Polysaccharides are synthesized from two forms of activated glucose molecules: UDP-glucose and ADP-glucose.
Describe the mechanism of polysaccharide biosynthesis and its importance in bacteria
- Uridine diphosphate glucose (UDP-glucose) is a nucleotide sugar. UDP-glucose consists of the pyrophosphate group, the pentose sugar ribose, glucose, and the nucleobase uracil. It is used as a substrate for enzymes called glucosyltransferases.
- UDP-glucose can also be used as a precursor of lipopolysaccharides, and peptidoglycan. ADP-glucose is usually the precursor for glycogen production in bacteria.
- When the cells are grown on a carbon source different than glucose, gluconeogenesis (abbreviated GNG) is the metabolic pathway used to produce glucose from non- carbohydrate carbon substrates such as phosphoenolpyruvate (PEP).
- Pathogenic bacteria can produce a thick, mucous-like, layer of polysaccharide. This “capsule” cloaks antigenic proteins on the bacterial surface. Bacteria and other microbes, secrete polysaccharides as an evolutionary adaptation to help them adhere to surfaces and to prevent them from drying out.
- Polysaccharides: Polysaccharides are long, carbohydrate molecules of repeated monomer units joined together by glycosidic bonds. They range in structure from linear to highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit.
- gluconeogenesis: Gluconeogenesis (abbreviated GNG) is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates such as pyruvate, lactate, glycerol, and glucogenic amino acids.
- glucosyltransferases: Glucosyltransferases are a type of glycosyltransferase that enable the transfer of glucose such as glycogen synthesis.
Polysaccharides are long carbohydrate molecules of repeated monomer units joined together by glycosidic bonds. They range in structure from linear to highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure, these macromolecules can have distinct properties from their monosaccharide building blocks. They may be amorphous or even insoluble in water.
One of the most common building block of polysaccharides is glucose. However, glucose has to be in its activated forms. There are two forms of activated glucose: UDP-glucose and ADP-glucose.
Uridine diphosphate glucose (uracil-diphosphate glucose, UDP-glucose) is a nucleotide sugar. Components UDP-glucose consists of the pyrophosphate group, the pentose sugar ribose, glucose, and the nucleobase uracil. It is used in nucleotide sugars metabolism as an activated form of glucose as a substrate for enzymes called glucosyltransferases. UDP-glucose can also be used as a precursor of lipopolysaccharides, and peptidoglycan. ADP-glucose is usually the precursor for glycogen production in bacteria.
When the cells are grown on a carbon source different than glucose, then polysaccharides are synthesized using a different pathway. Gluconeogenesis (abbreviated GNG) is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates such as phosphoenolpyruvate (PEP). PEP is formed from the decarboxylation of oxaloacetate and hydrolysis of one guanosine triphosphate molecule. This reaction is a rate-limiting step in gluconeogenesis.
Pathogenic bacteria commonly produce a thick, mucous-like, layer of polysaccharide. This “capsule” cloaks antigenic proteins on the bacterial surface that would otherwise provoke an immune response and thereby lead to the destruction of the bacteria. Bacteria and many other microbes, including fungi and algae, often secrete polysaccharides as an evolutionary adaptation to help them adhere to surfaces and to prevent them from drying out. Humans have developed some of these polysaccharides into useful products including xanthan gum, dextran, welan gum, gellan gum, diutan gum, and pullulan.
Many of the immune activating abilities of lipopolysaccharide can be attributed to the lipid A unit.
Outline the characteristics and processes of lipid biosynthesis, including:; lipogenesis and fatty acid biosynthesis
- Lipid A is a lipid component of an endotoxin held responsible for toxicity of Gram-negative bacteria.
- The synthesis of unsaturated fatty acids involves a desaturation reaction, whereby a double bond is introduced into the fatty acyl chain.
- In archaea, the mevalonate pathway produces reactive precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate from acetyl-CoA, while in plants and bacteria the non-mevalonate pathway uses pyruvate and glyceraldehyde 3-phosphate as substrates.
- lipid A: Lipid A is a lipid component of an endotoxin held responsible for toxicity of Gram-negative bacteria. It is the innermost of the three regions of the lipopolysaccharide (LPS, also called endotoxin) molecule, and its hydrophobic nature allows it to anchor the LPS to the outer membrane.
- endotoxin: Any toxin secreted by a microorganism and released into the surrounding environment only when it dies.
- lipogenesis: The biochemical production of fat, especially the conversion of carbohydrate into fat so that it may be stored as a long-term source of energy when food is scarce.
