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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Data for Rapid and Efficient in planta Genome Editing in Sorghum Using Foxtail Mosaic Virus-mediated sgRNA Delivery

The requirement of in vitro tissue culture for the delivery of gene editing reagents limits the application of gene editing to commercially relevant varieties of many crop species. To overcome this bottleneck, plant RNA viruses have been deployed as versatile tools for in planta delivery of recombinant RNA. Viral delivery of single-guide RNAs (sgRNAs) to transgenic plants that stably express CRISPR-associated (Cas) endonuclease has been successfully used for targeted mutagenesis in several dicotyledonous and few monocotyledonous plants. Progress with this approach in monocotyledonous plants is limited so far by the availability of effective viral vectors. We engineered a set of foxtail mosaic virus (FoMV) and barley stripe mosaic virus (BSMV) vectors to deliver the fluorescent protein AmCyan to track viral infection and movement in Sorghum bicolor . We further used these viruses to deliver and express sgRNAs to Cas9 and Green Fluorescent Protein (GFP) expressing transgenic sorghum lines, targeting Phytoene desaturase (PDS), Magnesium-chelatase subunit I (MgCh), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, orthologs of maize Lemon white1 (Lw1) or GFP. The recombinant BSMV did neither infect sorghum nor deliver or express AmCyan and sgRNAs. In contrast, the recombinant FoMV systemically spread throughout sorghum plants and induced somatic mutations with frequencies reaching up to 60%. This mutagenesis led to visible phenotypic changes, demonstrating the potential of FoMV for in planta gene editing and functional genomics studies in sorghum.

Feedstock Production↗
DOE OSTI2020

Epigenetic Footprints of CRISPR/Cas9-Mediated Genome Editing in Plants

CRISPR/Cas9 has been widely applied to various plant species accelerating the pace of plant genome editing and precision breeding in crops. Unintended effects beyond off-target nucleotide mutations are still somewhat unexplored. We investigated the degree and patterns of epigenetic changes after gene editing. We examined changes in DNA methylation in genome-edited promoters of naturally hypermethylated genes (AT1G72350 and AT1G09970) and hypomethylated genes (AT3G17320 and AT5G28770) from Arabidopsis. Transgenic plants were developed via Agrobacterium-mediated floral dip transformation. Homozygous edited lines were selected from segregated T2 plants by an in vitro digestion assay using ribonucleoprotein complex. Bisulfite sequencing comparisons were made between paired groups of edited and non-edited plants to identify changes in DNA methylation of the targeted loci. We found that directed mutagenesis via CRISPR/Cas9 resulted in no unintended morphological or epigenetic alterations. Phenotypes of wild-type, transgenic empty vector, and transgenic edited plants were similar. Epigenetic profiles revealed that methylation patterns of promoter regions flanking target sequences were identical among wild-type, transgenic empty vector, and transgenic edited plants. There was no effect of mutation type on epigenetic status. We also evaluated off-target mutagenesis effects in the edited plants. Potential off-target sites containing up to 4-bp mismatch of each target were sequenced. No off-target mutations were detected in candidate sites. Our results showed that CRISPR/Cas9 did not leave an epigenetic footprint on either the immediate gene-edited DNA and flanking DNA or introduce off-target mutations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production

Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Understanding the role of negative charge in the scaffold of an artificial enzyme for CO 2 hydrogenation on catalysis

Here, we have approached the construction of an artificial enzyme by employing a robust protein scaffold, lactococcal multidrug resistance regulator, LmrR, providing a structured secondary and outer coordination spheres around a molecular rhodium complex, [Rh I (P Et2 N gly P Et2 ) 2 ] - . Previously, we demonstrated a 2–3 fold increase in activity for one Rh-LmrR construct by introducing positive charge in the secondary coordination sphere. In this study, a series of variants was made through site-directed mutagenesis where the negative charge is located in the secondary sphere or outer coordination sphere, with additional variants made with increasingly negative charge in the outer coordination sphere while keeping a positive charge in the secondary sphere. Placing a negative charge in the secondary or outer coordination sphere demonstrates decreased activity by a factor of two compared to the wild-type Rh-LmrR. Interestingly, addition of positive charge in the secondary sphere, with the negatively charged outer coordination sphere restores activity. Vibrational and NMR spectroscopy suggest minimal changes to the electronic density at the rhodium center, regardless of inclusion of a negative or positive charge in the secondary sphere, suggesting another mechanism is impacting catalytic activity, explored in the discussion.

