Secondary metabolite pathway of SDG (secoisolariciresinol) was observed to trigger ROS scavenging system in response to Ca2+ stress in cotton.

Ca2+ is an essential nutrient for plants and animals which plays an important role in plant signal transduction. Although the function and regulation of mechanism of Ca2+ in alleviating various biotic and abiotic stresses in plants have been studied deeply, the molecular mechanism to adapt high Ca2+ stress is still unclear in cotton. In this study, 103 cotton accessions were germinated under 200 mM CaCl2 stress, and two extremely Ca2+-resistant (Zhong 9807, R) and Ca2+-sensitive (CRI 50, S) genotypes were selected from 103 cotton accessions. The two accessions were then germinated for 5 days in 0 mM CaCl2 and 200 mM CaCl2 respectively, after which they were sampled for transcriptome sequencing. Morphological and physiological analyses suggested that PLR2 specifically expressed in R may enhance the ability of cotton to scavenge ROS by promoting the synthesis of SDG. In conclusion, this study proposed the adaptation mechanisms to response to the high Ca2+ stress in cotton which can contribute to improve the stress resistance of cotton.

Techno-economic risk assessment, life cycle analysis and life cycle costing for poly(butylene succinate) and poly(lactic acid) production using renewable resources.

The sustainable production of poly(lactic acid) (PLA) or poly(butylene succinate) (PBS) from corn glucose syrup, corn stover and sugar beet pulp (SBP) have been assessed via process design, preliminary techno-economic evaluation, life cycle assessment and life cycle costing (LCC). Cost-competitive PLA and PBS production can be achieved in a SBP-based biorefinery, including separation of crude pectin-rich extract as co-product, leading to minimum selling prices of $1.14/kgPLA and $1.37/kgPBS. Acidification Potential, Eutrophication Potential and Human Toxicity Potential are lower when SBP is used. The LCC of PLA ($1.42/kgPLA) and PBS ($1.72/kgPBS) production from SBP are lower than biaxial oriented polypropylene (BOPP, $1.66/kg) and general purpose polystyrene (GPPS, $2.04/kg) at pectin-rich extract market prices of $3/kg and $4/kg, respectively. Techno-economic risk assessment via Monte-Carlo simulations showed that PLA and PBS could be produced from SBP at the market prices of BOPP ($1.4/kg) and GPPS ($1.72/kg) with 100% probability to achieve a positive Net Present Value at pectin-rich extract market prices of $3/kg and $4/kg, respectively. This study demonstrated that SBP-based biorefinery development ensures sustainable production of PLA and PBS as compared to fossil-derived counterparts and single product bioprocesses using glucose syrup and corn stover.

Assessment of a Small Molecule Synthetic Lignan in Enhancing Oxidative Balance and Decreasing Lipid Accumulation in Human Retinal Pigment Epithelia.

Visual function depends on the intimate structural, functional and metabolic interactions between the retinal pigment epithelium (RPE) and the neural retina. The daily phagocytosis of the photoreceptor outer segment tips by the overlaying RPE provides essential nutrients for the RPE itself and photoreceptors through intricate metabolic synergy. Age-related retinal changes are often characterized by metabolic dysregulation contributing to increased lipid accumulation and peroxidation as well as the release of proinflammatory cytokines. LGM2605 is a synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant and anti-inflammatory properties demonstrated in diverse in vitro and in vivo inflammatory disease models. In these studies, we tested the hypothesis that LGM2605 may be an attractive small-scale therapeutic that protects RPE against inflammation and restores its metabolic capacity under lipid overload. Using an in vitro model in which loss of the autophagy protein, LC3B, results in defective phagosome degradation and metabolic dysregulation, we show that lipid overload results in increased gasdermin cleavage, IL-1 ß release, lipid accumulation and decreased oxidative capacity. The addition of LGM2605 resulted in enhanced mitochondrial capacity, decreased lipid accumulation and amelioration of IL-1 ß release in a model of defective lipid homeostasis. Collectively, these studies suggest that lipid overload decreases mitochondrial function and increases the inflammatory response, with LGM2605 acting as a protective agent.

