Merging automation and fundamental discovery into the design–build–test–learn cycle of nontraditional microbes

Gurdo, Nicolás; Volke, Daniel C.; Nikel, Pablo I.

doi:10.1016/j.tibtech.2022.03.004

Cited by 29 publications

(17 citation statements)

References 92 publications

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“…[91] Apart from its value to gather fundamental knowledge and lead to exciting biotechnological developments, the implementation of the CBBc in new hosts can provide a platform for the directed evolution of RuBisCO in the hunt for variants with improved kinetic parameters or substrate specificity. [29] Although some efforts have been implemented in this direction, utilizing a combination of growth-coupled selection strategies, [43] in vivo directed mutagenesis, [90b,92] and high-throughput and automation routines [93] will offer new opportunities for improvement.…”

Section: Discussionmentioning

confidence: 99%

From Rags to Riches: Exploiting the Calvin‐Benson‐Bassham Cycle for Biomanufacturing

Federici,

Orsi,

Nikel

2023

ChemCatChem

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Industrial chemical production largely relies on fossil fuels, resulting in the unavoidable release of carbon dioxide (CO2) into the atmosphere. The concept of a circular carbon bioeconomy has been proposed to address this issue, wherein CO2 is captured and used as raw material for manufacturing new chemicals. Microbial cell factories and, in particular, autotrophic microorganisms capable of utilizing CO2 as the sole carbon source, emerged as potential catalysts for upcycling CO2 to valuable products. The Calvin‐Benson‐Bassham cycle (CBBc), the best‐known CO2 fixation pathway, is widely distributed in Nature. While extensively studied, microbial engineering programmes based on the CBBc remains relatively underexplored. In this review, we discuss avenues towards biotechnological exploitation of the CBBc to engineer CO2‐utilizing microbial cell factories, with a focus on chemically‐derived electron donors. We also highlight the advantages and challenges of implementing the CBBc in heterotrophic microbial hosts and its potential to advance a true circular carbon bioeconomy. Moreover, based on the pathway’s architecture, we argue about the ideal value‐added products to generate from this metabolic route. Studying and engineering the CBBc in both natural‐ and synthetic‐autotrophs will enhance our understanding on this CO2 fixation pathway, enabling further exploration of biomanufacturing avenues with CO2 as feedstock.

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Section: Discussionmentioning

confidence: 99%

From Rags to Riches: Exploiting the Calvin‐Benson‐Bassham Cycle for Biomanufacturing

Federici,

Orsi,

Nikel

2023

ChemCatChem

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“…Taken together, these results highlight the versatility of the CBE and ABE modules for engineering Gram-negative bacteria-demonstrated by a first-case example of site-directed mutation and reversal in the same strain. Moreover, the easily-curable vectors used for base-editing enable cycling of the genome modifications in a simple protocol that can be adapted to different purposes (including metabolic engineering and fundamental discovery of gene-to-function relationships) and amenable to automation [52]. This strategy eases complex strain engineering programs independently of homologous recombination and yields plasmid-free engineered cells.…”

Section: The Pablo•pcasso Toolset For Unconstrained Base-editing In G...mentioning

confidence: 99%

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

Kozaeva

Nielsen

Nikel

2023

Preprint

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Pseudomonasspecies are platforms for chemical production. To unleash their potential as cell factories, we designed synthetic biology tools based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) base-editors. A cytidine base-editor (CBE) and an adenine base-editor (ABE) were tailored for high-resolution genome engineering. CBE involves a cytidine deaminase fused to a nickase Cas9 fromStreptococcus pyogenes(SpnCas9) variant that converts cytidine to uracil, resulting in a C→T (or G→A) substitution. ABE relies on an adenine deaminase fused to a nickaseSpnCas9 variant to enable A→G (or T→C) editing. We established plasmid-borne, base-editing systems for efficient nucleotide substitution using a novel protospacer adjacent motif (PAM)-relaxedSpnCas9 (SpRY) variant. Base-editing was demonstrated by introducing prematureSTOPcodons in target genes (encoding both fluorescent proteins and metabolic functions), resulting in functional knockouts (using CBE). The wild-type genotype was recovered by eliminating the engineeredSTOPcodons (using ABE).P.putidacould be base-edited at ca. 75-95% efficiency for both types of editors. Furthermore, a set of induction-dependent vectors enabled efficient curing of the plasmid-based editing platform in a single, 8-h cultivation step. This strategy eases complex strain construction programs independently of homologous recombination and yields plasmid-free engineered cells.

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“…Indeed, it has paved the way for systematic exploration of the metabolic solution spaces required to produce target metabolites ( Wu et al, 2016 ; Gudmundsson and Nogales, 2021 ). Design-Build-Test-Learn (DBTL) iterative cycles stand up as popular examples of such multidisciplinary approaches to the production of a plethora of chemical compounds ( Gurdo et al, 2022 ). DBTL cycles are iterative designs combining the advances in systems and synthetic biology to deliver rational genetic modifications and high-throughput phenotyping of strains.…”

Section: Introductionmentioning

confidence: 99%

System metabolic engineering of Escherichia coli W for the production of 2-ketoisovalerate using unconventional feedstock

Carranza-Saavedra

Torres‐Bacete

Blázquez

et al. 2023

Front. Bioeng. Biotechnol.

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Replacing traditional substrates in industrial bioprocesses to advance the sustainable production of chemicals is an urgent need in the context of the circular economy. However, since the limited degradability of non-conventional carbon sources often returns lower yields, effective exploitation of such substrates requires a multi-layer optimization which includes not only the provision of a suitable feedstock but the use of highly robust and metabolically versatile microbial biocatalysts. We tackled this challenge by means of systems metabolic engineering and validated Escherichia coli W as a promising cell factory for the production of the key building block chemical 2-ketoisovalerate (2-KIV) using whey as carbon source, a widely available and low-cost agro-industrial waste. First, we assessed the growth performance of Escherichia coli W on mono and disaccharides and demonstrated that using whey as carbon source enhances it significantly. Second, we searched the available literature and used metabolic modeling approaches to scrutinize the metabolic space of E. coli and explore its potential for overproduction of 2-KIV identifying as basic strategies the block of pyruvate depletion and the modulation of NAD/NADP ratio. We then used our model predictions to construct a suitable microbial chassis capable of overproducing 2-KIV with minimal genetic perturbations, i.e., deleting the pyruvate dehydrogenase and malate dehydrogenase. Finally, we used modular cloning to construct a synthetic 2-KIV pathway that was not sensitive to negative feedback, which effectively resulted in a rerouting of pyruvate towards 2-KIV. The resulting strain shows titers of up to 3.22 ± 0.07 g/L of 2-KIV and 1.40 ± 0.04 g/L of L-valine in 24 h using whey in batch cultures. Additionally, we obtained yields of up to 0.81 g 2-KIV/g substrate. The optimal microbial chassis we present here has minimal genetic modifications and is free of nutritional autotrophies to deliver high 2-KIV production rates using whey as a non-conventional substrate.

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Merging automation and fundamental discovery into the design–build–test–learn cycle of nontraditional microbes

Cited by 29 publications

References 92 publications

From Rags to Riches: Exploiting the Calvin‐Benson‐Bassham Cycle for Biomanufacturing

From Rags to Riches: Exploiting the Calvin‐Benson‐Bassham Cycle for Biomanufacturing

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

System metabolic engineering of Escherichia coli W for the production of 2-ketoisovalerate using unconventional feedstock

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