Ancestral sporulation initiation

Members of the classes Bacilli and Clostridia are able to form endospores, with clostridia representing the ancestral phylogenetic line. In contrast to Bacillus subtilis, the process of sporulation initiation at the molecular level is less well understood in Clostridium acetobutylicum, the model organism of the clostridia. Especially the question of how the master regulator of sporulation, Spo0A, becomes phosphorylated, remained unsolved so far. Steiner et al. now provide compelling genetic and biochemical evidence for two different pathways of direct phosphorylation of Spo0A in C. acetobutylicum by three orphan sensor kinases. Cac0903 and Cac3319 act together, while the other pathway is only dependent on Cac0323. Abortion of sporulation initiation can be achieved by a kinase-like protein, Cac0437.     

Problems with the microbial production of butanol

With the incessant fluctuations in oil prices and increasing stress from environmental pollution, renewed attention is being paid to the microbial production of biofuels from renewable sources. As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hygroscopicity. A variety of cheap substrates have been successfully applied in the production of biobutanol, highlighting the commercial potential of biobutanol development. In this review, in order to better understand the process of acetone–butanol–ethanol production, traditional clostridia fermentation is discussed. Sporulation is probably induced by solvent formation, and the molecular mechanism leading to the initiation of sporulation and solventogenesis is also investigated. Different strategies are employed in the metabolic engineering of clostridia that aim to enhancing solvent production, improve selectivity for butanol production, and increase the tolerance of clostridia to solvents. However, it will be hard to make breakthroughs in the metabolic engineering of clostridia for butanol production without gaining a deeper understanding of the genetic background of clostridia and developing more efficient genetic tools for clostridia. Therefore, increasing attention has been paid to the metabolic engineering of E. coli for butanol production. The importation and expression of a non-clostridial butanol-producing pathway in E. coli is probably the most promising strategy for butanol biosynthesis. Due to the lower butanol titers in the fermentation broth, simultaneous fermentation and product removal techniques have been developed to reduce the cost of butanol recovery. Gas stripping is the best technique for butanol recovery found so far.     

Classification of the Genes Involved in the Two-Component System of the Moss Physcomitrella patens

Physcomitrella patens, belonging to bryopsida, is a basal lineage of land plants. To gain insight into the diversification of the two-component system (TCS), which is widely conserved from prokaryotes to eukaryotes, we compiled TCS-associated genes by employing P. patens genome databases. The moss has a set of His-kinases (HKs), including homologs of the cytokinin- and ethylene-receptors in seed plants. In addition, it has a number of coding-sequences specifying unique HKs. We found evidence that a putative cytokinin-receptor HK in P. patans serves as a sensor for this hormone, and that the HK activity of a putative ethylene-receptor homolog is regulated by ethylene, as observed for Arabidopsis thaliana.     

Metabolic pathways of clostridia for producing butanol

Worldwide demand for energy has been the impetus for research to produce alcohol biofuels from renewable resources. This review focuses on the biosynthesis of butanol, which is regarded to be superior to ethanol as a fuel. Although acetone/butanol fermentation is one of the oldest large-scale fermentation processes, butanol yield by anaerobic fermentation remains sub-optimal. Metabolic engineering provides a means for fermentation improvements. Consequently, a comprehensive assessment of the intermediary enzymes involved in butanol formation from carbohydrates by the saccharolytic bacterium, Clostridium acetobutylicum and other closely allied clostridia was performed to provide guidelines for potentially enhancing butanol productivity. The activity of the enzymes, their regulation and contribution to the metabolic pathways was reviewed. Published kinetic data for each important enzymatic reaction were assessed. For most enzymatic reactions, the systematic investigation of the kinetic data and the properties of the enzymes led to the development of rate equations that were able to describe activity as the function of the substrates, products, and allosteric effectors.     

Intracellular metabolism analysis of Clostridium cellulovorans via modeling integrating proteomics,metabolomics and fermentation

A consolidated bioprocess for cellulosic n-butanol production has been developed by engineering Clostridium cellulovorans to overexpress a bifunctional aldehyde/alcohol dehydrogenase. Rational metabolic engineering is important to further improve butanol production. This study aimed to investigate intracellular metabolism and identify the key regulators of cellulosic butanol formation in C. cellulovorans via integrated Omics and fermentation kinetics data analysis. First, comparative proteomics and metabolomics analyses of wild type and n-butanol producing mutant strain were conducted, which quantified 624 host cell proteins and 474 primary and secondary metabolites. Compared to wild type, most cellulases in cellulolysis were up-regulated, but three glycolysis enzymes and three enzymes in central pathway were down-regulated in the n-butanol producing strain. Second, a dynamic model integrating Omics and fermentation data was developed to identify key regulators in butanol biosynthesis, which were ranked by further metabolic control analysis. Finally, rational metabolic engineering was performed in C. cellulovorans by overexpressing two genes (thl and hbd) identified as important factors limiting butanol biosynthesis, which improved butanol yield and C4/C2 ratio. This study demonstrated a research approach to integrate multi-Omics and fermentation data of C. cellulovorans and guide its rational metabolic engineering, which can also be applied to other microorganisms.     

