CRISPR/Cas9 suppression of OsAT10, a rice BAHD acyltransferase, reduces p-coumaric acid incorporation into arabinoxylan without increasing saccharification.

Ester-linked hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA) play important roles in crosslinking within cell wall arabinoxylans (AX) and between AX and lignin in grass cell walls. The addition of hydroxycinnamates to AX, is mediated by the Mitchell clade of BAHD acyl-coenzyme A-utilizing transferases. Overexpression of OsAT10 (a Mitchell clade BAHD acyl transferase) in rice, has previously been shown to increase p-CA content in AX in leaves and stems, leading to increased cell wall digestibility, potentially associated with a concomitant decrease in FA content. To investigate the physiological role of OsAT10 we established CRISPR/Cas9 rice knock-out mutants devoid of OsAT10. Our analysis of hydroxycinnamic acid content in wild type plants revealed that AX associated p-CA is found almost exclusively in rice husks, with very little found in other tissues. Mutant plants were essentially devoid of ester-linked p-CA associated with AX, indicating that OsAT10 represents the major enzyme responsible for the addition of p-CA to arabinoxylan in rice plants. We found no change in the digestibility of rice husk lacking AX-associated p-CA, suggesting that the changes in digestibility seen in OsAT10 overexpressing plants were solely due to compensatory decreases in AX-associated FA.

Effect of harvest time on the compositional changes in essential oils, cannabinoids, and waxes of hemp (Cannabis sativa L.).

Demand for cannabinoid is growing, with the global market expected to reach $9.69 billion by 2025. Understanding how chemical composition changes in hemp at different harvest times is crucial to maximizing this industrial crop value. Important compositional changes in three different compound classes (essential oils, cannabinoids, and lipids) from inflorescences (tops), leaves, and stems of hemp (Cannabis sativa L., Finola variety) at different harvesting stages have been investigated. Over 85% of the total extracts from the tops were cannabinoids, while leaves demonstrated the greatest quantities of wax ester and sterols. Essential oil and cannabinoid increased in tops until full flowering (third harvest), reaching 2030 µg g-1 and 39 475 µg g-1, respectively. Cannabinoids decreased at seed maturity (final harvest) to 26 969 µg g-1. This demonstrates the importance of early harvesting to maximize cannabidiol (CBD), which is highly sought after for its therapeutic and pharmacological properties. A total of 21 161 µg g-1 of CBD was extracted from the tops at full flowering (third harvest); however, a significant increase (63%) in the banned psychoactive tetrahydrocannabinol (THC) was observed from budding (157 µg g-1 of biomass) to the full flowering (9873 µg g-1 of biomass). Harvesting the tops after budding is preferable due to the high CBD content and low amounts of THC.

Flexible and digestible wood caused by viral-induced alteration of cell wall composition.

Woody plant material represents a vast renewable resource that has the potential to produce biofuels and other bio-based products with favorable net CO2 emissions.1,2 Its potential has been demonstrated in a recent study that generated novel structural materials from flexible moldable wood.3 Apple rubbery wood (ARW) disease is the result of a viral infection that causes woody stems to exhibit increased flexibility.4 Although ARW disease is associated with the presence of an RNA virus5 known as apple rubbery wood virus (ARWV), how the unique symptoms develop is unknown. We demonstrate that the symptoms of ARWV infections arise from reduced lignification within the secondary cell wall of xylem fibers and result in increased wood digestibility. In contrast, the mid-lamellae region and xylem ray cells are largely unaffected by the infection. Gene expression and proteomic data from symptomatic xylem clearly show the downregulation of phenylalanine ammonia lyase (PAL), the enzyme catalyzing the first committed step in the phenylpropanoid pathway leading to lignin biosynthesis. A large increase in soluble phenolics in symptomatic xylem, including the lignin precursor phenylalanine, is also consistent with PAL downregulation. ARWV infection results in the accumulation of many host-derived virus-activated small interfering RNAs (vasiRNAs). PAL-derived vasiRNAs are among the most abundant vasiRNAs in symptomatic xylem and are likely the cause of reduced PAL activity. Apparently, the mechanism used by the virus to alter lignin exhibits similarities to the RNAi strategy used to alter lignin in genetically modified trees to generate comparable improvements in wood properties.6-8.

