Towards next-generation cell factories by rational genome-scale engineering

Yilmaz, Suzan; Nyerges, Ákos; Oost, John van der; Church, George M.; Claassens, Nico J.

doi:10.1038/s41929-022-00836-w

Cited by 20 publications

(11 citation statements)

References 144 publications

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“…ALE screens the host genome for serendipitous edits and relies on fitness improvement to select the best ones (Mavrommati et al, 2022; Sandberg et al, 2019). Genome-wide screens introduce genetic perturbations, generally with CRISPR, recombineering or transposable elements (Cain et al, 2020; McCarty et al, 2020; Yilmaz et al, 2022), and screen and sort the edited library using massively parallelized reporter assays and next-generation sequencing to identify enriched edits (Bock et al, 2022; Cain et al, 2020). While both ALE and genome-wide screens have been extremely powerful at generating improved strains, the mechanistic learnings they generate are limited.…”

Section: Discussionmentioning

confidence: 99%

Microbial cell factory optimisation using genome-wide host-pathway interaction screens

Cachera,

Kurt,

Røpke

et al. 2023

Preprint

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The ubiquity of genetic interactions in living cells challenges the concept of parts orthogonality, which is a cornerstone of synthetic biology. Parts, such as heterologously expressed genes, draw from shared pools of limited cellular resources and interactions between parts themselves and their host are inevitable. Instead of trying to eliminate or disregard these interactions, we propose to leverage them to promote desirable phenotypes. We recently described CRI-SPA, a method for high-throughput genome-wide gene delivery and screening of host:pathway interactions in Saccharomyces cerevisiae. In this study, we combine this method with biosensor-based high-throughput screening and high-density colony image analysis to identify lead engineering targets for optimising cis-cis-muconic acid (CCM) production in yeast cell factories. Using the biosensor screen, we phenotype >9,700 genotypes for their interaction with the heterologously expressed CCM biosynthesis pathway, including both gene knock-out and overexpression, and identify novel metabolic targets belonging to sulphur assimilation and methionine synthesis, as well as cellular redox homeostasis, positively impacting CCM biosynthesis by up to 280%. Our genome-wide exploration of host pathway interaction opens novel strategies for the metabolic engineering of yeast cell factories.

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Section: Discussionmentioning

confidence: 99%

Microbial cell factory optimisation using genome-wide host-pathway interaction screens

Cachera,

Kurt,

Røpke

et al. 2023

Preprint

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“…pET28a- fap plasmid was prepared according to previous reports( 2, 3 ). Briefly, cDNA encoding FAP from Chlorella variabilis ( Cvfap ) was cloned, amplified by excluding predicted targeting sequence for chloroplasts, and codon-optimized for expression in E. coli and ligated into the pLIC07 vector.…”

Section: Methodsmentioning

confidence: 99%

“…Synthetic biology and metabolic engineering provide greener solutions for the production of valuable chemicals compared to chemical-based approaches due to their less detrimental effects on the environment ( 1, 2 ). The approach of using microbial cell factories is also sustainable because it can convert renewable biomass (rather than petrochemicals or other non-renewable resources) into products of interest by fermentations ( 1, 3, 4 ).…”

Section: Introductionmentioning

confidence: 99%

A versatilein situcofactor enhancing system for meeting cellular demands for engineered metabolic pathways

Jaroensuk

Sutthaphirom

Phonbuppha

et al. 2023

Preprint

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Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase levels of a pool of sugar-phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells; demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase, GDH) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

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“…Recently, cutting-edge technologies, such as genome editing, (semi-)rational protein engineering, and other synthetic biology strategies, have been employed to overcome unknown metabolic regulatory challenges. , As a crucial strategy for enzyme engineering, the semi-rational design guided by molecular docking and simulation generates small, focused, but effective libraries for developing mutant enzymes with desirable properties. , Multivariate modular metabolic engineering is a powerful strategy for transposing metabolic flux toward interested pathways via the overexpression of rate-limiting enzymes and deregulation or knockout genes of the competing pathways . It can transform a complex pathway into simple distinct modules allowing parallel optimization .…”

Section: Introductionmentioning

confidence: 99%

Combinatorial Protein Engineering and Metabolic Engineering for Efficient Synthesis of l-Histidine in Corynebacterium glutamicum

Chai

Liu

et al. 2023

ACS Synth. Biol.

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l-Histidine is an essential proteinogenic amino acid in food with extensive applications in the pharmaceutical field. Herein, we constructed a Corynebacterium glutamicum recombinant strain for efficient biosynthesis of l-histidine. First, to alleviate the l-histidine feedback inhibition, the ATP phosphoribosyltransferase mutant HisGT235P‑Y56M was constructed based on molecular docking and high-throughput screening, resulting in the accumulation of 0.83 g/L of l-histidine. Next, we overexpressed rate-limiting enzymes including HisGT235P‑Y56M and PRPP synthetase and knocked out the pgi gene in the competing pathway, which increased the l-histidine production to 1.21 g/L. Furthermore, the energy status was optimized by decreasing the reactive oxygen species level and enhancing the supply of adenosine triphosphate, reaching a titer of 3.10 g/L in a shake flask. The final recombinant strain produced 5.07 g/L of l-histidine in a 3 L bioreactor, without the addition of antibiotics and chemical inducers. Overall, this study developed an efficient cell factory for l-histidine biosynthesis by combinatorial protein engineering and metabolic engineering.

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Towards next-generation cell factories by rational genome-scale engineering

Cited by 20 publications

References 144 publications

Microbial cell factory optimisation using genome-wide host-pathway interaction screens

Microbial cell factory optimisation using genome-wide host-pathway interaction screens

A versatilein situcofactor enhancing system for meeting cellular demands for engineered metabolic pathways

Combinatorial Protein Engineering and Metabolic Engineering for Efficient Synthesis of l-Histidine in Corynebacterium glutamicum

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