Engineering and evolution of the complete Reductive Glycine Pathway in Saccharomyces cerevisiae for formate and CO assimilation.

Using captured CO and C1-feedstocks like formate and methanol derived from electrochemical activation of CO are key solutions for transforming industrial processes towards a circular carbon economy. Engineering formate and CO-based growth in the biotechnologically relevant yeast Saccharomyces cerevisiae could boost the emergence of a formate-mediated circular bio-economy. This study adopts a growth-coupled selection scheme for modular implementation of the Reductive Glycine Pathway (RGP) and subsequent Adaptive Laboratory Evolution (ALE) to enable formate and CO assimilation for biomass formation in yeast.

PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in .

Metabolic engineering approaches do not exclusively require fine-tuning of heterologous genes but oftentimes also modulation or even induction of host gene expression, , in order to rewire metabolic fluxes. Here, we introduce the programmable red light switch PhiReX 2.0, which can rewire metabolic fluxes by targeting endogenous promoter sequences through single-guide RNAs (sgRNAs) and activate gene expression in upon red light stimulation.

Optimizing as a formatotrophic platform for bioproduction the reductive glycine pathway.

Microbial C1 fixation has a vast potential to support a sustainable circular economy. Hence, several biotechnologically important microorganisms have been recently engineered for fixing C1 substrates. However, reports about C1-based bioproduction with these organisms are scarce.

Unusual DNA-binding properties of the Arabidopsis thaliana WRKY50 transcription factor at target gene promoters.

WRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding. Based on the genome-wide prediction of WRKY50 target genes and the similarity of a WRKY50 binding site to WT-boxes in microbe-associated molecular pattern (MAMP)-responsive cis-regulatory modules (CRM), four WT-box containing CRMs from the promoter region of three WRKY50 target genes were investigated for their interaction with WRKY50.

The AssemblX Toolkit for Reliable and User-Friendly Multigene Assemblies.

The implementation of complex cloning projects covering the assembly of entire biological pathways or large genetic circuits poses a major challenge in the field of biotechnology and synthetic biology, as such projects can be costly and time-consuming. To overcome these difficulties, we developed the software-assisted AssemblX toolkit, which allows even unexperienced users to design, build, and subsequently test large DNA constructs. Currently, AssemblX allows the assembly of up to 25 functional units (e.

Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast.

One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate.

Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants.

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes.

Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in .

Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like depend on synthetic transcription factors (synTFs) and corresponding -regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs) with minimal sequence identity to the host's endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400.

PhiReX: a programmable and red light-regulated protein expression switch for yeast.

Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic pathways. To address this, we developed PhiReX, a novel red/far-red light-regulated protein expression system for use in Saccharomyces cerevisiae. PhiReX is based on the combination of a customizable synTALE DNA-binding domain, the VP64 activation domain and the light-sensitive dimerization of the photoreceptor PhyB and its interacting partner PIF3 from Arabidopsis thaliana.

Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae.

Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana.

AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies.

The assembly of large DNA constructs coding for entire pathways poses a major challenge in the field of synthetic biology. Here, we present AssemblX, a novel, user-friendly and highly efficient multi-gene assembly strategy. The software-assisted AssemblX process allows even unexperienced users to rapidly design, build and test DNA constructs with currently up to 25 functional units, from 75 or more subunits.

Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter.

Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity.

'In silico expression analysis', a novel PathoPlant web tool to identify abiotic and biotic stress conditions associated with specific cis-regulatory sequences.

Using bioinformatics, putative cis-regulatory sequences can be easily identified using pattern recognition programs on promoters of specific gene sets. The abundance of predicted cis-sequences is a major challenge to associate these sequences with a possible function in gene expression regulation. To identify a possible function of the predicted cis-sequences, a novel web tool designated 'in silico expression analysis' was developed that correlates submitted cis-sequences with gene expression data from Arabidopsis thaliana.

Identification of a novel type of WRKY transcription factor binding site in elicitor-responsive cis-sequences from Arabidopsis thaliana.

Using a combination of bioinformatics and synthetic promoters, novel elicitor-responsive cis-sequences were discovered in promoters of pathogen-upregulated genes from Arabidopsis thaliana. One group of functional sequences contains the conserved core sequence GACTTTT. This core sequence and adjacent nucleotides are essential for elicitor-responsive gene expression in a parsley protoplast system.

Inducible expression of p50 from TMV for increased resistance to bacterial crown gall disease in tobacco.

The dominant tobacco mosaic virus (TMV) resistance gene N induces a hypersensitive response upon TMV infection and protects tobacco against systemic spread of the virus. It has been proposed to change disease resistance specificity by reprogramming the expression of resistance genes or their corresponding avirulence genes. To reprogramme the resistance response of N towards bacterial pathogens, the helicase domain (p50) of the TMV replicase, the avirulence gene of N, was linked to synthetic promoters 4D and 2S2D harbouring elicitor-responsive cis-elements.

Differential expression of the TMV resistance gene N prevents a hypersensitive response in seeds and during germination.

The dominant tobacco mosaic virus (TMV) resistance gene N confers a hypersensitive response (HR) at the site of TMV infection and protects tobacco against systemic spread of the virus. To study N gene activity in seeds and early seedling development, the avirulence gene of N, the helicase domain (p50) of the TMV replicase, was constitutively expressed in a tobacco genotype without N (nn). Transgenic F1 expressing N and p50 were generated by crossing with an NN genotype.

Integration of bioinformatics and synthetic promoters leads to the discovery of novel elicitor-responsive cis-regulatory sequences in Arabidopsis.

A combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. With Arabidopsis (Arabidopsis thaliana) microarray data annotated to the PathoPlant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. Using the binding site estimation suite of tools, BEST, 407 conserved sequence motifs were identified in promoter regions of these coregulated gene sets.

Factors required for the high CO2 specificity of the anaerobically induced maize GapC4 promoter in transgenic tobacco.

Flooding, a natural cause of anaerobiosis, is often accompanied by high CO(2) concentrations in the flood water. Plants need to respond to these environmental conditions. Strong anaerobic reporter gene activity in tobacco (Nicotiana tabacum) controlled by the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GapC4) promoter from maize (Zea mays) depends on the presence of CO(2) and light.

Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision.