17 Search Results

Lipomyces starkeyi is an oleaginous yeast with a native metabolism well-suited for production of lipids and biofuels from complex lignocellulosic and waste feedstocks. Recent advances in genetic engineering tools have facilitated the development of L. starkeyi into a microbial chassis for biofuel and chemical production. However, the feasibility of redirecting L. starkeyi lipid flux away from lipids and towards other products remains relatively unexplored. Here, we engineer the native metabolism to produce malic acid by introducing the reductive TCA pathway and a C4-dicarboxylic acid transporter to the yeast. Heterogeneous expression of two genes, the Aspergillus oryzae malate transporter and malatemore » dehydrogenase, enabled L. starkeyi malic acid production. Overexpression of a third gene, the native pyruvate carboxylase, allowed titers to reach approximately 10 g/L during shaking flasks cultivations, with production of malic acid inhibited at pH values less than 4. Corn-stover hydrolysates were found to be well-tolerated, and controlled bioreactor fermentations on the real hydrolysate produced 26.5 g/L of malic acid. Proteomic, transcriptomic and metabolomic data from real and mock hydrolysate fermentations indicated increased levels of a S. cerevisiae hsp9/hsp12 homolog (proteinID: 101453), glutathione dependent formaldehyde dehydrogenases (proteinIDs: 2047, 278215), oxidoreductases, and expression of efflux pumps and permeases during growth on the real hydrolysate. Simultaneously, machine learning based medium optimization improved production dynamics by 18% on mock hydrolysate and revealed lower tolerance to boron (a trace element included in the standard cultivation medium) than other yeasts. Together, this work demonstrated the ability to produce organic acids in L. starkeyi with minimal byproducts. The fermentation characterization and omic analyses provide a rich dataset for understanding L. starkeyi physiology and metabolic response to growth in hydrolysates. Identified upregulated genes and proteins provide potential targets for overexpression for improving growth and tolerance to concentrated hydrolysates, as well as valuable information for future L. starkeyi engineering work.« less

The genus Aspergillus is diverse, including species of industrial importance, human pathogens, plant pests, and model organisms. Aspergillus includes species from sections Usti and Cavernicolus, which until recently were joined in section Usti, but have now been proposed to be non-monophyletic and were split by section Nidulantes, Aenei and Raperi. To learn more about these sections, we have sequenced the genomes of 13 Aspergillus species from section Cavernicolus (A. cavernicola, A. californicus, and A. egyptiacus), section Usti (A. carlsbadensis, A. germanicus, A. granulosus, A. heterothallicus, A. insuetus, A. keveii, A. lucknowensis, A. pseudodeflectus and A. pseudoustus), and section Nidulantes (A.more » quadrilineatus, previously A. tetrazonus). We compared these genomes with 16 additional species from Aspergillus to explore their genetic diversity, based on their genome content, repeat-induced point mutations (RIPs), transposable elements, carbohydrate-active enzyme (CAZyme) profile, growth on plant polysaccharides, and secondary metabolite gene clusters (SMGCs). All analyses support the split of section Usti and provide additional insights: Analyses of genes found only in single species show that these constitute genes which appear to be involved in adaptation to new carbon sources, regulation to fit new niches, and bioactive compounds for competitive advantages, suggesting that these support species differentiation in Aspergillus species. Sections Usti and Cavernicolus have mainly unique SMGCs. Section Usti contains very large and information-rich genomes, an expansion partially driven by CAZymes, as section Usti contains the most CAZyme-rich species seen in genus Aspergillus. Section Usti is clearly an underutilized source of plant biomass degraders and shows great potential as industrial enzyme producers.« less

Microbial conversion of lignocellulosic biomass represents an alternative route for production of biofuels and bioproducts. While researchers have mostly focused on engineering strains such as Rhodotorula toruloides for better bisabolene production as a sustainable aviation fuel, less is known about the impact of the feedstock heterogeneity on bisabolene production. Critical material attributes like feedstock composition, nutritional content, and inhibitory compounds can all influence bioconversion. Further, the given feedstocks can have a marked influence on selection of suitable pretreatment and hydrolysis technologies, optimizing the fermentation conditions, and possibly even modifying the microorganism's metabolic pathways, to better utilize the available feedstock. Here,more » this work aimed to examine and understand how variations in corn stover batches, anatomical fractions, and storage conditions impact the efficiency of bisabolene production by R. toruloides. All of these represent different facets of feedstock heterogeneity. Deacetylation, mechanical refining, and enzymatic hydrolysis of these variable feedstocks served as the basis of this research. The resulting hydrolysates were converted to bisabolene via fermentation, a sustainable aviation fuel precursor, using an engineered R. toruloides strain. This study showed that different sources of feedstock heterogeneity can influence microbial growth and product titer in counterintuitive ways, as revealed through global analysis of protein expression. The maximum bisabolene produced by R. toruloides was on the stalk fraction of corn stover hydrolysate (8.89 ± 0.47 g/L). Further, proteomics analysis comparing the protein expression between the anatomic fractions showed that proteins relating to carbohydrate metabolism, energy production, and conversion as well as inorganic ion transport metabolism were either significantly upregulated or downregulated. Specifically, downregulation of proteins related to the iron–sulfur cluster in stalk fraction suggests a coordinated response by R. toruloides to maintain overall metabolic balance, and this was corroborated by the concentration of iron in the feedstocks.« less