In vitro toxicity assessment of crosslinking agents used in hyaluronic acid dermal filler.

Hyaluronic acid (HA) dermal fillers are produced by crosslinking HA with agents, such as 1,4-butanediol diglycidyl ether (BDDE) and poly (ethylene glycol) diglycidyl ether (PEGDE) to acquire desired properties. Thus, the safety evaluation of these crosslinkers is needed at the cellular level. In the present study, cell viability, cytotoxicity, membrane integrity, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and inflammatory responses were evaluated in the human keratinocyte cell line, HaCaT and human dermal fibroblast cell line, HDF in response to treatment with the crosslinkers. In both the cell lines, BDDE significantly decreased cell viability at 100-1000 ppm, while PEGDE showed a decrease at 500-1000 ppm. In HaCaT cells, BDDE markedly increased cytotoxicity (lactate dehydrogenase release) at 100-1000 ppm, but PEGDE showed an increase at 500-1000 ppm. Cells treated with BDDE (100 ppm) caused alteration in the integrity of cell membrane and shape. In both the cell lines, BDDE-treated cells showed significantly higher ROS levels and MMP loss than PEGDE-treated cells. Also, BDDE-treated cells exhibited higher COX-2 expression at 100 ppm. Expression of inflammatory cytokines (TNF-α, and IL-1 ß) was higher in BDDE-treated cells. Taken together, PEGDE-treated cells showed markedly lower cytotoxicity, ROS production, and inflammatory responses than BDDE-treated cells. Our data suggest that PEGDE is safer than BDDE as a crosslinker in HA dermal fillers.

A newly isolated Enterobacter sp. strain produces 2,3-butanediol during its cultivation on low-cost carbohydrate-based substrates.

2,3-Butanediol (BDO) is an important platform chemical with a wide range of applications in various industries. In the present study, a newly isolated wild Enterobacter sp. strain (FMCC-208) was evaluated towards its ability to produce BDO on media composed of sugars derived from sucrose refinery plant. Optimum values of temperature and pH as well as substrate inhibition were determined through batch experiments. The ability of the strain to convert various monosaccharides was also investigated. Maximum BDO concentrations of 90.3 and 10 g l-1 of acetoin were obtained during a fed-batch bioreactor experiment with cane molasses and sucrose employed as substrates. A high volumetric productivity was noted in a fed-batch experiment using molasses and sucrose as carbon sources at T = 37°C, in which 73.0 g l-1 of BDO together with 12.4 g l-1 of acetoin was produced where 1.15 g l-1 h-1 of diol/acetoin was produced. In previously pasteurized media, 70.0 g l-1 of BDO and 5.0 g l-1 of acetoin were produced (yield = 0.39 g g-1). Finally, besides BDO production, growth on molasses was accompanied by non-negligible decolorization (25-35%) of the residue. Therefore, the strain is a promising candidate for the conversion of sucrose-based materials into BDO.

Improvement on bioprocess economics for 2,3-butanediol production from very high polarity cane sugar via optimisation of bioreactor operation.

This study focuses on the optimisation of 2,3-butanediol (BDO) production in fed-batch cultures carried out with the bacterial strain Enterobacter ludwigii using very high polarity (VHP) sugar from sugarcane mills. Various kLa values were evaluated using either complex or synthetic fermentation media demonstrating that the latter enhance BDO production efficiency with low by-product formation. The pH (6.3) and temperature (33.9 °C) employed in fed-batch bioreactor cultures has been optimised via experimental design. Fed-batch cultures carried out at the optimum temperature and pH and varying kLa values resulted in BDO concentration, yield and productivity of 86.8 g/L, 0.37 g/g and 3.95 g L-1 h-1. Using this fermentation efficiency, the minimum selling price of BDO for annual production capacities of 10,000 t and 50,000 t was estimated at $3.12/kg and $2.67/kg, respectively, for a VHP cane sugar market price of $0.4/kg.

Engineered E. coli W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis.

