Major Products Of Industrial Microbiology PDF

MAJOR PRODUCTS OF INDUSTRIAL MICROBIOLOGY 5.1 INTRODUCTION Once a suitable microorganism is obtained using selection and mutation processes, one can produce many compounds and transform a variety of substrates. The discipline responsible for such accomplishments is known as industrial microbiology...

MAJOR PRODUCTS OF INDUSTRIAL MICROBIOLOGY 5.1 INTRODUCTION Once a suitable microorganism is obtained using selection and mutation processes, one can produce many compounds and transform a variety of substrates. The discipline responsible for such accomplishments is known as industrial microbiology. These products include pharmaceutical and medical compounds (antibiotics, hormones, and transformed steroids), solvents, organic acids, chemical feedstocks, amino acids, and enzymes. Microorganisms also can be used to produce fuels and energy-related products. The economics of the production of these materials is constantly changing. Microbial products are classified as primary and secondary metabolites. Primary metabolites consist of compounds related to the synthesis of microbial cells and often are involved in the growth phase, or trophophase (Figs. 5.1 and 5.2). They include amino acids, nucleotides, and fer-mentation end products such as ethanol and organic acids. Industrially useful enzymes, either associated with the microbial cells or exoenzymes, often are synthesized by microorganisms during growth. These enzymes find many uses in food production and textile finishing. Secondary metabolites (Figs. 5.1 and 5.3) usually accumulate during the period that follows the active growth phase, often called the idiophase. Compounds produced in the idiophase have no direct re-lationship to the synthesis of cell materials and normal growth. Most antibiotics and the mycotoxins fall into this category. 78 Fig. 5.1: Depending on the particular organism, the desired product may be formed during or after growth. (a) Primary Metabolite. A primary metabolite like ethanol has a production curve that lags only slightly behind the line showing cell growth – and is formed during the trophophase (active growth). (b) Secondary Metabolite, A secondary metabolite like penicillin from mould begins to be produced only after the main growth phase of the cell is completed. The main production of a secondary metabolite occurs during the stationary phase of cell growth (idiophase).whereas secondary metabolites are formed after growth is completed, the idiophase (Tortora et al., 1994). Fig. 5.2: Primary metabolite is synthesized by microorganisms in the course of the metabolic processes that keep the cells growing. In a reactor vessel a primary metabolite accumulates in tandem with the accumulation of the cells that synthesize it. The graph shows the accumulation of yeast cells with the concomitant accumulation of ethanol. Fig. 5.3: Secondary metabolite is not formed as a direct result of the metabolism that keeps the cells alive. The accumulation of a secondary metabolite in a reactor vessel lags behind the growth of the cells that produce it. The graph shows the accumulation of mould cells and the subsequent accumulation of penicillin. The temperature and pH that are best for the growth of cells are seldom best for the synthesis of a secondary metabolite. In a batch process one seeks a compromise between the two sets of optimum conditions. 79 5.2 MICROBIAL SYNTHESIS OF PRIMARY METABOLITES Primary metabolites are microbial products made during the exponential phase of growth and whose synthesis is an integral part of the normal growth process. They include intermediates and end products of anabolic metabolism, which are used by the cell as building blocks for essential macromolecules (e.g. amino acids, nucleotides) or are converted to coenzymes (e.g., vitamins). On the other hand, primary metabolites of catabolic metabolism (e.g., citric acid, acetic acid, and ethanol) are not biosynthetic precursors but their production, which is related to energy production and substrate utilization, is essential for growth. Industrially, the most important primary metabolites are amino acids, nucleotides, vitamins, solvents, and organic acids. These are made by a diverse range of bacteria and fungi and have numerous applications in the food, chemical, pharmaceutical and nutraceutical industries. Many of these metabolites are manufactured by microbial fermentation rather than chemical synthesis because the fermentations are economically competitive and produce biologically active isomers. Several other industrially important chemicals could be manufactured via microbial fermentations (e.g., glycerol and other polyhydroxy alcohols) but are presently synthesized cheaply as petroleum by-products. However, since the cost of petroleum has skyrocketed recently, there is renewed interest in the microbial production of ethanol, organic acids, and solvents. 