SEARCH · bioRxiv Science

Results for “Synthetic Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,117 records · Page 62Linked to original sources

bioRxiv2021

Tailored extracellular electron transfer pathways enhance the electroactivity of Escherichia coli

Extracellular electron transfer (EET) engineering in Escherichia coli holds great potential for bioremediation, energy and electrosynthesis applications fueled by readily available organic substrates. Due to its vast metabolic capabilities and availability of synthetic biology tools to adapt strains to specific applications, E. coli is of advantage over native exoelectrogens, but limited in electron transfer rates. We enhanced EET in engineered strains through systematic expression of electron transfer pathways differing in cytochrome composition, localization and origin. While a hybrid pathway harboring components of an E. coli nitrate reductase and the Mtr complex from the exoelectrogen Shewanella oneidensis MR-1 enhanced EET, the highest efficiency was achieved by implementing the complete Mtr pathway from S. oneidensis MR1 in E. coli. We show periplasmic electron shuttling through overexpression of a small tetraheme cytochrome to be central to the electroactivity of this strain, leading to enhanced degradation of the pollutant methyl orange and significantly increased electrical current to graphite electrodes.

bioengineering↗

bioRxiv2021

Regulating expression of mistranslating tRNAs by readthrough RNA polymerase II transcription

Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II) transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4 or TetR-VP16 activated promoters downstream of a mistranslating tRNASer variant that mis-incorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.

molecular biology↗

bioRxiv2021

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

Efficient, targeted integration of large DNA constructs represent a significant hurdle in genetic engineering for the development of mouse models of human disease and synthetic biology research. To address this, we developed a system for efficient and precise, targeted single-copy integration of large transgenes directly into the zygote using multiple mouse genetic backgrounds. Conventional approaches, such as random transgenesis, CRISPR/Cas9-mediated homology-directed repair (HDR), lentivirus-based insertion, or DNA transposases all have significant limitations. Our strategy uses in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) to efficiently generate precise single-copy integrations of transgenes. This is achieved using a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, pre-positioned in the Gt(ROSA)26Sor safe harbor locus. Successful RMCE is achieved in att carrier zygotes using donor DNA carrying cognate attachment sites flanking the desired donor transgene microinjected along with Bxb1-integrase mRNA. This approach routinely achieves perfect vector-free integration of donor constructs at efficiencies as high as 43% and has generated transgenic animals containing inserts up to [~]43kb. Furthermore, when coupled with a nanopore-based Cas9-targeted sequencing (nCATS) approach, complete verification of the precise insertion sequence can be achieved. As a proof-of-concept we describe the creation and characterization of C57BL/6J and NSG Krt18-ACE2 transgenic mouse models for SARS-CoV2 research with verified heterozygous N1 animals available for experimental use in [~]4 months. In addition, we created a diverse series of mouse backgrounds carrying a single att site version of the landing pad allele in C57BL/6J, NSG, B6(Cg)-Tyrc-2J/J, FVB/NJ, PWK/PhJ, 129S1/SvImJ, A/J, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, and DBA/2J for rapid transgene insertion. Combined, this system enables predictable, rapid creation of precisely targeted transgenic animals across multiple genetic backgrounds, simplifying characterization, speeding expansion and use.

genetics↗

bioRxiv2021

A yeast with muscle does not run faster: full humanization of the glycolytic pathway in Saccharomyces cerevisiae

While transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper the humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of both glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light novel, context-dependent responses. Remarkably, human hexokinase 1 and 2, but not 4, required mutations in their catalytic or allosteric sites for functionality in yeast, while hexokinase 3 was unable to complement its yeast ortholog. Comparison with human tissues cultures showed the preservation of turnover numbers of human glycolytic enzymes in yeast and human cell cultures. This demonstration of transplantation of an entire, essential pathway paves the way to the establishment of species, tissue and disease-specific metazoan models. One Sentence SummaryThis work demonstrates the successful humanization of an entire pathway in Saccharomyces cerevisiae and establishes an attractive strategy to study (human) glycolysis architecture and regulation. HighlightsO_LIThe successful humanization of the entire glycolytic pathway in yeast offers new microbial models for both fundamental and applied studies. C_LIO_LIBoth glycolytic and moonlighting functions and turnover numbers of glycolytic enzymes are highly conserved between yeast and human. C_LIO_LIFunctionality of human hexokinases 1 and 2 in yeast requires mutations in the catalytic or allosteric binding sites. C_LIO_LICombination of single gene and full transplantation with laboratory evolution reveals context-dependent activity and evolution of glycolytic enzymes. C_LI

