Integrated platform for the accelerated engineering of microorganisms: application to industrial bioprocessing

Due to climate change and uncertainties in global fuel prices, there is a need to adopt biomass derived feed stocks for sustainable manufacturing of fuels, chemicals and pharmaceuticals. As a result, many major industrial manufacturers are now seeking routes to their products that are sustainable, more efficient, and less waste or energy intensive. While bioprocesses to produce compounds ranging from therapeutic drugs to fuels have already been widely implemented, the current microbes being employed are often relatively inefficient and limited in the feedstocks they can utilise. Inherent to successful bioprocess development is the ability to rapidly and predictably engineer microbes for the efficient flux of simple biomass towards compounds of industrial significance. Current iterative and empirical processes for microbial strain improvement are limited and therefore improved enabling technologies to accelerate these processes are required. To address these issues, this thesis describes the development of a platform for the rapid and predictable engineering of microbes for industrial bioprocesses. This has been achieved through complementing an accelerated DNA assembly technique for biosynthetic pathway construction with quantitative proteomics to identify pathway bottlenecks and guide subsequent rounds of pathway optimisation. Only through the ability to rapidly construct biosynthetic pathways and then assess the failure or success of the introduced pathways can microbes be engineered in an intuitive and predictable manner. A central theme of this thesis is the optimisation and implementation of a DNA assembly technique for the construction of multicomponent pathways. Despite being a fundamental aspect of strain engineering, DNA assembly is often unreliable and time consuming. One limitation of this technique is the reduced efficiency observed in the assembly of multiple DNA fragments (as is often the case when constructing a heterologous pathway). To overcome this issue a ‘nested’ DNA assembly methodology has been developed for the predictable construction of combinatorial vector libraries and complex vectors resulting in the successful assembly of up to 10 fragments. Appropriate shake flask and microtiter plate assays were additionally developed to characterise these constructs. A parallel strand of this work has been the optimisation of the methodology to maximise throughput and efficiency whilst also ensuring the method is amenable to process automation. To exemplify the power of proteomics in guiding strain engineering the reverse glyoxylate shunt was selected as a simple benchmark heterologous pathway in the commonly used host, Escherichia coli. This pathway allows for the conversion of tricarboxylic acid cycle intermediates malate and succinate to oxaloacetate and two molecules of acetyl-CoA. Strains have been engineered to overexpress the pathway genes and tryptic digestions of cell lysates carried out. Liquid chromatography, mass spectrometry and data analysis methods have been developed for the identification of over 100 proteins from these lysates. Work was then focused on developing quantitative acquisitions which will allow for the identification of pathway bottlenecks. The coupling of techniques for pathway engineering and pathway analysis will create a step change in the speed and predictability with which microbes can be engineered for industrial application

The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris

Catalase (KatA) and KatA-associated protein (KapA) are essential to persistent colonization in the Helicobacter pylori SS1 mouse model

Helicobacter pylori infects the human gastric mucosa and elicits an aggressive inflammatory response. Despite the severity of the inflammatory response, the bacterium is able to persist and cause a chronic infection. It is believed that antioxidant defence mechanisms enable this organism to persist. Wild-type H. pylori strain SS1, and KatA- and KapA-deficient mutants, were used to infect C57/BL6 mice to test this hypothesis. Neither KatA nor KapA was essential for the initial colonization of H. pylori SS1 in the murine model of infection. The wild-type SS1 colonized the gastric mucosa at significantly higher levels than both mutants throughout the 24-week experiment. Neither KatA- nor KapA-deficient mutants were able to maintain consistent ongoing colonization for the 24-week period, indicating the necessity of both KapA and KatA in sustaining a long-term infection. At 24 weeks, 5/10 mice inoculated with the KatA mutant and 2/10 mice inoculated with the KapA mutant were colonized, compared with 10/10 of the mice inoculated with the wild-type SS1. An increase in the severity of inflammation in the wild-type-inoculated mice appeared to correlate with the decline in colonization of animals inoculated with the mutants, suggesting that increased oxidative stress militated against continued infection by the mutants. These data indicate that KapA may be of equal or greater importance than KatA in terms of sustained infection on inflamed gastric mucosae

BJS commission on surgery and perioperative care post-COVID-19

Background: Coronavirus disease 2019 (COVID-19) was declared a pandemic by the WHO on 11 March 2020 and global surgical practice was compromised. This Commission aimed to document and reflect on the changes seen in the surgical environment during the pandemic, by reviewing colleagues experiences and published evidence. Methods: In late 2020, BJS contacted colleagues across the global surgical community and asked them to describe how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had affected their practice. In addition to this, the Commission undertook a literature review on the impact of COVID-19 on surgery and perioperative care. A thematic analysis was performed to identify the issues most frequently encountered by the correspondents, as well as the solutions and ideas suggested to address them. Results: BJS received communications for this Commission from leading clinicians and academics across a variety of surgical specialties in every inhabited continent. The responses from all over the world provided insights into multiple facets of surgical practice from a governmental level to individual clinical practice and training. Conclusion: The COVID-19 pandemic has uncovered a variety of problems in healthcare systems, including negative impacts on surgical practice. Global surgical multidisciplinary teams are working collaboratively to address research questions about the future of surgery in the post-COVID-19 era. The COVID-19 pandemic is severely damaging surgical training. The establishment of a multidisciplinary ethics committee should be encouraged at all surgical oncology centres. Innovative leadership and collaboration is vital in the post-COVID-19 era

BJS commission on surgery and perioperative care post-COVID-19

Coronavirus disease 2019 (COVID-19) was declared a pandemic by the WHO on 11 March 2020 and global surgical practice was compromised. This Commission aimed to document and reflect on the changes seen in the surgical environment during the pandemic, by reviewing colleagues' experiences and published evidence