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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Globally distributed Myxococcota with photosynthesis gene clusters illuminate the origin and evolution of a potentially chimeric lifestyle

Photosynthesis is a fundamental biogeochemical process, thought to be restricted to a few bacterial and eukaryotic phyla. However, understanding the origin and evolution of phototrophic organisms can be impeded and biased by the difficulties of cultivation. Here, we analyzed metagenomic datasets and found potential photosynthetic abilities encoded in the genomes of uncultivated bacteria within the phylum Myxococcota. A putative photosynthesis gene cluster encoding a type-II reaction center appears in at least six Myxococcota families from three classes, suggesting vertical inheritance of these genes from an early common ancestor, with multiple independent losses in other lineages. Analysis of metatranscriptomic datasets indicate that the putative myxococcotal photosynthesis genes are actively expressed in various natural environments. Furthermore, heterologous expression of myxococcotal pigment biosynthesis genes in a purple bacterium supports that the genes can drive photosynthetic processes. Given that predatory abilities are thought to be widespread across Myxococcota, our results suggest the intriguing possibility of a chimeric lifestyle (combining predatory and photosynthetic abilities) in members of this phylum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A Sesquiterpene Synthase from the Endophytic Fungus Serendipita indica Catalyzes Formation of Viridiflorol

Interactions between plant-associated fungi and their hosts are characterized by a continuous crosstalk of chemical molecules. Specialized metabolites are often produced during these associations and play important roles in the symbiosis between the plant and the fungus, as well as in the establishment of additional interactions between the symbionts and other organisms present in the niche. Serendipita indica, a root endophytic fungus from the phylum Basidiomycota, is able to colonize a wide range of plant species, conferring many benefits to its hosts. The genome of S. indica possesses only few genes predicted to be involved in specialized metabolite biosynthesis, including a putative terpenoid synthase gene (SiTPS). In our experimental setup, SiTPS expression was upregulated when the fungus colonized tomato roots compared to its expression in fungal biomass growing on synthetic medium. Heterologous expression of SiTPS in Escherichia coli showed that the produced protein catalyzes the synthesis of a few sesquiterpenoids, with the alcohol viridiflorol being the main product. To investigate the role of SiTPS in the plant-endophyte interaction, an SiTPS-over-expressing mutant line was created and assessed for its ability to colonize tomato roots. Although overexpression of SiTPS did not lead to improved fungal colonization ability, an in vitro growth-inhibition assay showed that viridiflorol has antifungal properties. Addition of viridiflorol to the culture medium inhibited the germination of spores from a phytopathogenic fungus, indicating that SiTPS and its products could provide S. indica with a competitive advantage over other plant-associated fungi during root colonization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Role of SaPCR2 in Zn Uptake in the Root Elongation Zone of the Zn/Cd Hyperaccumulator Sedum alfredii

Zn pollution is a potential toxicant for agriculture and the environment. Sedum alfredii is a Zn/Cd hyperaccumulator found in China and has been proven as a useful resource for the phytoremediation of Zn-contaminated sites. However, the molecular mechanism of Zn uptake in S. alfredii is limited. In this study, the function of SaPCR2 on Zn uptake in S. alfredii was identified by gene expression analysis, yeast function assays, Zn accumulation and root morphology analysis in transgenic lines to further elucidate the mechanisms of uptake and translocation of Zn in S. alfredii. The results showed that SaPCR2 was highly expressed in the root elongation zone of the hyperaccumulating ecotype (HE) S. alfredii, and high Zn exposure downregulated the expression of SaPCR2 in the HE S. alfredii root. The heterologous expression of SaPCR2 in yeast suggested that SaPCR2 was responsible for Zn influx. The overexpression of SaPCR2 in the non-hyperaccumulating ecotype (NHE) S. alfredii significantly increased the root uptake of Zn, but did not influence Mn, Cu or Fe. SR-μ-XRF technology showed that more Zn was distributed in the vascular buddle tissues, as well as in the cortex and epidermis in the transgenic lines. Root morphology was also altered after SaPCR2 overexpression, and a severe inhibition was observed. In the transgenic lines, the meristematic and elongation zones of the root were lower compared to the WT, and Zn accumulation in meristem cells was also reduced. These results indicate that SaPCR2 is responsible for Zn uptake, and mainly functions in the root elongation zone. This research on SaPCR2 could provide a theoretical basis for the use of genetic engineering technology in the modification of crops for their safe production and biological enhancement.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Metabolic engineering of the oleaginous yeast Yarrowia lipolytica PO1f for production of erythritol from glycerol

Abstract Background Sugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica , an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica . Results Y. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica . We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production. Conclusions We demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica . We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica . This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica . Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.

