of some heterogeneous proteins in Pichia pastoris is unsatisfactory [11,22]. Therefore, improvement of Pichia pastoris expression system is very important, such as the discovery of novel promoters, effective chaperones co-expression, optimization of transcriptional regulatory regions, optimization of target gene sequence, and selection of target gene copy number [23][24][25].…”