Glycerol production by microbial fermentation: a review

Microbial production of glycerol has been known for 150 years, and glycerol was produced commercially during World War I. Glycerol production by microbial synthesis subsequently declined since it was unable to compete with chemical synthesis from petrochemical feedstocks due to the low glycerol yields and the difficulty with extraction and purification of glycerol from broth. As the cost of propylene has increased and its availability has decreased especially in developing countries and as glycerol has become an attractive feedstock for production of various chemicals, glycerol production by fermentation has become more attractive as an alternative route. Substantial overproduction of glycerol by yeast from monosaccharides can be obtained by: (1) forming a complex between acetaldehyde and bisulfite ions thereby retarding ethanol production and restoring the redox balance through glycerol synthesis; (2) growing yeast cultures at pH values near 7 or above; or (3) using osmotolerant yeasts. In recent years, significant improvements have been made in the glycerol production using osmotolerant yeasts on a commercial scale in China. The most outstanding achievements include: (1) isolation of novel osmotolerant yeast strains producing up to 130 g/L glycerol with yields up to 63% and the productivities up to 32 g/(L day); (2) glycerol yields, productivities and concentrations in broth up to 58%, 30 g/(L day) and 110-120 g/L, respectively, in an optimized aerobic fermentation process have been attained on a commercial scale; and (3) a carrier distillation technique with a glycerol distillation efficiency greater than 90% has been developed. As glycerol metabolism has become better understood in yeasts, opportunities will arise to construct novel glycerol overproducing microorganisms by metabolic engineering.     

Enzyme synthesis of oligosaccharides using cashew apple juice as substrate

The use of agriculture substrates in industrial biotechnological processes has been increasing because of their low cost. In this work, the use of clarified cashew apple juice was investigated as substrate for enzyme synthesis of prebiotic oligosaccharide. The results showed that cashew apple juice is a good source of reducing sugars and can be used as substrate for the production of dextransucrase by Leuconostoc citreum B-742 for the synthesis of oligosaccharides using the crude enzyme. Optimal oligosaccharide yield (approximately 80%) was obtained for sucrose concentrations lower than 60 g/L and reducing sugar concentrations higher than 100 g/L.     

Synthesis of sugar fatty acid esters using a β-xylosidase from <Emphasis Type="Italic">Aspergillus awamori</Emphasis> for transxylosylation

Aspergillus awamori K4 β-xylosidase has broad acceptor specificity. It has been used to synthesize a sugar fatty acid ester via its transxylosylation activity. One xylosyl residue was initially transferred to hexamethylene glycol as a linker with a yield of 0.36 g/g xylobiose. Linoleic acid was subsequently linked to one terminal hydroxyl side of the transfer product hydroxyhexyl xyloside through an esterification reaction catalyzed by a lipase. The synthesis of hexyl linoleoyl xyloside was confirmed by TOF-MS analysis. The binding with a linker improved the esterification reaction because of the hydrophobic hexamethylene chain and also prevented steric hindrance by the xylosyl residue. This sugar fatty acid ester synthesis method using transglycosylation should facilitate the production of emulsifiers or surfactants with various functions.     

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.     

Studies on the dependence of chlorophyll synthesis on protein synthesis in Euglena gracilis,together with a nomogram for determination of chlorophyll concentration

Summary Experiments have been carried out to determine the basis for the dependence of chloroplast pigment synthesis on protein synthesis in dark-grown cells of Euglena gracilis greening in the light. The complete inhibition of chlorophyll synthesis brought about by actidione (10 g/ml) when added half way through the greening process was not relieved, even to the slightest extent, when 0.01 M -aminolaevulinic acid (ALA) was also present. The much smaller inhibition of chlorophyll synthesis brought about by chloramphenicol (2 mg/ml) was also relieved little, if at all, by the addition of ALA. It is concluded that the inhibition of chlorophyll synthesis by actidione can not be solely or primarily due to lack of ALA resulting from the decay of possibly labile enzymes of ALA synthesis, but could be due to inhibition of synthesis of the thylakoid structural protein. The results obtained with chloramphenicol are difficult to interpret because of the possibility that the drug, at high concentration, directly inhibits processes other than protein synthesis.Chlorophyll and carotenoid synthesis by E. gracilis were both markedly stimulated by the addition of ALA. It is suggested that the rate of chlorophyll synthesis in the greening cells is limited by the rate of formation of ALA. The stimulation of formation of carotenoids as well as chlorophyll may indicate that the cells have a mechanism for ensuring that the rate of carotenoid synthesis does not fall below a certain proportion of the rate of chlorophyll synthesis.A nomogram has been devised from which the concentrations of chlorophylls a and b, and total chlorophyll can be read off once the absorbances of an 80% acetone extract at 663 and 645 m have been determined.     

