Darren Nesbeth - Academia.edu (original) (raw)

Papers by Darren Nesbeth

Biotechnology Progress, May 6, 2016

Fab’ fragments have become an established class of biotherapeutic over the last two decades. Like... more 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

discovery.ucl.ac.uk

... Cell engineering for manufacturability; the use of ultra scale-down experimentation. Balasund... more ... Cell engineering for manufacturability; the use of ultra scale-down experimentation. Balasundaram, B and Nesbeth, D and Ward, JM and Keshavarz-Moore, E and Bracewell, DG Cell engineering for manufacturability; the use of ultra scale-down experimentation. ...

A novel experimental setup for measuring fluorescence in live trypanosome (FILT) cells was establ... more A novel experimental setup for measuring fluorescence in live trypanosome (FILT) cells was established as described in Borg (2015) and Borg et al. (2021), a study which will be linked to this dataset upon publication. The FILT set up was used to capture 60 fluorescent and 60 brightfield images of a field of vision which encompassed 88 cells of the recombinant Trypanosoma brucei brucei cell line, TbGOS02. All these images are provided in the file within this dataset labelled, "TbGOS02 All Frames 88 cells 140619.pdf". Each of the 88 cells in the image was enumerated as detailed in Borg et al. (2021). For cell number 08, 60 fluorescent and 60 brightfield cropped images featuring only this cell are provided in the file within this dataset labelled, " TbGOS02 All Frames Cell 08 140619.pdf ". Equivalent images for cells numbered 17, 63 and 76 are provided in the remaining three PDF files of this dateset, as summarised below. TbGOS0...

Sequence of the UbeK degron signal used to decrease the half life of GFP expressed in Tr... more Sequence of the UbeK degron signal used to decrease the half life of GFP expressed in Trypanosoma brucei brucei (T. b. brucei). The UbeK degron consists of 76 residues of native T. b. brucei ubiquitin protein, at the N-terminus and ending in a leucine residue to signal proteasomal degradation, followed by a short 'eK' region which featuring an additional proteasomal degradation signal of two lysine residues flanking an arginine.

A proprietary algorithm was applied, by a commercial service provider, to the coding DNA of <i... more A proprietary algorithm was applied, by a commercial service provider, to the coding DNA of Chromobacterium violaceum transaminase CV2025 gene, National Center for Biotechnology Information (NCBI) sequence reference WP_011135573.1, to optimise the codons used for expression in Komagataella phaffii (Pichia pastoris). The data sheet for the suggested codon optimisation is provided here, featuring 1. codon usage bias adjustment, 2. GC content adjustment, 3. restriction enzymes sites and cis-acting elements, 4. removed repeat sequences, 5. the optimized sequence and finally 6. an alignment of the starting and codon-optimized sequences.

Olusegun Folarin, The Advanced Centre for Biochemical Engineering, Department of Biochemical Engi... more Olusegun Folarin, The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, UK Darren Nesbeth, The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, UK Eli Keshavarz-Moore, The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, UK

Journal of Gene Medicine, 2004

Pichia pastoris (P. pastoris) is an attractive industrial host cell due to its ability to grow up... more Pichia pastoris (P. pastoris) is an attractive industrial host cell due to its ability to grow up to 60% wet cell weight (WCW) by volume, a far higher level of biomass than the typical values reached by Escherichia coli (E. coli) and Saccharomyces cerevisiae. This thesis seeks to explore how the genetic tractability and high cell densities characteristic of P. pastoris can be exploited to intensify whole-cell biocatalysis.

Frontiers in Bioengineering and Biotechnology, 2020

Synthetic biology has the potential to positively transform society in many application areas, in... more Synthetic biology has the potential to positively transform society in many application areas, including medicine. In common with all revolutionary new technologies, synthetic biology can also enable crime. Like cybercrime, that emerged following the advent of the internet, biocrime can have a significant effect on society, but may also impact on peoples' health. For example, the scale of harm caused by the SARS-CoV-2 pandemic illustrates the potential impact of future biocrime and highlights the need for prevention strategies. Systematic evidence quantifying the crime opportunities posed by synthetic biology has to date been very limited. Here, we systematically reviewed forms of crime that could be facilitated by synthetic biology with a view to informing their prevention. A total of 794 articles from four databases were extracted and a three-step screening phase resulted in 15 studies that met our threshold criterion for thematic synthesis. Within those studies, 13 exploits were identified. Of these, 46% were dependent on technologies characteristic of synthetic biology. Eight potential crime types emerged from the studies: bio-discrimination, cyber-biocrime, bio-malware, biohacking, at-home drug manufacturing, illegal gene editing, genetic blackmail, and neuro-hacking. 14 offender types were identified. For the most commonly identified offenders (>3 mentions) 40% were outsider threats. These observations suggest that synthetic biology presents substantial new offending opportunities. Moreover, that more effective engagement, such as ethical hacking, is needed now to prevent a crime harvest from developing in the future. A framework to address the synthetic biology crime landscape is proposed.