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

Uncovering novel mutational signatures by de novo extraction with SigProfilerExtractor

Mutational signature analysis is commonly performed in cancer genomic studies. Here, we present SigProfilerExtractor, an automated tool for de novo extraction of mutational signatures, and benchmark it against another 13 bioinformatics tools by using 34 scenarios encompassing 2,500 simulated signatures found in 60,000 synthetic genomes and 20,000 synthetic exomes. For simulations with 5% noise, reflecting high-quality datasets, SigProfilerExtractor outperforms other approaches by elucidating between 20% and 50% more true-positive signatures while yielding 5-fold less false-positive signatures. Applying SigProfilerExtractor to 4,643 whole-genome- and 19,184 whole-exome-sequenced cancers reveals four novel signatures. Two of the signatures are confirmed in independent cohorts, and one of these signatures is associated with tobacco smoking. In summary, this report provides a reference tool for analysis of mutational signatures, a comprehensive benchmarking of bioinformatics tools for extracting signatures, and several novel mutational signatures, including one putatively attributed to direct tobacco smoking mutagenesis in bladder tissues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

C/EBP-α induces autophagy by binding to Beclin1 through its own acetylation modification in activated hepatic stellate cells

The activation of hepatic stellate cells (HSCs) plays a key role in the occurrence of liver fibrosisand promoting the apoptosis of activated HSCs or reducing the number of activated HSCs can reverse the development of liver fibrosis. In our previous studies, we have demonstrated that the CCAAT/enhancer binding protein α (C/EBP-α) played an important role in promoting the apoptosis of activated HSCs, thereby exerting an anti-liver fibrosis effect. Unlike apoptosis, autophagy, as a caspase-independent programmed cell death, can promptly remove the abnormal accumulation of substances or damaged organelles in cells and play a key role in regulating the homeostasis of intracellular environment. However, it is still unclear whether C/EBP-α participates in the occurrence of autophagy in HSCs. Therefore, in this study, we firstly used the methods of Western blot and immunofluorescence to characterize the consequence of C/EBP-α overexpression on the expression of proteins LC3B, P62, ATG5 and Beclin1 which were related to autophagy in HSCs. Subsequently, we performed Western blot and site-directed mutagenesis methods to clarify the type and related mechanism of autophagy which was induced by C/EBP-α. Here we show that C/EBP-α promotes the occurrence of autophagy in HSCs and the autophagy induced by C/EBP-α belongs to mitophagy. The stability of C/EBP-α protein regulates the level of autophagy in HSCs. In addition, acetylation of C/EBP-α also regulates the occurrence of autophagy in HSCs. Acetylation of lysine at positions K298, K302 and K326 of C/EBP-α promotes its binding to Beclin1. In conclusion, our study uncovers the role of C/EBP-α in regulating autophagy in HSCs, thereby providing a new strategy for clinical treatment of liver fibrosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

DeCOIL: Optimization of Degenerate Codon Libraries for Machine Learning-Assisted Protein Engineering

With advances in machine learning (ML)-assisted protein engineering, models based on data, biophysics, and natural evolution are being used to propose informed libraries of protein variants to explore. Synthesizing these libraries for experimental screens is a major bottleneck, as the cost of obtaining large numbers of exact gene sequences is often prohibitive. Degenerate codon (DC) libraries are a cost-effective alternative for generating combinatorial mutagenesis libraries where mutations are targeted to a handful of amino acid sites. However, existing computational methods to optimize DC libraries to include desired protein variants are not well suited to design libraries for ML-assisted protein engineering. To address these drawbacks, we present DEgenerate Codon Optimization for Informed Libraries (DeCOIL), a generalized method which directly optimizes DC libraries to be useful for protein engineering: to sample protein variants that are likely to have both high fitness and high diversity in the sequence search space. Using computational simulations and wet-lab experiments, we demonstrate that DeCOIL is effective across two specific case studies, with potential to be applied to many other use cases. DeCOIL offers several advantages over existing methods, as it is direct, easy-to-use, generalizable, and scalable. With accompanying software, DeCOIL can be readily implemented to generate desired informed libraries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

LevSeq: Rapid Generation of Sequence-Function Data for Directed Evolution and Machine Learning