09 BIOMASS FUELS↗
DOE OSTI2021

Alternative pathways utilize or circumvent putrescine for biosynthesis of putrescine-containing rhizoferrin

The siderophore rhizoferrin (N 1 ,N 4 -dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Cross-kingdom expression of synthetic genetic elements promotes discovery of metabolites in the human microbiome

Small molecules encoded by biosynthetic pathways mediate cross-species interactions and harbor untapped potential, which has provided valuable compounds for medicine and biotechnology. Since studying biosynthetic gene clusters in their native context is often difficult, alternative efforts rely on heterologous expression, which is limited by host-specific metabolic capacity and regulation. Here, in this work, we describe a computational-experimental technology to redesign genes and their regulatory regions with hybrid elements for cross-species expression in Gram-negative and -positive bacteria and eukaryotes, decoupling biosynthetic capacity from host-range constraints to activate silenced pathways. These synthetic genetic elements enabled the discovery of a class of microbiome-derived nucleotide metabolites—tyrocitabines—from Lactobacillus iners. Tyrocitabines feature a remarkable orthoester-phosphate, inhibit translational activity, and invoke unexpected biosynthetic machinery, including a class of “Amadori synthases” and “abortive” tRNA synthetases. Our approach establishes a general strategy for the redesign, expression, mobilization, and characterization of genetic elements in diverse organisms and communities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

An orphan gene BOOSTER enhances photosynthetic efficiency and plant productivity

Organelle-to-nucleus DNA transfer is an ongoing process playing an important role in the evolution of eukaryotic life. Here, genome-wide association studies (GWAS) of non-photochemical quenching parameters in 743 Populus trichocarpa accessions identified a nuclear-encoded genomic region associated with variation in photosynthesis under fluctuating light. The identified gene, BOOSTER (BSTR), comprises three exons, two with apparent endophytic origin and the third containing a large fragment of plastid-encoded Rubisco large subunit. Higher expression of BSTR facilitated anterograde signaling between nucleus and plastid, which corresponded to enhanced expression of Rubisco, increased photosynthesis, and up to 35% greater plant height and 88% biomass in poplar accessions under field conditions. Overexpression of BSTR in Populus tremula × P. alba achieved up to a 200% in plant height. Similarly, Arabidopsis plants heterologously expressing BSTR gained up to 200% in biomass and up to 50% increase in seed.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, in this study, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three P. putida strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h -1 on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators gacS, gacA, and turA along with large duplication and intergenic deletion events that impacted the heterologously-expressed tphAB II catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of tphAB II and tpaK displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve P. putida growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Lipid membrane remodeling and metabolic response during isobutanol and ethanol exposure in Zymomonas mobilis

Abstract Background Recent engineering efforts have targeted the ethanologenic bacterium Zymomonas mobilis for isobutanol production. However, significant hurdles remain due this organism’s vulnerability to isobutanol toxicity, adversely affecting its growth and productivity. The limited understanding of the physiological impacts of isobutanol on Z. mobilis constrains our ability to overcome these production barriers. Results We utilized a systems-level approach comprising LC–MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis . Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis’ CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure. Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology—including cell swelling and formation of intracellular protein aggregates —when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis ’ primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates. Conclusions This study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis . These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars.