Systematic Reviews, 2020

Background When new technologies are developed, it is common for their crime and security implica... more Background When new technologies are developed, it is common for their crime and security implications to be overlooked or given inadequate attention, which can lead to a ‘crime harvest’. Potential methods for the criminal exploitation of biotechnology need to be understood to assess their impact, evaluate current policies and interventions and inform the allocation of limited resources efficiently. Recent studies have illustrated some of the security implications of biotechnology, with outcomes of misuse ranging from compromised computers using malware stored in synthesised DNA, infringement of intellectual property on biological matter, synthesis of new threatening viruses, ‘genetic genocide,’ and the exploitation of food markets with genetically modified crops. However, there exists no synthesis of this information, and no formal quality assessment of the current evidence. This review therefore aims to establish what current and/or predicted crimes have been reported as a result ...

Heliyon, 2019

We have engineered strain BG-10 of the methylotrophic yeast Komagataella phaffii for use as an ef... more We have engineered strain BG-10 of the methylotrophic yeast Komagataella phaffii for use as an effective whole cell biocatalyst. We introduced into the yeast a transgene encoding a Chromobacterium violaceum ω-transaminase for transcription in response to methanol induction. The strain was then assessed with respect to its growth performance and biotransformation of a fed ketoalcohol substrate to an amino-alcohol. In the resultant strain, BG-TAM, methanol induction did not compromise cell growth. Successful bioconversion of fed substrates to the byproduct, acetophenone, indicated transaminase activity in shake flask-cultivated BG-TAM cells. We then used bioreactor cultivation to exploit the high levels of biomass achievable by Komagataella phaffii. In a 900 μL reaction the BG-TAM strain at OD 600 ¼ 1024 achieved up to 0.41 mol mol À1 (mol product mol substrate À1) yield on substrate (Yp/ s) for production of 1-methyl-3-phenylpropylamine and a space time yield (STY) of 0.29 g L À1 h À1 for production of 2-amino-1,3,4-butanetriol. We have shown that transamination, an important step for bespoke synthesis of small molecule medicines, is biologically realisable using enzymes with a broad substrate range, such as ω-transaminases, within living yeast cells that are fed low-cost substrates for bioconversion.

Biotechnology Progress, 2019

Whole cell biocatalysis is an ideal tool for biotransformations that demand enzyme regeneration o... more Whole cell biocatalysis is an ideal tool for biotransformations that demand enzyme regeneration or robustness to fluctuating pH, osmolarity and biocontaminant load in feedstocks. The methylotrophic yeast Komagataella phaffii is an attractive alternative to Escherichia coli for whole cell biocatalysis due to its genetic tractability and capacity to grow to up to 60% wet cell weight by volume. We sought to exploit high cell density K. phaffii to intensify whole‐cell chiral amino‐alcohol (CAA) biosynthesis. We engineered two novel K. phaffii GS115 strains: one by inserting a Chromobacterium violaceum ω‐transaminase CV2025 transgene, for strain PpTAmCV708, and a second strain, PpTAm‐TK16, by also inserting the same CV2025 transgene plus a second transgene for a native transketolase. At high cell density, both strains tolerated high substrate concentrations. When fed three low cost substrates, 200 mM glycolaldehyde, 200 mM hydroxypyruvate and 150 mM methylbenzylamine, PpTAm‐TK16 whole cells achieved 0.29 g L−1 hr−1 space–time yield of the acetophenone by‐product, a 49‐fold increase of the highest levels reported for E. coli whole cells harboring the equivalent pathway. When fed only the low‐cost substrate, 150 mM methylbenzylamine, strain PpTAmCV708 achieved a 105‐fold increase of reported E. coli whole cell biocatalysis performance, with a space–time yield of 0.62 g L−1 hr−1 of the CAA, 2‐amino‐1,3,4‐butanetriol (ABT). The rapid growth and high biomass characteristics of K. phaffii were successfully exploited for production of ABT by whole‐cell biocatalysis at higher levels than the previously achieved with E. coli in the presence of the same substrates.

Enzyme and Microbial Technology, 2019

This study investigates how sorbitol/methanol mixed induction affects fermentation performance, d... more This study investigates how sorbitol/methanol mixed induction affects fermentation performance, dewatering characteristics of cells during harvesting and the profile of host cell proteins (HCP) in the process fluid when producing the target recombinant protein aprotinin. Compared to standard methanol induction, sorbitol/methanol (1:1, C-mol/C-mol) mixed induction improved cellular viability from 92.8±0.3% to 97.7±0.1% although resulted in a reduced product yield from 1.65±0.03 g•L-1 to 1.12±0.07 g•L-1. On the other hand, average oxygen consumption rate (OUR) dropped from 241.4±21.3 mmol•L-1 •h-1 to 145.5 ±6.7 mmol•L-1 •h-1. Cell diameter decreased over time in the mixed induction, resulting in a D50 value of 3.14 μm at harvest compared to 3.85 μm with methanol. The reduction in cell size enhanced the maximum dewatering efficiency from 78.1±3.9% to 84.5±3.3% as evaluated by using an established ultra scale-down methodology that models pilot and industrial scale disc stack centrifugation. Seventy host cell proteins (HCPs) were identified in clarified supernatant when using sorbitol/methanol mixed induction regimen. The total number of HCPs identified with standard methanol induction was nearly one hundred. The downstream process advantage of the mixed induction lies in improved product purity by reducing both cell mortality and level of released whole cell proteins. This needs to be balanced and optimised against the observed reduction in product yield during fermentation. Highlights Sorbitol/methanol induction increases cell viability and decreases oxygen consumption The mixed strategy halves the quantity of inflammable methanol needed at scaleup The mixed induction reduces number of host cell proteins co-released with the product The mixed induction improves the centrifugal dewatering of cell culture