systems biology↗

bioRxiv2021

AlphaDesign: A de novo protein design framework based on AlphaFold

De novo protein design is a longstanding fundamental goal of synthetic biology, but has been hindered by the difficulty in reliable prediction of accurate high-resolution protein structures from sequence. Recent advances in the accuracy of protein structure prediction methods, such as AlphaFold (AF), have facilitated proteome scale structural predictions of monomeric proteins. Here we develop AlphaDesign, a computational framework for de novo protein design that embeds AF as an oracle within an optimisable design process. Our framework enables rapid prediction of completely novel protein monomers starting from random sequences. These are shown to adopt a diverse array of folds within the known protein space. A recent and unexpected utility of AF to predict the structure of protein complexes, further allows our framework to design higher-order complexes. Subsequently a range of predictions are made for monomers, homodimers, heterodimers as well as higher-order homo-oligomers - trimers to hexamers. Our analyses also show potential for designing proteins that bind to a pre-specified target protein. Structural integrity of predicted structures is validated and confirmed by standard ab initio folding and structural analysis methods as well as more extensively by performing rigorous all-atom molecular dynamics simulations and analysing the corresponding structural flexibility, intramonomer and interfacial amino-acid contacts. These analyses demonstrate widespread maintenance of structural integrity and suggests that our framework allows for fairly accurate protein design. Strikingly, our approach also reveals the capacity of AF to predict proteins that switch conformation upon complex formation, such as involving switches from -helices to {beta}-sheets during amyloid filament formation. Correspondingly, when integrated into our design framework, our approach reveals de novo design of a subset of proteins that switch conformation between monomeric and oligomeric state.

bioinformatics↗

bioRxiv2021

Transfer of Knowledge from Model Organisms to Evolutionarily Distant Non-Model Organisms: The Coral Pocillopora damicornis Membrane Signaling Receptome

With the ease of gene sequencing and the technology available to study and manipulate non-model organisms, the need to translate our understanding of model organisms to non-model organisms has become an urgent problem. For example, mining of large coral and their symbiont sequence data is a challenge, but also provides an opportunity for understanding functionality and evolution of these and other non-model organisms. Much more information than for any other eukaryotic species is available for humans, especially related to signal transduction and diseases. However, the coral cnidarian host and human have diverged over 700 million years ago and homologies between proteins are therefore often in the gray zone or undetectable with traditional BLAST searches. We introduce a two-stage approach to identifying putative coral homologues of human proteins. First, through remote homology detection using Hidden Markov Models, we identify candidate human homologues in the cnidarian genome. However, for many proteins, the human genome alone contains multiple family members with similar or even more divergence in sequence. In the second stage, therefore, we filter the remote homology results based on the functional and structural plausibility of each coral candidate, shortlisting the coral proteins likely to be true human homologues. We demonstrate our approach with a pipeline for mapping membrane receptors in humans to membrane receptors in corals, with specific focus on the stony coral, P. damicornis. More than 1000 human membrane receptors mapped to 335 coral receptors, including 151 G protein coupled receptors (GPCRs). To validate specific sub-families, we chose opsin proteins, representative GPCRs that confer light sensitivity, and Toll-like receptors, representative non-GPCRs, which function in the immune response, and their ability to communicate with microorganisms. Through detailed structure-function analysis of their ligand-binding pockets and downstream signaling cascades, we selected those candidate remote homologues likely to carry out related functions in the corals. This pipeline may prove generally useful for other non-model organisms, such as to support the growing field of synthetic biology.

bioinformatics↗

bioRxiv2021

A simple and general approach to control the activity of DNA processing enzymes

DNA processing enzymes, such as DNA polymerases and endonucleases, have found many applications in biotechnology, molecular diagnostics, and synthetic biology, among others. The development of enzymes with controllable activity, such as hot-start or light-activatable versions, has boosted their applications and improved the sensitivity and specificity of the existing ones. However, current approaches to produce controllable enzymes are experimentally demanding to develop and case specific. Here, we introduce a simple and general method to design light-start DNA processing enzymes. In order to prove its versatility, we applied our method to three DNA polymerases commonly used in biotechnology, including the Phi29 (mesophilic), Taq and Pfu polymerases, and one restriction enzyme. Light-start enzymes showed suppressed polymerase, exonuclease and endonuclease activity until they were re-activated by an UV pulse. Finally, we applied our enzymes to common molecular biology assays, and showed comparable performance to commercial hot-start enzymes.

