In Situ Raman Methodology for Online Analysis of CO2 and H2O Loadings in a Water-Lean Solvent for CO2 Capture.

Carbon capture represents a key pathway to meeting climate change mitigation goals. Powerful next-generation solvent-based capture processes are under development by many researchers, but optimization and testing would be significantly aided by integrating in situ monitoring capability. Further, real-time water analysis in water-lean solvents offers the potential to maintain their water balance in operation. To explore data acquisition techniques in depth for this purpose, Raman spectra of CO2, H2O, and a single-component water-lean solvent, N-(2-ethoxyethyl)-3-morpholinopropan-1-amine (2-EEMPA) were collected at different CO2 and H2O concentrations using an in situ Raman cell. The quantification of CO2 and H2O loadings in 2-EEMPA was done by principal component regression and partial least squares methods with analysis of uncertainties. We conclude with discussions on how this simultaneous online analysis method to quantify CO2 and H2O loadings can be an important tool to enable the optimal efficiency of water-lean CO2 solvents while also maintaining the critical water balance under operating conditions relevant to post-combustion CO2 capture.

Evolving tolerance of Yarrowia lipolytica to hydrothermal liquefaction aqueous phase waste.

Hydrothermal liquefaction (HTL) is an emerging method for thermochemical conversion of wet organic waste and biomass into renewable biocrude. HTL also produces an aqueous phase (HTL-AP) side stream containing 2-4% light organic compounds that require treatment. Although anaerobic digestion (AD) of HTL-AP has shown promise, lengthy time periods were required for AD microbial communities to adapt to metabolic inhibitors in HTL-AP. An alternative for HTL-AP valorization was recently demonstrated using two engineered strains of Yarrowia lipolytica, E26 and Diploid TAL, for the overproduction of lipids and the polyketide triacetic acid lactone (TAL) respectively. These strains tolerated up to 10% HTL-AP (v/v) in defined media and up to 25% (v/v) HTL-AP in rich media. In this work, adaptive laboratory evolution (ALE) of these strains increased the bulk population tolerance for HTL-AP to up to 30% (v/v) in defined media and up to 35% (v/v) for individual isolates in rich media. The predominate organic acids within HTL-AP (acetic, butyric, and propionic) were rapidly consumed by the evolved Y. lipolytica strains. A TAL-producing isolate (strain 144-3) achieved a nearly 3-fold increase in TAL titer over the parent strain while simultaneously reducing the chemical oxygen demand (COD) of HTL-AP containing media. Fermentation with HTL-AP as the sole nutrient source demonstrated direct conversion of waste into TAL at 10% theoretical yield. Potential genetic mutations of evolved TAL production strains that could be imparting tolerance were explored. This work advances the potential of Y. lipolytica to biologically treat and simultaneously extract value from HTL wastewater. KEY POINTS: • Adaptive evolution of two Y. lipolytica strains enhanced their tolerance to waste. • Y. lipolytica reduces chemical oxygen demand in media containing waste. • Y. lipolytica can produce triacetic acid lactone directly from wastewater.

Integration of Proteomics and Metabolomics Into the Design, Build, Test, Learn Cycle to Improve 3-Hydroxypropionic Acid Production in Aspergillus pseudoterreus.