BACKGROUND: Efficient microbial production of chemicals is often hindered by the cytotoxicity of the products or by the pathogenicity of the host strains. Hence 2,3-butanediol, an important drop-in chemical, is an interesting alternative target molecule for microbial synthesis since it is non-cytotoxic. Metabolic engineering of non-pathogenic and industrially relevant microorganisms, such as Escherichia coli, have already yielded in promising 2,3-butanediol titers showing the potential of microbial synthesis of 2,3-butanediol. However, current microbial 2,3-butanediol production processes often rely on yeast extract as expensive additive, rendering these processes infeasible for industrial production. RESULTS: The aim of this study was to develop an efficient 2,3-butanediol production process with E. coli operating on the premise of using cost-effective medium without complex supplements, considering second generation feedstocks. Different gene donors and promoter fine-tuning allowed for construction of a potent E. coli strain for the production of 2,3-butanediol as important drop-in chemical. Pulsed fed-batch cultivations of E. coli W using microaerobic conditions showed high diol productivity of 4.5 g l-1 h-1. Optimizing oxygen supply and elimination of acetoin and by-product formation improved the 2,3-butanediol titer to 68 g l-1, 76% of the theoretical maximum yield, however, at the expense of productivity. Sugar beet molasses was tested as a potential substrate for industrial production of chemicals. Pulsed fed-batch cultivations produced 56 g l-1 2,3-butanediol, underlining the great potential of E. coli W as production organism for high value-added chemicals. CONCLUSION: A potent 2,3-butanediol producing E. coli strain was generated by considering promoter fine-tuning to balance cell fitness and production capacity. For the first time, 2,3-butanediol production was achieved with promising titer, rate and yield and no acetoin formation from glucose in pulsed fed-batch cultivations using chemically defined medium without complex hydrolysates. Furthermore, versatility of E. coli W as production host was demonstrated by efficiently converting sucrose from sugar beet molasses into 2,3-butanediol.

Screening of novel bacteria for the 2,3-butanediol production.

Biotechnologically produced 2,3-butanediol (2,3-BDO) is a potential starting material for industrial bulk chemicals such as butadiene or methyl ethyl ketone which are currently produced from fossil feedstocks. So far, the highest 2,3-BDO concentrations have been obtained with risk group 2 microorganisms. In this study, three risk group 1 microorganisms are presented that are so far unknown for an efficient production of 2,3-BDO. The strains Bacillus atrophaeus NRS-213, Bacillus mojavensis B-14698, and Bacillus vallismortis B-14891 were evaluated regarding their ability to produce high 2,3-BDO concentrations with a broad range of different carbon sources. A maximum 2,3-BDO concentration of 60.4 g/L was reached with the strain B. vallismortis B-14891 with an initial glucose concentration of 200 g/L within 55 h in a batch cultivation. Besides glucose, B. vallismortis B-14891 converts 14 different substrates that can be obtained from residual biomass sources to 2,3-BDO. Therefore B. vallismortis B-14891 is a promising candidate for the large-scale production of 2,3-BDO with low-cost substrates.

Strategies for efficient and economical 2,3-butanediol production: new trends in this field.

2,3-Butanediol (2,3-BD) is a promising bulk chemical with a potentially wide range of applications e.g., in the manufacture of printing inks, perfumes, synthetic rubber, fumigants, antifreeze agents, fuel additives, foodstuffs and pharmaceuticals. Its high heating value and ability to increase the octane number of fuels make 2,3-BD a promising drop-in fuel. It can also be converted to methyl-ethyl ketone (MEK), which is considered an effective liquid fuel additive. After combination with MEK and hydrogenation reaction, 2,3-BD can be converted to octane, which is used to produce high-quality aviation fuel. Currently 2,3-BD is mainly produced on an industrial scale by chemical methods. However, microbiological production of 2,3-BD offers a less expensive and more environmentally friendly alternative to traditional synthesis. This alcohol is generated from hexoses and pentoses mainly by bacterial strains of the genera Klebsiella, Bacillus, Serratia, and Enterobacter, which can convert waste products (such as glycerol and agricultural residues) and excess biomass (such as wood hydrolysates) to 2,3-BD. Recently, a significant improvement in microbial production has been achieved by the screening of efficient natural microbial strains, the application of alternative cost-effective substrates, and the genetic improvement of microbial producers. Furthermore, Klebsiella strains, which are regarded the most efficient natural 2,3-BD producers, have been subjected to genetic modifications aiming at the removal of pathogenic factors and the development of avirulent strains that could be used for the safe production of the diol. This review summarizes existing knowledge and experience concerning various strategies for efficient and economical microbial production of 2,3-BD.