The effect of fermentable carbohydrate on sporulation and butanol production by Clostridium acetobutylicum P262

Summary This study was conducted to determine whether or not a variation in the type of carbohydrate fermented by Clostridium acetobutylicum could be exploited to inhibit sporulation during the butanol-producing phase of fermentation and thus enhance butanol production. C. acetobutylicum P262 was found to ferment a wide variety of carbohydrates, but butanol production was not necessarily enhanced when percentage sporulation was low. Butanol concentration was more related to the total amount of acidic end-products (acetic and butyric acid) reutilized by the microorganism for solvent production and to the type and amount of carbohydrate utilized. Fermentation of cellobiose led to conditions resulting in complete acid reutilization and the highest butanol concentration (10.4–10.6 g/l). In cultures containing a mixture of glucose and cellobiose, glucose repression of cellobiose utilization resulted in lower butanol concentrations (6.6–7.5 g/l). Sporulation was dependent on the type of carbohydrate utilized by the microorganism. Glucose had a greater enhancing effect on the sporulation process (22–42%) than starch (9–12%) or cellobiose (22–34%). It was concluded that whereas the type of carbohydrate fermented had a specific effect on the extent of sporulation of a culture, conditions of low sporulation did not enhance butanol concentration unless carbohydrate utilization and the reutilization of acidic products were high.Correspondence to: W. M. Ingledew     

A eukaryotic LOV-histidine kinase with circadian clock function in the picoalga Ostreococcus

The marine environment has unique properties of light transmission, with an attenuation of long wavelengths within the first meters of the water column. Marine organisms have therefore evolved specific blue‐light receptors such as aureochromes to absorb shorter‐wavelength light. Here, we identify and characterize a light, oxygen, or voltage sensing (LOV) containing histidine kinase (LOV‐HK) that functions as a new class of eukaryotic blue‐light receptor in the pico‐phytoplanktonic cell Ostreococcus tauri. This LOV‐HK is related to the large family of LOV‐HKs found in prokaryotes. Phylogenetic analysis indicates that the LOV domains from LOV‐HKs, including O. tauri LOV‐HK, and phototropins (phot; plant and green algal LOV serine/threonine kinases) have different evolutionary histories. Photochemical analysis shows that the LOV domain of LOV‐HK binds a flavin cofactor and absorbs blue light with a fast photocycle compared with its prokaryotic counterparts. Ostreococcus tauri LOV‐HK expression is induced by blue light and is under circadian control. Further, both overexpression and downregulation of LOV‐HK result in arrhythmia of the circadian reporter CCA1:Luc under constant blue light. In contrast, photochemical inactivation of O. tauri LOV‐HK is without effect, demonstrating its importance for function of the circadian clock under blue light. Overexpression/downregulation of O. tauriLOV‐HK alters CCA1 rhythmicity under constant red light, irrespective of LOV‐HK’s photochemical reactivity, suggesting that O. tauri LOV‐HK also participates in regulation of the circadian clock independent of its blue‐light‐sensing property. Molecular characterization of O. tauri LOV‐HK demonstrates that this type of photoreceptor family is not limited to prokaryotes.     

Comparative Genomic Analysis of Two-Component Signal Transduction Systems in Probiotic Lactobacillus casei

Lactobacillus casei has traditionally been recognized as a probiotic, thus needing to survive the industrial production processes and transit through the gastrointestinal tract before providing benefit to human health. The two-component signal transduction system (TCS) plays important roles in sensing and reacting to environmental changes, which consists of a histidine kinase (HK) and a response regulator (RR). In this study we identified HKs and RRs of six sequenced L. casei strains. Ortholog analysis revealed 15 TCS clusters (HK–RR pairs), one orphan HKs and three orphan RRs, of which 12 TCS clusters were common to all six strains, three were absent in one strain. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. Some TCS clusters are involved with the response under the stress of the bile salts, acid, or oxidative, which contribute to survive the difficult journey through the human gastrointestinal tract. Computational predictions of 15 TCSs were verified by PCR experiments. This genomic level study of TCSs should provide valuable insights into the conservation and divergence of TCS proteins in the L. casei strains.     

