An inverse metabolic engineering strategy was used to select for Escherichia coli cells with an increased capability to N-glycosylate a specific target protein. We developed a screen for E. coli cells containing extra-chromosomal DNA fragments for improved ability to add precise sugar groups onto the AcrA protein using the glycosylation system from Campylobacter jejuni. Four different sized (1, 2, 4, and 8 kb) genomic DNA libraries were screened, and the sequences that conferred a yield advantage were determined. These advantageous genomic fragments were mapped onto the E. coli W3110 chromosome. Five candidate genes (identified across two or more libraries) were subsequently selected for forward engineering verification in E. coli CLM24 cells, utilizing a combination of internal standards for absolute quantitation and pseudo-selective reaction monitoring (pSRM) and Western blotting validation. An increase in glycosylated protein was quantified in cells overexpressing 4-α-glucantransferase and a phosphoenolpyruvate-dependent sugar phosphotransferase system, amounting to a 3.8-fold (engineered cells total = 5.3 mg L(-1) ) and 6.7-fold (engineered cells total = 9.4 mg L(-1) ) improvement compared to control cells, respectively. Furthermore, increased glycosylation efficiency was observed in cells overexpressing enzymes involved with glycosylation precursor synthesis, enzymes 1-deoxyxylulose-5-phosphate synthase (1.3-fold) and UDP-N-acetylglucosamine pyrophosphorylase (1.6-fold). To evaluate the wider implications of the engineering, we tested a modified Fc fragment of an IgG antibody as the target glycoprotein with two of our engineered cells, and achieved a ca. 75% improved glycosylation efficiency.

Highlights► Escherichia coli cells require modifying to make ideal hosts for producing glycoproteins. ► Codon optimising pglB leads to increased glycosylation efficiency. ► Lipid linked precursors do not appear to be limiting for E. coli N-glycosylation. ► Increasing expression of WecA improves E. coli N-glycosylation efficiency.

No abstract

Microbubbles have several applications in gas-liquid contacting operations. Conventional production of microbubbles is energetically unfavourable since surface energy required to generate the bubbles is inversely proportional to the size of the bubble generated. Fluidic oscillators have demonstrated a size decrease for a system with high throughput and low energetics but the achievable bubble size is limited due to coalescence. The hypothesis of this paper is that this limitation can be overcome by modifying bubble formation dynamics mediated by oscillatory flow. Frequency and amplitude are two easily controlled factors in oscillatory flow. The bubble can be formed at the displacement phase of the frequency cycle if the amplitude is sufficient to detach the bubble. If the frequency is too low, the conventional steady flow detachment mechanism occurs instead; if the frequency is too high, the bubbles coalesce. Our hypothesis proposes the existence of a resonant mode or ‘sweet-spot’ condition, via frequency modulation and increase in amplitude, to reduce coalescence and produce smallest bubble size with no additional energy input. This condition is identified for an exemplar system showing relative size changes, and a bubble size reduction from 650 µm for steady flow, to 120 µm for oscillatory-flow, and 60 µm for resonant condition (volume average) and 250 µm for steady-flow, 15 µm for oscillatory-flow, 7 µm for the resonant condition. A 10-fold reduction in bubble size with minimal increase in associated energetics results in a substantial reduction in energy requirements for all processes involving gas-liquid operations. The reduction in the energetic footprint of this method has widespread ramifications in all gas-liquid contacting operations including but not limited to wastewater aeration, desalination, flotation separation operations, and other operations.

Bubble measurement has been widely discussed in the literature and comparison studies have been widely performed to validate the results obtained for various forms of bubble size inferences. This paper explores three methods used to obtain a bubble size distribution—optical detection, laser diffraction and acoustic inferences—for a bubble cloud. Each of these methods has advantages and disadvantages due to their intrinsic inference methodology or design flaws due to lack of specificity in measurement. It is clearly demonstrated that seeing bubbles and hearing them are substantially and quantitatively different. The main hypothesis being tested is that for a bubble cloud, acoustic methods are able to detect smaller bubbles compared to the other techniques, as acoustic measurements depend on an intrinsic bubble property, whereas photonics and optical methods are unable to “see” a smaller bubble that is behind a larger bubble. Acoustic methods provide a real-time size distribution for a bubble cloud, whereas for other techniques, appropriate adjustments or compromises must be made in order to arrive at robust data. Acoustic bubble spectrometry consistently records smaller bubbles that were not detected by the other techniques. The difference is largest for acoustic methods and optical methods, with size differences ranging from 5–79% in average bubble size. Differences in size between laser diffraction and optical methods ranged from 5–68%. The differences between laser diffraction and acoustic methods are less, and range between 0% (i.e., in agreement) up to 49%. There is a wider difference observed between the optical method, laser diffraction and acoustic methods whilst good agreement between laser diffraction and acoustic methods. The significant disagreement between laser diffraction and acoustic method (35% and 49%) demonstrates the hypothesis, as there is a higher proportion of smaller bubbles in these measurements (i.e., the smaller bubbles ‘hide’ during measurement via laser diffraction). This study, which shows that acoustic bubble spectrometry is able to detect smaller bubbles than laser diffraction and optical techniques. This is supported by heat and mass transfer studies that show enhanced performance due to increased interfacial area of microbubbles, compared to fine bubbles.