Sequence-function data provides valuable information about the protein functional landscape but is rarely obtained during directed evolution campaigns. Here, we present Long-read every variant Sequencing (LevSeq), a pipeline that combines a dual barcoding strategy with nanopore sequencing to rapidly generate sequence-function data for entire protein-coding genes. LevSeq integrates into existing protein engineering workflows and comes with open-source software for data analysis and visualization. The pipeline facilitates data-driven protein engineering by consolidating sequence-function data to inform directed evolution and provide the requisite data for machine learning-guided protein engineering (MLPE). LevSeq enables quality control of mutagenesis libraries prior to screening, which reduces time and resource costs. Simulation studies demonstrate LevSeq’s ability to accurately detect variants under various experimental conditions. Lastly, we show LevSeq’s utility in engineering protoglobins for new-to-nature chemistry. Widespread adoption of LevSeq and sharing of the data will enhance our understanding of protein sequence-function landscapes and empower data-driven directed evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Mechanism of tethered agonist-mediated signaling by polycystin-1

Mutations of polycystin-1 (PC1) are the major cause (85% of cases) of autosomal dominant polycystic kidney disease (ADPKD), which is the fourth leading cause of kidney failure. PC1 is thought to function as an atypical G protein-coupled receptor, yet the mechanism by which PC1 regulates G-protein signaling remains poorly understood. A significant portion of ADPKD mutations of PC1 encode a protein with defects in maturation or reduced function that may be amenable to functional rescue. In this work, we have combined complementary biochemical and cellular assay experiments and accelerated molecular simulations, which revealed an allosteric transduction pathway in activation of the PC1 C-terminal fragment. Our findings will facilitate future rational drug design efforts targeting the PC1 signaling function. Polycystin-1 (PC1) is an important unusual G protein-coupled receptor (GPCR) with 11 transmembrane domains, and its mutations account for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple characteristics with Adhesion GPCRs. These include a GPCR proteolysis site that autocatalytically divides these proteins into extracellular, N-terminal, and membrane-embedded, C-terminal fragments (CTF), and a tethered agonist (TA) within the N-terminal stalk of the CTF that is suggested to activate signaling. However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined functional cellular signaling experiments of PC1 CTF expression constructs encoding wild type, stalkless, and three different ADPKD stalk variants with all-atom Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated signaling activation. Correlations of residue motions and free-energy profiles calculated from the GaMD simulations correlated with the differential signaling abilities of wild type and stalk variants of PC1 CTF. They suggested an allosteric mechanism involving residue interactions connecting the stalk, Tetragonal Opening for Polycystins (TOP) domain, and putative pore loop in TA-mediated activation of PC1 CTF. Key interacting residues such as N3074–S3585 and R3848–E4078 predicted from the GaMD simulations were validated by mutagenesis experiments. Together, these complementary analyses have provided insights into a TA-mediated activation mechanism of PC1 CTF signaling, which will be important for future rational drug design targeting PC1.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Trapping conformational states of a flavin-dependent N-monooxygenase in crystallo reveals protein and flavin dynamics

The siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase from Aspergillus fumigatus is a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is an N -monooxygenase that catalyzes the NADPH-dependent hydroxylation of l -ornithine through a multistep oxidative mechanism, utilizing a C4a-hydroperoxyflavin intermediate. Here we present four new crystal structures of SidA in various redox and ligation states, including the first structure of oxidized SidA without NADP(H) or l -ornithine bound (resting state). The resting state structure reveals a new out active site conformation characterized by large rotations of the FAD isoalloxazine around the C1–'C2' and N10–C1' bonds, coupled to a 10-Å movement of the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD to the in conformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) and l -ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of the out conformation. These calculations identified two potential ligand-binding sites. Altogether, our results provide new information about conformational dynamics in flavin-dependent monooxygenases. Overall, understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Heritable, multinucleotide deletions in plants using viral delivery of a repair exonuclease and guide RNAs