Melatonin as a central molecule connecting neural development and calcium signaling

Melatonin (MEL) is a neuroendocrine hormone secreted by the pineal gland in association with the suprachiasmatic nucleus and peripheral tissues. MEL has been observed to play a critical role in the reproductive process and in the fetomaternal interface. Extrapineal synthesis has been reported in mammalian models during pregnancy, especially by the placenta tissue. MEL can regulate intracellular processes (e.g., G-proteins) and the activity of second messengers (e.g., cAMP, IP_3, Ca²⁺). During neurodevelopment, these activities regulated by melatonin have an important role as an intracellular signaling for gene expression regulation. To review the role of MEL in neurodevelopment, we built interactome networks of different proteins that act in these processes using systems biology tools. The analyses of interactome networks revealed that MEL could modulate neurodevelopment through the regulation of Ca²⁺ intracellular levels and influencing BMP/SMAD signaling, thus affecting neural gene responses and neuronal differentiation.     

Interrelationships between water and cellular metabolism in Artemia cysts. VI. RNA and protein synthesis

Using ¹⁴CO₂ as a labelled precursor the relationship between the initiation of protein and RNA synthesis, and water concentration, has been examined in cysts (encysted embryos) of the brine shrimp, Artemia salina. Although incorporation of radioactivity into amino acids and nucleotides occurred in cysts at hydrations as low as 0.3 g H₂O/g dried cysts, incorporation into proteins and RNA was not measurable until the cysts had achieved a hydration in the range of 0.6–0.6 g/g. In no case was radioactivity detected in DNA of unemerged cysts. Fully hydrated cysts (about 1.3 g/g) that were actively synthesizing proteins and RNA, stopped doing so when dehydrated to levels below the same hydration range: thus, the hydration dependence does not involve appreciable hysteresis. The hydration range required to initiate synthesis of these macromolecules is essentially the same as that previously shown to initiate embryonic development.     

DNA SYNTHESIS AND MITOSIS IN MERISTEMS: REQUIREMENTS FOR RNA AND PROTEIN SYNTHESIS

Following provision of sucrose to starved, stationary phase pea root meristems, G₁ and G₂ cells enter DNA synthesis and mitosis, respectively. Puromycin (450 μg/ml) and cycloheximide (5 μg/ml) completely prevent this initiation of progression through the cell cycle. Actinomycin D (10 μg/ml) has no effect on the initial entry of G₁ and G₂ cells into S and mitosis, although later entry is prevented. The resistance of the cells to actinomycin D is lost slowly with time in medium without sucrose, suggesting that an RNA required for the resumption of proliferative activity is being gradually lost. The effects of the inhibitors on transitional and proliferative phase meristem cells indicate that such dividing cells do indeed have sufficient of the requisite RNA for 8-12 hr progression through the cycle, but that protein synthesis is required continuously. It is suggested that this RNA is the one lost slowly during starvation, allowing starved cells to reinitiate progression through the cycle in the presence of actinomycin D.     

Designer biocatalysts for direct incorporation of exogenous galactose into globotriose

Galactose is ubiquitous. The synthesis of galactose-containing oligosaccharides using Leloir galactosyltransferase requires uridine diphosphate (UDP)-galactose as the precursor. Of all UDP-galactose synthesis pathways developed for in vitro synthesis, the salvage pathway represents the simplest route. In this study, for the first time, we designed and constructed an Escherichia coli strain to use salvage pathway for UDP-galactose synthesis, demonstrating effective and direct incorporation of exogenous galactose into globotriose (Gb3). Successful establishment of salvage pathway enabled a complete delineation of carbon and energy source. Consequently, the designed biocatalyst was able to achieve high yield synthesis from galactose (0.95 moles of Gb3/moles galactose consumed) and a high product titer (2 g/L) in shaker flask within 24 hr. Elimination of limitation in acceptor sugar via homologous overexpression of LacY, the transporter for lactose, further improved the synthesis, raising Gb3 titer to 6 g/L in 24 hr and 7.5 g/L in 48 hr. The design principles successfully demonstrated in this study could be broadly applied for synthesis of other galactose-containing oligosaccharides. This study also illustrates a valid strategy to overcome limitation in the transport of acceptor sugar. As lactose is one of the most important basal structures, the significant improvement in synthesis through its enhanced transport could be emulated in numerous other lactose-based oligosaccharides.     