09 BIOMASS FUELS↗
DOE OSTI2023

Functional analysis of Salix purpurea genes support roles for ARR17 and GATA15 as master regulators of sex determination

Abstract The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regulating sex in the genus Salix have been limited to genome resequencing and differential gene expression, which are mostly descriptive in nature, and functional validation of candidate sex determination genes has not yet been conducted. Here, we used Arabidopsis to functionally characterize a suite of previously identified candidate genes involved in sex determination and sex dimorphism in the bioenergy shrub willow Salix purpurea . Six candidate master regulator genes for sex determination were heterologously expressed in Arabidopsis, followed by floral proteome analysis. In addition, 11 transcription factors with predicted roles in mediating sex dimorphism downstream of the SDR were tested using DAP‐Seq in both male and female S. purpurea DNA. The results of this study provide further evidence to support models for the roles of ARR17 and GATA15 as master regulator genes of sex determination in S. purpurea , contributing to a regulatory system that is notably different from that of its sister genus Populus . Evidence was also obtained for the roles of two transcription factors, an AP2 / ERF family gene and a homeodomain‐like transcription factor, in downstream regulation of sex dimorphism.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Catalytic Bias of NADH-Dependent Reduced Ferredoxin: NADP+ Oxidoreductase (Nfn) and its Relevance to Ethanol Production in Thermoanaerobacterium Saccharolyticum

NADH-dependent reduced ferredoxin: NADP+ oxidoreductase (Nfn) enzyme catalyzes an energy-conserving flavin-based electron bifurcation (FBEB) reaction. In microbial metabolism, Nfn links redox pools of three electron carriers - ferredoxin (Fd), NAD(H), and NADP(H) - through the following FBEB reaction: 2 NADPH + NAD+ + 2 Fdox 2 NADP+ + NADH + 2 Fdred + H+ The forward reaction is termed electron bifurcation, and the reverse reaction is electron confurcation. Catalytic bias describes an enzyme's tendency to favor one direction of a reversible reaction over the other and is expressed as the ratio of activities in the two directions. Thermoanaerobacterium saccharolyticum (Tsac) is a thermophilic, ethanologenic bacterium that ferments hemicellulose to ethanol at yields above 90%. Its Nfn enzyme is known to support ethanol production, presumably by operating in the confurcating direction to balance cellular cofactors, but this has not previously been demonstrated. To investigate the Tsac Nfn further, we heterologously expressed, purified, and reconstituted the proteins NfnS (NfnA), NfnL (NfnB), and the putative partner Fd with iron-sulfur cluster and/or FAD cofactors. Activity assays monitoring the oxidation or reduction of Fd showed that, across pH 5-10, Tsac Nfn is catalytically biased towards the confurcating direction, favoring NADPH generation over NADPH oxidation by at least fivefold. Using protein electrochemistry, we also determined the reduction potentials of the cofactors in NfnL and Fd. These results indicate that the energetic landscape of FBEB in Tsac Nfn is similar to that in an ortholog. However, Tsac Fd has redox properties distinct from previously assayed Fds, suggesting that the identity and redox properties of Fd may help determine the catalytic bias of Nfn. Additionally, we confirmed the standalone ferredoxin: NADP+ oxidoreductase (FNOR) activity of NfnL but found it to be low and likely insignificant for in vivo redox conversion. We also show how the catalytic bias of Nfn integrates with the hydrogen cycling mechanism proposed in Tsac to better explain cofactor balancing for ethanol production. Our work advances the understanding of electron transfer processes within metabolic networks for the generation of valuable bioproducts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Expression of divergent methyl/alkyl coenzyme M reductases from uncultured archaea

Abstract Methanogens and anaerobic methane-oxidizing archaea (ANME) are important players in the global carbon cycle. Methyl-coenzyme M reductase (MCR) is a key enzyme in methane metabolism, catalyzing the last step in methanogenesis and the first step in anaerobic methane oxidation. Divergent mcr and mcr -like genes have recently been identified in uncultured archaeal lineages. However, the assembly and biochemistry of MCRs from uncultured archaea remain largely unknown. Here we present an approach to study MCRs from uncultured archaea by heterologous expression in a methanogen, Methanococcus maripaludis . Promoter, operon structure, and temperature were important determinants for MCR production. Both recombinant methanococcal and ANME-2 MCR assembled with the host MCR forming hybrid complexes, whereas tested ANME-1 MCR and ethyl-coenzyme M reductase only formed homogenous complexes. Together with structural modeling, this suggests that ANME-2 and methanogen MCRs are structurally similar and their reaction directions are likely regulated by thermodynamics rather than intrinsic structural differences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Tetratricopeptide Repeat 2 Is a Quantitative Trait Locus That Controls Seed Size