molecular biology↗

bioRxiv2021

Mitochondria transplantation between living cells

Mitochondria and the complex endomembrane system are hallmarks of eukaryotic cells. To date, it has been difficult to manipulate organelle structures within single live cells. We developed a FluidFM-based approach to extract, inject and transplant organelles from and into living cells with subcellular spatial resolution. The approach enabled the transfer of controlled quantities of mitochondria into cells while maintaining their viability and monitoring their fate in new host cells. Transplantation of healthy and drug-impaired mitochondria into primary keratinocytes allowed real-time tracking of mitochondrial subpopulation rescue. Fusion with the mitochondrial network of recipient cells occurred 20 min after transplantation and continued for over 16 hours. After transfer of mitochondria and cell propagation over generations, we show that donor mtDNA was replicated in recipient cells without the need for selection pressure. The approach opens new prospects for the study of organelle physiology and homeostasis, but also for mechanobiology, synthetic biology, and therapy.

cell biology↗

bioRxiv2021

Long-lasting and responsive DNA/enzyme-based programs in serum-supplemented extracellular media

DNA molecular programs are emerging as promising pharmaceutical approaches due to their versatility for biomolecular sensing and actuation. However, the implementation of DNA programs has been mainly limited to serum-deprived in vitro assays due to the fast deterioration of the DNA reaction networks by the nucleases present in the serum. Here, we show that DNA/enzyme programs are functional in serum for 24h but are latter disrupted by nucleases that give rise to parasitic amplification. To overcome this, we implement 3-letter code networks that suppress autocatalytic parasites while still conserving the functionality of DNA/enzyme programs for at least 3 days in the presence of 10% serum. In addition, we define a new buffer that further increases the biocompatibility and conserves responsiveness to changes in molecular composition across time. Finally, we demonstrate how serum-supplemented extracellular DNA molecular programs remain responsive to molecular inputs in the presence of living cells, having responses 6-fold faster than cellular division rate and are sustainable for at least 3 cellular divisions. This demonstrates the possibility of implementing in situ biomolecular characterization tools for serum-demanding in vitro models. We foresee that the coupling of chemical reactivity to our DNA programs by aptamers or oligonucleotide conjugations will allow the implementation of extracellular synthetic biology tools, which will offer new biomolecular pharmaceutical approaches and the emergence of complex and autonomous in vitro models.

biophysics↗

bioRxiv2021

Optogenetic operated probiotics to regulate host metabolism by mimicking enteroendocrine

The enteroendocrine system plays an important role in metabolism. The gut microbiome regulates enteroendocrine in an extensive way, arousing attention in biomedicine. However, conventional strategies of enteroendocrine regulation via gut microbiome are usually non-specific or imprecise. Here, an optogenetic operated probiotics system was developed combining synthetic biology and flexible electronics to achieve in situ controllable secretion to mimic enteroendocrine. Firstly, optogenetic engineered Lactococcus lactis (L. lactis) were administrated in the intestinal tract. A wearable optogenetic device was designed to control optical signals remotely. Then, L. lactis could secrete enteroendocrine hormone according to optical signals. As an example, optogenetic L. lactis could secrete glucagon-like peptide-1(GLP-1) under the control of the wearable optogenetic device. To improve the half-life of GLP-1 in vivo, the Fc domain from immunoglobulin was fused. Treated with this strategy, blood glucose, weight and other features were relatively well controlled in rats and mice models. Furthermore, up-conversion microcapsules were introduced to increase the excitation wavelength of the optogenetic system for better penetrability. This strategy has biomedical potential in metabolic diseases therapy by mimicking enteroendocrine.

bioengineering↗

bioRxiv2021

Engineering RNA viruses with unnatural amino acid to evoke adjustable immune response in mice

Ribonucleic acid (RNA) viruses pose heavy burdens on public-health systems. Synthetic biology holds great potential for artificially controlling their replication, a strategy that could be used to attenuate infectious viruses but is still in the exploratory stage. Herein, we used the genetic-code expansion technique to convert Enterovirus 71 (EV71), a model of RNA virus, into a controllable EV71 strain carrying the unnatural amino acid (UAA) N{varepsilon}-2-azidoethyloxycarbonyl-L-lysine (NAEK), which we termed an EV71-NAEK virus. EV71-NAEK could recapitulate an authentic NAEK time- and dose-dependent infection in vitro and in vivo, which could serve as a novel method to manipulate virulent viruses in conventional laboratories. We further validated the prophylactic effect of EV71-NAEK in two mouse models. In susceptible parent mice, vaccination with EV71-NAEK elicited a strong immune response and potentially protected their neonatal offspring from lethal challenge similar to that of commercial vaccines. Meanwhile, in transgenic mice harboring a PylRS-tRNAPyl pair, substantial elements of genetic-code expansion technology, EV71-NAEK evoked an adjustable neutralizing-antibody response in a strictly external NAEK dose-dependent manner. These findings suggested that EV71-NAEK could be the basis of a feasible immunization program for populations with different levels of immunity. Moreover, we expanded the strategy to generate controllable coxsackieviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for conceptual verification. In combination, these results could underlie a competent strategy for attenuating viruses and priming the immune system via artificial control, which might be a promising direction for the development of amenable vaccine candidates and be broadly applied to other RNA viruses.

