Improving Fab' fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA.

OBJECTIVES: To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. RESULTS: We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease. CONCLUSIONS: We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media.

Measuring E. coli and bacteriophage DNA in cell sonicates to evaluate the CAL1 reaction as a synthetic biology standard for qPCR.

We measured the impact of the presence of total Escherichia coli (E. coli) cellular material on the performance of the Linear Regression of Efficiency (LRE) method of absolute quantitative PCR (LRE qPCR), which features the putatively universal CAL1 calibration reaction, which we propose as a synthetic biology standard. We firstly used a qPCR reaction in which a sequence present in the lone genomic BirA locus is amplified. Amplification efficiency for this reaction, a key metric for many quantitative qPCR methods, was inhibited by cellular material from bioreactor cultivation to a greater extent than material from shake flask cultivation. We then compared LRE qPCR to the Standard Curve method of absolute qPCR (SC qPCR). LRE qPCR method matched the performance of the SC qPCR when used to measure 417-4.17 × 107 copies of the BirA target sequence present in a shake flask-derived cell sonicates sample, and for 97-9.7 × 105 copies in the equivalent bioreactor-derived sample. A plasmid-encoded T7 bacteriophage sequence was next used to compare the methods. In the presence of cell sonicates from samples of up to OD600 = 160, LRE qPCR outperformed SC qPCR in the range of 1.54 × 108-1.54 × 1010 copies of the T7 target sequence and matched SC qPCR over 1.54 × 104-1.54 × 107 copies. These data suggest the CAL1 standard, combined with the LRE qPCR method, represents an attractive choice as a synthetic biology qPCR standard that performs well even when unpurified industrial samples are used as the source of template material.

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 Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic 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.