Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort

Jeschek, Markus; Gerngross, Daniel; Panke, Sven

doi:10.1038/ncomms11163

Cited by 127 publications

(115 citation statements)

References 49 publications

(69 reference statements)

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“…Our results highlight the potential effect of transcriptional balancing on the flux through extrinsic pathways and nicely complement similar findings for violacein production in E. coli. [54,55] We sequenced strains with different production efficiencies ( Figure 1D) and correlated the identified promoters to their previously measured expression strengths [33] to further investigate the cause of the variations in ectoine production and to better understand why some mutants exhibited particular high production. The strongest promoter (H36) occurred at a frequency of only 4% in the ten best producers, whereas medium and weak promoters were much more prominent ( Figure 1D).…”

Section: Transcriptional Balancing Of the Ectoine Genes Is Crucial Fomentioning

confidence: 99%

Metabolic Engineering of Corynebacterium glutamicum for High‐Level Ectoine Production: Design, Combinatorial Assembly, and Implementation of a Transcriptionally Balanced Heterologous Ectoine Pathway

Gießelmann

Dietrich

Jungmann

et al. 2019

Biotechnology Journal

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Ectoine is formed in various bacteria as cell protectant against all kinds of stress. Its preservative and protective effects have enabled various applications in medicine, cosmetics, and biotechnology, and ectoine therefore has high commercial value. Industrially, ectoine is produced in a complex high‐salt process, which imposes constraints on the costs, design, and durability of the fermentation system. Here, Corynebacterium glutamicum is upgraded for the heterologous production of ectoine from sugar and molasses. To overcome previous limitations, the ectoine pathway taken from Pseudomonas stutzeri is engineered using transcriptional balancing. An expression library with 185,193 variants is created, randomly combining 19 synthetic promoters and three linker elements. Strain screening discovers several high‐titer mutants with an improvement of almost fivefold over the initial strain. High production thereby particularly relies on a specifically balanced ectoine pathway. In an optimized fermentation process, the new top producer C. glutamicum ectABCopt achieves an ectoine titer of 65 g L−1 and a specific productivity of 120 mg g−1 h−1. This process is the first reported example of a simple fermentation process under low‐salt conditions using well‐established feedstocks to produce ectoine with industrial efficiency. There is a compelling case for more intensive implementation of transcriptional balancing in future metabolic engineering of C. glutamicum.

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Section: Transcriptional Balancing Of the Ectoine Genes Is Crucial Fomentioning

confidence: 99%

Metabolic Engineering of Corynebacterium glutamicum for High‐Level Ectoine Production: Design, Combinatorial Assembly, and Implementation of a Transcriptionally Balanced Heterologous Ectoine Pathway

Gießelmann

Dietrich

Jungmann

et al. 2019

Biotechnology Journal

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“…The violacein biosynthesis pathway also produces deoxyviolacein (DV) as a by-product, and the coloured properties of violacein and deoxyviolacein make them interesting targets for natural product pathway engineering. For example, high-throughput combinatorial promoter library screening 3 , CRISPR-based multiplex transcriptional regulation 4 or diverting pathway flux from deoxyviolacein to violacein via RBS engineering 5 .…”

Section: Introductionmentioning

confidence: 99%

A GenoChemetic strategy for derivatization of the violacein natural product scaffold

Lai¹,

Obled²,

Chee³

et al. 2017

Preprint

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The next frontier in drug discovery could be the semi-synthesis of non-natural, xenobiotic compounds combining both natural product biosynthesis and synthetic chemistry. However, the required tools and underlying engineering principles are yet to be fully understood. One way to investigate non-natural product biosynthesis is to probe the substrate promiscuity of a clinically relevant biosynthesis pathway. Violacein is a bisindole compound produced by the VioABCDE biosynthesis pathway using L-tryptophan as the starting substrate. Previous studies have shown that violacein exhibits antimicrobial properties, and synthetic analogues of violacein might give rise to new targets for therapeutic development to combat antimicrobial resistance. By adding seven types of tryptophan analogues available commercially, 62 new violacein or deoxyviolacein analogues were generated with a synthetic violacein biosynthesis pathway expressed in Escherichia coli, demonstrating the promiscuity of violacein biosynthesis enzymes. Growth inhibition assays against Bacillus subtilis, a Gram-positive bacterium, were carried out to measure growth inhibitory activity of violacein analogues compared to violacein. In addition, we show that four new 7-chloro analogues of violacein or deoxyviolacein can be generated in vivo by combining the rebeccamycin and violacein biosynthesis pathways and purified 7-chloro violacein was found to have similar growth inhibitory activity compared to violacein. Structural studies of VioA revealed active site residues that are important for catalytic activity, and further pathway recombination with VioA homologues in related bisindole pathways may lead to more efficient enzymes that would accept tryptophan analogues more readily.

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“…Effective strategies are needed first to design the granularity and distribution of fractional sampling of large pathway design spaces given a priori information, and then to iteratively optimise designs by moving through design space towards progressively better pathway variants. Such strategies have become the main subject of various recent studies (31,(73)(74)(75) . This is a significant development reflecting the evolving role of systematic synthetic biology approaches in underpinning increasingly effective metabolic engineering and industrial biotechnology.…”

Section: Discussionmentioning

confidence: 99%

Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering

Taylor¹,

Mordaka²,

Heap³

2018

Preprint

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DNA assembly allows individual DNA constructs or designed mixtures to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable 'scar' sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in E. coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.

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Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort

Cited by 127 publications

References 49 publications

Metabolic Engineering of Corynebacterium glutamicum for High‐Level Ectoine Production: Design, Combinatorial Assembly, and Implementation of a Transcriptionally Balanced Heterologous Ectoine Pathway

Metabolic Engineering of Corynebacterium glutamicum for High‐Level Ectoine Production: Design, Combinatorial Assembly, and Implementation of a Transcriptionally Balanced Heterologous Ectoine Pathway

A GenoChemetic strategy for derivatization of the violacein natural product scaffold

Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering

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