Effect of glucose availability on glucose transport in bovine mammary epithelial cells

Primary bovine mammary epithelial cells (BMEC) were cultured in media containing varying concentrations of glucose, to determine the effects of glucose availability on glucose transport and its mechanism in bovine mammary gland. The BMEC incubated with 10 and 20 mM glucose had twofold greater glucose uptake than that with 2.5 mM glucose (P < 0.05). Increased glucose availability enhanced the cell proliferation (P < 0.05). As the glucose uptake is mediated by facilitative glucose transporters (GLUTs), the expression of GLUT mRNA was investigated. Compared with the control (2.5 mM), 5 and 10 mM glucose did not influence the abundance of GLUT1 mRNA (P < 0.05), whereas 20 mM glucose decreased the GLUT1 mRNA expression in the BMEC (P < 0.05). The expression of GLUT8 mRNA was not affected by any concentration of glucose (P > 0.05). As GLUTs are coupled with hexokinases (HKs) in regulating glucose uptake, the expression of HKs and their activities were also studied. The HK activity was greater in 5, 10 and 20 mM glucose than that in 2.5 mM glucose (P < 0.05). The expression of HK2 mRNA rather than HK1 mRNA was detected in the BMEC; however, the abundance of HK2 mRNA was not elevated by any concentrations of glucose compared with control (P > 0.05). Furthermore, addition of 3-bromopyruvate (30, 50 or 70 μM), an inhibitor of HK2, resulted in the decrease of glucose uptake and cell proliferation at both 2.5 and 10 mM glucose (P < 0.05). Therefore, the glucose concentrations may affect glucose uptake partly by altering the activity of HKs, and HK2 may play an important role in the regulation of glucose uptake in the BMEC.     

Efficient butanol production under aerobic conditions by coculture of Clostridium acetobutylicum and Nesterenkonia sp. strain F

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone–butanol–ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful “coculture” for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE.     

Engineering alternative butanol production platforms in heterologous bacteria

Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.     

Streptomyces ghanaensis pleiotropic regulatory gene wblA(gh) influences morphogenesis and moenomycin production

The wblA _gh gene, encoding a homologue of the WhiB-family of proteins, was identified in the sequenced genome of moenomycin producer Streptomyces ghanaensis. Deletion of the gene blocked aerial mycelium sporulation and caused a 230% increase in moenomycins production. S. ghanaensis overexpressing SSFG-01620: a homologue of extracellular protease inhibitor SCO0762, whose expression in Streptomyces coelicolor is down-regulated by wblA: showed deficiencies in sporulation similar to that of wblA _gh knockout strain. The wblA _gh gene of S. ghanaensis appears to play a negative role in the control of moenomycin biosynthesis and is essential for sporulation.     

Isozymes of plant hexokinase: occurrence, properties and functions

Hexokinase (HK) occurs in all phyla, as an enzyme of the glycolytic pathway. Its importance in plant metabolism has emerged with compelling evidence that its preferential substrate, glucose, is both a nutrient and a signal molecule that controls development and expression of different classes of genes. A variety of plant tissues and organs have been shown to express multiple HK isoforms with different kinetic properties and subcellular localizations. Although plant HK is known to fulfill a catalytic function and act as a glucose sensor, the physiological relevance of plural isoforms and their contribution to either function are still poorly understood. We review here the current knowledge and hypotheses on the physiological roles of plant HK isoforms that have been identified and characterized. Recent findings provide hints on how the expression patterns, biochemical properties and subcellular localizations of HK isoforms may relate to their modes of action. Special attention is devoted to kinetic, mutant and transgenic data on HKs from Arabidopsis thaliana and the Solanaceae potato, tobacco, and tomato, as well as HK gene expression data from Arabidopsis public DNA microarray resources. Similarities and differences to known properties of animal and yeast HKs are also discussed as they may help to gain further insight into the functional adaptations of plant HKs.     

Controlled-ph batch butanol-acetone fermentation by low acid producing Clostridium acetobutylicum B18

Summary C. acetobutylicum B18 produced a large amount of butanol over a wide range of pH (4.5–6.0). At pH 6.0 fermentation and cell growth were most active at pH 6.0, and the highest values of glucose consumption rate (4.37 g/L-h), butanol productivity (1.0 g/L-h), butyric acid recycle rate (0.31 g/L-h), and cell growth rate (0.2 h^-1) were obtained. There existed a critical pH between 6.0 and 6.5 above which cells switched to organic acid producing mode. Clostridial stage appeared essential for solvent production by strain B18 but sporulation was not necessary for solvent formation.     

