Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

García‐García, Jorge Donato; Joshi, Jaya; Patterson, Jenelle A.; Rodriguez, Lidimarie Trujillo; Reisch, Christopher R; Javanpour, Alex A.; Liu, Chang C.; Hanson, Andrew D.

doi:10.3390/life10090179

Cited by 25 publications

(44 citation statements)

References 68 publications

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“…With orthogonal in vivo evolution machineries at hand, any trait that can be coupled to growth (e.g. antibiotic resistance, tolerance to cultivation conditions and/or complementation of auxotrophies) enables facile identification of improved target genes without need for direct screening (Esvelt et al ., 2011 ; Ravikumar et al ., 2014 ; García‐García et al ., 2020 ; Rix et al ., 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast

Damgaard‐Møller

Ambri

Bendtsen

et al. 2021

Microbial Biotechnology

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SummaryDirected evolution is a powerful method to optimize proteins and metabolic reactions towards user‐defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence‐spaces is a labour‐intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high‐throughput using a transcription factor‐based biosensor. We demonstrate the strengths of this workflow by evolving a rate‐limiting enzymatic reaction of the biosynthetic pathway for cis,cis‐muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high‐throughput sorting of best‐performing variants using a transcription factor‐based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13‐fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.

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Section: Introductionmentioning

confidence: 99%

Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast

Damgaard‐Møller

Ambri

Bendtsen

et al. 2021

Microbial Biotechnology

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“…It depends on coupling cell growth to the activity of the target enzyme and, in most cases, on being able to sharply shut off target expression, thereby locking subsequent growth to the number of catalytic cycles that the target mediates before inactivation. We are seeking proof-of-concept by evolving the suicide enzyme THI4 (CCR = 1) for higher CCR and function in mild conditions ( 64 , 65 ).…”

Section: Discussionmentioning

confidence: 99%

The number of catalytic cycles in an enzyme’s lifetime and why it matters to metabolic engineering

Hanson

McCarty

Henry

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part’s working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100–200 enzymes each from Lactococcus lactis, yeast, and Arabidopsis. CCRs in these organisms had similar ranges (<103 to >107) but different median values (3–4 × 104 in L. lactis and yeast versus 4 × 105 in Arabidopsis). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible.

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“…Besides the nature of the metal inserted, there may well be other determinants of aerotolerance. 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.…”

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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Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

Cited by 25 publications

References 68 publications

Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast

Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast

The number of catalytic cycles in an enzyme’s lifetime and why it matters to metabolic engineering

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

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