Bio-Producing Bacterial Cellulose Filaments through Co-Designing with Biological Characteristics.

The need for circular textiles has led to an interest in the production of biologically derived materials, generating new research into the bioproduction of textiles through design and interdisciplinary approaches. Bacterial cellulose has been produced directly from fermentation into sheets but not yet investigated in terms of producing filaments directly from fermentation. This leaves a wealth of material qualities unexplored. Further, by growing the material directly into filaments, production such as wet spinning are made redundant, thus reducing textile manufacturing steps. The aim of this study was to grow the bio-material, namely bacterial cellulose directly into a filament. This was achieved using a method of co-designing with the characteristics of biological materials. The method combines approaches of material-driven textile design and human-centred co-design to investigate co-designing with the characteristics of living materials for biological material production. The project is part of a wider exploration of bio-manufacturing textiles from waste. The practice-based approach brought together biological sciences and material design through a series of iterative experiments. This, in turn, resulted in designing with the inherent characteristics of bacterial cellulose, and by doing so filaments were designed to be fabricated directly from fermentation. In this investigation, creative exploration was encouraged within a biological laboratory space, showing how interdisciplinary collaboration can offer innovative alternative bioproduction routes for textile filament production.

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.

Responses of Lotus corniculatus to environmental change 3: The sensitivity of phenolic accumulation to growth temperature and light intensity and effects on tissue digestibility.

MAIN CONCLUSION: Growth temperature and light intensity are major drivers of phenolic accumulation in Lotus corniculatus resulting in major changes in carbon partitioning which significantly affects tissue digestibility and forage quality. The response of plant growth, phenolic accumulation and tissue digestibility to light and temperature was determined in clonal plants of three genotypes of Lotus corniculatus (birdsfoot trefoil) cv Leo, with low, intermediate or high levels of proanthocyanidins (condensed tannins). Plants were grown from 10 °C to 30 °C, or at light intensities from 20 to 500 µm m-2 s-1. Plants grown at 25 °C had the highest growth rate and highest digestibility, whereas the maximum tannin concentration was found in plants grown at 15 °C. Approximately linear increases in leaf flavonol glycoside levels were found with increasing growth temperature in the low tannin genotype. Tannin hydroxylation increased with increasing growth temperature but decreased with increasing light intensity. The major leaf flavonols were kaempferol glycosides of which kaempferol-3-glucoside and kaempferol-3,7-dirhamnoside were the major components. Increases in both tannin and total flavonol concentrations in leaves were linearly related to light intensity and were preceded by a specific increase in the transcript level of a non-legume type chalcone isomerase. Changes in growth temperature and light intensity, therefore, result in major changes in the partitioning of carbon into phenolics, which significantly affects tissue digestibility and nutritional quality with a high correlation between tannin concentration and leaf digestibility.

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.

Redirection of flux through the phenylpropanoid pathway by increased glucosylation of soluble intermediates.

The phenylpropanoid pathway is used in biosynthesis of a wide range of soluble secondary metabolites including hydroxycinnamic acid esters, flavonoids and the precursors of lignin and lignans. In Arabidopsis thaliana a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases (GTs) that glucosylate phenylpropanoids in vitro. This study explores the effect of constitutively over-expressing two of these GTs (UGT72E1 and E3) in planta using the CaMV-35S promoter to determine whether phenylpropanoid homeostasis can be altered in a similar manner to that achieved by over-expression of UGT72E2 as previously reported. The data show that impact of over-expressing UGT72E3 in leaves is highly similar to that of UGT72E2 in that the production of massive levels of coniferyl and sinapyl alcohol 4-O-glucosides and a substantial loss in sinapoyl malate. In contrast, the over-expression of UGT72E1 in leaves led only to minimal changes in coniferyl alcohol 4-O-glucoside and no effect was observed on sinapoyl malate levels. In roots, over-expression of both UGTs led to some increase in the accumulation of the two glucosides. The cell specificity expression of the whole UGT72E gene cluster was investigated and interestingly only UGT72E3 was found to be wound and touch responsive.

The glucosyltransferase UGT72E2 is responsible for monolignol 4-O-glucoside production in Arabidopsis thaliana.

