Sarah W. Harcum

Emergent properties of reduced-genome Escherichia coli

With the use of synthetic biology, we reduced the Escherichia coli K-12 genome by making planned, precise deletions. The multiple-deletion series (MDS) strains, with genome reductions up to 15%, were designed by identifying nonessential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving good growth profiles and protein production. Genome reduction also led to unanticipated beneficial properties: high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Eradication of stress-induced transposition evidently stabilized the MDS genomes and provided some of the new properties.

Xanthan gum production from waste sugar beet pulp

The feasibility of using waste sugar beet pulp (WSBP) as a supplemental substrate for xanthan gum production from Xanthomonas campestris was investigated. For the range of incubation periods and contact times investigated (1 to 5 days), there were no differences in the mean WSBP degradation. The mean WSBP degradation was significantly greater for incubation temperatures of 28°C as compared to incubation temperatures of 32°C. WSBP degradation was insensitive to the contact temperatures evaluated. These results indicate that optimal cell growth might optimize WSBP degradation. Xanthan gum production from the WSBP supplemented cultures was significantly greater than the unsupplemented production medium. Based on a preliminary analysis, the use of WSBP for xanthan gum production has the potential to be a cost-effective supplemental substrate to produce non-food grade xanthan gum.

Heat-shock and stringent responses have overlapping protease activity in Escherichia coli : Implications for heterologous protein yield

The cellular response of a heat-shocked controlled chemostat of Escherichia coli JM105 [pSH101] was characterized and compared to that of a similar culture induced by isopropyl-β-d-thiogalactopyranoside (IPTG). The proteases elicited by the IPTG pulse were previously shown to be upregulated by the stringent stress response and were shown here to be upregulated by heat shock, although to a lesser extent. Owing to the apparent overlap between these responses, a relaxed mutant (rel−, devoid of the stringent response; JM109) was examined for its response to both a chemically imposed stringent response and to IPTG induction in controlled chemostats. There was no significant upregulation of protease activity under either imposed stress. More important, a nine-fold increase of chloramphenicol acetyltransferase (CAT) activity was found for the IPTG-induced relaxed mutant culture. Additionally, the responses from heat shock and IPTG induction were examined in batch cultures. The culture that was simultaneously IPTG-induced and heat-shocked was observed to have the highest CAT activity as well as the most rapid loss in activity after a maximum. Control experiments indicated that the heat shock did not affect loss of CAT activity; instead, the loss of activity correlated with the amount of CAT synthesized. Furthermore, an increase in CAT expression was found during heat shock. Results indicated that heat shock and, alternatively, the use of stringent response-mutant hosts could both be used to facilitate increased recombinant protein yields in the E. coli expression system.

Genomics in mammalian cell culture bioprocessing.

Explicitly identifying the genome of a host organism including sequencing, mapping, and annotating its genetic code has become a priority in the field of biotechnology with aims at improving the efficiency and understanding of cell culture bioprocessing. Recombinant protein therapeutics, primarily produced in mammalian cells, constitute a

Global transcriptome response of recombinant Escherichia coli to heat-shock and dual heat-shock recombinant protein induction

108 billion global market. The most common mammalian cell line used in biologic production processes is the Chinese hamster ovary (CHO) cell line, and although great improvements have been made in titer production over the past 25 years, the underlying molecular and physiological factors are not well understood. Confident understanding of CHO bioprocessing elements (e.g. cell line selection, protein production, and reproducibility of process performance and product specifications) would significantly improve with a well understood genome. This review describes mammalian cell culture use in bioprocessing, the importance of obtaining CHO cell line genetic sequences, and the current status of sequencing efforts. Furthermore, transcriptomic techniques and gene expression tools are presented, and case studies exploring genomic techniques and applications aimed to improve mammalian bioprocess performance are reviewed. Finally, future implications of genomic advances are surmised.