Seed size is a key trait affecting evolution and agronomic performance by influencing seedling establishment in natural populations and crop yields. The Arabidopsis thaliana Seed Size QTL1 (SSQ1) locus explains 10–15% of the variation in seed size. We report here that the causal gene for this locus is Tetratricopeptide Repeat Protein 2 (TPR2), which encodes a co-chaperone. Expressing TPR2 across ecotypes and genotypes showed consistent dosage effects. Each additional TPR2Col-0 allele increased seed mass and volume by 10–14% with high reliability in Col-0, Sha, Tsu-1, and tsu2 genetic backgrounds. Reciprocal genetic crosses indicated that this locus acts maternally, consistent with female sporophytic or female gametophytic mutations. To elucidate how TPR2 regulates seed size, the biomass composition of seeds was measured. While oil content remained unchanged, sucrose levels were markedly elevated in TPR2Col-0 transformant lines and reduced in tpr2 mutants. Interestingly, heterologous expression of TPR2Col-0 across genetic backgrounds increased seed protein accumulation by 18% on average. Based on these changes in sucrose and protein levels, potential modes of action for TPR2 are discussed.

Biochemistry & Molecular Biology↗
DOE OSTI2026

Biosynthesis of Minimal C-Phycocyanin Chromophore Assemblies in E. coli Provides a Platform to Dissect Protein-Mediated Tuning of Exciton Transfer

Cyanobacteria are arguably among the most evolutionarily successful organisms on Earth, inhabiting a wide range of ocean, freshwater, soil, and even desert environments on every continent. The cyanobacterial phycobilisome consists of stacks of disk-like light-collecting moieties, allophycocyanin (APC) and phycocyanin (CPC), with covalently bound phycocyanobilin (PCB) pigments. The ways in which the energies of the specific chromophores in these complexes are tuned by the protein to achieve its highly efficient and directional energy transfer are not fully understood, as complex combinations of decay pathways are occurring simultaneously and competitively through this elaborate light-harvesting system. This makes it difficult to extract information about isolated protein-pigment interactions. We provide herein a description of a useful new experimental platform in which we have recombinantly expressed a fully functioning CPC complex and selectively created minimal chromophore sets to study their individual contributions to the overall CPC spectra. Structural and computational analysis of this protein system have provided a greater understanding of how the protein environment serves to alter the photophysics of each of these chromophores. Introduction of a quencher into various positions within CPC confirmed the ability of the protein environment to tune the directionality of energy transport in this assembly. Further mutational analysis suggested the roles of key amino acids surrounding the chromophores, showcasing the utility of heterologous expression techniques for understanding the effects of structure on EET mechanisms in the phycobilisome.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli

Regulation of metabolic gene expression is crucial for maximizing bioproduction titers. Recent engineering tools including CRISPR/Cas9, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) have enabled effective knock-out, knock-down, and overexpression of endogenous pathway genes, respectively, for advanced strain engineering. CRISPRi in particular has emerged as a powerful tool for gene repression through the use of a deactivated Cas9 (dCas9) protein and target guide RNA (gRNA). By constructing gRNA arrays, CRISPRi has the capacity for multiplexed gene downregulation across multiple orthogonal pathways for enhanced bioproduction titers. In this study, we harnessed CRISPRi to downregulate 32 essential and non-essential genes in E. coli strains heterologously expressing either the original mevalonate pathway or isopentenyl diphosphate (IPP) bypass pathway for isoprenol biosynthesis. Isoprenol remains a candidate bioproduct both as a drop-in blend additive and as a precursor for the high-performance sustainable aviation fuel, 1,4-dimethylcyclooctane (DMCO). Of the 32 gRNAs targeting genes associated with isoprenol biosynthesis, a subset was found to vastly improve product titers. Construction of a multiplexed gRNA library based on single guide RNA (sgRNA) performance enabled simultaneous gene repression, yielding a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) on M9-MOPS minimal medium. We then scaled the best performing CRISPRi strain to 2-L fed-batch cultivation and demonstrated translatable titer improvements, ultimately obtaining 12.4 ± 1.3 g/L isoprenol. Our strategy further establishes CRISPRi as a powerful tool for tuning metabolic flux in production hosts and that titer improvements are readily scalable with potential for applications in industrial bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Group of O-Acetyltransferases Catalyze Xyloglucan Backbone Acetylation and Can Alter Xyloglucan Xylosylation Pattern and Plant Growth When Expressed in Arabidopsis

