Biology subjects

TerAvest, M. A.

Publications and source records attributed to TerAvest, M. A..

14 recordsLinked to original sources

bioRxiv2026

OxyR regulates the oxidative stress response in Zymomonas mobilis during oxic growth and anoxic biofuel fermentation

The bacterium Zymomonas mobilis is widely studied for its potential as an industrial biofuel producer. Anoxic fermentation by Z. mobilis in lignocellulosic hydrolysate can generate bioethanol from renewable plant biomass. In this study, we deleted a gene from the Z. mobilis genome encoding a homolog of OxyR, a transcription factor that activates an oxidative stress response in bacteria to reduce reactive oxygen species (ROS). Deletion of this transcription factor inhibited growth of Z. mobilis in oxic, but not anoxic, conditions in laboratory media. A ROS probe revealed that the oxyR response is required to reduce intracellular ROS during oxic growth. Importantly for biofuel production, the absence of oxyR inhibited growth and delayed ethanol production during anoxic hydrolysate fermentation. To determine the source of oxidative stress in hydrolysate, we grew{Delta} oxyR in a synthetic hydrolysate containing known inhibitors found in hydrolysate. There was no growth defect in{Delta} oxyR in the synthetic hydrolysate, indicating that known inhibitory compounds are not the source of anoxic oxidative stress. We determined that Ammonia-Fiber Expansion (AFEX) switchgrass hydrolysate contains significant peroxide concentrations. Addition of catalase to hydrolysate improves growth of both{Delta} oxyR and wild-type Z. mobilis in hydrolysate. This study uncovers an important source of stress to Z. mobilis during biofuel fermentation. ImportanceFermentation of non-food biomass is a promising avenue for sustainable production of fuels and chemicals, but several challenges currently limit applicability of this technology. One major hurdle is that when biomass is deconstructed into a fermentable form, many byproducts are generated that inhibit microbial fermentation. Here, we investigated how a fermentative bacterium, Zymomonas mobilis, experiences oxidative stress during anoxic biomass fermentation, and identified genes important in this response. These findings provide a better understanding of the stresses faced by Z. mobilis during biofuel production. Fully understanding the effects of hydrolysate on biofuel-producing microbes is crucial for optimizing production and making carbon-neutral fuel a reality.

microbiology↗

bioRxiv2026

Multi-lab, Multi-enzyme Study Demonstrates the Versatility of Bacterial Microcompartment Shells as a Modular Platform for Confined Biocatalysis

Bacterial microcompartments (BMCs) are proteinaceous organelles that spatially organize metabolic reactions in bacteria and represent an attractive scaffold for pathway engineering. Here, we present a proof-of-concept in vitro study demonstrating a simple, scalable, and modular BMC shell-based platform for enzyme encapsulation using the SpyCatcher-SpyTag (SC-ST) covalent conjugation system. To evaluate the generality of this approach, 16 dehydrogenases were selected, of which 13 were successfully expressed and purified as SC-tagged enzymes in E. coli by five research groups working in parallel. Twelve of these efficiently conjugated to ST-fused BMC-T1 proteins, and addition of urea-solubilized BMC-H triggered rapid self-assembly of HT1 shells, resulting in successful encapsulation of all conjugated enzymes. The only enzyme lacking detectable activity after encapsulation was also inactive in its free SC-fused form, indicating that encapsulation retained enzymatic activity for all tested enzymes. Encapsulation modulated enzymatic activity and kinetic parameters in an enzyme-dependent manner, likely arising from variations in catalytic mechanism, structural flexibility affected by immobilization, and sensitivity to the local microenvironment created by encapsulation. Functional characterization of a subset of encapsulated enzymes revealed enhanced thermal stability up to [~]50 {degrees}C and improved storage stability relative to free SC-fused enzymes. Enzyme-loaded shells could be lyophilized and reconstituted without loss of structural integrity or activity. Finally, we demonstrate co-encapsulation of two enzymes within a single shell and their cooperative function through cofactor recycling. Together, these results establish engineered BMCs as a robust and modular platform for organizing multi-enzyme pathways, enabling rapid assembly, stabilization, and functional integration of enzymes for diverse metabolic engineering applications. HighlightsA single strategy enables encapsulation of 12 diverse dehydrogenases in BMCs. SpyCatcher-SpyTag interactions drive rapid enzyme assembly in BMCs. Encapsulated enzymes are active and show improved thermal stability. The platform enables scalable construction of synthetic metabolic modules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712704v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d34f8dorg.highwire.dtl.DTLVardef@10bba9corg.highwire.dtl.DTLVardef@10868b7org.highwire.dtl.DTLVardef@112cb03_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

