Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species.

The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications. .

Context matters: On the road to responsible biosafety technologies in synthetic biology.

Biosafety is a major challenge for developing for synthetic organisms. An early focus on application and their context could assist with the design of appropriate genetic safeguards.

Exploring the Impact of Tensions in Stakeholder Norms on Designing for Value Change: The Case of Biosafety in Industrial Biotechnology.

Synthetic biologists design and engineer organisms for a better and more sustainable future. While the manifold prospects are encouraging, concerns about the uncertain risks of genome editing affect public opinion as well as local regulations. As a consequence, biosafety and associated concepts, such as the Safe-by-design framework and genetic safeguard technologies, have gained notoriety and occupy a central position in the conversation about genetically modified organisms. Yet, as regulatory interest and academic research in genetic safeguard technologies advance, the implementation in industrial biotechnology, a sector that is already employing engineered microorganisms, lags behind. The main goal of this work is to explore the utilization of genetic safeguard technologies for designing biosafety in industrial biotechnology. Based on our results, we posit that biosafety is a case of a changing value, by means of further specification of how to realize biosafety. Our investigation is inspired by the Value Sensitive Design framework, to investigate scientific and technological choices in their appropriate social context. Our findings discuss stakeholder norms for biosafety, reasonings about genetic safeguards, and how these impact the practice of designing for biosafety. We show that tensions between stakeholders occur at the level of norms, and that prior stakeholder alignment is crucial for value specification to happen in practice. Finally, we elaborate in different reasonings about genetic safeguards for biosafety and conclude that, in absence of a common multi-stakeholder effort, the differences in informal biosafety norms and the disparity in biosafety thinking could end up leading to design requirements for compliance instead of for safety.

Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida.

The inclusion of biosafety strategies into strain engineering pipelines is crucial for safe-by-design biobased processes. This in turn might enable a more rapid regulatory acceptance of bioengineered organisms in both industrial and environmental applications. For this reason, we equipped the industrially relevant microbial chassis Pseudomonas putida KT2440 with an effective biocontainment strategy based on a synthetic dependency on phosphite, which is generally not readily available in the environment. The produced PSAG-9 strain was first engineered to assimilate phosphite through the genome-integration of a phosphite dehydrogenase and a phosphite-specific transport complex. Subsequently, to deter the strain from growing on naturally assimilated phosphate, all native genes related to its transport were identified and deleted generating a strain unable to grow on media containing any phosphorous source other than phosphite. PSAG-9 exhibited fitness levels with phosphite similar to those of the wild type with phosphate, and low levels of escape frequency. Beyond biosafety, this strategy endowed P. putida with the capacity to be cultured under non-sterile conditions using phosphite as the sole phosphorous source with a reduced risk of contamination by other microbes, while displaying enhanced NADH regenerative capacity. These industrially beneficial features complement the metabolic advantages for which this species is known for, thereby strengthening it as a synthetic biology chassis with potential uses in industry, with suitability towards environmental release.

Identification of Aspergillus niger Aquaporins Involved in Hydrogen Peroxide Signaling.

Aspergillus niger is a robust microbial cell factory for organic acid production. However, the regulation of many industrially important pathways is still poorly understood. The regulation of the glucose oxidase (Gox) expression system, involved in the biosynthesis of gluconic acid, has recently been uncovered. The results of that study show hydrogen peroxide, a by-product of the extracellular conversion of glucose to gluconate, has a pivotal role as a signaling molecule in the induction of this system. In this study, the facilitated diffusion of hydrogen peroxide via aquaporin water channels (AQPs) was studied. AQPs are transmembrane proteins of the major intrinsic proteins (MIPs) superfamily. In addition to water and glycerol, they may also transport small solutes such as hydrogen peroxide. The genome sequence of A. niger N402 was screened for putative AQPs. Seven AQPs were found and could be classified into three main groups. One protein (AQPA) belonged to orthodox AQP, three (AQPB, AQPD, and AQPE) were grouped in aquaglyceroporins (AQGP), two (AQPC and AQPF) were in X-intrinsic proteins (XIPs), and the other (AQPG) could not be classified. Their ability to facilitate diffusion of hydrogen peroxide was identified using yeast phenotypic growth assays and by studying AQP gene knock-outs in A. niger. The X-intrinsic protein AQPF appears to play roles in facilitating hydrogen peroxide transport across the cellular membrane in both Saccharomyces cerevisiae and A. niger experiments.

ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida.

Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9-mediated counterselection built upon the minimal PAM 5'-NNG-3' of the Streptococcus canis Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis Pseudomonas putida by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in P. putida, opening promising avenues for genome editing and applications thereof in this bacterium and beyond.

From Innovation to Application: Bridging the Valley of Death in Industrial Biotechnology.

Few biotechnology innovations make it through the Valley of Death to markets. Based on our experience with academia, technology transfer offices, and industry, we provide insights into differences in operating levels, how to best traverse the Valley of Death, and ways to foster more innovation towards market implementation.

A navigation guide of synthetic biology tools for Pseudomonas putida.

Pseudomonas putida is a microbial chassis of huge potential for industrial and environmental biotechnology, owing to its remarkable metabolic versatility and ability to sustain difficult redox reactions and operational stresses, among other attractive characteristics. A wealth of genetic and in silico tools have been developed to enable the unravelling of its physiology and improvement of its performance. However, the rise of this microbe as a promising platform for biotechnological applications has resulted in diversification of tools and methods rather than standardization and convergence. As a consequence, multiple tools for the same purpose have been generated, whilst most of them have not been embraced by the scientific community, which has led to compartmentalization and inefficient use of resources. Inspired by this and by the substantial increase in popularity of P. putida, we aim herein to bring together and assess all currently available (wet and dry) synthetic biology tools specific for this microbe, focusing on the last 5 years. We provide information on the principles, functionality, advantages and limitations, with special focus on their use in metabolic engineering. Additionally, we compare the tool portfolio for P. putida with those for other bacterial chassis and discuss potential future directions for tool development. Therefore, this review is intended as a reference guide for experts and new 'users' of this promising chassis.

Genetic Safeguards for Safety-by-design: So Close Yet So Far.

Safety-by-design (SbD) is paramount for risk management in synthetic biology, with genetic safeguards being a key technology for its implementation. While attractive in theory, the integration of genetic safeguards into SbD strategies is rarely exercised in practice, despite technological advances. Here we question why and what might be done about it.