Biological engineering of microorganisms to produce value-added chemicals is a promising route to sustainable manufacturing. However, overproduction of metabolic intermediates at high titer, rate, and yield from inexpensive substrates is challenging in non-model systems where limited information is available regarding metabolic flux and its control in production conditions. Integrated multi-omic analyses of engineered strains offers an in-depth look at metabolites and proteins directly involved in growth and production of target and non-target bioproducts. Here we applied multi-omic analyses to overproduction of the polymer precursor 3-hydroxypropionic acid (3HP) in the filamentous fungus Aspergillus pseudoterreus. A synthetic pathway consisting of aspartate decarboxylase, beta-alanine pyruvate transaminase, and 3HP dehydrogenase was designed and built for A. pseudoterreus. Strains with single- and multi-copy integration events were isolated and multi-omics analysis consisting of intracellular and extracellular metabolomics and targeted and global proteomics was used to interrogate the strains in shake-flask and bioreactor conditions. Production of a variety of co-products (organic acids and glycerol) and oxidative degradation of 3HP were identified as metabolic pathways competing with 3HP production. Intracellular accumulation of nitrogen as 2,4-diaminobutanoate was identified as an off-target nitrogen sink that may also limit flux through the engineered 3HP pathway. Elimination of the high-expression oxidative 3HP degradation pathway by deletion of a putative malonate semialdehyde dehydrogenase improved the yield of 3HP by 3.4 × after 10 days in shake-flask culture. This is the first report of 3HP production in a filamentous fungus amenable to industrial scale biomanufacturing of organic acids at high titer and low pH.

Valorizing a hydrothermal liquefaction aqueous phase through co-production of chemicals and lipids using the oleaginous yeast Yarrowia lipolytica.

Hydrothermal liquefaction is a promising technology to upgrade wet organic waste into a biocrude oil for diesel or jet fuel; however, this process generates an acid-rich aqueous phase which poses disposal issues. This hydrothermal liquefaction aqueous phase (HTL-AP) contains organic acids, phenol, and other toxins. This work demonstrates that Y. lipolytica as a unique host to valorize HTL-AP into a variety of co-products. Specifically, strains of Y. lipolytica can tolerate HTL-AP at 10% in defined media and 25% in rich media. The addition of HTL-AP enhances production of the polymer precursor itaconic acid by 3-fold and the polyketide triacetic acid lactone at least 2-fold. Additional co-products (lipids and citric acid) were produced in these fermentations. Finally, bioreactor cultivation enabled 21.6 g/L triacetic acid lactone from 20% HTL-AP in mixed sugar hydrolysate. These results demonstrate the first use of Y. lipolytica in HTL-AP valorization toward production of a portfolio of value-added compounds.

Transcriptomic analysis of the oleaginous yeast Lipomyces starkeyi during lipid accumulation on enzymatically treated corn stover hydrolysate.

BACKGROUND: Efficient and economically viable production of biofuels from lignocellulosic biomass is dependent on mechanical and chemical pretreatment and enzymatic hydrolysis of plant material. These processing steps yield simple sugars as well as plant-derived and process-added organic acids, sugar-derived dehydration products, aldehydes, phenolics and other compounds that inhibit the growth of many microorganisms. Lipomyces starkeyi is an oleaginous yeast capable of robust growth on a variety of sugars and lipid accumulation on pretreated lignocellulosic substrates making it attractive as an industrial producer of biofuels. Here, we examined gene expression during batch growth and lipid accumulation in a 20-L bioreactor with either a blend of pure glucose and xylose or pretreated corn stover (PCS) that had been enzymatically hydrolyzed as the carbon sources. RESULTS: We monitored sugar and ammonium utilization as well as biomass accumulation and found that growth of L. starkeyi is inhibited with PCS hydrolysate as the carbon source. Both acetic acid and furfural are present at concentrations toxic to L. starkeyi in PCS hydrolysate. We quantified gene expression at seven time-points for each carbon source during batch growth and found that gene expression is similar at physiologically equivalent points. Analysis of promoter regions revealed that gene expression during the transition to lipid accumulation is regulated by carbon and nitrogen catabolite repression, regardless of carbon source and is associated with decreased expression of the translation machinery and suppression of the cell cycle. We identified 73 differentially expressed genes during growth phase in the bioreactor that may be involved in detoxification of corn stover hydrolysate. CONCLUSIONS: Growth of L. starkeyi is inhibited by compounds present in PCS hydrolysate. Here, we monitored key metabolites to establish physiologically equivalent comparisons during a batch bioreactor run comparing PCS hydrolysate and purified sugars. L. starkeyi's response to PCS hydrolysate is primarily at the beginning of the run during growth phase when inhibitory compounds are presumably at their highest concentration and inducing the general detoxification response by L. starkeyi. Differentially expressed genes identified herein during growth phase will aid in the improvement of industrial strains capable of robust growth on substrates containing various growth inhibitory compounds.

