Optimizing E. coli as a formatotrophic platform for bioproduction via 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. Here, we describe the optimization of a previously engineered formatotrophic Escherichia coli strain. Short-term adaptive laboratory evolution enhanced biomass yield and accelerated growth of formatotrophic E. coli to 3.3 g-CDW/mol-formate and 6 h doubling time, respectively. Genome sequence analysis revealed that manipulation of acetate metabolism is the reason for better growth performance, verified by subsequent reverse engineering of the parental E. coli strain. Moreover, the improved strain is capable of growing to an OD600 of 22 in bioreactor fed-batch experiments, highlighting its potential use for industrial bioprocesses. Finally, demonstrating the strain’s potential to support a sustainable, formate-based bioeconomy, lactate production from formate was engineered. The optimized strain generated 1.2 mM lactate —10% of the theoretical maximum— providing the first proof-of-concept application of the reductive glycine pathway for bioproduction

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. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain

Identification of a novel WRKY transcription factor binding site in bioinformatically identified pathogen-responsive cis-elements from Arabidopsis thaliana

Pflanzen sind einer Vielzahl von Phytopathogenen ausgesetzt und besitzen komplexe Resistenz-und Abwehrmechanismen. Ein wichtiger Bestandteil der Pathogenabwehr ist die massive transkriptionelle Reprogrammierung. Dafür sind unter anderem Transkriptionsfaktorbindungsstellen (TFBS) in den Promotoren der Zielgene unverzichtbar. Bioinformatische Analysen haben in der Vergangenheit zur Identifikation neuer potentieller TFBS in Pathogen-responsiven Promotoren aus A. thaliana geführt. Viele dieser cis-Sequenzen können als synthetische Promotoren in Petersilie-Protoplasten eine Responsivität auf das MAMP (microbe-associated molecular pattern) Pep25 vermitteln. Zwei Sequenzen aus einer Gruppe Pep25-responsiver Sequenzen mit einer hoch konservierten [T/G]ACTTTT-Kernsequenz wurden in der vorliegenden Arbeit näher analysiert. Es konnte gezeigt werden, dass in beiden Sequenzen die Kernsequenz und weitere benachbarte Nukleotide unverzichtbar für die Pep25-Responsivität sind. Untersuchungen an transgenen A. thaliana-Linien belegen, dass beide Sequenzen eine Responsivität auf Infektion mit Botrytis cinerea vermitteln. Yeast One-Hybrid-Screenings und Transaktivierungsassays in Protoplasten führten zur Identifizierung von WRKY70 als Aktivator der untersuchten Sequenzen. WRKY70 kann zwar die beiden hier untersuchten cis-Sequenzen transaktivieren, eine TFBS und die direkte in vitro Interaktion konnte aber nur mit einer der Sequenzen gezeigt werden. Die identifizierte TFBS hat die für WRKY-Faktoren untypische Sequenz CGACTTTT. Mutationen innerhalb der TFBS führen zum Verlust der Pep25-Responsivität, der Transaktivierbarkeit und der Bindung durch WRYK70. Bindungsstudien mit weiteren eng verwandten Pep25-responsiven Sequenzen legen nahe, dass die Konsensussequenz der WRKY70-TFBS als YGACTTTT beschrieben werden kann. Bioinformatische Analysen zeigen, dass diese Sequenz in WRKY70-induzierten Genen angereichert ist und dass potentielle WRKY70-Zielgene in die Pathogenantwort involviert sind.Plants have evolved complex resistance mechanisms to facilitate disease resistance. A major part of these mechanisms is transcriptional induction of defense genes. This requires the presence of defined transcription factor binding sites (TFBS) in target gene promoters. Recently, bioinformatic analyses led to the identification of potential TFBS in pathogen-responsive promoters from Arabidopsis thaliana. When used as synthetic promoters in parsley protoplasts, many of these cis-regulatory elements confer responsiveness toward the microbe-associated molecular pattern (MAMP) Pep25. Two of these cis-elements, belonging to a group of Pep25-responsive sequences with a highly conserved [T/G]ACTTTT core, have been analyzed in the present work. Mutational analyses revealed that the conserved core in both sequences is indispensable for Pep25-responsivness. However, in both sequences nucleotides adjacent to the core sequence are also essential for induction by Pep25. Pathogen infections of transgenic A. thaliana demonstrate that both sequences mediate responsiveness toward infection with Botrytis cinerea. WRKY70 was identified as transcriptional activator for both sequences using yeast one-hybrid screening and trans-activation assays in protoplasts. Both sequences investigated in the present work are trans-activated by WRKY70. A direct in vitro interaction between WRKY70 and a TFBS within the bioinformatically identified sequence occurs only with one of the two sequences. The WRKY70 TFBS (CGACTTTT) identified in this work is atypical for WRKY TFs. Mutations within this TFBS abolish trans-activation and binding of WRKY70. In vitro binding studies with closely related Pep25-responsive sequences suggest a YGACTTTT consensus sequence for the WRKY70 TFBS: Bioinformatic analyses demonstrate enrichment of this consensus sequence in WRKY70-induced gene promoters and suggest a role in defense responses for potential WRKY70 target genes

Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs) and corresponding cis-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. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems

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. 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 one-hybrid 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.</p

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

<div><p>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. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included <i>ccd</i>B counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent <i>Escherichia coli</i>. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to <i>Nicotiana benthamiana</i> in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.</p></div