Integration of Aspergillus niger transcriptomic profile with metabolic model identifies potential targets to optimise citric acid production from lignocellulosic hydrolysate.

BACKGROUND: Citric acid is typically produced industrially by Aspergillus niger-mediated fermentation of a sucrose-based feedstock, such as molasses. The fungus Aspergillus niger has the potential to utilise lignocellulosic biomass, such as bagasse, for industrial-scale citric acid production, but realising this potential requires strain optimisation. Systems biology can accelerate strain engineering by systematic target identification, facilitated by methods for the integration of omics data into a high-quality metabolic model. In this work, we perform transcriptomic analysis to determine the temporal expression changes during fermentation of bagasse hydrolysate and develop an evolutionary algorithm to integrate the transcriptomic data with the available metabolic model to identify potential targets for strain engineering. RESULTS: The novel integrated procedure matures our understanding of suboptimal citric acid production and reveals potential targets for strain engineering, including targets consistent with the literature such as the up-regulation of citrate export and pyruvate carboxylase as well as novel targets such as the down-regulation of inorganic diphosphatase. CONCLUSIONS: In this study, we demonstrate the production of citric acid from lignocellulosic hydrolysate and show how transcriptomic data across multiple timepoints can be coupled with evolutionary and metabolic modelling to identify potential targets for further engineering to maximise productivity from a chosen feedstock. The in silico strategies employed in this study can be applied to other biotechnological goals, assisting efforts to harness the potential of microorganisms for bio-based production of valuable chemicals.

Characterisation of the enzyme transport path between shipworms and their bacterial symbionts.

BACKGROUND: Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal's gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. RESULTS: Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. CONCLUSION: Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm's mouth and digestive tract, where they aid in wood digestion.

Accessory enzymes of hypercellulolytic Penicillium funiculosum facilitate complete saccharification of sugarcane bagasse.

BACKGROUND: Sugarcane bagasse (SCB) is an abundant feedstock for second-generation bioethanol production. This complex biomass requires an array of carbohydrate active enzymes (CAZymes), mostly from filamentous fungi, for its deconstruction to monomeric sugars for the production of value-added fuels and chemicals. In this study, we evaluated the repertoire of proteins in the secretome of a catabolite repressor-deficient strain of Penicillium funiculosum, PfMig188, in response to SCB induction and examined their role in the saccharification of SCB. RESULTS: A systematic approach was developed for the cultivation of the fungus with the aim of producing and understanding arrays of enzymes tailored for saccharification of SCB. To achieve this, the fungus was grown in media supplemented with different concentrations of pretreated SCB (0-45 g/L). The profile of secreted proteins was characterized by enzyme activity assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 280 proteins were identified in the secretome of PfMig188, 46% of them being clearly identified as CAZymes. Modulation of the cultivation media with SCB up to 15 g/L led to sequential enhancement in the secretion of hemicellulases and cell wall-modifying enzymes, including endo-ß-1,3(4)-glucanase (GH16), endo-α-1,3-glucanase (GH71), xylanase (GH30), ß-xylosidase (GH5), ß-1,3-galactosidase (GH43) and cutinase (CE5). There was ~ 122% and 60% increases in ß-xylosidase and cutinase activities, respectively. There was also a 36% increase in activities towards mixed-linked glucans. Induction of these enzymes in the secretome improved the saccharification performance to 98% (~ 20% increase over control), suggesting their synergy with core cellulases in accessing the recalcitrant region of SCB. CONCLUSION: Our findings provide an insight into the enzyme system of PfMig188 for degradation of complex biomass such as SCB and highlight the importance of adding SCB to the culture medium to optimize the secretion of enzymes specific for the saccharification of sugarcane bagasse.

Lytic Polysaccharide Monooxygenases as Chitin-Specific Virulence Factors in Crayfish Plague.

