Overexpression of a Prefoldin β subunit gene reduces biomass recalcitrance in the bioenergy crop Populus.

Prefoldin (PFD) is a group II chaperonin that is ubiquitously present in the eukaryotic kingdom. Six subunits (PFD1-6) form a jellyfish-like heterohexameric PFD complex and function in protein folding and cytoskeleton organization. However, little is known about its function in plant cell wall-related processes. Here, we report the functional characterization of a PFD gene from Populus deltoides, designated as PdPFD2.2. There are two copies of PFD2 in Populus, and PdPFD2.2 was ubiquitously expressed with high transcript abundance in the cambial region. PdPFD2.2 can physically interact with DELLA protein RGA1_8g, and its subcellular localization is affected by the interaction. In P. deltoides transgenic plants overexpressing PdPFD2.2, the lignin syringyl/guaiacyl ratio was increased, but cellulose content and crystallinity index were unchanged. In addition, the total released sugar (glucose and xylose) amounts were increased by 7.6% and 6.1%, respectively, in two transgenic lines. Transcriptomic and metabolomic analyses revealed that secondary metabolic pathways, including lignin and flavonoid biosynthesis, were affected by overexpressing PdPFD2.2. A total of eight hub transcription factors (TFs) were identified based on TF binding sites of differentially expressed genes in Populus transgenic plants overexpressing PdPFD2.2. In addition, several known cell wall-related TFs, such as MYB3, MYB4, MYB7, TT8 and XND1, were affected by overexpression of PdPFD2.2. These results suggest that overexpression of PdPFD2.2 can reduce biomass recalcitrance and PdPFD2.2 is a promising target for genetic engineering to improve feedstock characteristics to enhance biofuel conversion and reduce the cost of lignocellulosic biofuel production

Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production

Background Lignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, “absent technological breakthroughs”, it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022. We here describe the properties of switchgrass (Panicum virgatum) biomass that has been genetically engineered to increase the cellulosic ethanol yield by more than 2-fold. Results We have increased the cellulosic ethanol yield from switchgrass by 2.6-fold through overexpression of the transcription factor PvMYB4. This strategy reduces carbon deposition into lignin and phenolic fermentation inhibitors while maintaining the availability of potentially fermentable soluble sugars and pectic polysaccharides. Detailed biomass characterization analyses revealed that the levels and nature of phenolic acids embedded in the cell-wall, the lignin content and polymer size, lignin internal linkage levels, linkages between lignin and xylans/pectins, and levels of wall-bound fucose are all altered in PvMYB4-OX lines. Genetically engineered PvMYB4-OX switchgrass therefore provides a novel system for further understanding cell wall recalcitrance. Conclusions Our results have demonstrated that overexpression of PvMYB4, a general transcriptional repressor of the phenylpropanoid/lignin biosynthesis pathway, can lead to very high yield ethanol production through dramatic reduction of recalcitrance. MYB4-OX switchgrass is an excellent model system for understanding recalcitrance, and provides new germplasm for developing switchgrass cultivars as biomass feedstocks for biofuel production. Keywords: Switchgrass; Bioenergy; Biofuel; Feedstock; Cellulosic ethanol; PvMYB4; Transcription factor; Cell wall; Recalcitrance; Lignin; Hemicellulose; Pecti

Down-regulation of the caffeic acid \u3cem\u3eO\u3c/em\u3e-methyltransferase gene in switchgrass reveals a novel monolignol analog

Background Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors. Results GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples. Conclusions Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures

Combining loss of function of FOLYLPOLYGLUTAMATE SYNTHETASE1 and CAFFEOYL-COA 3-O-METHYLTRANSFERASE1 for lignin reduction and improved saccharification efficiency in Arabidopsis thaliana

This article tests if lignin content can be further reduced by combining genetic mutations in C1 metabolism and the lignin biosynthetic pathway by generating and functionally characterizing fpgs1ccoaomt1 double mutants. The observations demonstrate that additional reduction in lignin content and improved sugar release can be achieved by simultaneous downregulation of a gene in the C1 (FPGS1) and lignin biosynthetic (CCOAOMT) pathways

Determination of hydroxyl groups in biorefinery resources via quantitative 31P NMR spectroscopy

The analysis of chemical structural characteristics of biorefinery product streams (such as lignin and tannin) has advanced substantially over the past decade, with traditional wet-chemical techniques being replaced or supplemented by NMR methodologies. Quantitative 31P NMR spectroscopy is a promising technique for the analysis of hydroxyl groups because of its unique characterization capability and broad potential applicability across the biorefinery research community. This protocol describes procedures for (i) the preparation/solubilization of lignin and tannin, (ii) the phosphitylation of their hydroxyl groups, (iii) NMR acquisition details, and (iv) the ensuing data analyses and means to precisely calculate the content of the different types of hydroxyl groups. Compared with traditional wet-chemical techniques, the technique of quantitative 31P NMR spectroscopy offers unique advantages in measuring hydroxyl groups in a single spectrum with high signal resolution. The method provides complete quantitative information about the hydroxyl groups with small amounts of sample (~30 mg) within a relatively short experimental time (~30-120 min)