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels.

The filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis.IMPORTANCETrichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance.

Increased production of fatty acids and triglycerides in Aspergillus oryzae by enhancing expressions of fatty acid synthesis-related genes.

Microbial production of fats and oils is being developed as a means of converting biomass to biofuels. Here we investigate enhancing expression of enzymes involved in the production of fatty acids and triglycerides as a means to increase production of these compounds in Aspergillus oryzae. Examination of the A. oryzae genome demonstrates that it contains two fatty acid synthases and several other genes that are predicted to be part of this biosynthetic pathway. We enhanced the expression of fatty acid synthesis-related genes by replacing their promoters with the promoter from the constitutively highly expressed gene tef1. We demonstrate that by simply increasing the expression of the fatty acid synthase genes we successfully increased the production of fatty acids and triglycerides by more than two-fold. Enhancement of expression of the fatty acid pathway genes ATP-citrate lyase and palmitoyl-ACP thioesterase increased productivity to a lesser extent. Increasing expression of acetyl-CoA carboxylase caused no detectable change in fatty acid levels. Increases in message level for each gene were monitored using quantitative real-time reverse transcription polymerase chain reaction. Our data demonstrate that a simple increase in the abundance of fatty acid synthase genes can increase the detectable amount of fatty acids.

A versatile toolkit for high throughput functional genomics with Trichoderma reesei.

BACKGROUND: The ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies. RESULTS: Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414. CONCLUSIONS: Using this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing.

Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.

ELISA-BASE: an integrated bioinformatics tool for analyzing and tracking ELISA microarray data.

SUMMARY: ELISA-BASE is an open source database for capturing, organizing and analyzing enzyme-linked immunosorbent assay (ELISA) microarray data. ELISA-BASE is an extension of the BioArray Software Environment (BASE) database system. AVAILABILITY: http://www.pnl.gov/statistics/ProMAT/ELISA-BASE.stm.

Optimization of aptamer microarray technology for multiple protein targets.

Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl2 and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.

Production and processing of aptamer microarrays.

Aptamers are nucleic acid species that are selected in vitro for their specific binding properties. We describe methods for the production and processing of aptamer microarrays, including detailed procedures for the high-throughput, enzymatic synthesis of 5' RNA biotinylated aptamers and for arraying them onto streptavidin-coated glass slides. Also presented are methods for processing the aptamer microarrays, including blocking, washing, drying, and scanning. Examples are shown for the specific capture of fluorescently labeled target proteins either alone in binding buffer or in competition with labeled intracellular proteins from cell lysates. Consideration is given to the challenges involved in producing multiplex aptamer chips composed of aptamers taken from disparate literature sources, and to the development of standardized methods for characterizing the performance of capture reagents used in biosensors.

Functional RNA microarrays for high-throughput screening of antiprotein aptamers.

High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.

The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs.

BACKGROUND: Comparative analysis of RNA sequences is the basis for the detailed and accurate predictions of RNA structure and the determination of phylogenetic relationships for organisms that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-organized, and processed collections of RNA sequences. This data, starting with the sequences organized into a database management system and aligned to reveal their higher-order structure, and patterns of conservation and variation for organisms that span the phylogenetic tree, has been collected and analyzed. This type of information can be fundamental for and have an influence on the study of phylogenetic relationships, RNA structure, and the melding of these two fields. RESULTS: We have prepared a large web site that disseminates our comparative sequence and structure models and data. The four major types of comparative information and systems available for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2) Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4) Data Access Systems. CONCLUSIONS: This online RNA sequence and structure information, the result of extensive analysis, interpretation, data collection, and computer program and web development, is accessible at our Comparative RNA Web (CRW) Site http://www.rna.icmb.utexas.edu. In the future, more data and information will be added to these existing categories, new categories will be developed, and additional RNAs will be studied and presented at the CRW Site.