The oomycete pathogen Aphanomyces astaci, also known as "crayfish plague", is an obligate fungal-like parasite of freshwater crustaceans and is considered responsible for the ongoing decline of native European crayfish populations. A. astaci is thought to secrete a wide array of effectors and enzymes that facilitate infection, however their molecular mechanisms have been poorly characterized. Here, we report the identification of AA15 lytic polysaccharide monooxygenases (LPMOs) as a new group of secreted virulence factors in A. astaci. We show that this enzyme family has greatly expanded in A. astaci compared to all other oomycetes, and that it may facilitate infection through oxidative degradation of crystalline chitin, the most abundant polysaccharide found in the crustacean exoskeleton. These findings reveal new roles for LPMOs in animal-pathogen interactions, and could help inform future strategies for the protection of farmed and endangered species.

Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes.

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.

A multi-omics approach to lignocellulolytic enzyme discovery reveals a new ligninase activity from Parascedosporium putredinis NO1.

Lignocellulose, the structural component of plant cells, is a major agricultural byproduct and the most abundant terrestrial source of biopolymers on Earth. The complex and insoluble nature of lignocellulose limits its conversion into value-added commodities, and currently, efficient transformation requires expensive pretreatments and high loadings of enzymes. Here, we report on a fungus from the Parascedosporium genus, isolated from a wheat-straw composting community, that secretes a large and diverse array of carbohydrate-active enzymes (CAZymes) when grown on lignocellulosic substrates. We describe an oxidase activity that cleaves the major ß-ether units in lignin, thereby releasing the flavonoid tricin from monocot lignin and enhancing the digestion of lignocellulose by polysaccharidase mixtures. We show that the enzyme, which holds potential for the biorefining industry, is widely distributed among lignocellulose-degrading fungi from the Sordariomycetes phylum.

Gel-permeation chromatography-enzyme-linked immunosorbent assay method for systematic mass distribution profiling of plant cell wall matrix polysaccharides.

Cell walls are dynamic and multi-component materials that play important roles in many areas of plant biology. The composition and interactions of the structural elements give rise to material properties, which are modulated by the activity of wall-related enzymes. Studies of the genes and enzymes that determine wall composition and function have made great progress, but rarely take account of potential compensatory changes in wall polymers that may accompany and accommodate changes in other components, particularly for specific polysaccharides. Here, we present a method that allows the simultaneous examination of the mass distributions and quantities of specific cell wall matrix components, allowing insight into direct and indirect consequences of cell wall manipulations. The method employs gel-permeation chromatography fractionation of cell wall polymers followed by enzyme-linked immunosorbent assay to identify polymer types. We demonstrate the potential of this method using glycan-directed monoclonal antibodies to detect epitopes representing xyloglucans, heteromannans, glucuronoxylans, homogalacturonans (HGs) and methyl-esterified HGs. The method was used to explore compositional diversity in different Arabidopsis organs and to examine the impacts of changing wall composition in a number of previously characterized cell wall mutants. As demonstrated in this article, this methodology allows a much deeper understanding of wall composition, its dynamism and plasticity to be obtained, furthering our knowledge of cell wall biology.

Mechanistic strategies of microbial communities regulating lignocellulose deconstruction in a UK salt marsh.

BACKGROUND: Salt marshes are major natural repositories of sequestered organic carbon with high burial rates of organic matter, produced by highly productive native flora. Accumulated carbon predominantly exists as lignocellulose which is metabolised by communities of functionally diverse microbes. However, the organisms that orchestrate this process and the enzymatic mechanisms employed that regulate the accumulation, composition and permanence of this carbon stock are not yet known. We applied meta-exo-proteome proteomics and 16S rRNA gene profiling to study lignocellulose decomposition in situ within the surface level sediments of a natural established UK salt marsh. RESULTS: Our studies revealed a community dominated by Gammaproteobacteria, Bacteroidetes and Deltaproteobacteria that drive lignocellulose degradation in the salt marsh. We identify 42 families of lignocellulolytic bacteria of which the most active secretors of carbohydrate-active enzymes were observed to be Prolixibacteracea, Flavobacteriaceae, Cellvibrionaceae, Saccharospirillaceae, Alteromonadaceae, Vibrionaceae and Cytophagaceae. These families secreted lignocellulose-active glycoside hydrolase (GH) family enzymes GH3, GH5, GH6, GH9, GH10, GH11, GH13 and GH43 that were associated with degrading Spartina biomass. While fungi were present, we did not detect a lignocellulolytic contribution from fungi which are major contributors to terrestrial lignocellulose deconstruction. Oxidative enzymes such as laccases, peroxidases and lytic polysaccharide monooxygenases that are important for lignocellulose degradation in the terrestrial environment were present but not abundant, while a notable abundance of putative esterases (such as carbohydrate esterase family 1) associated with decoupling lignin from polysaccharides in lignocellulose was observed. CONCLUSIONS: Here, we identify a diverse cohort of previously undefined bacteria that drive lignocellulose degradation in the surface sediments of the salt marsh environment and describe the enzymatic mechanisms they employ to facilitate this process. Our results increase the understanding of the microbial and molecular mechanisms that underpin carbon sequestration from lignocellulose within salt marsh surface sediments in situ and provide insights into the potential enzymatic mechanisms regulating the enrichment of polyphenolics in salt marsh sediments. Video Abstract.

