Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1–2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.     

Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents

Lignin is known to impede conversion of lignocellulose into ethanol. In this study, forage sorghum plants carrying brown midrib (bmr) mutations, which reduce lignin contents, were evaluated as bioenergy feedstocks. The near-isogenic lines evaluated were: wild type, bmr-6, bmr-12, and bmr-6 bmr-12 double mutant. The bmr-6 and bmr-12 mutations were equally efficient at reducing lignin contents (by 13% and 15%, respectively), and the effects were additive (27%) for the double mutant. Reducing lignin content was highly beneficial for improving biomass conversion yields. Sorghum biomass samples were pretreated with dilute acid and recovered solids washed and hydrolyzed with cellulase to liberate glucose. Glucose yields for the sorghum biomass were improved by 27%, 23%, and 34% for bmr-6, bmr-12, and the double mutant, respectively, compared to wild type. Sorghum biomass was also pretreated with dilute acid followed by co-treatment with cellulases and Saccharomyces cerevisiae for simultaneous saccharification and fermentation (SSF) into ethanol. Conversion of cellulose to ethanol for dilute-acid pretreated sorghum biomass was improved by 22%, 21%, and 43% for bmr-6, bmr-12, and the double mutant compared to wild type, respectively. Electron microscopy of dilute-acid treated samples showed an increased number of lignin globules in double-mutant tissues as compared to the wild-type, suggesting the lignin had become more pliable. The mutations were also effective for improving ethanol yields when the (degrained) sorghum was pretreated with dilute alkali instead of dilute acid. Following pretreatment with dilute ammonium hydroxide and SSF, ethanol conversion yields were 116 and 130 mg ethanol/g dry biomass for the double-mutant samples and 98 and 113 mg/g for the wild-type samples.     

Vesicle formation and cellulose degradation in Bacteroides succinogenes cultures: ultrastructural aspects

In 3-day-old cultures of Bacteroides succinogenes grown on filter paper, no cell division was observed. When grown on cellulosic substrate, bacteria exhibited vesicles clustered within cell wall pockets. In 2 day-old filter paper cultures, cells adhered tightly to the substrate. Twenty to 30% of them were dividing. There were cell wall pockets in about 25% of the bacteria, but no vesicles. Whether they adhered to the cellulosic substrate or not, and irrespective of the age of the bacteria, storage polysaccharides were found in the form of dense granules in the cytoplasm. It would appear that vesicles are not essential for cellulose degradation, but are rather a sign of ageing of the cells.     

Map-based cloning and expression analysis of BMR-6 in sorghum

Journal of Genetics - Brown midrib mutants in sorghum are associated with reduced lignin content and increased cell wall digestibility. In this study, we characterized a bmr-6 sorghum mutant, which...     

Biomass and Cellulosic Ethanol Production of Forage Sorghum Under Limited Water Conditions

This study presents results from a 2-year evaluation of biomass and cellulosic ethanol (EtOH) production potential of forage sorghum (Sorghum bicolor L. Moench) cultivars differing in brown midrib trait (i.e., bmr12) under dryland (no irrigation) and limited irrigation (2.88 mm?day^?1; subsurface drip) in the semiarid Southern High Plains of the USA. Commercial cultivar Sorghum Partners 1990 (SP 1990, conventional non-bmr) produced significantly more biomass (29–62 %) than a bmr12 cultivar PaceSetter bmr (PS bmr) under irrigated and dryland conditions during both years of this study. However, PS bmr biomass had higher cellulosic EtOH conversion efficiency than SP 1990 in both years according to simultaneous saccharification and fermentation analysis. Irrigation resulted in 26–49 % more biomass and 28–72 % more cellulosic EtOH production during both growing seasons, indicating that limited irrigation had favorable effects on both biomass and biofuel production. In the first year, when precipitation was below average, both cultivars produced similar amounts of cellulosic EtOH. During the second year, when precipitation was above average, higher biomass production of SP 1990 resulted in 28 % higher cellulosic EtOH production than PS bmr when averaged across both irrigated and dryland conditions. The large range of cellulosic EtOH production (1,600 to 3,380 L?ha^?1) during the 2 years of this study was primarily driven by differences in water availability that resulted from precipitation and irrigation. Our findings indicates that chemical composition and biomass yield potential of sorghum cultivars are critical factors that affect biomass and biofuel production under limited water conditions.     

Ethanol metabolism in a peroxisome-deficient mutant of the yeast Hansenula polymorpha

Abstract: This paper describes ethanol metabolism in a peroxisome-deficient (PER) mutant of Hansenula polymorpha . The PER mutant was able to use ethanol as sole-carbon source but showed reduced growth rates compared to wild-type cells together with a reduced rate of ethanol utilization under μ_max conditions. In chemostat cultures at low-dilution rates, the activities of alcohol dehydrogenase, isocitrate lyase and malate synthase were comparable in wild-type and PER cells. In PER cells the two latter enzymes, exclusively microbody-bound in wild-type cells, were active in the cytosol. The possible advantage of intact microbodies in the intermediary metabolism of ethanol in H. polymorpha is discussed.     

Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures

A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.     

Altered biological properties of cell membranes in Escherichia coli dnaA and seqA mutants

We present evidence that biological properties of cell membranes are altered in dnaA and seqA mutants of Escherichia coli relative to wild-type bacteria. We found that bacteriophage λ forms extremely large plaques on the dnaA seqA double mutants. On the single mutants, dnaA and seqA, the plaques are also bigger than those formed on the wild-type host. However, no significant differences in intracellular phage λ development were observed between wild-type and mutant hosts, indicating that differences in burst size do not account for the observed differences in plaque size. On the other hand, more efficient release of the phage lytic proteins and/or higher sensitivity of the cell membranes to these proteins may result in more efficient cell lysis. We found that the efficiency of adsorption of bacteriophage λ to the dnaA seqA mutant cells is decreased at 0°?C , but not at 30°?C, relative to the wild-type strain. A considerable increase in the permeability of membranes of the mutant cells for β-galactosidase is demonstrated. The dnaA and seqA mutants are more sensitive to ethanol (an organic solvent) than wild-type bacteria, and the seqA strain and the double mutant dnaA seqA are very sensitive to deoxycholate (a detergent). We conclude that lesions in the genes dnaA and seqA result in alterations in cell membranes, such that the permeability and possibly also other properties of the membranes are significantly altered relative to wild-type bacteria.     

The effect of ethanol on the phase behavior of membrane lipids extracted from Clostridium thermocellum strains

The phase behavior of aqueous dispersions of extracted lipids from Clostridium thermocellum wild-type and ethanol-tolerant C919 cells has been examined by DSC. The optimum growth temperature of this anaerobe is 60°C. The wild-type lipids exhibit a broad phase transition centered at 30°C; the C919 mutant lipids show a 10°C lower T_m. The direct addition of growth inhibiting concentrations of ethanol has no significant effect on T_m or headgroup mobility (monitored by ²H-NMR) of either set of lipids. In contrast, wild-type cells adapted to growth in ethanol exhibit a broadened and lower T_m (15–25°C plateau); C919 membrane lipids do not exhibit significantly altered phase behavior when adapted to growth in ethanol. Both wild-type and mutant membranes have fatty acid composition changes upon growth in ethanol, which increases lower-melting components. It is concluded that fatty acid changes which occur upon adaptation of the organism to growth in ethanol are secondary responses and not necessarily direct responses to alter membrane fluidity.     

Genomic inversion caused by gamma irradiation contributes to downregulation of a WBC11 homolog in bloomless sorghum

Epicuticular wax (bloom) plays important roles in protecting the tissues of sorghum (Sorghum bicolor (L.) Moench) plants from abiotic stresses. However, reducing wax content provides resistance to greenbug and sheath blight—a useful trait in agricultural crops. We generated a sorghum bloomless (bm) mutant by gamma irradiation. One bm population segregated for individuals with and without epicuticular wax at a frequency of 72:22, suggesting that the bm mutation was under the control of a single recessive nuclear gene. Genes differentially expressed in the wild-type and the bm mutant were identified by RNA-seq technology. Of the 31 downregulated genes, Sb06g023280 was the most differentially expressed and was similar to WBC11, which encodes an ABC transporter responsible for wax secretion in Arabidopsis. An inversion of about 1.4 Mb was present in the region upstream of the Sb06g023280 gene in the bm mutant; it is likely that this inversion changed the promoter sequence of the Sb06g023280 gene. Using genomic PCR, we confirmed that six independent F₂ bm mutant-phenotype plants carried the same inversion. Therefore, we concluded that the inversion involving the Sb06g023280 gene inhibited wax secretion in the bloomless sorghum.     

Switchgrass (Panicum virgatum L.) cell suspension cultures: Establishment, characterization, and application

Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable attention as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell suspension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using protoplast isolation. A high protoplast isolation rate of up to 10⁶ protoplasts/1.0 g of cells was achieved for the fine milky culture, whereas only a few protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of protoplast isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research.     

Developmental and genetic characterization of a short leaf mutant of sorghum (Sorghum bicolor L. (Moench.))

Changes in plant architecture, specifically conversion to compact canopy for cereal crops, have resulted in significant increases in grain yield for wheat (Triticum aestivum) and rice (Oryza sativa). For sorghum (Sorghum bicolor L. Moench.) a versatile crop with an open canopy, plant architecture is an important feature that merits strong consideration for modification. Here, we report the genetic, developmental, and physiological characterization of a sorghum genetic stock, KFS2061, a stable mutant (in the Western Black Hull Kafir background) which exhibit short and erect leaves resulting in compact plant architecture. Genetic study of an F₂ population derived from the cross of KFS2061 to BTx623 showed that the short leaf is recessive and appeared to be controlled by a single gene. The expression of the short leaf trait commenced with the 3rd leaf and is propagated through the entire leaf hierarchy of the canopy. The short leaf mutant exhibited consistent steep leaf angle, 43° (with the main culm as reference), and greener leaves than wild type. Biochemical analyses indicated significantly higher chlorophyll and cellulose content per leaf area in the mutant than wild type. Histological studies revealed reduction in cell length along the longitudinal axis and enlargement of bulliform cells in the adaxial surface of the mutant leaf. Further evaluation of agronomic traits indicated that this mutation could increase harvest index. This study provides information on a short leaf genetic stock that could serve as a vital resource in understanding how to manipulate plant canopy architecture of sorghum.     