Abstract CRISPR/Cas9-mediated mutagenesis typically results in short insertion/deletion mutations, which are often too small to disrupt the function of cis-acting regulatory elements. Here, we describe a highly efficient in planta gene editing approach called VirTREX2-HLDel that achieves heritable multinucleotide deletions in both protein-coding genes and noncoding DNA regulatory elements. VirTREX2-HLDel uses RNA viruses to deliver both the 3 prime repair exonuclease 2 (TREX2) and single-guide RNAs. Our method enables recovery of multiplexed heritable deletions and increases the heritable gene editing frequency at poorly edited sites. We identified functional conservation and divergence of MICRORNA164 (miR164) in Nicotiana benthamiana and tomato (Solanum lycopersicum) using VirTREX2-HLDel and observed previously uncharacterized phenotypes in plants with large deletions at this locus. Our viral delivery method reduces the need for tissue culture and will accelerate the understanding of protein-coding and regulatory regions in plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Ni( II )-binding affinity of Cc NikZ-II and its homologs: the role of the HH -prong and variable loop revealed by structural and mutational studies

Extracytoplasmic Ni(II)-binding proteins (NiBPs) are molecular shuttles involved in cellular nickel uptake. Here, we determined the crystal structure of apo CcNikZ-II at 2.38 Å, which revealed a Ni(II)-binding site comprised of the double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Mutagenesis of the site identified Glu60 and His511 as critical for high affinity Ni(II)-binding. Phylogenetic analysis showed 15 protein clusters with two groups containing the HH-prong. Metal-binding assays with 11 purified NiBPs containing this feature yielded higher Ni(II)-binding affinities. Replacement of the wild type v-loop with those from other NiBPs improved the affinity by up to an order of magnitude. This work provides molecular insights into the determinants for Ni(II) affinity and paves way for NiBP engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Promising approaches for the assembly of the catalytically active, recombinant Desulfomicrobium baculatum hydrogenase with substitutions at the active site

Background: Hydrogenases (H2ases) are metalloenzymes capable of the reversible conversion of protons and electrons to molecular hydrogen. Exploiting the unique enzymatic activity of H2ases can lead to advancements in the process of biohydrogen evolution and green energy production. Results: Here we created of a functional, optimized operon for rapid and robust production of recombinant [NiFe] Desulfomicrobium baculatum hydrogenase (Dmb H2ase). The conversion of the [NiFeSe] Dmb H2ase to [NiFe] type was performed on genetic level by site-directed mutagenesis. The native dmb operon includes two structural H2ase genes, coding for large and small subunits, and an additional gene, encoding a specific maturase (protease) that is essential for the proper maturation of the enzyme. Dmb, like all H2ases, needs intricate bio-production machinery to incorporate its crucial inorganic ligands and cofactors. Strictly anaerobic, sulfate reducer D. baculatum bacteria are distinct, in terms of their biology, from E. coli. Thus, we introduced a series of alterations within the native dmb genes. As a result, more than 100 elements, further compiled into 32 operon variants, were constructed. The initial requirement for a specific maturase was omitted by the artificial truncation of the large Dmb subunit. The assembly of the produced H2ase subunit variants was investigated both, in vitro and in vivo. This approach resulted in 4 recombinant [NiFe] Dmb enzyme variants, capable of H 2 evolution. The aim of this study was to overcome the gene expression, protein biosynthesis, maturation and ligand loading bottlenecks for the easy, fast, and cost-effective delivery of recombinant [NiFe] H2ase, using a commonly available E. coli strains. Conclusion: The optimized genetic constructs together with the developed growth and purification procedures appear to be a promising platform for further studies toward fully-active and O 2 tolerant, recombinant [NiFeSe] Dmb H2ase, resembling the native Dmb enzyme. It could likely be achieved by selective cysteine to selenocysteine substitution within the active site of the [NiFe] Dmb variant.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Single Amino Acid Change in Nramp6 from Sedum Alfredii Hance Affects Cadmium Accumulation

SaNramp6 in Sedum alfredii encodes a membrane-localized metal transporter. We isolated the SaNramp6h allele from the hyperaccumulating ecotype (HE) of S. alfredii . When this allele was expressed in transgenic yeast and Arabidopsis thaliana , it enhanced their cadmium (Cd) sensitivity by increased Cd transport and accumulation. We isolated another allele, SaNramp6n , from a nonhyperaccumulating ecotype (NHE) of S. alfredii . Amino acid sequence comparisons revealed three amino acid differences between SaNramp6h and SaNramp6n. We investigated the Cd transport activity of the Nramp6 allele, and determined which residues are essential for the transport activity. We conducted structure-function analyses of SaNramp6 based on site-directed mutagenesis and functional assays of the mutants in yeast and Arabidopsis . The three residues that differed between SaNramp6h and SaNramp6n were mutated. Only the L157P mutation of SaNramp6h impaired Cd transport. The other mutations, S218N and T504A, did not affect the transport activity of SaNramp6h, indicating that these residues are not essential for metal selectivity. Transgenic plants overexpressing SaNramp6h L157P showed altered metal accumulation in shoots and roots. Our results suggest that the conserved site L157 is essential for the high metal transport activity of SaNramp6h. This information may be useful for limiting or increasing Cd transport by other plant natural resistance associated macrophage protein (NRAMP) proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