Overcoming the trade-off between grain weight and number in wheat by the ectopic expression of expansin in developing seeds leads to increased yield potential.

Wheat is the most widely grown crop globally, providing 20% of all human calories and protein. Achieving step changes in genetic yield potential is crucial to ensure food security, but efforts are thwarted by an apparent trade-off between grain size and number. Expansins are proteins that play important roles in plant growth by enhancing stress relaxation in the cell wall, which constrains cell expansion. Here, we describe how targeted overexpression of an α-expansin in early developing wheat seeds leads to a significant increase in grain size without a negative effect on grain number, resulting in a yield boost under field conditions. The best-performing transgenic line yielded 12.3% higher average grain weight than the control, and this translated to an increase in grain yield of 11.3% in field experiments using an agronomically appropriate plant density. This targeted transgenic approach provides an opportunity to overcome a common bottleneck to yield improvement across many crops.

Design of experiments driven optimization of alkaline pretreatment and saccharification for sugarcane bagasse.

To maximize the sugar release from sugarcane bagasse, a high-resolution Fractional Factorial Design (FFD) was combined with a Central Composite Orthogonal (CCO) design to simultaneously evaluate a wide range of variables for alkaline pretreatment (NaOH: 0.1-1 mol/L, temperature: 100-220 °C, and time: 20-80 min) and enzymatic saccharification (enzyme loading: 2.5-17.5%, and reaction volume: 550-850 µL). A total of 46 experimental conditions were evaluated and the maximum sugar yield (423 mg/g) was obtained after 18 h enzymatic hydrolysis under optimized conditions (0.25 mol/L NaOH at 202 °C for 40 min, with 12.5% of enzyme loading). Biomass compositional analyses showed that the pretreatments strongly removed lignin (up to 70%), silica (up to 80%) and promoted cellulose enrichment (25-110%). This robust design of experiments resulted in maximizing enzymatic hydrolysis efficiency of sugarcane bagasse and further indicated that this combined approach is versatile for other lignocellulosic biomasses.

Biomass composition of the golden tide pelagic seaweeds Sargassum fluitans and S. natans (morphotypes I and VIII) to inform valorisation pathways.

Massive strandings of the pelagic brown algae Sargassum have occurred in the Caribbean, and to a lesser extent, in western Africa, almost every year since 2011. These events have major environmental, health, and economic impacts in the affected countries. Once on the shore, Sargassum is mechanically harvested and disposed of in landfills. Existing commercial applications of other brown algae indicate that the pelagic Sargassum could constitute a valuable feedstock for potential valorisation. However, limited data on the composition of this Sargassum biomass was available to inform on possible application through pyrolysis or enzymatic fractionation of this feedstock. To fill this gap, we conducted a detailed comparative biochemical and elemental analysis of three pelagic Sargassum morphotypes identified so far as forming Atlantic blooms: Sargassum natans I (SnI), S. fluitans III (Sf), and S. natans VIII (SnVIII). Our results showed that SnVIII accumulated a lower quantity of metals and metalloids compared to SnI and Sf, but it contained higher amounts of phenolics and non-cellulosic polysaccharides. SnVIII also had more of the carbon storage compound mannitol. No differences in the content and composition of the cell wall polysaccharide alginate were identified among the three morphotypes. In addition, enzymatic saccharification of SnI produced more sugars compared to SnVIII and Sf. Due to high content of arsenic, the use of pelagic Sargassum is not recommended for nutritional purposes. In addition, low yields of alginate extracted from this biomass, compared with brown algae used for industrial production, limit its use as viable source of commercial alginates. Further work is needed to establish routes for future valorisation of pelagic Sargassum biomass.

