Identification of a specific exporter that enables high production of aconitic acid in Aspergillus pseudoterreus.

Aconitic acid is an unsaturated tricarboxylic acid that is attractive for its potential use in manufacturing 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, the aconitic acid transporter gene (aexA) was identified using comparative global discovery proteomics analysis between the wild-type and cadA deletion strains. The protein AexA belongs to the Major Facilitator Superfamily (MFS). Deletion of aexA almost abolished aconitic acid secretion, while its overexpression led to a significant increase in aconitic acid production. Transportation of aconitic acid across the plasma membrane is a key limiting step in its production. In vitro, proteoliposome transport assay further validated AexA's function and substrate specificity. This research provides new approaches to efficiently pinpoint and characterize exporters of fungal organic acids and accelerate metabolic engineering to improve secretion capability and lower the cost of bioproduction.

Deletion analysis of the itaconic acid biosynthesis gene cluster components in Aspergillus pseudoterreus ATCC32359.

The filamentous fungus Aspergillus terreus has been successfully used for industrial production of itaconic acid (IA) for many years. The IA biosynthesis pathway has recently been characterized at a molecular genetic level as an IA gene cluster by a clone-based transcriptomic approach. The cluster consists of four genes, including genes for cis-aconitic acid decarboxylase (cadA), a predicted transcription factor (tf), a mitochondrial organic acid transporter (mttA) and an MFS (major facilitator superfamily) type transporter (mfsA). In this research, we performed expressed sequence tag (EST) analysis and systematic gene deletions to further investigate the role of those genes during IA biosynthesis in A. pseudoterreus ATCC32359. EST analysis showed a similar expression pattern among those four genes that were distinct from neighboring genes and further confirmed that they belong to the same biosynthesis cluster. Systematic gene deletion analysis demonstrated that tf, cadA, mttA and mfsA genes in the cluster are essential for IA production; deletion of any of them will either completely abolish the IA production or dramatically decrease the amount of IA produced. The tf gene plays a regulatory role in this cluster. Deletion of tf led to decreased expression levels of cadA, mttA and mfsA. More importantly, a significant amount of aconitic acid was detected in the cadA deletion strain but not in the other deletion strains. Therefore, by deleting only one gene, the cadA, we established a novel microbial host for the production of aconitic acid and other value-added chemicals from sugars in lignocellulosic biomass.

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species.

BACKGROUND: 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. RESULTS: In this study, the 3-HP ß-alanine pathway consisting of aspartate decarboxylase, ß-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control 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 ß-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 ß-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. CONCLUSIONS: 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.

Solvent-Induced Selectivity of Isoprene From Bio-Derived Prenol.

In this work we demonstrate the selective catalytic conversion of prenol, which is an allylic alcohol that can be prepared from renewable resources to isoprene. The catalyst is an inexpensive molybdenum complex (Molyvan L) designed and used as an additive for lubricants. Isoprene is generated under relatively mild reaction parameters at 130-150°C, for 2 h, under vapor pressure conditions that do not exceed 50 psi. Two cases were studied: one in which Molyvan L was dissolved in a base oil at 1% concentration (weight/weight) and then mixed with a solvent and prenol and the other in which neat Molyvan L was introduced in the reaction and the base oil was replaced with the solvent and prenol. We investigated the selectivity of the reaction using the following solvents in both cases: dodecane, dodecanol, isododecane, octane, blendstock for oxygenate blending (BOB3), a fuel surrogate, a polyalphaolefin (PAO4), and methoxy polyethylene glycol (methoxy PEG350). Although conversion of prenol was above 94% in all experiments, isoprene was formed with various degrees of efficiency alongside a prenol isomeric alcohol, diprenyl ether and mixed ether via intramolecular and intermolecular dehydration reactions. Dodecane appeared to have the highest level of selectivity initially in base oil so we studied the effect of various dodecane-like solvents on isoprene yield and product profile. Surprisingly, octane (similar to dodecane) and isododecane (branched alkane) generated insignificant amounts of byproducts, essentially providing the highly desired isoprene with a very high selectivity. Branching of the solvent does not appear to have an effect on selectivity. Another advantage of this catalyst is the low loadings required to effect the transformation; that is, 0.25% (weight/volume) in the cases using neat Molyvan L and 0.5% (weight/volume) in the cases using Molyvan L dissolved in the base oil. Provided that prenol can be produced in large scale from bioresources, this work would enable the sustainable production of isoprene, in good yield, and with very high selectivity.

