Engineered reduction of S-adenosylmethionine alters lignin in sorghum.

BACKGROUND: Lignin is an aromatic polymer deposited in secondary cell walls of higher plants to provide strength, rigidity, and hydrophobicity to vascular tissues. Due to its interconnections with cell wall polysaccharides, lignin plays important roles during plant growth and defense, but also has a negative impact on industrial processes aimed at obtaining monosaccharides from plant biomass. Engineering lignin offers a solution to this issue. For example, previous work showed that heterologous expression of a coliphage S-adenosylmethionine hydrolase (AdoMetase) was an effective approach to reduce lignin in the model plant Arabidopsis. The efficacy of this engineering strategy remains to be evaluated in bioenergy crops. RESULTS: We studied the impact of expressing AdoMetase on lignin synthesis in sorghum (Sorghum bicolor L. Moench). Lignin content, monomer composition, and size, as well as biomass saccharification efficiency were determined in transgenic sorghum lines. The transcriptome and metabolome were analyzed in stems at three developmental stages. Plant growth and biomass composition was further evaluated under field conditions. Results evidenced that lignin was reduced by 18% in the best transgenic line, presumably due to reduced activity of the S-adenosylmethionine-dependent O-methyltransferases involved in lignin synthesis. The modified sorghum features altered lignin monomer composition and increased lignin molecular weights. The degree of methylation of glucuronic acid on xylan was reduced. These changes enabled a ~20% increase in glucose yield after biomass pretreatment and saccharification compared to wild type. RNA-seq and untargeted metabolomic analyses evidenced some pleiotropic effects associated with AdoMetase expression. The transgenic sorghum showed developmental delay and reduced biomass yields at harvest, especially under field growing conditions. CONCLUSIONS: The expression of AdoMetase represents an effective lignin engineering approach in sorghum. However, considering that this strategy potentially impacts multiple S-adenosylmethionine-dependent methyltransferases, adequate promoters for fine-tuning AdoMetase expression will be needed to mitigate yield penalty.

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid.

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

Neurospora intermedia from a traditional fermented food enables waste-to-food conversion.

Fungal fermentation of food and agricultural by-products holds promise for improving food sustainability and security. However, the molecular basis of fungal waste-to-food upcycling remains poorly understood. Here we use a multi-omics approach to characterize oncom, a fermented food traditionally produced from soymilk by-products in Java, Indonesia. Metagenomic sequencing of samples from small-scale producers in Western Java indicated that the fungus Neurospora intermedia dominates oncom. Further transcriptomic, metabolomic and phylogenomic analysis revealed that oncom-derived N. intermedia utilizes pectin and cellulose degradation during fermentation and belongs to a genetically distinct subpopulation associated with human-generated by-products. Finally, we found that N. intermedia grew on diverse by-products such as fruit and vegetable pomace and plant-based milk waste, did not encode mycotoxins, and could create foods that were positively perceived by consumers outside Indonesia. These results showcase the traditional significance and future potential of fungal fermentation for creating delicious and nutritious foods from readily available by-products.

Systematic engineering for production of anti-aging sunscreen compound in Pseudomonas putida.

Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.

Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit.

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

Deciphering triterpenoid saponin biosynthesis by leveraging transcriptome response to methyl jasmonate elicitation in Saponaria vaccaria.

Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria. This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP-D-fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.

Improved polyketide production in C. glutamicum by preventing propionate-induced growth inhibition.

Corynebacterium glutamicum is a promising host for production of valuable polyketides. Propionate addition, a strategy known to increase polyketide production by increasing intracellular methylmalonyl-CoA availability, causes growth inhibition in C. glutamicum. The mechanism of this inhibition was unclear before our work. Here we provide evidence that accumulation of propionyl-CoA and methylmalonyl-CoA induces growth inhibition in C. glutamicum. We then show that growth inhibition can be relieved by introducing methylmalonyl-CoA-dependent polyketide synthases. With germicidin as an example, we used adaptive laboratory evolution to leverage the fitness advantage of polyketide production in the presence of propionate to evolve improved germicidin production. Whole-genome sequencing revealed mutations in germicidin synthase, which improved germicidin titer, as well as mutations in citrate synthase, which effectively evolved the native glyoxylate pathway to a new methylcitrate pathway. Together, our results show that C. glutamicum is a capable host for polyketide production and we can take advantage of propionate growth inhibition to drive titers higher using laboratory evolution or to screen for production of polyketides.

Complete integration of carbene-transfer chemistry into biosynthesis.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

Expanding Extender Substrate Selection for Unnatural Polyketide Biosynthesis by Acyltransferase Domain Exchange within a Modular Polyketide Synthase.

Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.

Lignin deconstruction by anaerobic fungi.

Lignocellulose forms plant cell walls, and its three constituent polymers, cellulose, hemicellulose and lignin, represent the largest renewable organic carbon pool in the terrestrial biosphere. Insights into biological lignocellulose deconstruction inform understandings of global carbon sequestration dynamics and provide inspiration for biotechnologies seeking to address the current climate crisis by producing renewable chemicals from plant biomass. Organisms in diverse environments disassemble lignocellulose, and carbohydrate degradation processes are well defined, but biological lignin deconstruction is described only in aerobic systems. It is currently unclear whether anaerobic lignin deconstruction is impossible because of biochemical constraints or, alternatively, has not yet been measured. We applied whole cell-wall nuclear magnetic resonance, gel-permeation chromatography and transcriptome sequencing to interrogate the apparent paradox that anaerobic fungi (Neocallimastigomycetes), well-documented lignocellulose degradation specialists, are unable to modify lignin. We find that Neocallimastigomycetes anaerobically break chemical bonds in grass and hardwood lignins, and we further associate upregulated gene products with the observed lignocellulose deconstruction. These findings alter perceptions of lignin deconstruction by anaerobes and provide opportunities to advance decarbonization biotechnologies that depend on depolymerizing lignocellulose.

MEP pathway products allosterically promote monomerization of deoxy-D-xylulose-5-phosphate synthase to feedback-regulate their supply.

Isoprenoids are a very large and diverse family of metabolites required by all living organisms. All isoprenoids derive from the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids. It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase (DXS), a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway. Here we provide experimental insights into the underlying mechanism. Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro, causing a size shift. In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer. Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells. Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply, whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance (e.g., by a blockage in their conversion to downstream isoprenoids). Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.

Engineering isoprenoids production in metabolically versatile microbial host Pseudomonas putida.

With the increasing need for microbial bioproduction to replace petrochemicals, it is critical to develop a new industrial microbial workhorse that improves the conversion of lignocellulosic carbon to biofuels and bioproducts in an economically feasible manner. Pseudomonas putida KT2440 is a promising microbial host due to its capability to grow on a broad range of carbon sources and its high tolerance to xenobiotics. In this study, we engineered P. putida KT2440 to produce isoprenoids, a vast category of compounds that provide routes to many petrochemical replacements. A heterologous mevalonate (MVA) pathway was engineered to produce potential biofuels isoprenol (C5) and epi-isozizaene (C15) for the first time in P. putida. We compared the difference between three different isoprenoid pathways in P. putida on isoprenol production and achieved 104 mg/L of isoprenol production in a batch flask experiment through optimization of the strain. As P. putida can natively consume isoprenol, we investigated how to prevent this self-consumption. We discovered that supplementing L-glutamate in the medium can effectively prevent isoprenol consumption in P. putida and metabolomics analysis showed an insufficient energy availability and an imbalanced redox status during isoprenol degradation. We also showed that the engineered P. putida strain can produce isoprenol using aromatic substrates such as p-coumarate as the sole carbon source, and this result demonstrates that P. putida is a valuable microbial chassis for isoprenoids to achieve sustainable biofuel production from lignocellulosic biomass.

Evaluation of engineered low-lignin poplar for conversion into advanced bioproducts.

BACKGROUND: Lignocellulosic resources are promising feedstocks for the manufacture of bio-based products and bioenergy. However, the inherent recalcitrance of biomass to conversion into simple sugars currently hinders the deployment of advanced bioproducts at large scale. Lignin is a primary contributor to biomass recalcitrance as it protects cell wall polysaccharides from degradation and can inhibit hydrolytic enzymes via non-productive adsorption. Several engineering strategies have been designed to reduce lignin or modify its monomeric composition. For example, expression of bacterial 3-dehydroshikimate dehydratase (QsuB) in poplar trees resulted in a reduction in lignin due to redirection of metabolic flux toward 3,4-dihydroxybenzoate at the expense of lignin. This reduction was accompanied with remarkable changes in the pools of aromatic compounds that accumulate in the biomass. RESULTS: The impact of these modifications on downstream biomass deconstruction and conversion into advanced bioproducts was evaluated in the current study. Using ionic liquid pretreatment followed by enzymatic saccharification, biomass from engineered trees released more glucose and xylose compared to wild-type control trees under optimum conditions. Fermentation of the resulting hydrolysates using Rhodosporidium toruloides strains engineered to produce α-bisabolene, epi-isozizaene, and fatty alcohols showed no negative impact on cell growth and yielded higher titers of bioproducts (as much as + 58%) in the case of QsuB transgenics trees. CONCLUSION: Our data show that low-recalcitrant poplar biomass obtained with the QsuB technology has the potential to improve the production of advanced bioproducts.