Association mapping identifies quantitative trait loci (QTL) for digestibility in rice straw.

BACKGROUND: The conversion of lignocellulosic biomass from agricultural waste into biofuels and chemicals is considered a promising way to provide sustainable low carbon products without compromising food security. However, the use of lignocellulosic biomass for biofuel and chemical production is limited by the cost-effectiveness of the production process due to its recalcitrance to enzymatic hydrolysis and fermentable sugar release (i.e., saccharification). Rice straw is a particularly attractive feedstock because millions of tons are currently burned in the field each year for disposal. The aim of this study was to explore the underlying natural genetic variation that impacts the recalcitrance of rice (Oryza sativa) straw to enzymatic saccharification. Ultimately, we wanted to investigate whether we could identify genetic markers that could be used in rice breeding to improve commercial cultivars for this trait. Here, we describe the development and characterization of a Vietnamese rice genome-wide association panel, high-throughput analysis of rice straw saccharification and lignin content, and the results from preliminary genome-wide association studies (GWAS) of the combined data sets. We identify both QTL and plausible candidate genes that may have an impact on the saccharification of rice straw. RESULTS: We assembled a diversity panel comprising 151 rice genotypes (Indica and Japonica types) from commercial, historical elite cultivars, and traditional landraces grown in Vietnam. The diversity panel was genotyped using genotype by sequencing (GBS) methods yielding a total of 328,915 single nucleotide polymorphisms (SNPs). We collected phenotypic data from stems of these 151 genotypes for biomass saccharification and lignin content. Using GWAS on the indica genotypes over 2 years we identified ten significant QTL for saccharification (digestibility) and seven significant QTL for lignin. One QTL on chromosome 11 occurred in both GWAS for digestibility and for lignin. Seven QTL for digestibility, on CH2, CH6, CH7, CH8, and CH11, were observed in both years of the study. The QTL regions for saccharification include three potential candidate genes that have been previously reported to influence digestibility: OsAT10; OsIRX9; and OsMYB58/63-L. CONCLUSIONS: Despite the difficulties associated with multi-phasic analysis of complex traits in novel germplasm, a moderate resolution GWAS successfully identified genetic associations encompassing both known and/or novel genes involved in determining the saccharification potential and lignin content of rice straw. Plausible candidates within QTL regions, in particular those with roles in cell wall biosynthesis, were identified but will require validation to confirm their value for application in rice breeding.

Eco-evolutionary Dynamics Set the Tempo and Trajectory of Metabolic Evolution in Multispecies Communities.

The eco-evolutionary dynamics of microbial communities are predicted to affect both the tempo and trajectory of evolution in constituent species [1]. While community composition determines available niche space, species sorting dynamically alters composition, changing over time the distribution of vacant niches to which species adapt [2], altering evolutionary trajectories [3, 4]. Competition for the same niche can limit evolutionary potential if population size and mutation supply are reduced [5, 6] but, alternatively, could stimulate evolutionary divergence to exploit vacant niches if character displacement results from the coevolution of competitors [7, 8]. Under more complex ecological scenarios, species can create new niches through their exploitation of complex resources, enabling others to adapt to occupy these newly formed niches [9, 10]. Disentangling the drivers of natural selection within such communities is extremely challenging, and it is thus unclear how eco-evolutionary dynamics drive the evolution of constituent taxa. We tracked the metabolic evolution of a focal species during adaptation to wheat straw as a resource both in monoculture and in polycultures wherein on-going eco-evolutionary community dynamics were either permitted or prevented. Species interactions accelerated metabolic evolution. Eco-evolutionary dynamics drove increased use of recalcitrant substrates by the focal species, whereas greater exploitation of readily digested substrate niches created by other species evolved if on-going eco-evolutionary dynamics were prevented. Increased use of recalcitrant substrates was associated with parallel evolution of tctE, encoding a carbon metabolism regulator. Species interactions and species sorting set, respectively, the tempo and trajectory of evolutionary divergence among communities, selecting distinct ecological functions in otherwise equivalent ecosystems.