Encapsulation in a bacterial microcompartment shell improves thermal stability of a glycolytic enzyme

Selective encapsulation of target enzymes is an increasingly well studied field with a host of potential applications for biotechnology. Natively, many bacteria utilize bacterial microcompartments (BMCs) for enzyme encapsulation to enhance catalysis. BMCs are protein shells that enable selective localization of targeted metabolic enzymes and may improve catalytic rates by co-localizing pathway enzymes and/or serve to sequester toxic or volatile intermediates. The microcompartment shell of Haliangium ochraceum (HO) is a notable BMC chassis because of its modularity and versatility; it is easily expressed and assembled outside its native host and can accept a wide array of cargo. Recently, it was demonstrated that assembly of HO BMC shells can be easily achieved in vitro. Following up on our previous work on in vivo assembly of HO-BMCs with triose phosphate isomerase (TPI) as model enzyme cargo, here we have demonstrated the advantages of in vitro assembly (IVA) for targeted enzyme encapsulation. We achieved variable loading of BMC shells with targeted amounts of TPI and demonstrated enhanced thermal stability of encapsulated TPI versus free TPI up to 62{degrees}C.

synthetic biology↗

bioRxiv2024

Structure Characterization of Bacterial Microcompartment Shells via X-ray Scattering and Coordinate Modeling: Evidence for adventitious capture of cytoplasmic proteins

Bacterial microcompartments (BMCs) are self-assembling, protein shell structures that are widely investigated across a broad range of biological and abiotic chemistry applications. A central challenge in BMC research is the targeted capture of enzymes during shell assembly. While crystallography and cryo-EM techniques have been successful in determining BMC shell structures, there has been only limited success in visualizing the location of BMC-captured enzyme cargo. Here, we demonstrate the opportunity to use small angle X-ray scattering (SAXS) and pair density distribution function (PDDF) measurements combined with quantitative comparison to coordinate structure models as an approach to characterize BMC shell structures in solution conditions directly relevant to biochemical function. Using this approach, we analyzed BMC shells from Haliangium ochraceum that were isolated following expression in E. coli. The analysis allowed BMC shell structures and the extent of encapsulated enzyme cargo to be identified. Notably, the results demonstrate that HO-BMC shells adventitiously capture significant amounts of cytoplasmic cargo during assembly in E. coli. Our findings highlight the utility of SAXS/PDDF analysis for evaluating BMC architectures and enzyme encapsulation, offering valuable insights for designing BMC shells as platforms for biological and abiotic catalyst capture within confined environments.

biophysics↗

bioRxiv2024

Controlled enzyme cargo loading in engineered bacterial microcompartment shells

Bacterial microcompartments (BMCs) are nanometer-scale organelles with a protein-based shell that serve to co-localize and encapsulate metabolic enzymes. They may provide a range of benefits to improve pathway catalysis, including substrate channeling and selective permeability. Several groups are working toward using BMC shells as a platform for enhancing engineered metabolic pathways. The microcompartment shell of Haliangium ochraceum (HO) has emerged as a versatile and modular shell system that can be expressed and assembled outside its native host and with non-native cargo. Further, the HO shell has been modified to use the engineered protein conjugation system SpyCatcher-SpyTag for non-native cargo loading. Here, we used a model enzyme, triose phosphate isomerase (Tpi), to study non-native cargo loading into four HO shell variants and begin to understand maximal shell loading levels. We also measured activity of Tpi encapsulated in the HO shell variants and found that activity was determined by the amount of cargo loaded and was not strongly impacted by the predicted permeability of the shell variant to large molecules. All shell variants tested could be used to generate active, Tpi-loaded versions, but the simplest variants assembled most robustly. We propose that the simple variant is the most promising for continued development as a metabolic engineering platform.

biochemistry↗

bioRxiv2024

Electrochemical cofactor recycling of bacterial microcompartments

Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B12) reductase and, curiously, found it in many unrelated BMC types that do not employ B12 cofactors. We propose NAD+ regeneration as a new function of this enzyme and name it MNdh, for Metabolosome NADH dehydrogenase. Its partner shell protein BMC-TSE assists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, EPR spectroscopy, protein voltammetry and structural modeling verified with X-ray footprinting. This discovery represents a new paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.

microbiology↗

bioRxiv2024

A small number of point mutations confer formate tolerance in Shewanella oneidensis