Correction to "Expression of Dehydroshikimate Dehydratase in Sorghum Improves Biomass Yield, Accumulation of Protocatechuate, and Biorefinery Economics".

[This corrects the article DOI: 10.1021/acssuschemeng.2c01160.].

Engineering sorghum for higher 4-hydroxybenzoic acid content.

Engineering bioenergy crops to accumulate coproducts in planta can increase the value of lignocellulosic biomass and enable a sustainable bioeconomy. In this study, we engineered sorghum with a bacterial gene encoding a chorismate pyruvate-lyase (ubiC) to reroute the plastidial pool of chorismate from the shikimate pathway into the valuable compound 4-hydroxybenzoic acid (4-HBA). A gene encoding a feedback-resistant version of 3-deoxy-d-arabino-heptulonate-7-phosphate synthase (aroG) was also introduced in an attempt to increase the carbon flux through the shikimate pathway. At the full maturity and senesced stage, two independent lines that co-express ubiC and aroG produced 1.5 and 1.7 dw% of 4-HBA in biomass, which represents 36- and 40-fold increases compared to the titer measured in wildtype. The two transgenic lines showed no obvious phenotypes, growth defects, nor alteration of cell wall polysaccharide content when cultivated under controlled conditions. In the field, when harvested before grain maturity, transgenic lines contained 0.8 and 1.2 dw% of 4-HBA, which represent economically relevant titers based on recent technoeconomic analysis. Only a slight reduction (11-15%) in biomass yield was observed in transgenics grown under natural environment. This work provides the first metabolic engineering steps toward 4-HBA overproduction in the bioenergy crop sorghum to improve the economics of biorefineries by accumulating a value-added coproduct that can be recovered from biomass and provide an additional revenue stream.

Engineering site-selective incorporation of fluorine into polyketides.

Although natural products and synthetic small molecules both serve important medicinal functions, their structures and chemical properties are relatively distinct. To expand the molecular diversity available for drug discovery, one strategy is to blend the effective attributes of synthetic and natural molecules. A key feature found in synthetic compounds that is rare in nature is the use of fluorine to tune drug behavior. We now report a method to site-selectively incorporate fluorine into complex structures to produce regioselectively fluorinated full-length polyketides. We engineered a fluorine-selective trans-acyltransferase to produce site-selectively fluorinated erythromycin precursors in vitro. We further demonstrated that these analogs could be produced in vivo in Escherichia coli on engineering of the fluorinated extender unit pool. By using engineered microbes, elaborate fluorinated compounds can be produced by fermentation, offering the potential for expanding the identification and development of bioactive fluorinated small molecules.

Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule.

Production of natural rubber by Parthenium argentaum (guayule) requires increased yield for economic sustainability. An RNAi gene silencing strategy was used to engineer isoprenoid biosynthesis by downregulation of squalene synthase (SQS), such that the pool of farnesyl diphosphate (FPP) substrate might instead be available to initiate natural rubber synthesis. Downregulation of SQS resulted in significantly reduced squalene and slightly increased rubber, but not in the same tissues nor to the same extent, partially due to an apparent negative feedback regulatory mechanism that downregulated mevalonate pathway isoprenoid production, presumably associated with excess geranyl pyrophosphate levels. A detailed metabolomics analysis of isoprenoid production in guayule revealed significant differences in metabolism in different tissues, including in active mevalonate and methylerythritol phosphate pathways in stem tissue, where rubber and squalene accumulate. New insights and strategies for engineering isoprenoid production in guayule were identified.

Nitrogen Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing.

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

Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4.

Rhamnolipids (RLs) are well-studied biosurfactants naturally produced by pathogenic strains of Pseudomonas aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH4) provides an intriguing alternative as a substrate for RL production because it is low cost and may mitigate greenhouse gas emissions. Here, we demonstrate RL production from CH4 by Methylotuvimicrobium alcaliphilum DSM19304. RLs are inhibitory to M. alcaliphilum growth (<0.05 g/l). Adaptive laboratory evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/l of RLs. Metabolomics and proteomics of the adapted strain grown on CH4 in the absence of RLs revealed metabolic changes, increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared with the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 µM of RLs, a 600-fold increase compared with the parent strain, solely from CH4. This work has promise for developing technologies to produce fatty acid-derived bioproducts, including biosurfactants, from CH4.