5.2.1 CONTROL OF PRIMARY METABOLISM Microbial metabolism is a conservative process that usually does not expend energy or nutrients to make compounds already available in the environment and does not overproduce compounds of intermediary metabolism. Coordination of metabolic functions ensures that at any given moment only the necessary enzymes, and the correct amount of each, are made. Once a sufficient quantity of material is made the enzymes concerned with its formation are no longer synthesized and the activities of preformed enzymes are curbed by a number of specific regulatory control mechanisms, such as feedback inhibition and covalent modifications. 5.2.1.1 Feedback regulation The most important mechanism responsible for regulation of anabolic enzymes involved in the biosynthesis of amino acids, nucleotides and vitamins is not induction or nutrient repression but feedback regulation. This category of regulation functions at two levels: enzyme action (feedback inhibition) and enzyme synthesis (feedback repression and attenuation). In feedback inhibition, the final metabolite of a pathway when present in sufficient quantity, inhibits the action of the first enzyme of the pathway by binding to the enzyme at an allosteric site resulting in the interference of binding of the enzyme and substrate. This prevents the synthesis of unwanted intermediates on the one hand and the wasting of energy on the other. Feedback repression involves the turning off of enzyme synthesis when the amount of the product has been made in sufficient quantities to satisfy the biosynthetic demands (Fig. 5.4). The end product of the pathway acts as a co-repressor. The apo-repressor specified by the regulator gene is inactive in the absence of its co-repressor and is unable to bind to the operator region. However, in the presence of a co- repressor, the inactive apo-repressor is converted to an active repressor that binds to the operator region, thus preventing the binding of RNA polymerase to the promoter region, which in turn brings enzyme biosynthesis to a halt. Feedback inhibition may be seen as a rapid control that switches off the biosynthesis of an end 80 product and feedback repression as a mechanism that then switches off the synthesis of temporarily redundant enzymes. Control in pathways producing only one end product (linear/unbranched pathways) is normally due to the inhibition of the first enzyme in the pathway by the end product and repression of synthesis of all enzymes in the pathway by the end product (Fig. 5.4). Control in pathways yielding many end products (branched pathways) is more complex (Figs 5.5-5.9). Several mechanisms have been involved to control the various end products so that the cell is not deprived of necessary intermediates. Concerted/multivalent feedback control (Fig. 5.5): more than one end product exerts control – the first enzyme is inhibited or repressed when all the end products are in excess. Cooperative feedback control (Fig. 5.6): weak control may be exhibited by each end product independently but when all are in excess, synergistic/cooperative inhibition or repression occurs. When one end product is in excess, for efficient control, there is control immediately after the branch point to that end product. Cumulative feedback control (Fig. 5.7): Each of the end products inhibits the first enzyme to a certain percentage independently of the other end products. Sequential feedback control (Fig. 5.8): Each end product of the pathway controls the enzyme immediately after the branch point to the product. The intermediates which build up as a result of this then control earlier enzymes in the pathway. Isoenzyme control (Fig. 5.9): Isoenzymes are enzymes that catalyse the same reaction but differ in their control characteristics. If a critical control reaction is catalysed by more than one isoenzyme, the different isoenzymes may be controlled by the different end products. Fig. 5.4: Control of linear biosynthetic pathway converting precursor A to end product E via the intermediates B, C and D (Stanbury et al., 1984). Fig. 5.5: Control of a branched biosynthetic pathway by the concerted effects of products D and F on the first enzyme in the pathway (Stanbury et al., 1984). Fig. 5.6: Control of a branched biosynthetic pathway by the co-operative control by end products D and F. (Stanbury et al., 1984). Fig. 5.7: Control of a branched biosynthetic pathway by the cumulative control of end products D and F. (Stanbury et al., 1984). 81 5.3 APPROACHES TO STRAIN IMPROVEMENTS The ideal industrial microorganism however needs to produce amounts of product greater than that required for growth. Thus, these organisms should have a de-regulated metabolism with regards to the product of interest. Such organisms may be modified (mutants) as follows: 1 The end products which control the key enzymes of the pathway are lost from the cell due to some abnormality in permeability. 