bims-mricoa 2023-02-05 papers

Issue of 2023‒02‒05
three papers selected by
Merve Yavuz
Bilkent University

PLoS One. 2023 ;18(2): e0280499

Robust performance of a live bacterial therapeutic chassis lacking the colibactin gene cluster.

Aida Kalantari, Michael J James, Lauren A Renaud, Mylene Perreault, Catherine E Monahan, Mary N McDonald, David L Hava, Vincent M Isabella.

E. coli Nissle (EcN) is a non-pathogenic probiotic bacterium of the Enterobacteriaceae family that has been used for over a century to promote general gut health. Despite the history of safe usage of EcN, concerns have been raised regarding the presence of the pks gene cluster, encoding the genotoxin colibactin, due to its association with colorectal cancer. Here, we sought to determine the effect of pks island removal on the in vitro and in vivo robustness and activity of EcN and EcN-derived strains. A deletion of the pks island (Δpks) was constructed in wild type and engineered strains of EcN using lambda red recombineering. Mass spectrometric measurement of N-myristoyl-D-asparagine, released during colibactin maturation, confirmed that the pks deletion abrogated colibactin production. Growth curves were comparable between Δpks strains and their isogenic parents, and wild type EcN displayed no competitive advantage to the Δpks strain in mixed culture. Deletion of pks also had no effect on the activity of strains engineered to degrade phenylalanine (SYNB1618 and SYNB1934) or oxalate (SYNB8802). Furthermore, 1:1 mixed dosing of wild type and Δpks EcN in preclinical mouse and nonhuman primate models demonstrated no competitive disadvantage for the Δpks strain with regards to transit time or colonization. Importantly, there was no significant difference on in vivo strain performance between the clinical-stage strain SYNB1934 and its isogenic Δpks variant with regards to recovery of the quantitative strain-specific biomarkers d5- trans-cinnamic acid, and d5-hippuric acid. Taken together, these data support that the pks island is dispensable for Synthetic Biotic fitness and activity in vivo and that its removal from engineered strains of EcN will not have a deleterious effect on strain efficacy.

DOI: https://doi.org/10.1371/journal.pone.0280499

Nat Rev Chem. 2023 Jan 23. 1-18

DNA synthesis technologies to close the gene writing gap.

Alex Hoose, Richard Vellacott, Marko Storch, Paul S Freemont, Maxim G Ryadnov.

Synthetic DNA is of increasing demand across many sectors of research and commercial activities. Engineering biology, therapy, data storage and nanotechnology are set for rapid developments if DNA can be provided at scale and low cost. Stimulated by successes in next generation sequencing and gene editing technologies, DNA synthesis is already a burgeoning industry. However, the synthesis of >200 bp sequences remains unaffordable. To overcome these limitations and start writing DNA as effectively as it is read, alternative technologies have been developed including molecular assembly and cloning methods, template-independent enzymatic synthesis, microarray and rolling circle amplification techniques. Here, we review the progress in developing and commercializing these technologies, which are exemplified by innovations from leading companies. We discuss pros and cons of each technology, the need for oversight and regulatory policies for DNA synthesis as a whole and give an overview of DNA synthesis business models.

Keywords: DNA; Synthetic biology

DOI: https://doi.org/10.1038/s41570-022-00456-9

Biotechnol Bioeng. 2023 Jan 29.

Bacterial Delivery of Therapeutic Proteins to the Nuclei of Cancer Cells.

Shoshana M K Bloom, Nicholas O'Hare, Neil S Forbes.

Targeting nucleic targets with therapeutic proteins would enhance the treatment of hard-to-treat cancers. However, exogenous proteins are excluded from the nucleus by both the cellular and nuclear membranes. We have recently developed Salmonella that deliver active proteins into the cytoplasm of cancer cells. Here, we hypothesized that bacterially delivered proteins accumulate within nuclei, nuclear localization sequences increase delivery, and bacterially delivered proteins kill cancer cells. To test this hypothesis, we developed intranuclear delivering (IND) Salmonella and quantified the delivery of three model proteins. IND Salmonella delivered both ovalbumin and GFP to nuclei of MCF7 cancer cells. The amount of protein in nuclei was linearly dependent on the amount delivered to the cytoplasm. The addition of a nuclear localization sequence increased both the amount of protein in each nucleus and the number of nuclei that received protein. Delivery of Omomyc, a protein inhibitor of the nuclear transcript factor, Myc, altered cell physiology and significantly induced cell death. These results show that IND Salmonella deliver functional proteins to the nucleus of cancerous cells. Extending this method to other transcription factors will increase the number of accessible targets for cancer therapy. This article is protected by copyright. All rights reserved.

Keywords: Bacterial cancer therapy; Intracellular delivering Salmonella; Nuclear delivery; Omomyc; Synthetic biology; Therapeutic; proteins

DOI: https://doi.org/10.1002/bit.28340