Aspergillus terreus has attracted interest due to its application in industrial biotechnology, particularly for the production of itaconic acid and bioactive secondary metabolites. As related species also seem to possess a prosperous secondary metabolism, they are of high interest for genome mining and exploitation. Here, we present draft genome sequences for six species from Aspergillus section Terrei and one species from Aspergillus section Nidulantes. Whole-genome phylogeny confirmed that section Terrei is monophyletic. Genome analyses identified between 70 and 108 key secondary metabolism genes in each of the genomes of section Terrei, the highest rate found in the genus Aspergillus somore » far. The respective enzymes fall into 167 distinct families with most of them corresponding to potentially unique compounds or compound families. Moreover, 53% of the families were only found in a single species, which supports the suitability of species from section Terrei for further genome mining. Intriguingly, this analysis, combined with heterologous gene expression and metabolite identification, suggested that species from section Terrei use a strategy for UV protection different to other species from the genus Aspergillus. Section Terrei contains a complete plant polysaccharide degrading potential and an even higher cellulolytic potential than other Aspergilli, possibly facilitating additional applications for these species in biotechnology.« less

The Lipomyces clade contains oleaginous yeast species with advantageous metabolic features for biochemical and biofuel production. Limited knowledge about the metabolic networks of the species and limited tools for genetic engineering have led to a relatively small amount of research on the microbes. Here, a genome-scale metabolic model (GSM) of Lipomyces starkeyi NRRL Y-11557 was built using orthologous protein mappings to model yeast species. Phenotypic growth assays were used to validate the GSM (66% accuracy) and indicated that NRRL Y-11557 utilized diverse carbohydrates but had more limited catabolism of organic acids. The final GSM contained 2,193 reactions, 1,909 metabolites, andmore » 996 genes and was thus named iLst996. The model contained 96 of the annotated carbohydrate-active enzymes. iLst996 predicted a flux distribution in line with oleaginous yeast measurements and was utilized to predict theoretical lipid yields. Twenty-five other yeasts in the Lipomyces clade were then genome sequenced and annotated. Sixteen of the Lipomyces species had orthologs for more than 97% of the iLst996 genes, demonstrating the usefulness of iLst996 as a broad GSM for Lipomyces metabolism. Pathways that diverged from iLst996 mainly revolved around alternate carbon metabolism, with ortholog groups excluding NRRL Y-11557 annotated to be involved in transport, glycerolipid, and starch metabolism, among others. Overall, this study provides a useful modeling tool and data for analyzing and understanding Lipomyces species metabolism and will assist further engineering efforts in Lipomyces.« less

Aconitic acid is an unsaturated tricarboxylic acid that is attractive for its potential use in the manufacture of biodegradable and biocompatible polymers, plasticizers, and surfactants. Previously Aspergillus pseudoterreus was engineered as a platform to produce aconitic acid by deleting the cadA (cis-aconitic acid decarboxylase) gene in the itaconic acid biosynthetic pathway. In this study aconitic acid transporter gene (aexA) was identified using comparative global discovery proteomics analysis between the wild-type and cadA deletion strains. Deletion of aexA almost eliminated aconitic acid secretion, while its overexpression led to a significant increase in aconitic acid production. Transportation of aconitic acid across themore » plasma membrane is a key limiting step. In vitro proteoliposome transport assay further validated AexA’s function and its substrate specificity. This research provides new approaches to efficiently pinpoint and characterize exporters of fungal organic acids and accelerate the metabolic engineering to improve secretion capability and lower cost for bioproduction.« less

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals. This study focuses on establishing and optimizing the production of 3HP in R. toruloides. As R. toruloides naturally has a high metabolic flux towards malonyl-CoA, we exploited this pathway to produce 3HP. Upon finding the yeast capable of catabolizing 3HP, we then implemented functional genomicsmore » and metabolomic analysis to identify the catabolic pathways. Deletion of a putative malonate semialdehyde dehydrogenase gene encoding an oxidative 3HP pathway was found to significantly reduce 3HP degradation. We further explored monocarboxylate transporters to promote 3HP transport and identified a novel 3HP transporter in Aspergillus pseudoterreus by RNA-seq and proteomics. Combining these engineering efforts with media optimization in a fed-batch fermentation resulted in 45.4 g/L 3HP production. This represents one of the highest 3HP titers reported, and the highest titer produced from lignocellulosic hydrolysate to the best of our knowledge. This work demonstrates microbial production of 3HP in R. toruloides from lignocellulosic hydrolysate at high titers, and it represents a significant step toward enabling industrial production of 3HP in the future.« less

Multidimensional measurements using state-of-the-art separations and mass spectrometry provide advantages in untargeted metabolomics analyses for studying biological and environmental bio-chemical processes. However, the lack of rapid analytical methods and robust algorithms for these heterogeneous data has limited its application. Here, we develop and evaluate a sensitive and high-throughput analytical and computational workflow to enable accurate metabolite profiling. Our workflow combines liquid chromatography, ion mobility spectrometry and data-independent acquisition mass spectrometry with PeakDecoder, a machine learning-based algorithm that learns to distinguish true co-elution and co-mobility from raw data and calculates metabolite identification error rates. We apply PeakDecoder for metabolite profiling ofmore » various engineered strains of Aspergillus pseudoterreus, Aspergillus niger, Pseudomonas putida and Rhodosporidium toruloides. Results, validated manually and against selected reaction monitoring and gas-chromatography platforms, show that 2683 features could be confidently annotated and quantified across 116 microbial sample runs using a library built from 64 standards.« less

Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the controlmore » of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol^-1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol^-1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol^-1 sugars and resulted in a final titer of 36.0 g/L 3-HP. The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.« less

Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitromore » biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.« less