Continuous bioprocessing: The real thing this time?: 10th Annual bioProcessUK Conference,December 3–4, 2013, London,UK

The Annual bioProcessUK Conference has acted as the key networking event for bioprocess scientists and engineers in the UK for the past 10 years. The following article is a report from the sessions that focused on continuous bioprocessing during the 10^th Annual bioProcessUK Conference (London, December 2013). These sessions were organized by the ‘EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies’ hosted at University College London. A plenary lecture and workshop provided a forum for participants to debate topical issues in roundtable discussions with industry and academic experts from institutions such as Genzyme, Janssen, Novo Nordisk, Pfizer, Merck, GE Healthcare and University College London. The aim of these particular sessions was to understand better the challenges and opportunities for continuous bioprocessing in the bioprocessing sector.     

Recruitment of naphthalene dissimilatory enzymes for the oxidation of 1,4-dichloronaphthalene to 3,6-dichlorosalicylate, a precursor for the herbicide dicamba.

Pseudomonas putida expresses plasmid-encoded enzymes and regulatory proteins for the dissimilation of naphthalene through salicylate and the alpha-keto acid pathway. A strain of P. putida (NAH:Tn5/G67) defective in salicylate hydroxylase (nahG) was assessed for its ability to oxidize 1,4-dichloronaphthalene. Washed cell suspensions were shown to accumulate 3,6-dichlorosalicylate, which, after further chemical treatment, yields the herbicide dicamba (3,6-dichloro-2-methoxybenzoate). However, the rate of dichlorosalicylate formation from dichloronaphthalene was less than 1% of the rate of salicylate formation from unsubstituted naphthalene.     

The reaction of cytochromes c and c2 with the Rhodospirillum rubrum reaction center involves the heme crevice domain

In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 X 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 X 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.     

Ecology of heath communities dominated by Cassiope tetragona at Alexandra Fiord, Ellesmere Island, Canada

At a high arctic lowland on Ellesmere Island, heath communities dominated by Cassiope tetragona were widespread, and occurred in a variety of habitats that differred in time of snowmelt, relative site moisture, soil thaw depth, and air and soil temperature. Cover, standing crop, and production were dominated by woody plants, notably Cassiope tetragona and Dryas integrifolia . The Cassiope -dominated heaths were similar in composition to those at other Canadian high arctic localities, but were less similar to localities in Greenland and Spitzbergen. Compositional relationships among Cassiope -dominated heaths in different habitats at the study site may be largely determined by two interrelated environmental factors, time of snowmelt and site moisture.
Aboveground vascular plant biomass was concentrated near the ground surface, resulting in simple vertical structure that takes advantage of relatively warm soil and air temperatures in summer, and a protective snow cover in winter. Aboveground vascular plant standing crop was largely comprised of attached dead tissue, and there were equal proportions of above- and belowground biomass. Low community production was due to dominance by long-lived, slow-growing species, and to short growing seasons and cold temperatures. Overall character of Cassiope -dominated heaths reflects the conservative, stress tolerant growth strategy of the dominant species, Cassiope tetragona .     

Strain Improvement of Rhodotorula graminis for Production of a Novel l-Phenylalanine Ammonia-Lyase

l-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Rhodotorula rubra has been used in the commercial manufacture of l-phenylalanine from trans-cinnamic acid. In this study, R. graminis PAL was investigated. Mutant strain GX6000 was isolated after ethyl methanesulfonate mutagenesis of wild-type R. graminis GX5007 by selecting for resistance to phenylpropiolic acid, an analog of trans-cinnamic acid. Mutant strain GX6000 produced inducible PAL at levels four- to fivefold higher than had wild-type R. graminis. Furthermore, this strain had several other physiological traits that make it more commercially useful than R. rubra. For example, during fermentation, the PAL half-life was three- to fivefold longer, PAL specific activity was six to seven times higher, and PAL synthesis was significantly less inhibited by temperatures above 30 degrees C. Induction of PAL in strain GX6000 appeared to be less tightly regulated; l-leucine acted synergistically with l-phenylalanine, the physiological inducer, to increase the PAL specific activity and titer to 165 U/g (dry weight) and 3,000 U/liter, respectively, a 40% increase over the effect of l-phenylalanine alone. Strain GX6000 PAL showed significantly greater stability in bioreactors for the synthesis of l-phenylalanine, a finding that is consistent with the stability properties observed during fermentation.     

In vivo environmental temperature and the in vitro pattern of luminal acidification in turtle bladders. Evidence for HCO3 ion reabsorption

In this study, it is shown how to transfer tared aliquots of (HCO3 + CO2)-containing luminal fluids directly into the mercury-sealed chamber of a modified Van Slyke apparatus and how to obtain direct as well as indirect manometric determinations of dissolved CO2 ([CO2]f) in each aliquot of such fluids. It is next shown that the pattern of in vitro luminal acidification in an isolated turtle bladder sac depends upon the prior in vivo ambient temperature to which the donor turtle had become adapted. Under in vivo conditions, the food intake, physical activity, and acid excretion of 32 degrees C-adapted turtles are greater than those of 21 degrees C or 26 degrees C-adapted turtles. Under in vitro conditions of incubating isolated bladder sacs (from 21, 26, and 32 degrees C turtles) in (HCO3 + CO2)-containing Ringer media at a single temperature (21 degrees C), the patterns of luminal acidification are as follows: (a) The rate of depletion of luminal [HCO3] is greatest in bladders from the 32 degrees C-adapted turtles. (b) Concomitant decreases in luminal [CO2]f, [HCO3], and pH (the 'CO2-decreasing patterns' of luminal acidification) develop in all bladders from 32 degrees C turtles, in half of those from 26 degrees C turtles, but in less than one-fifth of those from 21 degrees C-adapted turtles: and (c) a CO2-increasing pattern of luminal acidification is found in most of the bladders from 21 degrees C-adapted turtles. A postulated bicarbonate ion-reabsorbing pump is consistent with all of these patterns of luminal acidification.