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.

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.

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.

On-Line Raman Measurement of the Radiation-Enhanced Reaction of Cellobiose with Hydrogen Peroxide.

Production of a chemical feedstock as a secondary product from a commercial nuclear reactor can increase the economic viability of the reactor and enable the deployment of nuclear energy as part of the low-carbon energy grid. Currently, commercial nuclear reactors produce underutilized energy in the form of neutrons and gamma photons. This excess energy can be exploited to drive chemical reactions, increasing the fraction of utilized energy in reactors and providing a valuable secondary product from the reactor. Gamma degradation of cellulosic biomass has been studied previously. However, real-time, on-line monitoring of the breakdown of biomass materials under gamma radiation has not been demonstrated. Here, we demonstrate on-line monitoring of the reaction of cellobiose with hydrogen peroxide under gamma radiation using Raman spectroscopy, providing in situ quantification of organic and inorganic system components.

Toward the Practical Use of Cobalt-Free Lithium-Ion Batteries by an Advanced Ether-Based Electrolyte.

The criticality of cobalt (Co) has been motivating the quest for Co-free positive electrode materials for building lithium (Li)-ion batteries (LIBs). However, the LIBs based on Co-free positive electrode materials usually suffer from relatively fast capacity decay when coupled with conventional LiPF6-organocarbonate electrolytes. To address this issue, a 1,2-dimethoxyethane-based localized high-concentration electrolyte (LHCE) was developed and evaluated in a Co-free Li-ion cell chemistry (graphite||LiNi0.96Mg0.02Ti0.02O2). Extraordinary capacity retentions were achieved with the LHCE in coin cells (95.3%), single-layer pouch cells (79.4%), and high-capacity loading double-layer pouch cells (70.9%) after being operated within the voltage range of 2.5-4.4 V for 500 charge/discharge cycles. The capacity retentions of counterpart cells using the LiPF6-based conventional electrolyte only reached 61.1, 57.2, and 59.8%, respectively. Mechanistic studies reveal that the superior electrode/electrolyte interphases formed by the LHCE and the intrinsic chemical stability of the LHCE account for the excellent electrochemical performance in the Co-free Li-ion cells.

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.