The phenylpropanoid pathway in plants leads to the synthesis of a wide range of soluble secondary metabolites, many of which accumulate as glycosides. In Arabidopsis, a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases shown to glucosylate several phenylpropanoids in vitro, including monolignols, hydroxycinnamic acids and hydroxycinnamic aldehydes. The role of these genes in planta has now been investigated through genetically downregulating the expression of individual genes or silencing the entire cluster. Analysis of these transgenic Arabidopsis plants showed that the levels of coniferyl and sinapyl alcohol 4-O-glucosides that accumulate in light-grown roots were significantly reduced. A 50% reduction in both glucosides was observed in plants in which UGT72E2 was downregulated, whereas silencing the three genes led to a 90% reduction, suggesting some redundancy of function within the cluster. The gene encoding UGT72E2 was constitutively overexpressed in transgenic Arabidopsis to determine whether increased glucosylation of monolignols could influence flux through the soluble phenylpropanoid pathway. Elevated expression of UGT72E2 led to increased accumulation of monolignol glucosides in root tissues and also the appearance of these glucosides in leaves. In particular, coniferyl alcohol 4-O-glucoside accumulated to massive amounts (10 micromol g(-1) FW) in root tissues of these plants. Increased glucosylation of other phenylpropanoids also occurred in plants overexpressing this glycosyltransferase. Significantly changing the pattern of glycosides in the leaves also led to a pronounced change in accumulation of the hydroxycinnamic ester sinapoyl malate. The data demonstrate the plasticity of phenylpropanoid metabolism and the important role that glucosylation of secondary metabolites can play in cellular homeostasis.

Co-ordinate regulation of sterol biosynthesis enzyme activity during accumulation of sterols in developing rape and tobacco seed.

The activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol methyl transferase 1 and sterol acyltransferase, key enzymes involved in phytosterol biosynthesis were shown to be co-ordinately regulated during oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.) seed development. In both plants, enzyme activities were low during the initial stages of seed development, increasing towards mid-maturation where they remained stable for a time, before declining rapidly as the oilseeds reached maturity. During seed development, the level of total sterols increased 12-fold in tobacco and 9-fold in rape, primarily due to an increase in steryl ester production. In both seed tissues, stages of maximum enzyme activity coincided with periods of high rates of sterol production, indicating developmental regulation of the enzymes to be responsible for the increases in the sterol content observed during seed development. Consistent with previous studies the data presented suggest that sterol biosynthesis is regulated by two key steps, although there may be others. The first is the regulation of carbon flux into the isoprenoid pathway to cycloartenol. The second is the flux from cycloartenol to Delta(5)-end-product sterols. The implications of the results in terms of enhancing seed sterol levels by genetic modification are also discussed.

Enhancement of seed phytosterol levels by expression of an N-terminal truncated Hevea brasiliensis (rubber tree) 3-hydroxy-3-methylglutaryl-CoA reductase.

Dietary intake of phytosterols (plant sterols) has been shown to be effective in reducing blood cholesterol levels, thereby reducing the risk of cardiovascular disease. Phytosterols are most commonly sourced from vegetable oils, where they are present as minor components. We report here the generation of transgenic tobacco seeds substantially enhanced in phytosterol content by the expression of a modified form of one of the key sterol biosynthetic enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). The constitutive expression of an N-terminal truncated Hevea brasiliensis HMGR (t-HMGR), lacking the membrane binding domain, enhanced seed HMGR activities by 11-fold, leading to increases in total seed sterol of 2.4-fold. Seed-specific expression of t-HMGR enhanced total seed sterol levels by 3.2-fold, to 1.36% dry weight or 3.25% of oil. 4-desmethylsterols were increased by 2.2-fold, whilst certain sterol biosynthetic intermediates, in particular cycloartenol and 24-ethylidene lophenol, also accumulated. The additional sterol in seed tissue was present in the form of fatty acid esters. Constitutive expression of t-HMGR increased leaf phytosterol sterol levels by 10-fold, representing 1.8% dry weight, and the sterol was sequestered, in acyl ester form, as cytoplasmic 'oil droplets'. These studies establish HMGR as a key enzyme controlling overall flux into the sterol biosynthesis pathway in seed tissue, but the accumulation of certain intermediates suggests additional slow steps in the pathway. The expression of an N-truncated HMGR activity has generated novel phytosterol-enriched raw materials that may provide the basis of new sourcing opportunities for this important class of cholesterol-lowering actives.