Function of Trehalose and Glycogen in Cell Cycle Progression and Cell Viability in Saccharomyces cerevisiae

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO₂ production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.     

Candida albicans lacking the gene encoding the regulatory subunit of protein kinase A displays a defect in hyphal formation and an altered localization of the catalytic subunit

The fungal pathogen Candida albicans switches from a yeast-like to a filamentous mode of growth in response to a variety of environmental conditions. We examined the morphogenetic behavior of C. albicans yeast cells lacking the BCY1 gene, which encodes the regulatory subunit of protein kinase A. We cloned the BCY1 gene and generated a bcy1 tpk2 double mutant strain because a homozygous bcy1 mutant in a wild-type genetic background could not be obtained. In the bcy1 tpk2 mutant, protein kinase A activity (due to the presence of the TPK1 gene) was cyclic AMP independent, indicating that the cells harbored an unregulated phosphotransferase activity. This mutant has constitutive protein kinase A activity and displayed a defective germinative phenotype in N-acetylglucosamine and in serum-containing medium. The subcellular localization of a Tpk1-green fluorescent protein (GFP) fusion protein was examined in wild-type, tpk2 null, and bcy1 tpk2 double mutant strains. The fusion protein was observed to be predominantly nuclear in wild-type and tpk2 strains. This was not the case in the bcy1 tpk2 double mutant, where it appeared dispersed throughout the cell. Coimmunoprecipitation of Bcy1p with the Tpk1-GFP fusion protein demonstrated the interaction of these proteins inside the cell. These results suggest that one of the roles of Bcy1p is to tether the protein kinase A catalytic subunit to the nucleus.     

An Induced Sorghum Mutant Population Suitable for Bioenergy Research

The sorghum [Sorghum bicolor (L.) Moench] inbred line BTx623 has served as a parent for development of several mapping populations, also providing a source for the generation of DNA libraries for physical mapping, and as the inbred line selected for sorghum genome sequencing. Since genetic mapping, physical mapping and genome sequencing are all based on the same inbred line, these genetic resources have made the genome study of sorghum very efficient. However, in comparison with other model species, there is one important genetic resource still missing in the sorghum research community, a mutant population. A systematically annotated mutant population will facilitate many avenues of research, especially those focusing on functional genomics and bioenergy research. Here we report the generation of a sorghum mutant population derived from the inbred line BTx623 by treatment with the chemical agent ethyl methanesulfonate (EMS). The mutant population consists of 1,600 pedigreed M₃ families; each of them was derived from an independent M₁ seed. Many lines displayed traits such as brown midrib (bmr), erect leaves (erl), multiple tillers (mtl), and late flowering (lfl), characteristics useful for bioenergy research. Results from our phenotyping and genotyping studies indicate that this mutant population will be a valuable and useful genetic resource for both sorghum functional genomics and bioenergy research.     

Isolation of Copper Biochelates from Methylosinus trichosporium OB3b and Soluble Methane Monooxygenase Mutants

Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMO^c) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 μM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 × 10¹⁶ M⁻¹. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMO^c phenotype is probably related to the mutant’s inability to take up CBL-complexed copper, not to a defective CBL structure.     

Distribution of cellulosic wall in the anthers of Arabidopsis during microsporogenesis

Key message

Cellulose-specific staining revealed that tapetal cells and microsporocytes lose cellulosic walls before the onset of meiosis. Cellulosic wall degradation in microsporocytes might be independent of tapetal cells (or TPD1).

Abstract

Some cell types in a variety of angiosperms have been reported to lack cell walls. Here, we report that the tapetal cells of the anther of Arabidopsis thaliana did not appear to have a cellulosic wall based on staining with Calcofluor and Renaissance 2200. During sporogenous cell formation, cellulosic wall was present in all anther tissues. However, before meiosis it was almost absent on the tapetal cells and on the microsporocytes. In a sporocyteless/nozzle (spl/nzz) mutant, which lacks several components (microsporocytes, tapetum, middle layer and endothecium), cellulosic wall was detected in all anther cells. In another mutant, tapetum determinant1 (tpd1), which lacks tapetum and has more microsporocytes, cellulosic wall was almost absent on the microsporocytes before meiosis, similar to the wild type. These results suggest that the tapetum cells and microsporocytes lose cellulosic walls during microsporocyte formation, and that cell wall degradation occurs downstream of SPL/NZZ and is independent of TPD1.