Microbial electrosynthesis (MES) is a sustainable approach to chemical production from CO2 and clean electricity. However, limitations in electron transfer efficiency and gaps in understanding of electron transfer pathways in MES systems prevent full realization of this technology. Shewanella oneidensis could serve as an MES biocatalyst because it has a well-studied, efficient transmembrane electron transfer pathway. A key first step in MES in this organism could be CO2 reduction to formate. However, wild-type S. oneidensis does not tolerate high levels of formate. In this work, we created and characterized formate-tolerant strains of S. oneidensis for further engineering and future use in MES systems through adaptive laboratory evolution. Two different point mutations in a sodium-dependent bicarbonate transporter and a DUF2721-contianing protein separately confer formate tolerance to S. oneidensis. The mutations were further evaluated to understand their role in improving formate tolerance. We also show that the wild-type and mutant versions of the S. oneidensis sodium-dependent bicarbonate transporter improves formate tolerance of Zymomonas mobilis, indicating the potential for the transfer of this formate tolerance phenotype to other organisms. ImportanceShewanella oneidensis is a bacterium with a well-studied, efficient extracellular electron transfer pathway. This capability could make this organism a suitable host for microbial electrosynthesis using CO2 or formate feedstocks. However, formate is toxic to S. oneidensis, limiting the potential for its use in these systems. In this work, we evolve several strains of S. oneidensis that have improved formate tolerance and we investigate some mutations that confer this phenotype. The phenotype is confirmed to be attributed to several single point mutations by transferring the wild- type and mutant versions of each gene to the unmutated strain. Finally, the formate tolerance mechanism of one mutation is studied using structural modeling and expression in another host. This study therefore presents a simple method for conferring formate tolerance to bacterial hosts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/601276v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@111c7a7org.highwire.dtl.DTLVardef@4acc78org.highwire.dtl.DTLVardef@b60062org.highwire.dtl.DTLVardef@d35dca_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

The Electron Transport Chain of Shewanella oneidensis MR-1 can Operate Bidirectionally to Enable Microbial Electrosynthesis

Extracellular electron transfer (EET) is a process by which bacterial cells can exchange electrons with a redox active material located outside of the cell. In Shewanella oneidensis, this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive microbial electrosynthesis of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (Bdh). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via Bdh. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and O2 reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical systems (BES), and the translocation of protons across the membrane during O2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death. ImportanceMicrobial electrosynthesis is increasingly employed for the generation of specialty chemicals such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of bioelectrochemical systems design.

biochemistry↗

bioRxiv2023

The Genetics of Aerotolerant Growth in a Naturally Reduced Genome Alphaproteobacterium

Reduced genome bacteria are genetically simplified systems that facilitate biological study and industrial use. The free-living Alphaproteobacterium, Zymomonas mobilis, has a naturally reduced genome containing fewer than 2000 protein coding genes. Despite its small genome, Z. mobilis thrives in diverse conditions including the presence or absence of atmospheric oxygen. However, insufficient characterization of essential and conditionally essential genes has limited broader adoption of Z. mobilis as a model Alphaproteobacterium. Here, we use genome-scale CRISPRi-seq to systematically identify and characterize Z. mobilis genes that are conditionally essential for aerotolerant or anaerobic growth, or are generally essential across both conditions. Comparative genomics revealed that the essentiality of most "generally essential" genes was shared between Z. mobilis and other Alphaproteobacteria, validating Z. mobilis as reduced genome model. Among conditionally essential genes, we found that the DNA repair gene, recJ, was critical only for aerobic growth but reduced the mutation rate under both conditions. Further, we show that genes encoding the F1FO ATP synthase and Rnf respiratory complex are required for anaerobic growth of Z. mobilis. Combining CRISPRi partial knockdowns with metabolomics and membrane potential measurements, we determined that the ATP synthase generates membrane potential that is consumed by Rnf to power downstream processes. Rnf knockdown strains accumulated isoprenoid biosynthesis intermediates, suggesting a key role for Rnf in powering essential biosynthetic reactions. Our work establishes Z. mobilis as a streamlined model for alphaproteobacterial genetics, has broad implications in bacterial energy coupling, and informs Z. mobilis genome manipulation for optimized production of valuable isoprenoid-based bioproducts. ImportanceThe inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the Alphaproteobacterium, Zymomonas mobilis, have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts.