Membrane fouling by lysozyme: Effect of local interaction

Molecular dynamics simulations were performed to understand the adsorption of positive-charged lysozyme onto negative-charged polyvinylidene fluoride membrane at three pH and two ionic strengths. The lysozyme was initialized at six orientations at 10 Å from the membrane and adsorption was deemed to occur when the lysozyme is less than 4 Å from the membrane. Local interactions are clearly important: (i) despite opposite net charges, no adsorption occurs in some cases throughout; (ii) lysozyme-membrane separation distances differ among the initial orientations of lysozyme; and (iii) the correlation between lysozyme-membrane interaction energy and adsorption probability is poor. Correspondingly, seven key local adsorption sites on lysozyme were identified. Of the two most common sites, one anchors due to electrostatics, while the other is not electrostatics-based. Also, different sites are dominant in different feed conditions and have different interaction energies. These results reveal the importance of local interactions in membrane fouling by proteins in different feed conditions.Agency for Science, Technology and Research (A*STAR)Economic Development Board (EDB)Ministry of Education (MOE)This work was supported by the Singapore GSK (GlaxoSmithKline)—EDB (Economic Development Board, Singapore) Trust Fund, A*STAR (Singapore) Advanced Manufacturing and Engineering (AME) under its Pharma Innovation Programme Singapore (PIPS) program (A20B3a0070) and Individual Research Grant (IME) program (A2083c0049), and the Singapore Ministry of Education Tier 1 Grant (2019-T1-002-065). The computations were fully performed on resources of the National Super-computing Centre, Singapore (https://www.nscc.sg)

Internal membrane fouling by proteins during microfiltration

The current study aimed to understand both external and internal membrane fouling by three proteins with different net charges, namely, negatively charged pepsin and bovine serum albumin (BSA), as well as positively charged lysozyme. Polycarbonate track-etched (PCTE) membranes were used. Per electrostatic attraction, the flux decline was the worst for lysozyme, which is attributed by the fouling model to the greatest pore blockage (α) and pore constriction (β), and by field-emission scanning electron microscope (FESEM) and optical coherence tomography (OCT) to the most extensive external fouling. Between pepsin and BSA, BSA gave worse flux decline despite its more negative net charge. The fouling model indicates that BSA gave greater pore blockage (α) and denser internal cake (Rc/Rm), while the quartz crystal microbalance with dissipation (QCM-D) indicates a rigid cake structure. Notably, despite monotonic flux decline with filtration, the OCT fouling voxel trends show significant fluctuations, which has not been reported before and thus signify the unique behavior of protein foulants in straight-through pores. Specifically, the trends below and above the −4.5 μm layer (i.e., 4.5 μm below the feed-membrane interface) are perfectly opposite, indicating the non-uniform protein deposits slipping downwards in the membrane pores as filtration progressed. The dynamic movements of the protein cakes unveiled here warrant more understanding in future studies.Agency for Science, Technology and Research (A*STAR)Economic Development Board (EDB)Ministry of Education (MOE)We acknowledge funding from the Singapore GSK (GlaxoSmithKline)-EDB (Economic Development Board) Trust Fund, A*STAR (Singapore) Advanced Manufacturing and Engineering (AME) under its Pharma Innovation Programme Singapore (PIPS) program (A20B3a0070), A*STAR (Singapore) Advanced Manufacturing and Engineering (AME) under its Individual Research Grant (IRG) program (A2083c0049), the Singapore Ministry of Education Academic Research Fund Tier 1 Grant (2019-T1-002-065; RG100/19), and the Singapore Ministry of Education Tier 2 Academic Research Fund (MOE-MOET2EP10120-0001)

A review of membrane fouling by proteins in ultrafiltration and microfiltration

Fouling of ultrafiltration (UF) and microfiltration (MF) membranes by proteins is a major challenge in the bioprocessing and dairy industries, as well as in surface and wastewater treatment applications. This review attempts at presenting a comprehensive state-of-the-art understanding on protein fouling of membranes. Effects of operating conditions, along with properties of proteins and membranes, are discussed. Various tools and techniques used to characterize and monitor fouling are described. Different mitigation techniques and cleaning methods used are also presented. Two main factors have been identified as playing important roles in governing protein fouling, namely, ratio of protein size to membrane pore size and interfacial interactions (i.e., protein-protein and protein-membrane). Some directions for future research are suggested: (1) explore a wider range of proteins and their mixtures with respect to their fouling tendencies; and (2) create a comprehensive dataset that can be used to develop machine-learning models to enhance both predictive capabilities and mechanistic understanding.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)This study was supported by A*STAR (Singapore) Advanced Manufacturing and Engineering (AME) under its Pharma Innovation Programme Singapore (PIPS) (A20B3a0070); A*STAR (Singapore) Advanced Manufacturing and Engineering (AME) under its Individual Research Grant (IRG) program (A2083c0049); the Singapore Ministry of Education Academic Research Tier 1 Grant (2019-T1-002-065; RG100/ 19) and the Singapore Ministry of Education Academic Research Tier 2 Grant (MOE-MOET2EP10120-0001)