Development of a commercial scale process for production of 1,4-butanediol from sugar.

A sustainable bioprocess for the production of 1,4-butanediol (BDO) from carbohydrate feedstocks was developed. BDO is a chemical intermediate that goes into a variety of products including automotive parts, electronics, and apparel, and is currently manufactured commercially through energy-intensive petrochemical processes using fossil raw materials. This review highlights the development of an Escherichia coli strain and an overall process that successfully performed at commercial scale for direct production of bio-BDO from dextrose. Achieving such high level performance required an integrated technology platform enabling detailed engineering of enzyme, pathway, metabolic network, and organism, as well as development of effective fermentation and downstream recovery processes.

Techno-economic evaluation of a complete bioprocess for 2,3-butanediol production from renewable resources.

This study presents the techno-economic evaluation of 2,3-butanediol (BDO) production via fermentation using glycerol, sucrose and sugarcane molasses as carbon sources. Literature-cited experimental data were used to design the fermentation stage, whereas downstream separation of BDO was based on reactive extraction of BDO employing an aldehyde to convert BDO into an acetal that is immiscible with water. The selected downstream process can be used in all fermentations employed. Sensitivity analysis was carried out targeting the estimation of the minimum selling price (MSP) of BDO at different plant capacities and raw material purchase costs. In all cases, the MSP of BDO is higher than 1 $/kg that is considered as the target in order to characterize a fermentation product as platform chemical. The complex nutrient supplements, the raw material market price and the fermentation efficiency were identified as the major reasons for the relatively high MSP observed.

Chitosan/polyester-based scaffolds for cartilage tissue engineering: assessment of extracellular matrix formation.

Naturally derived polymers have been extensively used in scaffold production for cartilage tissue engineering. The present work aims to evaluate and characterize extracellular matrix (ECM) formation in two types of chitosan-based scaffolds, using bovine articular chondrocytes (BACs). The influence of these scaffolds' porosity, as well as pore size and geometry, on the formation of cartilagineous tissue was studied. The effect of stirred conditions on ECM formation was also assessed. Chitosan-poly(butylene succinate) (CPBS) scaffolds were produced by compression moulding and salt leaching, using a blend of 50% of each material. Different porosities and pore size structures were obtained. BACs were seeded onto CPBS scaffolds using spinner flasks. Constructs were then transferred to the incubator, where half were cultured under stirred conditions, and the other half under static conditions for 4 weeks. Constructs were characterized by scanning electron microscopy, histology procedures, immunolocalization of collagen type I and collagen type II, and dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Both materials showed good affinity for cell attachment. Cells colonized the entire scaffolds and were able to produce ECM. Large pores with random geometry improved proteoglycans and collagen type II production. However, that structure has the opposite effect on GAG production. Stirred culture conditions indicate enhancement of GAG production in both types of scaffold.

Development of an industrial medium for economical 2,3-butanediol production through co-fermentation of glucose and xylose by Klebsiella oxytoca.

An industrial medium containing urea as a sole nitrogen source, low levels of corn steep liquor and mineral salts as nutrition factors to retain high 2,3-butanediol production through co-fermentation of glucose and xylose (2:1, wt/wt) by Klebsiella oxytoca was developed. Urea and corn steep liquor were identified as the most significant factors by the two-level Plackett-Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors and a central composite design was employed to determine their optimal levels. Under the optimal medium, the yield of 2,3-butanediol plus acetoin relative to glucose and xylose was up to 0.428 g/g, which was 85.6% of theoretical value. The cheap nitrogen source and nutrition factors combining the co-fermentation process using lignocellulose derived glucose and xylose as the carbon source in the developed medium would be a potential solution to improve the economics of microbial 2,3-butanediol production.

