Search

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. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast onehybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA. [ABSTRACT FROM AUTHOR]

Phenylpropenes are a class of natural products that are synthesised by a vast range of plant species and hold considerable promise in the flavour and fragrance industries. Many in vitro studies have been carried out to elucidate and characterise the enzymes responsible for the production of these volatile compounds. However, there is a scarcity of studies demonstrating the in vivo production of phenylpropenes in microbial cell factories. In this study, we engineered Escherichia coli to produce methylchavicol, methyleugenol and isoeugenol from their respective phenylacrylic acid precursors. We achieved this by extending and modifying a previously optimised heterologous pathway for the biosynthesis of chavicol and eugenol. We explored the potential of six S -adenosyl l-methionine (SAM)-dependent O- methyltransferases to produce methylchavicol and methyleugenol from chavicol and eugenol, respectively. Additionally, we examined two isoeugenol synthases for the production of isoeugenol from coniferyl acetate. The best-performing strains in this study were able to achieve titres of 13 mg L -1 methylchavicol, 59 mg L -1 methyleugenol and 361 mg L -1 isoeugenol after feeding with their appropriate phenylacrylic acid substrates. We were able to further increase the methyleugenol titre to 117 mg L -1 by supplementation with methionine to facilitate SAM recycling. Moreover, we report the biosynthesis of methylchavicol and methyleugenol from l-tyrosine through pathways involving six and eight enzymatic steps, respectively., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Selenzyme is a retrobiosynthesis tool that suggests candidate enzymes for user query reactions. Enzyme suggestions are based on identical reactions, as well as similar reactions, since enzymes are often capable of promiscuous substrate binding. Selenzyme is a user-friendly, widely used web-tool for ranking enzymes based on reaction similarity and additional features, including the phylogenetic distance between the source species of the enzyme and the intended host. While Selenzyme has proved invaluable in assisting with enzyme selection for known reactions, as well as many novel or orphan reactions, weaknesses have been exposed in its ability to rank functionally related enzymes. Within this update, we introduce a new reaction similarity scoring algorithm, which is used in conjunction with the previous similarity calculation, to improve the accuracy of enzyme suggestions based on non-identical similar reactions, across a range of EC reaction classes. This allows enzymes to be suggested for reactions not found within the database, even if the reaction is unbalanced. A database update was also carried out, to ensure that reaction and enzyme knowledge remains current. This update can be accessed at http://selenzymeRF.synbiochem.co.uk/., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Cupriavidus necator H16 is one of the most researched carbon dioxide (CO 2 )-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO 2 utilising precursors derived from the CBB cycle., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

CRISPR technologies have become standard laboratory tools for genetic manipulations across all kingdoms of life. Despite their origins in bacteria, the development of CRISPR tools for engineering bacteria has been slower than for eukaryotes; nevertheless, their function and application for genome engineering and gene regulation via CRISPR interference (CRISPRi) has been demonstrated in various bacteria, and adoption has become more widespread. Here, we provide simple plasmid-based systems for genome editing (gene knockouts/knock-ins, and genome integration of large DNA fragments) and CRISPRi in E. coli using a CRISPR-Cas12a system. The described genome engineering protocols allow markerless deletion or genome integration in just seven working days with high efficiency (> 80% and 50%, respectively), and the CRISPRi protocols allow robust transcriptional repression of target genes (> 90%) with a single cloning step. The presented minimized plasmids and their associated design and experimental protocols provide efficient and effective CRISPR-Cas12 genome editing, genome integration and CRISPRi implementation. These simple-to-use systems and protocols will allow the easy adoption of CRISPR technology by any laboratory., (© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Cellular functions are often controlled by small molecular weight molecules such as metabolites. Microorganisms, mainly prokaryotes, have evolved sensing and regulatory mechanisms based on transcriptional regulators (TRs) that are able to activate gene expression in response to changes in intra- and extracellular metabolite (ligand) concentrations. To understanding control mechanisms and cell factory development in synthetic biology applications, high throughput analytical procedures are required. In this chapter, we outline a methodological pipeline to design and build reporter constructs enabling the characterization of metabolite-responsive inducible gene expression systems. As an example, we present the design, cloning and characterization of the itaconate-inducible system which is composed of the LysR-type transcriptional regulator ItcR and the promoter P ccl from Yersinia pseudotuberculosis. Fluorescence-based plate reader and flow cytometry assays are described and the steps for performing data analysis are provided., (© 2019 Elsevier Inc. All rights reserved.)

Medium- and long-chain methyl ketones are fatty acid-derived compounds that can be used as biofuel blending agents, flavors and fragrances. However, their large-scale production from sustainable feedstocks is currently limited due to the lack of robust microbial biocatalysts. The oleaginous yeast Yarrowia lipolytica is a promising biorefinery platform strain for the production of methyl ketones from renewable lignocellulosic biomass due to its natively high flux towards fatty acid biosynthesis. In this study, we report the metabolic engineering of Y. lipolytica to produce long- and very long-chain methyl ketones. Truncation of peroxisomal β-oxidation by chromosomal deletion of pot1 resulted in the biosynthesis of saturated, mono-, and diunsaturated methyl ketones in the C 13 -C 23 range. Additional overexpression and peroxisomal targeting of a heterologous bacterial methyl ketone biosynthesis pathway yielded an initial titer of 151.5 mg/L of saturated methyl ketones. Dissolved oxygen concentrations in the cultures were found to substantially impact cell morphology and methyl ketone biosynthesis. Bioreactor cultivation under optimized conditions resulted in a titer of 314.8 mg/L of total methyl ketones, representing more than a 6000-fold increase over the parental strain. This work highlights the potential of Y. lipolytica to serve as chassis organism for the biosynthesis of acyl-thioester derived long- and very long-chain methyl ketones., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)