Cell wall remodeling under salt stress: Insights into changes in polysaccharides, feruloylation, lignification, and phenolic metabolism in maize.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC-MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.

Robust microorganisms for biofuel and chemical production from municipal solid waste.

BACKGROUND: Worldwide 3.4 billion tonnes of municipal solid waste (MSW) will be produced annually by 2050, however, current approaches to MSW management predominantly involve unsustainable practices like landfilling and incineration. The organic fraction of MSW (OMSW) typically comprises ~ 50% lignocellulose-rich material but is underexplored as a biomanufacturing feedstock due to its highly inconsistent and heterogeneous composition. This study sought to overcome the limitations associated with studying MSW-derived feedstocks by using OMSW produced from a realistic and reproducible MSW mixture on a commercial autoclave system. The resulting OMSW fibre was enzymatically hydrolysed and used to screen diverse microorganisms of biotechnological interest to identify robust species capable of fermenting this complex feedstock. RESULTS: The autoclave pre-treated OMSW fibre contained a polysaccharide fraction comprising 38% cellulose and 4% hemicellulose. Enzymatic hydrolysate of OMSW fibre was high in D-glucose (5.5% w/v) and D-xylose (1.8%w/v) but deficient in nitrogen and phosphate. Although relatively low levels of levulinic acid (30 mM) and vanillin (2 mM) were detected and furfural and 5-hydroxymethylfurfural were absent, the hydrolysate contained an abundance of potentially toxic metals (0.6% w/v). Hydrolysate supplemented with 1% yeast extract to alleviate nutrient limitation was used in a substrate-oriented shake-flask screen with eight biotechnologically useful microorganisms (Clostridium saccharoperbutylacetonicum, Escherichia coli, Geobacillus thermoglucosidasius, Pseudomonas putida, Rhodococcus opacus, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Zymomonas mobilis). Each species' growth and productivity were characterised and three species were identified that robustly and efficiently fermented OMSW fibre hydrolysate without significant substrate inhibition: Z. mobilis, S. cerevisiae and R. opacus, respectively produced product to 69%, 70% and 72% of the maximum theoretical fermentation yield and could theoretically produce 136 kg and 139 kg of ethanol and 91 kg of triacylglycerol (TAG) per tonne of OMSW. CONCLUSIONS: Developing an integrated biorefinery around MSW has the potential to significantly alleviate the environmental burden of current waste management practices. Substrate-oriented screening of a representative and reproducible OMSW-derived fibre identified microorganisms intrinsically suited to growth on OMSW hydrolysates. These species are promising candidates for developing an MSW biorefining platform and provide a foundation for future studies aiming to valorise this underexplored feedstock.

In silico evolution of Aspergillus niger organic acid production suggests strategies for switching acid output.

BACKGROUND: The fungus Aspergillus niger is an important industrial organism for citric acid fermentation; one of the most efficient biotechnological processes. Previously we introduced a dynamic model that captures this process in the industrially relevant batch fermentation setting, providing a more accurate predictive platform to guide targeted engineering. In this article we exploit this dynamic modelling framework, coupled with a robust genetic algorithm for the in silico evolution of A. niger organic acid production, to provide solutions to complex evolutionary goals involving a multiplicity of targets and beyond the reach of simple Boolean gene deletions. We base this work on the latest metabolic models of the parent citric acid producing strain ATCC1015 dedicated to organic acid production with the required exhaustive genomic coverage needed to perform exploratory in silico evolution. RESULTS: With the use of our informed evolutionary framework, we demonstrate targeted changes that induce a complete switch of acid output from citric to numerous different commercially valuable target organic acids including succinic acid. We highlight the key changes in flux patterns that occur in each case, suggesting potentially valuable targets for engineering. We also show that optimum acid productivity is achieved through a balance of organic acid and biomass production, requiring finely tuned flux constraints that give a growth rate optimal for productivity. CONCLUSIONS: This study shows how a genome-scale metabolic model can be integrated with dynamic modelling and metaheuristic algorithms to provide solutions to complex metabolic engineering goals of industrial importance. This framework for in silico guided engineering, based on the dynamic batch growth relevant to industrial processes, offers considerable potential for future endeavours focused on the engineering of organisms to produce valuable products.