丙酮丁醇梭菌的基因编辑工具及代谢工程改造

丙酮丁醇梭菌作为极具潜力的新型生物燃料丁醇的生产菌,受到各国研究学者的广泛关注。通过丙酮丁醇梭菌(ABE)发酵生产丁醇,由于生产成本高,限制了其工业化应用。随着基因组学和分子生物学的快速发展,适用于丙酮丁醇的基因编辑工具不断发展并应用于提高菌株的发酵性能。本文对丙酮丁醇梭菌基因编辑工具和代谢工程改造取得的进展进行综述。     

Integration of biocatalyst and process engineering for sustainable and efficient n‐butanol production

Recent environmental economic developments generate a need for sustainable and cost‐effective (microbial) processes for the production of high‐volume, low‐priced bulk chemicals. As an example, n‐butanol has, as a second‐generation biofuel, beneficial characteristics compared to ethanol in liquid transportation fuel applications. The industrial revival of the classic n‐butanol (ABE) fermentation requires process and strain engineering solutions for overcoming the main process limitations: product toxicity and low space–time yield. Reaction intensification on the biocatalyst, fermentation, and bioprocess level can be based on economic and ecologic evaluations using quantifiable constraints. This review describes the means of process intensification for biotechnological processes. A quantitative approach is then used for the comparison of the massive literature on n‐butanol fermentation. A comprehensive literature study—including key fermentation performance parameters—is presented and the results are visualized using the window of operation methodology. The comparison allowed the identification of the key constraints, high cell densities, high strain stability, high specific production rate, cheap in situ product removal, high n‐butanol tolerance, to operate in situ product removal efficiently, and cheap carbon source. It can thus be used as a guideline for the bioengineer during the combined biocatalyst, fermentation, and bioprocess development and intensification.     

Effect of lentivirus-mediated shRNA inactivation of HK1, HK2, and HK3 genes in colorectal cancer and melanoma cells

Background

The switch from oxidative phosphorylation to glycolysis in proliferating cancer cells, even under aerobic conditions, has been shown first in 1926 by Otto Warburg. Today this phenomenon is known as the “Warburg effect” and recognized as a hallmark of cancer. The metabolic shift to glycolysis is associated with the alterations in signaling pathways involved in energy metabolism, including glucose uptake and fermentation, and regulation of mitochondrial functions. Hexokinases (HKs), which catalyze the first step of glycolysis, have been identified to play a role in tumorigenesis of human colorectal cancer (CRC) and melanoma. However, the mechanism of action of HKs in the promotion of tumor growth remains unclear.

Results

The purpose of the present study was to investigate the effect of silencing of hexokinase genes (HK1, HK2, and HK3) in colorectal cancer (HT-29, SW 480, HCT-15, RKO, and HCT 116) and melanoma (MDA-MB-435S and SK-MEL-28) cell lines using short hairpin RNA (shRNA) lentiviral vectors. shRNA lentiviral plasmid vectors pLSLP-HK1, pLSLP-HK2, and pLSLP-HK3 were constructed and then transfected separately or co-transfected into the cells. HK2 inactivation was associated with increased expression of HK1 in colorectal cancer cell lines pointing to the compensation effect. Simultaneous attenuation of HK1 and HK2 levels led to decreased cell viability. Co-transfection with shRNA vectors against HK1, HK2, and HK3 mRNAs resulted in a rapid cell death via apoptosis.

Conclusions

We have demonstrated that simultaneous inactivation of HK1 and HK2 was sufficient to decrease proliferation and viability of melanoma and colorectal cancer cells. Our results suggest that HK1 and HK2 could be the key therapeutic targets for reducing aerobic glycolysis in examined cancers.

     

Increased hexokinase activity, of either ectopic or endogenous origin, protects renal epithelial cells against acute oxidant-induced cell death.

Glucose (Glc) metabolism protects cells against oxidant injury. By virtue of their central position in both Glc uptake and utilization, hexokinases (HKs) are ideally suited to contribute to these effects. Compatible with this hypothesis, endogenous HK activity correlates inversely with injury susceptibility in individual renal cell types. We recently reported that ectopic HK expression mimics the anti-apoptotic effects of growth factors in cultured fibroblasts, but anti-apoptotic roles for HKs have not been examined in other cell types or in a cellular injury model. We therefore evaluated HK overexpression for the ability to mitigate acute oxidant-induced cell death in an established epithelial cell culture injury model. In parallel, we examined salutary heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) treatment for the ability to 1) increase endogenous HK activity and 2) mimic the protective effects of ectopic HK expression. Both HK overexpression and HB-EGF increased Glc-phosphorylating capacity and metabolism, and these changes were associated with markedly reduced susceptibility to acute oxidant-induced apoptosis. The uniform Glc dependence of these effects suggests an important adaptive role for Glc metabolism, and for HK activity in particular, in the promotion of epithelial cell survival. These findings also support the contention that HKs contribute to the protective effects of growth factors.