Kevlar-like Aramid Polymers from Mixed PET Waste.

This work describes the synthetic approaches, spectroscopic and thermal characterization of aramid polymers prepared from waste polyethylene terephthalate (PET) via sustainable and scalable processes. Direct depolymerization of PET with aliphatic diamines under melt conditions resulted in decomposition without substantial formation of any aramid polymer. The Higashi-Ogata methodology or direct polycondensation of terephthalic acid (TPA) derived from PET waste and p-phenylenediamine, resulted in oligomerization and formation of aramids with a low degree of polymerization. The highest molecular weight polymers were obtained via the acid chloride of TPA, the traditional method. A proprietary solvent enabled the dissolution of most polymers and subsequent size exclusion chromatography analysis in the same solvent. We emphasize that although the soluble polymer compounds are prepared via the traditional route, they are novel. The apparent molecular weights of the soluble polymers ranged between 10-35 kDa (M n ) and 28-81 kDa (M w ). All analogues were prepared with commercially available diamines and diamine combinations. The obtained solid powders were dissolved in D2SO4 and analyzed spectroscopically to qualitatively evaluate the degrees of polymerization, while the solids were characterized via thermogravimetric analysis and differential scanning calorimetry. Many reaction conditions were employed to improve the solution polycondensation reaction, and it was found that addition of pyridine (2 eq) to the NMP reaction medium was crucial in preventing the precipitation of the polymer. Contrary to conventional wisdom, CaCl2 did not play a crucial role in the molecular weight increase of the polymer when oxydianiline was used. Our data indicated that the temperature and absence of CaCl2 provided a boost in molecular weight. Both room temperature and 0 °C reactions generated similar polymers as suggested by nuclear magnetic resonance; however, the cold conditions enhanced gel formation, an important attribute in the future processing of these materials to obtain fibers. All analogues had a high degradation temperature at 5 and 10% weight loss (5% and T10%), above 400 °C, along with high percent char values. A glass transition (T g) was not detected in any of the analogues prepared.

Itaconic acid production is regulated by LaeA in Aspergillus pseudoterreus.

The global regulator LaeA controls secondary metabolism in diverse Aspergillus species. Here we explored its role in regulation of itaconic acid production in Aspergillus pseudoterreus. To understand its role in regulating metabolism, we deleted and overexpressed laeA, and assessed the transcriptome, proteome, and secreted metabolome prior to and during initiation of phosphate limitation induced itaconic acid production. We found that secondary metabolite clusters, including the itaconic acid biosynthetic gene cluster, are regulated by laeA and that laeA is required for high yield production of itaconic acid. Overexpression of LaeA improves itaconic acid yield at the expense of biomass by increasing the expression of key biosynthetic pathway enzymes and attenuating the expression of genes involved in phosphate acquisition and scavenging. Increased yield was observed in optimized conditions as well as conditions containing excess nutrients that may be present in inexpensive sugar containing feedstocks such as excess phosphate or complex nutrient sources. This suggests that global regulators of metabolism may be useful targets for engineering metabolic flux that is robust to environmental heterogeneity.

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.

Anaerobic digestion of organic fraction from hydrothermal liquefied algae wastewater byproduct.