Improvement in the titer of echinocandin-type antibiotics: A magnesium-limited medium supporting the biphasic production of pneumocandins A0 and B0

Summary We have developed a liquid fermentation medium for the submerged culture of the fungus,Zalerion arboricola, which supports the rapid production of an echinocandin-type antibiotic, pneumocandin A₀ (formerly L-671, 329), in yields increased at least 4-fold over those reported previously. The improvements were achieved through medium simplification, substitution of high levels of mannitol for glycerol as the major source of carbon, and restriction of available magnesium. Antibiotic formation in batch cultures with this mannitol-based medium is not confined to the idiophase; rather production appears to be biphasic, with synthesis beginning during growth (i.e., at day 3) and increasing in rate at day 11, well after rapid growth has ended. Accumulation of antibiotic continues beyond 14 days, and by 21 days titers greater than 500 g/ml are attained. For the synthesis of a related compound, pneumocandin B₀, by a mutant strain ofZ. arboricola, the medium gives similar production kinetics and a titer of 800 g/ml. Although supplementation of the medium with magnesium ions stimulates growth, it decreases titer by preferentially affecting the second phase of antibiotic synthesis. This decline in synthesis in the magnesium-supplemented medium is explained by the depletion of mannitol before the second phase of synthesis can begin. In contrast, mannitol in the magnesium-limited medium is used more slowly with approximately half still available at day 11 to support continued antibiotic formation.     

Alginate biosynthesis by <Emphasis Type="Italic">Azotobacter</Emphasis> bacteria

The ability of representatives of various species of the bacterial genus Azotobacter (A. chroococcum 7B, A. chroococcum 12B, A. chroococcum 12BS, A. agile 12, A. indicum 8, A. vinelandii 17, and A. vinelandii 5B) to alginate synthesis has been studied. It has been shown that all tested bacterial strains have this ability to different extents. Capsular alginate comprises from 2.6 to 32% of the total amount of synthesized alginate in various bacterial species. Strains that are able to active synthesis of alginate have been selected; the effect of the medium composition on their biosynthesis has been studied. The optimal conditions for alginate synthesis by the A. chroococcum 12BS producer strain include the presence of mannitol (40 g/L), yeast extract (1%), and low concentration of phosphates (KH₂PO₄—0.008 g/L, K₂HPO₄—0.032 g/L) in the medium; alginate production under these conditions is 4.5 g/L. The effect of aeration on polymer biosynthesis has been revealed: an increase in aeration causes an increase in alginate synthesis, while its decrease promotes the synthesis of poly-3-hydroxybutirate. It has been shown by IR spectroscopy that alginates obtained under various conditions of cultivation contain different ratios of residues of mannuronic and guluronic acids (M/G from 70/30 to 80/20) in the polymer chain and also differ in the amount of acetyl groups (from 10 to 25%) in the polyme structure.     

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.     

The metabolism in vivo of glycine and serine in eight areas of the rat central nervous system

Abstract— Results of studies designed to estimate the rates at which glycine is derived from various possible sources in discrete areas of the rat CNS are reported. These results suggest that glycine is derived predominantly by de novo synthesis, presumably via the established pathways leading from glucose through serine to glycine. The content of glycine ranged from a low of approximately 0-6 μmol/g in the cerebellum and telencephalon to a high of 5·5 μmol/g in the spinal cord grey matter; however, based on its estimated rate of synthesis from serine, there appeared to be no correlation between the content of glycine and its rate of synthesis in the various areas studied. The flux of glycine from blood into the CNS was slower (0·03-0·15 μmol/g/h depending on the CNS structure) than that of serine (0·15-0·23 μmol/g/h) and both amino acids entered various CNS areas at rates unrelated to their respective tissue contents. These data have been discussed with regard to the putative transmitter function of glycine in the spinal cord and brainstem.     

Asbestos-stimulated changes in nitric oxide and iron metabolism in rats

Under intratracheal asbestos fibers installation it has been investigated NO synthesis in the lung and liver tissues of Wistar rats by EPR method. Asbestos A6-45, sifted through the sieve with size 0.1 mm, has been administrated in a dose of 5 mg/kg. To evaluate the NO synthesis EPR and NO-trap methods have been used. The amplitude of EPR signal "trap-NO" in the lung samples was 12, 16 and 14 times greater than in controls on the 3th, 6th and 10th days after asbestos installation and was corresponding to NO rate of about 2 mkmol/(g x h). In the liver samples of asbestos-stimulated animals the NO level contained in the non-heme iron nitrosyl complexes was about 2 mkmol/g. Thus, the asbestos fibers stimulate NO synthesis not only in the lung tissue, but also in other organs. The obtained data shows that under NO hyperproduction certain changes in iron metabolism take place, such as: the decrease of transferrin iron and the accumulation of ferric iron not bound with transferrin. The accumulation of ferric iron not shielded by proteins is one of the oxidative stress triggers.     