Abstract Xyloglucan is a major hemicellulose in plant cell walls and exists in two distinct types, XXXG and XXGG. While the XXXG-type xyloglucan from dicot species only contains O-acetyl groups on side-chain galactose (Gal) residues, the XXGG-type xyloglucan from Poaceae (grasses) and Solanaceae bears O-acetyl groups on backbone glucosyl (Glc) residues. Although O-acetyltransferases responsible for xyloglucan Gal acetylation have been characterized, the biochemical mechanism underlying xyloglucan backbone acetylation remains to be elucidated. In this study, we showed that recombinant proteins of a group of DUF231 members from rice and tomato were capable of transferring acetyl groups onto O-6 of Glc residues in cello-oligomer acceptors, indicating that they are xyloglucan backbone 6-O-acetyltransferases (XyBATs). We further demonstrated that XyBAT-acetylated cellohexaose oligomers could be readily xylosylated by AtXXT1 (Arabidopsis xyloglucan xylosyltransferase 1) to generate acetylated, xylosylated cello-oligomers, whereas AtXXT1-xylosylated cellohexaose oligomers were much less effectively acetylated by XyBATs. Heterologous expression of a rice XyBAT in Arabidopsis led to a severe reduction in cell expansion and plant growth and a drastic alteration in xyloglucan xylosylation pattern with the formation of acetylated XXGG-type units, including XGG, XGGG, XXGG, XXGG,XXGGG and XXGGG (G denotes acetylated Glc). In addition, recombinant proteins of two Arabidopsis XyBAT homologs also exhibited O-acetyltransferase activity toward cellohexaose, suggesting their possible role in mediating xyloglucan backbone acetylation in vivo. Our findings provide new insights into the biochemical mechanism underlying xyloglucan backbone acetylation and indicate the importance of maintaining the regular xyloglucan xylosylation pattern in cell wall function.

Cell Biology↗
DOE OSTI2025

Archaeal protein containing domain of unknown function 2193 undergoes oligomeric reconfiguration upon iron–sulfur cluster binding

Methanogenic archaea are particularly rich in iron–sulfur proteins, yet their roles remain largely enigmatic. Here, we characterized aMethanococcus voltae(Mvo) protein from the domain of unknown function (DUF) 2193 family, a group of proteins present primarily in archaea and characterized by a conserved cysteine‐rich C‐terminal motif.MvoDUF2193 was heterologously expressed and characterized by a range of spectroscopic and analytical methods. The results demonstrate thatMvoDUF2193 binds a single [4Fe–4S] cluster per subunit and that cluster occupancy regulates the transition from an apo tetramer to a [4Fe–4S] monomeric form. We hypothesize thatMvoDUF2193 serves a regulatory role in the cell, mediated by [Fe–S] cluster binding and changes in oligomeric state.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Discovery of the Azaserine Biosynthetic Pathway Uncovers a Biological Route for α‐Diazoester Production

Abstract Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α‐diazoester functional group. Herein, we report the discovery of the azaserine ( aza ) biosynthetic gene cluster from Glycomyces harbinensis . Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)‐producing enzymes, implicating HYAA in α‐diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2‐electron oxidation of a hydrazonoacetyl intermediate is required for α‐diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for α‐diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene‐mediated biosynthetic transformations in vivo.

Van Cura, Devon↗