Systems for in vivo hypermutation: a quest for scale and depth in directed evolution

Rix, Gordon; Liu, Chang C.

doi:10.1016/j.cbpa.2021.02.008

Cited by 32 publications

(31 citation statements)

References 41 publications

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“…In fact, we estimate that, given a new SARS-CoV-2 RBD variant 37 , we will be able to quickly discover multiple high-affinity binders. Overall, we believe that AHEAD's core functionality for rapid and parallelizable antibody generation through autonomous hypermutation, along with the growing ecosystem of autonomous hypermutation systems in synthetic biology 48,49 , has the potential to broadly upgrade antibody generation, supporting all areas of biomedicine.…”

Section: Discussionmentioning

confidence: 99%

Rapid generation of potent antibodies by autonomous hypermutation in yeast

et al. 2021

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The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe 'autonomous hypermutation yeast surface display' (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large.

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Section: Discussionmentioning

confidence: 99%

Rapid generation of potent antibodies by autonomous hypermutation in yeast

et al. 2021

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“…We hope to identify such determinants, and to gain insight into the metal cofactor, from ongoing continuous directed evolution experiments [ 62 ] to improve the complementing activity of native non-Cys THI4s. If — as seems likely [ 63 ] — such improvement is possible, there is a realistic prospect of replacing suicidal plant THI4s with catalytic THI4s that work well in aerobic conditions and thus slash the energy cost of thiamin synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…We hope to identify such determinants, and to gain insight into the metal cofactor, from ongoing continuous directed evolution experiments [62] to improve the complementing activity of native non-Cys THI4s. Ifas seems likely [63] such improvement is possible, there is a realistic prospect of replacing suicidal plant THI4s with Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210565/919315/bcj-2021-0565.pdf by guest on 30 August 2021 Biochemical Journal. This is an Accepted Manuscript.…”

Section: Discussionmentioning

confidence: 99%

Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

Joshi

García‐García

et al. 2021

Biochemical Journal

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Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multiple-turnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been biochemically characterized – but only from archaeal methanogens that are anaerobic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

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“…We hope to identify such determinants, and to gain insight into the metal cofactor, from ongoing continuous directed evolution experiments [62] to improve the complementing activity of native non-Cys THI4s. If -as seems likely [63] -such improvement is possible, there is a realistic prospect of replacing suicidal plant THI4s with catalytic THI4s that work well in aerobic conditions and thus slash the energy cost of thiamin synthesis. iron-mediated sulfur transfer in archael and yeast thiazole synthases.…”

Section: Discussionmentioning

confidence: 99%

Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

Joshi

García‐García

et al. 2021

Preprint

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Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multipleturnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been characterized – but only from archaeal methanogens that are anaerobic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

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Systems for in vivo hypermutation: a quest for scale and depth in directed evolution

Cited by 32 publications

References 41 publications

Rapid generation of potent antibodies by autonomous hypermutation in yeast

Rapid generation of potent antibodies by autonomous hypermutation in yeast

Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

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