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, and others. The main biological functions of lipids include energy storage, as structural components of cell membranes, and as important signaling molecules.
Lipids may be broadly defined as hydrophobic or amphiphilic small molecules. The amphiphilic nature of some lipids allows them to form structures such as vesicles, liposomes, or membranes in an aqueous environment. Biological lipids originate entirely or in part from two distinct types of biochemical subunits or “building-blocks”: ketoacyl and isoprene groups. Using this approach, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides (derived from condensation of ketoacyl subunits); and sterol lipids and prenol lipids (derived from condensation of isoprene subunits).
Although humans and other mammals use various biosynthetic pathways to both break down and synthesize lipids, some essential lipids cannot be made this way and must be obtained from the diet.
In animals, when there is an oversupply of dietary carbohydrate, the excess carbohydrate is converted to triglycerides. This involves the synthesis of fatty acids from acetyl-CoA and the esterification of fatty acids in the production of triglycerides, a process called lipogenesis. Fatty acids are made by fatty acid synthases that polymerize and then reduce acetyl-CoA units. The acyl chains in the fatty acids are extended by a cycle of reactions that add the acetyl group, reduce it to an alcohol, dehydrate it to an alkene group and then reduce it again to an alkane group.
The enzymes of fatty acid biosynthesis are divided into two groups, in animals and fungi all these fatty acid synthase reactions are carried out by a single multifunctional protein, while in plant plastids and bacteria separate enzymes perform each step in the pathway. The fatty acids may be subsequently converted to triglycerides that are packaged in lipoproteins and secreted from the liver.
One important reaction that uses these activated isoprene donors is steroid biosynthesis. Here, the isoprene units are joined together to make squalene and then folded up and formed into a set of rings to make lanosterol. Lanosterol can then be converted into other steroids such as cholesterol and ergosterol.
Regulation by Biosynthetic Enzymes
Attenuation is a regulatory feature found throughout Archaea and Bacteria domains which causes premature termination of transcription.
Discuss how attenuation can regulate expression of biosynthetic enzymes
- Attenuators may be classified according to the type of molecule that induces the change in RNA structure.
- Attenuator is a nucleotide sequence in DNA that can lead to premature termination of transcription.
- An example of attenuation is the trp gene in bacteria.
- transcription: The synthesis of RNA under the direction of DNA.
- termination: The process of terminating or the state of being terminated.
- Attenuation: Attenuation (in genetics) is a proposed mechanism of control in some bacterial operons that results in premature termination of transcription. It is based on the fact that, in bacteria, transcription and translation can and do proceed simultaneously.
Attenuation (in genetics) is a proposed mechanism of control in some bacterial operons that results in premature termination of transcription. It is based on the fact that, in bacteria, transcription and translation can and do proceed simultaneously. Attenuation involves a provisional stop signal (attenuator), located in the DNA segment that corresponds to the leader sequence of mRNA. During attenuation, the ribosome becomes stalled (delayed) in the attenuator region in the mRNA leader. Depending on the metabolic conditions, the attenuator either stops transcription at that point or allows read-through to the structural gene part of the mRNA and synthesis of the appropriate protein.
Attenuation is a regulatory feature found throughout Archaea and Bacteria causing premature termination of transcription. Attenuators are 5′-cis acting regulatory regions that fold into one of two alternative RNA structures that determine the success of transcription. The folding is modulated by a sensing mechanism producing either a Rho-independent terminator, resulting in interrupted transcription and a non-functional RNA product; or an anti-terminator structure, resulting in a functional RNA transcript. There are now many equivalent examples where the translation, not transcription, is terminated by sequestering the Shine-Dalgarno sequence (ribosomal binding site) in a hairpin-loop structure. While not meeting the previous definition of (transcriptional) attenuation, these are now considered to be variants of the same phenomena and are included in this article. Attenuation is an ancient regulatory system, prevalent in many bacterial species providing fast and sensitive regulation of gene operons and is commonly used to repress genes in the presence of their own product (or a downstream metabolite). What is an attenuator? Attenuator is a nucleotide sequence in DNA that can lead to premature termination of transcription.
Attenuators may be classified according to the type of molecule which induces the change in RNA structure. It is likely that transcription-attenuation mechanisms developed early, perhaps prior to the archaea/bacteria separation and have since evolved to use a number of different sensing molecules (the tryptophan biosynthetic operon has been found to use three different mechanisms in different organisms.)