De Novo Design of High‐Affinity Miniprotein Binders Targeting Francisella Tularensis Virulence Factor

Abstract Francisella tularensis poses considerable public health risk due to its high infectivity and potential for bioterrorism. Francisella‐like lipoprotein (Flpp3), a key virulence factor unique to Francisella, plays critical roles in infection and immune evasion, making it a promising target for therapeutic development. However, the lack of well‐defined binding pockets and structural information on native interactions has hindered structure‐guided ligand discovery against Flpp3. Here, we used a combination of physics‐based and deep‐learning methods to design high‐affinity miniprotein binders targeting two distinct sites on Flpp3. We identified four binders for site I with binding affinities ranging between 24–110 nM. For the second site, an initial binder showed a dissociation constant ( K D ) of 81 nM, and subsequent site saturation mutagenesis yielded variants with sub‐nanomolar affinities. Circular dichroism confirmed the topology of designed miniproteins. The X‐ray crystal structure of Flpp3 in complex with a site I binder is nearly identical to the design model (Cα root‐mean‐square deviation (RMSD): 0.9 Å). These designed miniproteins provide research tools to explore the roles of Flpp3 in tularemia and should enable the development of new therapeutic candidates.

Gokce‐Alpkilic, Gizem [Molecular Engineering and S↗
DOE OSTI2020

A mass spectrometry-based high-throughput screening method for engineering fatty acid synthases with improved production of medium-chain fatty acids

Microbial cell factories have been extensively engineered to produce free fatty acids (FFAs), key components of crucial nutrients, soaps, industrial chemicals, and fuels. Yet, our ability to control the specificity of microbially synthesized FFAs is still limited for medium-chain fatty acids (MCFAs), which is mainly due to lack of high-throughput approaches for FFA analysis. Here we report a mass spectrometry (MS)-based method for rapid profiling of MCFAs in Saccharomyces cerevisiae by using membrane lipids as a proxy. Specially, matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS was used to detect shorter acyl chain phosphatidylcholines from membrane lipids and the m/z peak ratio at 730 and 758 was used as an index for improved MCFA production. This yeast colony-based method can be performed at a rate of ~2 second per sample, which is much faster than the gold standard gas chromatography (GC)-MS (typically > 30 min per sample). To demonstrate the power of this method, we performed site-saturation mutagenesis of the yeast fatty acid synthase and identified 9 mutants that resulted in improved MCFA production relative to the wild-type strain. This colony-based MALDI-ToF MS screening method offers an alternative way for engineering microbial fatty acid compositions in a high-throughput manner.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Carbohydrate‐binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity

Abstract Chemoenzymatic approaches using carbohydrate‐active enzymes (CAZymes) offer a promising avenue for the synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a “non‐catalytic” lectin‐like domain or carbohydrate‐binding modules (CBMs) in the catalytic step for glycosidic bond synthesis using β‐cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. This study provides evidence for the direct participation of CBMs in the catalytic reaction step for β‐glucan glycosidic bonds synthesis enhancing activity for CBM‐based CAZyme chimeras by >140‐fold over CDs alone. Dynamic intradomain interactions that facilitate this poorly understood reaction mechanism were further revealed by small‐angle X‐ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation‐type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBM‐based CAZyme chimeras for the synthesis of bespoke oligosaccharides using simple activated sugar monomers.

Bandi, Chandra Kanth↗
DOE OSTI2026

Engineering of 2‐ketoacid Decarboxylases for Production of Isobutanol and Other Fusel Alcohols in Saccharomyces cerevisiae

Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2-ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2-ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2-ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae (S. cerevisiae) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell-based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi-site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2-ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.

2-ketoacid decarboxylase↗