bioengineering↗

bioRxiv2021

STRAIGHT-IN: A platform for high-throughput targeting of large DNA payloads into human pluripotent stem cells

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR/Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation) by: (i) inserting various combinations of fluorescent reporters into hiPSCs to assess excitation-contraction coupling cascade in derivative cardiomyocytes, and; (ii) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorder into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically-matched panels of hiPSC lines efficiently and cost-effectively.

cell biology↗

bioRxiv2021

Nitrogen metabolism in Pseudomonas putida: functional analysis using random barcode transposon sequencing

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop- out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCEUnderstanding the genetic basis of P. putidas diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators--which are of particular interest for metabolic engineering.

microbiology↗

bioRxiv2022

Circuit-seq: Circular reconstruction of cut in vitro transposed plasmids using Nanopore sequencing

Recombinant DNA is a fundamental tool in biotechnology and medicine. Validation of the resulting plasmid sequence is a critical and time-consuming step, which has been dominated for the last 35 years by Sanger sequencing. As plasmid sequences grow more complex with new DNA synthesis and cloning techniques, we need new approaches that address the corresponding validation challenges at scale. Here we prototype a high-throughput plasmid sequencing approach using DNA transposition and Oxford Nanopore sequencing. Our method, Circuit-seq, creates robust, full-length, and accurate plasmid assemblies without prior knowledge of the underlying sequence for approximately $1.50 per plasmid. We demonstrate the power of Circuit-seq across a wide range of plasmid sizes and complexities, generating accurate and contiguous plasmid maps. We then leverage our long read-data to characterize epigenetic marks and estimate plasmid contamination levels. Circuit-seq scales to large numbers of samples at a lower cost than commercial Sanger sequencing, accelerating a key step in synthetic biology, with low startup costs make it practical for individual laboratories.

molecular biology↗

bioRxiv2022

DisGUVery: a versatile open-source software for high-throughput image analysis of Giant Unilamellar Vesicles

Giant Unilamellar Vesicles (GUVs) are cell-sized aqueous compartments enclosed by a phospholipid bilayer. Due to their cell-mimicking properties, GUVs have become a widespread experimental tool in synthetic biology to study membrane properties and cellular processes. In stark contrast to the experimental progress, quantitative analysis of GUV microscopy images has received much less attention. Currently, most analysis is performed either manually or with custom-made scripts, which makes analysis time-consuming and results difficult to compare across studies. To make quantitative GUV analysis accessible and fast, we present DisGUVery, an open-source, versatile software that encapsulates multiple algorithms for automated detection and analysis of GUVs in microscopy images. With a performance analysis, we demonstrate that DisGUVerys three vesicle detection modules successfully identify GUVs in images obtained with a wide range of imaging sources, in various typical GUV experiments. Multiple pre-defined analysis modules allow the user to extract properties such as membrane fluorescence, vesicle shape and internal fluorescence from large populations. A new membrane segmentation algorithm facilitates spatial fluorescence analysis of non-spherical vesicles. Altogether, DisGUVery provides an accessible tool to enable high-throughput automated analysis of GUVs, and thereby to promote quantitative data analysis in GUV research.

biophysics↗

bioRxiv2022

Imaging minimal bacteria at the nanoscale: a reliable and versatile process to perform Single Molecule Localization Microscopy in mycoplasmas