An example is the trp gene in bacteria. When there is a high level of tryptophan in the region, it is inefficient for the bacterium to synthesize more. When the RNA polymerase binds and transcribes the trp gene, the ribosome will start translating. (This differs from eukaryotic cells, where RNA must exit the nucleus before translation starts.) The attenuator sequence, which is located between the mRNA leader sequence (5′ UTR) and trp operon gene sequence, contains four domains, where domain 3 can pair with domain 2 or domain 4.
The attenuator sequence at domain 1 contains instruction for peptide synthesis that requires tryptophans. A high level of tryptophan will permit ribosomes to translate the attenuator sequence domains 1 and 2, allowing domains 3 and 4 to form a hairpin structure, which results in termination of transcription of the trp operon. Since the protein coding genes are not transcribed due to rho independent termination, no tryptophan is synthesized.
In contrast, a low level of tryptophan means that the ribosome will stall at domain 1, causing the domains 2 and 3 to form a different hairpin structure that does not signal termination of transcription. Therefore, the rest of the operon will be transcribed and translated, so that tryptophan can be produced. Thus, domain 4 is an attenuator. Without domain 4, translation can continue regardless of the level of tryptophan. The attenuator sequence has its codons translated into a leader peptide, but is not part of the trp operon gene sequence. The attenuator allows more time for the attenuator sequence domains to form loop structures, but does not produce a protein that is used in later tryptophan synthesis.
Polyhydroxyalkanoates or PHAs are linear polyesters produced in nature by bacterial fermentation of sugar or lipids.
Summarize the process of PHA production and its applications
- To produce PHA, a culture of a micro- organism such as Alcaligenes eutrophus is placed in a suitable medium and fed appropriate nutrients so that it multiplies rapidly.
- PHA synthases are the key enzymes of PHA biosynthesis.
- There are potential applications for PHA produced by micro-organisms within the medical and pharmaceutical industries, primarily due to their biodegradability.
- Polyhydroxyalkanoates: Polyhydroxyalkanoates or PHAs are linear polyesters produced in nature by bacterial fermentation of sugar or lipids.
- fermentation: Any of many anaerobic biochemical reactions in which an enzyme (or several enzymes produced by a microorganism) catalyses the conversion of one substance into another; especially the conversion (using yeast) of sugars to alcohol or acetic acid with the evolution of carbon dioxide.
- biodegradability: The capacity of a material to decompose over time as a result of biological activity, especially to be broken down by microorganisms
Polyhydroxyalkanoates, or PHAs, are linear polyesters produced in nature by bacterial fermentation of sugar or lipids. They are produced by the bacteria to store carbon and energy. More than 150 different monomers can be combined within this family to give materials extremely diverse properties. These plastics are biodegradeable and are used in the production of bioplastics. They can be either thermoplastic or elastomeric materials, with melting points ranging from 40 to 180°C.
The mechanical qualities and biocompatibility of PHA can also be changed by blending, modifying the surface or combining PHA with other polymers, enzymes and inorganic materials, making it possible for a wider range of applications.
PROCESS OF PHA PRODUCTION
To produce PHA, a culture of a micro-organism such as Alcaligenes eutrophus is placed in a suitable medium and fed appropriate nutrients so that it multiplies rapidly. The biosynthesis of PHA is usually caused by certain deficiency conditions (e.g. lack of macro elements such as phosphorus, nitrogen, trace elements, or lack of oxygen) and the excess supply of carbon sources. Recombinants Bacillus subtilis str. pBE2C1 and Bacillus subtilis str. pBE2C1AB were used in production of polyhydroxyalkanoates (PHA) and it was shown that they could use malt waste as carbon source for lower cost of PHA production. As raw material for the fermentation, carbohydrates such as glucose and sucrose can be used, but also vegetable oil or glycerine from biodiesel production. Researchers in the industry are working on methods with which transgenic crops will be developed that express PHA synthesis routes from bacteria and so produce PHA as energy storage in their tissues. Another group of researchers at Micromidas is working to develop methods of producing PHA from municipal waste water. Another even larger scale synthesis can be done with the help of soil organisms. For lack of nitrogen and phosphorus they produce a kilogram of PHA from three kilograms of sugar.
Polyesters are deposited in the form of highly refractive granules in the cells. Depending upon the microorganism and the cultivation conditions, homo- or copolyesters with different hydroxyalkanic acids are generated. PHAs granules are then recovered by disrupting the cells. In the industrial production of PHA, the polyester is extracted and purified from the bacteria by optimizing the conditions of microbial fermentation of sugar or glucose. Once the population has reached a substantial level, the nutrient composition is changed to force the micro-organism to synthesize PHA. The yield of PHA obtained from the intracellular inclusions can be as high as 80% of the organism’s dry weight.