Enzymes and binding proteins affecting retinoic acid concentrations

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alchol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low K_m substrate.     

LipidII: Just Another Brick in the Wall?

Nearly all bacteria contain a peptidoglycan cell wall. The peptidoglycan precursor molecule is LipidII, containing the basic peptidoglycan building block attached to a lipid. Although the suitability of LipidII as an antibacterial target has long been recognized, progress on elucidating the role(s) of LipidII in bacterial cell biology has been slow. The focus of this review is on exciting new developments, both with respect to antibacterials targeting LipidII as well as the emerging role of LipidII in organizing the membrane and cell wall synthesis. It appears that on both sides of the membrane, LipidII plays crucial roles in organizing cytoskeletal proteins and peptidoglycan synthesis machineries. Finally, the recent discovery of no less than three different categories of LipidII flippases will be discussed.Peptidoglycan (PG), the main component of the cell wall, is a structure unique to bacteria. Currently, over 50% of the antibiotics in use target bacterial cell wall synthesis, and thus PG synthesis is considered the Achilles’ heel of bacteria [1]. The precursor of PG is LipidII, a lipid-linked disaccharide with a pentapeptide side chain. Linkage of the disaccharide to a growing glycan strand results in release of the lipid anchor and leaves the pentapeptide free for crosslinking to peptides on other glycan strands or for processing. Various excellent reviews describe the synthesis of LipidII, the incorporation of LipidII into PG, and the use of LipidII as a target for antibacterials [2–6]. LipidII’s conserved structure makes it difficult for pathogens to develop resistance against LipidII targeting molecules. This review focuses on the latest findings on antibacterials targeting LipidII, such as teixobactin [7], and on new LipidII biology (summarized in Fig 1). It is becoming more and more evident that LipidII is not just a passive brick that is being added to the cell wall but rather plays a key role in organization of the membrane.Open in a separate windowFig 1Organization of cell wall synthesis by LipidII.Overview of recent work that highlights various new insights about the role of LipidII; for example, (1) in the identification of novel antibacterials that target LipidII (including teixobactin and bacteriocins), (2) how LipidII is translocated over the membrane by different families of flippases (such as FtsW or RodA, MurJ, and Amj), (3) how it is recruited to regions of increased fluidity (RIFs) and how it organizes attachment of MreB(-like) filaments, and (4) how cell wall synthesis enzymes (penicillin-binding proteins [PBPs]) are recruited to LipidII.     

Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed “starch-mannose-fermentation” biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.     

Integrating ecology,psychology and neurobiology within a food-hoarding paradigm

Many animals regularly hoard food for future use, which appears to be an important adaptation to a seasonally and/or unpredictably changing environment. This food-hoarding paradigm is an excellent example of a natural system that has broadly influenced both theoretical and empirical work in the field of biology. The food-hoarding paradigm has played a major role in the conceptual framework of numerous fields from ecology (e.g. plant–animal interactions) and evolution (e.g. the coevolution of caching, spatial memory and the hippocampus) to psychology (e.g. memory and cognition) and neurobiology (e.g. neurogenesis and the neurobiology of learning and memory). Many food-hoarding animals retrieve caches by using spatial memory. This memory-based behavioural system has the inherent advantage of being tractable for study in both the field and laboratory and has been shaped by natural selection, which produces variation with strong fitness consequences in a variety of taxa. Thus, food hoarding is an excellent model for a highly integrative approach to understanding numerous questions across a variety of disciplines. Recently, there has been a surge of interest in the complexity of animal cognition such as future planning and episodic-like-memory as well as in the relationship between memory, the environment and the brain. In addition, new breakthroughs in neurobiology have enhanced our ability to address the mechanisms underlying these behaviours. Consequently, the field is necessarily becoming more integrative by assessing behavioural questions in the context of natural ecological systems and by addressing mechanisms through neurobiology and psychology, but, importantly, within an evolutionary and ecological framework. In this issue, we aim to bring together a series of papers providing a modern synthesis of ecology, psychology, physiology and neurobiology and identifying new directions and developments in the use of food-hoarding animals as a model system.