Glycine to glutamic acid misincorporation observed in a recombinant protein expressed by Escherichia coli cells

A novel amino acid misincorporation, in which the intended glycine (Gly) residues were replaced by a glutamic acid (Glu), was observed in a recombinant protein expressed by Escherichia coli. The misincorporation was identified by peptide mapping and liquid chromatography-tandem mass spectrometric analysis on proteolyzed peptides of the protein and verified using the corresponding synthetic peptides containing the misincorporated residues. Analysis of the distribution of the misincorporated residues and their codon usage shows strong correlation between this misincorporation and the use of rarely used codon within the E. coli expression system. Results in this study suggest that the usage of the rare codon GGA has resulted in a Glu for Gly misincorporation.     

Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose

Five reactor systems (free cell batch, free cell continuous, entrapped cell immobilized, adsorbed cell packed bed, and cell recycle membrane reactors) were compared for ethanol production from xylose using Escherichia coli FBR5. In the free cell batch and free cell continuous reactors (continuous stirred tank reactor‐CSTR) productivities of 0.84 gL^?1 h^?1 and 1.77 gL^?1 h^?1 were achieved, respectively. A cell recycle membrane reactor resulted in the highest productivity of 55.56 gL^?1 h^?1, which is an increase of 66‐fold (e.g., 6614%) over the batch reactor. Calcium alginate gel CSTR resulted in a productivity of 2.04 gL^?1 h^?1 whereas adsorbed cell packed bed reactor resulted in a productivity of 4.39 gL^?1 h^?1. In the five reactor systems, ethanol concentrations ranged from 18.9 to 40.30 gL^?1 with metabolic yields from 0.44 to 0.51. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Exacting predictions by cybernetic model confirmed experimentally: Steady state multiplicity in the chemostat

We demonstrate strong experimental support for the cybernetic model based on maximizing carbon uptake rate in describing the microorganism's regulatory behavior by verifying exacting predictions of steady state multiplicity in a chemostat. Experiments with a feed mixture of glucose and pyruvate show multiple steady state behavior as predicted by the cybernetic model. When multiplicity occurs at a dilution (growth) rate, it results in hysteretic behavior following switches in dilution rate from above and below. This phenomenon is caused by transient paths leading to different steady states through dynamic maximization of the carbon uptake rate. Thus steady state multiplicity is a manifestation of the nonlinearity arising from cybernetic mechanisms rather than of the nonlinear kinetics. The predicted metabolic multiplicity would extend to intracellular states such as enzyme levels and fluxes to be verified in future experiments. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Improved E. coli erythromycin A production through the application of metabolic and bioprocess engineering

In this report, small-scale culture and bioreactor experiments were used to compare and improve the heterologous production of the antibiotic erythromycin A across a series of engineered prototype Escherichia coli strains. The original strain, termed BAP1(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7), was designed to allow full erythromycin A biosynthesis from the exogenous addition of propionate. This strain was then compared against two alternatives hypothesized to increase final product titer. Strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) is a derivative of BAP1 designed to increase biosynthetic pathway carbon flow as a result of a ygfH deletion; whereas, strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4-2, pGro7) provided an extra copy of a key deoxysugar glycosyltransferase gene. Production was compared across the three strains with TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) showing significant improvement in erythronolide B (EB), 3-mycarosylerythronolide B (MEB), and erythromycin A titers. This strain was further tested in the context of batch bioreactor production experiments with time-course titers leveling at 4 mg/L, representing an approximately sevenfold increase in final erythromycin A titer.     

Nonrecovery of varying proportions of viable bacteria during spread plating governed by the extent of spreader usage and proposal for an alternate spotting-spreading approach to maximize the CFU

Aims: To elucidate the cause of high variations and inconsistencies in bacterial CFU observed within and between different experiments while assessing viable bacterial counts through spread plating (SP). Methods and Results: Following the inconsistent results, CFU estimations were undertaken through conventional SP using the spreader, or a modified approach that did not use spreader employing four organisms. The latter approach involving spotting‐and‐tilt‐spreading of inoculum on agar surface [spotting spreading (SS)] yielded higher CFU by 11–120% over the weighted average depending on the organism and diluent. The adverse effect owing to the spreader was the most obvious in Escherichia coli followed by Staphylococcus epidermidis, Enterobacter cloacae and Bacillus pumilus. Plate attributes that determined the surface moisture levels of agar medium and the spreading practice adopted by the personnel formed two other major influencing factors. Plating for shorter periods (<60 s) using fresh 15/20 ml plates caused loss of 3–12% CFU owing to inoculum adhesion to spreader irrespective of glass or polypropylene make. On the other hand, prolonging the plating brought down the CFU significantly. Spreader movement on agar surface subsequent to the exhaustion of free moisture, which was marked by the experiencing of some friction to smooth spreader movement, was detrimental to vegetative cells, while Bacillus spores were less affected. Conclusions: The study brings out that the way SP is carried out exerts significant effects on CFU influenced by plate conditions. Prolonged use of spreader on dry agar surface could be highly detrimental to bacterial cells. A mild use of spreader accounting for spreader‐adhering inoculum or the practice of SS not involving the spreader is recommended. Significance and Impact of the Study: This study unravels the effects owing to the spreader on bacterial cells and the CFU and recommends an alternate approach of SS to minimize CFU inconsistencies and to maximize the viable bacterial counts.