PHA synthases are the key enzymes of PHA biosynthesis. They use the coenzyme A – thioester of (r)-hydroxy fatty acids as substrates. The two classes of PHA synthases differ in the specific use of hydroxyfattyacids of short or medium chain length. The resulting PHA is of the two types: Poly (HA SCL) from hydroxy fatty acids with short chain lengths including three to five carbon atoms are synthesized by numerous bacteria, including Ralstonia eutropha and Alcaligenes latus (PHB). Poly (HA MCL) from hydroxy fatty acids with middle chain lengths including six to 14 carbon atoms, can be made for example, by Pseudomonas putida. A few bacteria, including Aeromonas hydrophila and Thiococcus pfennigii, synthesize copolyester from the above two types of hydroxy fatty acids. The simplest and most commonly occurring form of PHA is the fermentative production of poly-beta-hydroxybutyrate) (poly-3-hydroxybutyrate, P3HB), which consists of 1000 to 30000 hydroxy fatty acid monomers.
PHAs are processed mainly via injection molding, extrusion and extrusion bubbles into films and hollow bodies. A PHA copolymer called PHBV (poly(3-hydroxybutyrate-co-3-hydroxyvalerate)) is less stiff and tougher, and it may be used as packaging material. There are also applications for PHA produced by micro-organisms within the medical and pharmaceutical industries, primarily due to their biodegradability. Some of the fixation and orthopaedic applications that have been devised for these polymers include:
- sutures and suture fasteners
- meniscus repair and regeneration devices
- rivets, tacks, staples, and screws
- bone plates and bone plating systems
- surgical mesh, repair patches, and cardiovascular patches
- vein valves, bone marrow scaffolds
- ligament and tendon grafts
- ocular cell implants
- skin substitutes, bone graft substitutes, and wound dressings
Polyketides are secondary metabolites produced from bacteria, fungi, plants, and animals.
Describe the characteristics associated with polyketides, including: type I, II and III polyketides
- Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism.
- Polyketides are usually biosynthesized through the decarboxylative condensation of malonyl-CoA derived extender units in a similar process to fatty acid biosynthesis.
- Polyketides are structurally a very diverse family of natural products with diverse biological activities and pharmacological properties.
- Polyketides: Polyketides are secondary metabolites from bacteria, fungi, plants, and animals. Polyketides are usually biosynthesized through the decarboxylative condensation of malonyl-CoA derived extender units in a similar process to fatty acid synthesis (a Claisen condensation).
- metabolites: Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, catalytic activity of their own (usually as a cofactor to an enzyme), defense, and interactions with other organisms (e.g. pigments, odorants, and pheromones).
- biosynthesized: Biosynthesis (also called biogenesis or “anabolism”) is an enzyme-catalyzed process in cells of living organisms by which substrates are converted to more complex products. The biosynthesis process often consists of several enzymatic steps in which the product of one step is used as substrate in the following step.
Polyketides are secondary metabolites produced from bacteria, fungi, plants, and animals.
Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. Unlike primary metabolites, the absence of secondary metabolites does not result in immediate death, but rather in long-term impairment of the organism’s survivability, fecundity, or aesthetics, or perhaps in no significant change at all. Secondary metabolites are often restricted to a narrow set of species within a phylogenetic group. Secondary metabolites often play an important role in plant defense against herbivory and other interspecies defenses. Humans use secondary metabolites as medicines, flavorings, and recreational drugs.
Polyketides are usually biosynthesized through the decarboxylative condensation of malonyl-CoA derived extender units in a similar process to fatty acid biosynthesis (a Claisen condensation). The polyketide chains produced by a minimal polyketide synthase are often further derivitized and modified into bioactive natural products.
Polyketides are structurally a very diverse family of natural products with diverse biological activities and pharmacological properties. They are broadly divided into three classes: type I polyketides (often macrolides produced by multimodular megasynthases), type II polyketides (often aromatic molecules produced by the iterative action of dissociated enzymes ), and type III polyketides (often small aromatic molecules produced by fungal species). Polyketide antibiotics, antifungals, cytostatics, anticholesteremic, antiparasitics, coccidiostats, animal growth promoters, and natural insecticides are in commercial use.
Examples of polyketides include: Macrolides; Pikromycin, the first isolated macrolide; the antibiotics erythromycin A; clarithromycin, and azithromycin; the immunosuppressant tacrolimus; Radicicol and Pochonin family (HSP90 inhibitor); Polyene antibiotics; Amphotericin; Tetracyclines and the tetracycline family of antibiotics.
Polyketides are synthesized by one or more specialized and highly complex polyketide synthase (PKS) enzymes.