Biogeography of dinoflagellate cysts in northwest Atlantic estuaries

Few biogeographic studies of dinoflagellate cysts include the near‐shore estuarine environment. We determine the effect of estuary type, biogeography, and water quality on the spatial distribution of organic‐walled dinoflagellate cysts from the Northeast USA (Maine to Delaware) and Canada (Prince Edward Island). A total of 69 surface sediment samples were collected from 27 estuaries, from sites with surface salinities >20. Dinoflagellate cysts were examined microscopically and compared to environmental parameters using multivariate ordination techniques. The spatial distribution of cyst taxa reflects biogeographic provinces established by other marine organisms, with Cape Cod separating the northern Acadian Province from the southern Virginian Province. Species such as Lingulodinium machaerophorum and Polysphaeridinium zoharyi were found almost exclusively in the Virginian Province, while others such as Dubridinium spp. and Islandinium? cezare were more abundant in the Acadian Province. Tidal range, sea surface temperature (SST), and sea surface salinity (SSS) are statistically significant parameters influencing cyst assemblages. Samples from the same type of estuary cluster together in canonical correspondence analysis when the estuaries are within the same biogeographic province. The large geographic extent of this study, encompassing four main estuary types (riverine, lagoon, coastal embayment, and fjord), allowed us to determine that the type of estuary has an important influence on cyst assemblages. Due to greater seasonal variations in SSTs and SSSs in estuaries compared to the open ocean, cyst assemblages show distinct latitudinal trends. The estuarine context is important for understanding present‐day species distribution, the factors controlling them, and to better predict how they may change in the future.     

Promoter engineering to optimize recombinant periplasmic Fab′ fragment production in Escherichia coli

Fab’ fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab’ fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab’ fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the P_tac promoter, present in the plasmid, pTTOD‐A33 Fab’, to a P_tic promoter which has been shown by others to direct expression at a 35% reduced rate compared to P_tac. We characterized the resultant production trains in which either P_tic or P_tac promoters direct Fab’ fragment expression. The P_tic promoter strain showed a 25?30% reduction in Fab’ expression relative to the original P_tac strain. Reduced Fab’ leakage and increased viability over the course of a fed‐batch fermentation were also observed for the P_tic promoter strain. We conclude that cell design steps such as the P_tac to P_tic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab’ fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840–847, 2016     

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016     

A mutant dec-1 transgene induces dominant female sterility in Drosophila melanogaster

The Drosophila dec-1 gene produces three proproteins required for female fertility and eggshell assembly. The three proproteins are distinguished by their C termini. Fc106, the most abundant proprotein, is cleaved within the vitelline membrane to three mature derivatives in a developmentally regulated manner. To define sequences within fc106 that are critical for its function, we created wild-type and mutant versions of an fc106 cDNA transgene. The functional consequences of the mutations were assessed in dec-14, a female-sterile splicing mutant that does not produce the fc106 isoform. The fertility of dec-14 females was restored by the introduction of either a wild-type transgene or a transgene bearing a C-terminal deletion that included fc106-specific sequences. Surprisingly, the removal of internal coding sequences created an aberrant DEC-1 proprotein that induced female sterility when introduced into wild-type flies. Dominant female sterility was not associated with larger deletions that included the fc106 N terminus, suggesting that abnormal juxtaposition of N- and C-terminal sequences in the aberrant proprotein interfered with endogenous DEC-1 proteins. Changes in the fractionation behavior of the endogenous fc106 C-terminal derivative, s60, and morphological changes in the endochorion in response to expression of the aberrant proprotein support this interpretation.