Microbial production of 2,3-butanediol from Jerusalem artichoke tubers by Klebsiella pneumoniae.

2,3-Butanediol is one of the promising bulk chemicals with wide applications. Its fermentative production has attracted great interest due to the high end concentration. However, large-scale production of 2,3-butanediol requires low-cost substrate and efficient fermentation process. In the present study, 2,3-butanediol production by Klebsiella pneumoniae from Jerusalem artichoke tubers was successfully performed, and various technologies, including separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), were investigated. The concentration of target products reached 81.59 and 91.63 g/l, respectively after 40 h in batch and fed-batch SSF processes. Comparing with fed-batch SHF, the fed-batch SSF provided 30.3% higher concentration and 83.2% higher productivity of target products. The results showed that Jerusalem artichoke tuber is a favorable substrate for 2,3-butanediol production, and the application of fed-batch SSF for its conversion can result in a more cost-effective process.

Hemicellulose bioconversion.

Various agricultural residues, such as corn fiber, corn stover, wheat straw, rice straw, and sugarcane bagasse, contain about 20-40% hemicellulose, the second most abundant polysaccharide in nature. The conversion of hemicellulose to fuels and chemicals is problematic. In this paper, various pretreatment options as well as enzymatic saccharification of lignocellulosic biomass to fermentable sugars is reviewed. Our research dealing with the pretreatment and enzymatic saccharification of corn fiber and development of novel and improved enzymes such as endo-xylanase, beta-xylosidase, and alpha- l-arabinofuranosidase for hemicellulose bioconversion is described. The barriers, progress, and prospects of developing an environmentally benign bioprocess for large-scale conversion of hemicellulose to fuel ethanol, xylitol, 2,3-butanediol, and other value-added fermentation products are highlighted.

Synthesis and characterization of composite nucleic acids containing 2', 5'-oligoriboadenylate linked to antisense DNA.

Composite nucleic acids, known as 2-5A antisense chimeras, cause the 2-5A-dependent ribonuclease (RNase L) to catalyze the specific cleavage of RNA in cell free systems and in intact cells. Such 2-5A antisense chimeras are 5'-monophosphorylated, 2,'5'-linked oligoadenylates covalently attached to antisense 3',5'-oligodeoxyribonucleotides by means of a linker containing two residues of 1,4-butanediol phosphate. Here we report a fully automated synthesis of 2-5A antisense chimeras on a solid support using phosphoramidite methodology with specific coupling time modifications and their subsequent purification by reverse-phase ion-pair and anion exchange HPLC. Purified 2-5A antisense chimeras were characterized by [1H]NMR and [31P]NMR, MALDIMS, and capillary gel electrophoresis. The synthetic 2',5'-linked oligoadenylate showed no phosphodiester isomerization to 3',5' during or after synthesis. In addition, we have developed facile methodologies to characterize the chimeras using digestion with various hydrolytic enzymes including snake venom phosphodiesterase I and nuclease P1. Finally, Maxam-Gilbert chemical sequencing protocols have been developed to confirm the entire sequence of these chimeric oligonucleotides.

Surface area-independent assessment of lung microvascular permeability with an amphipathic tracer.

A combination of an amphipathic-indicator-dilution (ID) diffusing tracer 1,4[14C]butanediol (B) and a hydrophilic tracer ([14C]urea) (U) was hypothesized to provide a capillary surface area- (S) independent assessment of lung microvascular permeability (P). We performed ID studies on isolated perfused dog lungs and administered randomly two interventions, increasing P by alloxan infusion and reduction in S by lobar ligation. The ratio of PS product of U (PSU) to that for butanediol (PSB) was sensitive to changes in P yet insensitive to changes in S. We performed ID studies in which the dependence of PSU and PSB on flow, hematocrit, and plasma protein binding were examined. Measurements of PSU and PSB after flow and hematocrit were changed suggested that these factors have no significant independent effects. From ID and in vitro studies we also found that no significant binding of B to plasma proteins (albumin) occurred. We concluded that ID techniques using B and U provide a consistent measure of P, despite changes in S, hematocrit, plasma protein concentration, and recruitment.