genetics↗

bioRxiv2023

Respiration is essential for aerobic growth of Zymomonas mobilis ZM4

Zymomonas mobilis is an alpha-proteobacterium that is a promising platform for industrial scale production of biofuels or valuable products due to its efficient ethanol fermentation and low biomass generation. Z. mobilis has also intriguing physiology, sometimes difficult to explain by the rules and strategies commonly observed in other bacteria. One of the most mysterious features of Z. mobilis is its growth in oxic conditions. Z. mobilis is an aerotolerant bacterium that encodes a complete respiratory electron transport chain but the benefit of respiration for growth in oxic conditions has never been confirmed, despite decades of research. Quite the opposite, growth and ethanol production of WT Z. mobilis is poor in oxic conditions indicating that it does not benefit from oxidative phosphorylation. Additionally, in previous studies, aerobic growth improved significantly when respiratory genes were disrupted (ndh) or acquired point mutations (cydA, cydB) even if respiration was significantly reduced by these changes. Here, we obtained clean deletions of respiratory genes ndh and cydAB, individually and in combination, and showed, for the first time, that deletion of cydAB completely inhibited O2 respiration and dramatically reduced growth in oxic conditions. Both respiration and aerobic growth were restored by expressing a heterologous, water-forming NADH oxidase, noxE. This result shows that the main role of the electron transport chain in Z. mobilis is reducing the toxicity of molecular oxygen, helping to explain why it is beneficial for Z. mobilis to use electron transport chain complexes that contribute little to oxidative phosphorylation. ImportanceA key to producing next generation biofuels is to engineer microbes that efficiently convert non-food materials into drop-in fuels and to engineer microbes effectively we must understand their metabolism thoroughly. Zymomonas mobilis is a bacterium that is a promising candidate biofuel producer but its metabolism remains poorly understood, especially its metabolism when exposed to oxygen. Although Z. mobilis respires with oxygen, its aerobic growth is poor and disruption of genes related to respiration counterintuitively improves aerobic growth. This unusual result has sparked decades of research and debate regarding the function of respiration in Z. mobilis. Here, we used a new set of mutants to determine that respiration is essential for aerobic growth and likely protects the cells from oxidative damage caused by molecular oxygen. These results indicate that respiration has a non-canonical function in Z. mobilis and expand our understanding of the role of respiration in metabolism and oxidative stress.

microbiology↗

bioRxiv2021

Zymomonas mobilis ZM4 utilizes an acetaldehyde dehydrogenase to produce acetate

Zymomonas mobilis is a promising bacterial host for biofuel production but further improvement has been hindered because some aspects of its metabolism remain poorly understood. For example, one of the main byproducts generated by Z. mobilis is acetate but the pathway for acetate production is unknown. Acetaldehyde oxidation has been proposed as the major source of acetate and an acetaldehyde dehydrogenase was previously isolated from Z. mobilis via activity guided fractionation, but the corresponding gene has never been identified. We determined that the locus ZMO1754 (also known as ZMO_RS07890) encodes an NADP+-dependent acetaldehyde dehydrogenase that is responsible for acetate production by Z. mobilis. Deletion of this gene from the chromosome resulted in a growth defect in oxic conditions, suggesting that acetaldehyde detoxification is an important role of acetaldehyde dehydrogenase. The deletion strain also exhibited a near complete abolition of acetate production, both in typical laboratory conditions and during lignocellulosic hydrolysate fermentation. Our results show that ZMO1754 encodes the major acetaldehyde dehydrogenase in Z. mobilis and we therefore rename the gene aldB based on functional similarity to the Escherichia coli acetaldehyde dehydrogenase. ImportanceBiofuel production from non-food crops is an important strategy for reducing carbon emissions from the transportation industry but it has not yet become commercially viable. An important avenue to improve biofuel production is to enhance the characteristics of fermentation organisms by genetic engineering. To make genetic modifications successful, we must gain sufficient information about the genome and metabolism of the organism to enable rational design and engineering. Here, we improved understanding of Zymomonas mobilis, a promising biofuel producing bacterium, by identifying a metabolic pathway and associated gene that lead to byproduct formation. This information may be used in the future for genetic engineering to reduce byproduct formation during biofuel production.

microbiology↗

bioRxiv2019

NADH dehydrogenases contribute to extracellular electron transfer by Shewanella oneidensis MR-1 in bioelectrochemical systems