The wastewater stream from hydrothermal liquefaction (HTL) process used in biofuel production, contains a large amounts of organic compounds where several can be regarded as environmentally hazardous and requires significant treatment before disposal. In this study, semi-continuous anaerobic digestion is used to degrade the organic fraction of wastewater streams from HTL of the algae Tetraselmis (AgTet) and Chlorella (AgChlr). Results indicated high methane yields at 20-30% (v/v) HTL wastewater together with clarified manure, producing 327.2mL/gVSin (or volatile solids in feed) for AgTet and 263.4mL/gVSin for AgChlr. There was a significant reduction in methane production at concentrations higher than 40% (v/v) HTL wastewater in the feed, possibly due to the accumulation of chloride salts or inhibitory compounds such as pyridines, piperidines and pyrrolidines. This was further confirmed by comparing COD, salt and the ammonia concentrations of the effluents after anaerobic digestion at different concentrations of wastewater in manure.

Biological conversion of the aqueous wastes from hydrothermal liquefaction of algae and pine wood by Rhodococci.

In this study, R. opacus PD630, R. jostii RHA1, R. jostii RHA1 VanA-, and their co-culture were employed to convert hydrothermal liquefaction aqueous waste (HTLAW) into lipids. After 11days, the COD reduction of algal-HTLAW reached 93.4% and 92.7% by R. jostii RHA1 and its mutant VanA-, respectively. Woody-HTLAW promoted lipid accumulation of 0.43glipid/gcell dry weight in R. opacus PD630 cells. Additionally, the total number of chemicals in HTLAW decreased by over 1/3 after 7days of coculture, and 0.10g/L and 0.46g/L lipids were incrementally accumulated in the cellular mass during the fermentation of wood- and algal-HTLAW, respectively. The GC-MS data supported that different metabolism pathways were followed when these Rhodococci strains degraded algae- and woody-HTLAW. These results indicated promising potential of bioconversion of under-utilized carbon and toxic compounds in HTLAW into useful products by selected Rhodococci.

Characterization of lignin derived from water-only and dilute acid flowthrough pretreatment of poplar wood at elevated temperatures.

BACKGROUND: Flowthrough pretreatment of biomass is a critical step in lignin valorization via conversion of lignin derivatives to high-value products, a function vital to the economic efficiency of biorefinery plants. Comprehensive understanding of lignin behaviors and solubilization chemistry in aqueous pretreatment such as water-only and dilute acid flowthrough pretreatment is of fundamental importance to achieve the goal of providing flexible platform for lignin utilization. RESULTS: In this study, the effects of flowthrough pretreatment conditions on lignin separation from poplar wood were reported as well as the characteristics of three sub-sets of lignin produced from the pretreatment, including residual lignin in pretreated solid residues (ReL), recovered insoluble lignin in pretreated liquid (RISL), and recovered soluble lignin in pretreatment liquid (RSL). Both the water-only and 0.05 % (w/w) sulfuric acid pretreatments were performed at temperatures from 160 to 270 °C on poplar wood in a flowthrough reactor system for 2-10 min. Results showed that water-only flowthrough pretreatment primarily removed syringyl (S units). Increased temperature and/or the addition of sulfuric acid enhanced the removal of guaiacyl (G units) compared to water-only pretreatments at lower temperatures, resulting in nearly complete removal of lignin from the biomass. Results also suggested that more RISL was recovered than ReL and RSL in both dilute acid and water-only flowthrough pretreatments at elevated temperatures. NMR spectra of the RISL revealed significant ß-O-4 cleavage, α-ß deoxygenation to form cinnamyl-like end groups, and slight ß-5 repolymerization in both water-only and dilute acid flowthrough pretreatments. CONCLUSIONS: Elevated temperature and/or dilute acid greatly enhanced lignin removal to almost 100 % by improving G unit removal besides S unit removal in flowthrough system. Only mild lignin structural modification was caused by flowthrough pretreatment. A lignin transformation pathway was proposed to explain the complexity of the lignin structural changes during hot water and dilute acid flowthrough pretreatment.Graphical abstractLignin transformations in water-only and dilute acid flowthrough pretreatment at elevated temperatures.