Mycoplasmas are the smallest free-living organisms. These bacteria are important models for both fundamental and Synthetic Biology, owing to their highly reduced genomes. They are also relevant in the medical and veterinary fields, as they are pathogenic of both humans and most livestock species. Mycoplasma cells have minute sizes, often in the 300-800 nanometers range. As these dimensions are close to the diffraction limit of visible light, fluorescence imaging in mycoplasmas is often poorly informative. Recently developed "Super-Resolution Imaging" techniques can break this diffraction limit, improving the imaging resolution by an order of magnitude and offering a new nanoscale vision of the organization of these bacteria. These techniques have however not been applied to mycoplasmas before. Here, we describe an efficient and reliable protocol to perform Single-Molecule Localization Microscopy (SMLM) imaging in mycoplasmas. We provide a polyvalent transposon-based system to express the photo-convertible fluorescent protein mEos3.2, enabling Photo-Activated Localization Microscopy (PALM) in most Mycoplasma species. We also describe the application of direct STochastic Optical Reconstruction Microscopy (dSTORM). We showcase the potential of these techniques by studying the subcellular localization of two proteins of interest. Our work highlights the benefits of state-of-the-art microscopy techniques for mycoplasmology and provides an incentive to further the development SMLM strategies to study these organisms in the future. ImportanceMycoplasmas are important models in biology, as well as highly problematic pathogens in the medical and veterinary fields. The very small size of these bacteria, well below the micron, limits the usefulness of traditional fluorescence imaging methods as their resolution limit is similar to the dimensions of the cells. Here, to bypass this issue, we established a set of state-of-the-art "Super-Resolution Microscopy" techniques in a wide range of Mycoplasma species. We describe two strategies: PALM, based on the expression of a specific photo-convertible fluorescent protein; and dSTORM, based on fluorophore-coupled antibody labeling. With these methods, we successfully performed single-molecule imaging of proteins of interest at the surface of the cells and in the cytoplasm, at lateral resolutions well below 50 nanometers. Our work paves the way toward a better understanding of mycoplasmas biology through imaging of subcellular structures at the nanometer scale.

microbiology↗

bioRxiv2022

Data-driven dynamical modelling of a pathogen-infected plant gene regulatory network: a comparative analysis

Recent advances in synthetic biology have enabled the design of genetic feedback control circuits that could be implemented to build resilient plants against pathogen attacks. To facilitate the proper design of these genetic feedback control circuits, an accurate model that is able to capture the vital dynamical behaviour of the pathogen-infected plant is required. In this study, using a data-driven modelling approach, we develop and compare four dynamical models (i.e. linear, Michaelis-Menten, standard S-System and extended S-System) of a pathogen-infected plant gene regulatory network (GRN). These models are then assessed across several criteria, i.e. ease of identifying the type of gene regulation, the predictive capability, Akaike Information Criterion (AIC) and the robustness to parameter uncertainty to determine its viability of modelling the pathogen-infected plant GRN. Using our defined ranking score, our analyses show that while the extended S-System model ranks highest in the overall comparison, the performance of the linear model is more consistent throughout the comparison, making it the preferred model for this pathogen-infected plant GRN.

systems biology↗

bioRxiv2022

Enhanced photosynthetic efficiency for increased carbon assimilation and woody biomass production in hybrid poplar INRA 717-1B4

Increasing CO2 levels in the atmosphere and the resulting negative impacts on climate change have compelled global efforts to achieve carbon neutrality or negativity. Most such efforts focus on carbon sequestration through chemical or physical approaches. We aim to harness the power of synthetic biology to enhance plants natural ability to draw down and sequester carbon, thereby positively affecting climate change. Past decades of scientific progress have shed light on strategies to overcome the intrinsic limitations of carbon drawdown and fixation through photosynthesis, particularly in row crops in hopes of improving agricultural productivity for food security. Incorporating a photorespiration bypass in C3 plants has shown promising results of increased biomass and grain yield. Despite their globally dominant role in atmospheric carbon flux, the drawdown rates of most trees are currently limited by their C3 photosynthetic metabolism, and efforts to improve the photosynthetic capacity of trees, such as by reducing energy loss in photorespiration, are currently lacking. Here, we selected a photorespiration bypass pathway and tested its effectiveness on photosynthetic enhancement in hybrid poplar INRA717-IB4. The design includes a RNAi strategy to reduce the transportation of the photorespiration byproduct, glycolate, out of chloroplast and a shunt pathway to metabolize the retained glycolate back to CO2 for fixation through the Calvin-Benson cycle. Molecular and physiological data collected from two repeated growth experiments indicates that transgenic plants expressing genes in the photorespiration bypass pathway have increased photosynthetic efficiency, leading to faster plant growth and elevated biomass production. One lead transgenic event accumulated 53% more above-ground dry biomass over a five month growth period in a controlled environment. Pilot projects with photosynthesis-enhanced trees in the field are in progress. Our results provide a proof-of-concept for engineering trees to help combat climate change.

plant biology↗