Temperature Effects on Product-Quality-Related Enzymes in Batch CHO Cell Cultures Producing Recombinant tPA

Recombinant Escherichia coli cultures are used to manufacture numerous therapeutic proteins and industrial enzymes, where many of these processes use elevated temperatures to induce recombinant protein production. The heat-shock response in wild-type E. coli has been well studied. In this study, the transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a dual heat-shock recombinant protein induction were examined. Most classical heat-shock protein genes were identified as regulated in both conditions. The major transcriptome differences between the recombinant and reported wild-type cultures were heavily populated by hypothetical and putative genes, which indicates recombinant cultures utilize many unique genes to respond to a heat-shock. Comparison of the dual stressed culture data with literature recombinant protein induced culture data revealed numerous differences. The dual stressed response encompassed three major response patterns: induced-like, in-between, and greater than either individual stress response. Also, there were no genes that only responded to the dual stress. The most interesting difference between the dual stressed and induced cultures was the amino acid-tRNA gene levels. The amino acid-tRNA genes were elevated for the dual cultures compared to the induced cultures. Since, tRNAs facilitate protein synthesis via translation, this observed increase in amino acid-tRNA transcriptome levels, in concert with elevated heat-shock chaperones, might account for improved productivities often observed for thermo-inducible systems. Most importantly, the response of the recombinant cultures to a heat-shock was more profound than wild-type cultures, and further, the response to recombinant protein induction was not a simple additive response of the individual stresses.

Optimal nutrient feed policies for heterologous protein production

Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors. Previous studies have demonstrated that enzymes, such as proteases and sialidases, accumulate in batch bioreactors. These enzymes are known to be detrimental to the quality of recombinant glycoproteins. Bioreactor temperature has been used to control cell growth and recombinant protein production rates. However, the effect of culture temperature on the production of proteases and sialidases has not been investigated. In this study, Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 °C over 8 days and with a constant temperature of 37 °C. Analysis of extracellular protease activity indicated that two major proteases were present (50 and 69 kDa). The 50 kDa protease activity decreased similarly with time for both culture conditions. The 69 kDa protease activity increased with time for both culture conditions. The constant‐temperature cultures had significantly lower 69 kDa protease levels compared to the ramped‐temperature cultures in the early stationary phase. Intracellular sialidase activity was present in both cultures. The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells were inoculated into fresh medium. The initial peak in intracellular sialidase activity was followed by a first‐order decay. The intracellular sialidase activities for the two culture conditions were not significantly different. The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions. Thus, the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.

Detection, quantification, and characterization of proteases in recombinant Escherichia coli

RecombinantEscherichia coli, which overproduce heterologous protein, redirect endogenous metabolic activity to that mediated by the recombinant expression vector. Consequently, cells may experience perturbations in the biosynthetic reaction network, including the amino acid biosynthesis pathways. These cells are characterized by decreased growth rate, decreased cell mass yield, and increased heterologous protein degradation. This study investigates the dynamics of chloramphenicol-acetyl-transferase (CAT) synthesis and degradation inE. coli JM105 grown on minimal media, and correlates the observed phenomena with induction strength. Coordinated amino acid feeding was shown to increase the heterologous protein yield. Rational design of nutrient feeding possibilities is explored.

Dynamic transcriptional response of Escherichia coli to inclusion body formation

Electrophoresis of crude cell extracts on PAGE gels in the presence of SDS copolymerized with a nonspecific protease substrate has been used to detect, characterize, and quantify intracellular proteases in recombinant Escherichia coli. After electrophoresis, the gels are incubated, SDS is removed, and protease activity is revealed by clear zones on the stained gel due to proteolysis of the nonspecific protease substrate (gelatin or casein). The method differentiates proteases based on activity and molecular weight.

Differential display identifies genes in chinese hamster ovary cells sensitive to elevated ammonium

Escherichia coli is used intensively for recombinant protein production, but one key challenge with recombinant E. coli is the tendency of recombinant proteins to misfold and aggregate into insoluble inclusion bodies (IBs). IBs contain high concentrations of inactive recombinant protein that require recovery steps to salvage a functional recombinant protein. Currently, no universally effective method exists to prevent IB formation in recombinant E. coli. In this study, DNA microarrays were used to compare the E. coli gene expression response dynamics to soluble and insoluble recombinant protein production. As expected and previously reported, the classical heat‐shock genes had increased expression due to IB formation, including protein folding chaperones and proteases. Gene expression levels for protein synthesis‐related and energy‐synthesis pathways were also increased. Many transmembrane transporter and corresponding catabolic pathways genes had decreased expression for substrates not present in the culture medium. Additionally, putative genes represented over one‐third of the genes identified to have significant expression changes due to IB formation, indicating many important cellular responses to IB formation still need to be characterized. Interestingly, cells grown in 3% ethanol had significantly reduced gene expression responses due to IB formation. Taken together, these results indicate that IB formation is complex, stimulates the heat‐shock response, increases protein and energy synthesis needs, and streamlines transport and catabolic processes, while ethanol diminished all of these responses. Biotechnol. Biotechnol. Bioeng. 2014;111: 980–999.