Shewanella oneidensis MR-1 is quickly becoming a synthetic biology workhorse for bioelectrochemical technologies due to a high level of understanding of its interaction with electrodes. Transmembrane electron transfer via the Mtr pathway has been well characterized, however, the role of NADH dehydrogenases in feeding electrons to Mtr has been only minimally studied in S. oneidensis MR-1. Four NADH dehydrogenases are encoded in the genome, suggesting significant metabolic flexibility in oxidizing NADH under a variety of conditions. Strains containing in-frame deletions of each of these dehydrogenases were grown in anodic bioelectrochemical systems with N-acetylglucosamine or D,L-lactate as the carbon source to determine impact on extracellular electron transfer. A strain lacking the two dehydrogenases essential for aerobic growth exhibited a severe growth defect with an anode (+0.4 VSHE) or Fe(III)-NTA as the terminal electron acceptor. Our study reveals that the same NADH dehydrogenase complexes are utilized under oxic conditions or with a high potential anode. Understanding the role of NADH in extracellular electron transfer may help improve biosensors and give insight into other applications for bioelectrochemical systems.\n\nTOC Graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC=\"FIGDIR/small/657668v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@163db4org.highwire.dtl.DTLVardef@2cf477org.highwire.dtl.DTLVardef@165cb63org.highwire.dtl.DTLVardef@c0bc5_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2018

Reversing an extracellular electron transfer pathway for electrode-driven NADH generation

Microbial electrosynthesis is an emerging technology with the potential to simultaneously store renewably generated energy, fix carbon dioxide, and produce high-value organic compounds. However, limited understanding of the route of electrons into the cell remains an obstacle to developing a robust microbial electrosynthesis platform. To address this challenge, we engineered an inward electron transfer pathway in Shewanella oneidensis MR-1. The pathway uses native Mtr proteins to transfer electrons from an electrode to the inner membrane quinone pool. Subsequently, electrons are transferred from quinones to NAD+ by native NADH dehydrogenases. This reverse functioning of NADH dehydrogenases is thermodynamically unfavorable, therefore we have added a light-driven proton pump (proteorhodopsin) to generate proton-motive force to drive this activity. Finally, we use reduction of acetoin to 2,3-butanediol via a heterologous butanediol dehydrogenase (Bdh) as an electron sink. Bdh is an NADH-dependent enzyme, therefore, observation of acetoin reduction supports our hypothesis that cathodic electrons are transferred to intracellular NAD+. Multiple lines of evidence indicate proper functioning of the engineered electrosynthesis system: electron flux from the cathode is influenced by both light and acetoin availability; and 2,3-butanediol production is highest when both light and a poised electrode are present. Using a hydrogenase-deficient S. oneidensis background strain resulted in a stronger correlation between electron transfer and 2,3-butanediol production, suggesting that hydrogen production is an off-target electron sink in the wild-type background. This system represents a promising genetically engineered microbial electrosynthesis platform and will enable a new focus on synthesis of specific compounds using electrical energy.

synthetic biology↗

bioRxiv2018

Current production as a rapid response expression reporter under micro-oxic and anoxic conditions

Inducible gene expression is crucial for regulating cellular processes and production of compounds within cellular pathways. Yet, inducing gene expression is only the first step to utilizing cellular processes for an applied purpose such as biosensors. Detecting when gene expression occurs is central to understanding the overall mechanism of the process as well as maximizing the process. Fluorescent proteins have been established as the primary tool for detecting gene expression in inducible systems. This study proposes electricity production as an alternate tool in reporting gene expression. Using a model organism for electricity production, Shewanella oneidensis MR-1, current was shown to be an efficient reporter for gene expression and comparable to superfolder green fluorescent protein (GFP). Through regulation of the lac operator and T7 promoter, current production was induced by isopropyl {beta}-D-1-thiogalactopyranoside (IPTG) addition. IPTG addition induced translation of GFP and the MtrB protein, which complemented a {triangleup}mtrB strain of S. oneidensis MR-1 and restored current production. This inducible system generated reproducible current in 18 minutes in both micro-oxic and anoxic conditions. These results show that current is a fast reporter for gene expression.\n\nFinancial DisclosureThe team was supported by the following departments and colleges at Michigan State University: College of Natural Science, College of Engineering, Biochemistry and Molecular Biology Department and Plant Research Laboratory. The team also received support from the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494) and startup funding from the Department of Molecular Biology and Biochemistry, Michigan State University and support from Michigan State University AgBioResearch (MICL02454) (to B.H.). This work was also supported by NSF CAREER (Award #1254238) to T.A.W. MSU Alpha Chi Sigma also supported the team.\n\nCompeting InterestsThe authors declare that no competing interests exist.\n\nEthics StatementN/A\n\nData AvailabilityAll data will be supplied upon request by the corresponding author.\n\nThis work was assessed during the iGEM/PLOS Realtime Peer Review Jamboree on 23rd February 2018 and has been revised in response to the reviewers comments.

synthetic biology↗