2 The end product that control the key enzymes in the pathway are not produced. 3 The organism does not recognize the presence of inhibiting or suppressing levels of the normal control metabolites. Mutation and Selection: Organisms used today for industrial production of primary metabolites have been developed by programs of intensive mutagenesis followed by screening and selection of overproducers. Such efforts often start with organisms having some capacity to make the desired product but that require multiple mutations leading to deregulation in a particular biosynthetic pathway before high productivity can be obtained. The enhancement of a metabolic path by gene overexpression can be the result of a point mutation in the promoter regions or an increase in the copy number of single genes or a whole gene cluster. The prevention of by-products can be achieved by searching for auxotrophic mutants. (Figs. 5.10 and 5.11). Fig. 5.9: Control of two isoenzymes catalyzing the conversion of A to B by end products D and F. (Stanbury et al., 1984). Fig. 5.8: Control of a branched biosynthetic pathway by sequential control (Stanbury et al., 1984). Fig. 5.11: Use of auxotrophic mutation in a branched pathway. The auxotrophic mutant will require primary metabolite 1 for growth. When non-excessive levels of 1 are used to support growth, primary metabolite 2 will be overproduced. Fig. 5.10: Use of auxotrophic mutation in a linear pathway. Feedback inhibition by the end product interferes with the activity of enzyme a and feedback repression interferes with the formation of enzymes a and b. By making a genetic block (mutation) at enzyme c, an auxotrophic mutant is made which cannot grow unless E is added to the medium. As long as the amount of E present is not excessive, there will be no feedback effects and C will be overproduced. 82 5.3.1 MODIFICATION OF PERMEABILITY The best example is that of the glutamic acid fermentation by Corynebacterium glutamicum. Permeability is modified by biotin: when biotin levels were below 5 μg/ml, glutamate was excreted and at concentrations optimum for growth, lactate was produced. The increased permeability of the biotin-limited cells is due to a change in the fatty acid and phospholipid content of the cell envelope. The crucial factor is the synthesis of membranes deficient in phospholipids. The inclusion of penicillin or fatty acid derivatives, such as Tween 80, during exponential growth under biotin limitation also causes excretion of glutamate. In addition, a glycerol requiring auxotrophic mutant in which phospholipid synthesis is controlled by glycerol supply also excretes glutamate. Thus, an understanding of the mode of biotin limitation has led to the use of genetic modification of permeability as a method to overcome the organism’s normal control mechanisms. 5.3.2 MUTANTS LACKING FEEDBACK INHIBITORS AND REPRESSORS In mutants that do not produce repressors and inhibitors, control of the pathway is lifted. However, since the control factors are essential for growth, they must be incorporated into the medium at concentrations which will allow growth to proceed but will not evoke the normal control reactions. Feedback inhibition by the end product interferes with the activity of enzyme a and feedback repression interferes with the formation of enzymes a and b. By making a genetic block (mutation) at enzyme c, an auxotrophic mutant is made which cannot grow unless E is added to the medium. As long the amount of E present is not excessive, there will be no feedback effects and C will be overproduced. Fig. 5.12(2) illustrates the removal of concerted inhibition - mutant is auxotrophic for E, provided E is supplied in non-inhibiting concentrations, C will be overproduced. Fig. 5.12(3) is similar to Fig. 5.12(2), except that the mutant is auxotrophic for both F and G. In Fig. 5.12(4), the mutant is a double mutant with the second mutation between F and G resulting in the accumulation of F. In Fig. 5.12(5) the mutant is auxotrophic for I and G and is an example of the deregulation of concerted inhibition and an accumulation of E. The sequential mutations ensure that nutrients are channelled efficiently to the appropriate products without significant deviation to other pathways. These mutations involve not only release of feedback control but also enhancement of the formation of pathway precursors and intermediates. This approach to strain improvement has been remarkably successful in producing organisms that make industrially significant concentrations of primary metabolite. However, some of the problems with this "brute force" approach include (i) the necessity of screening large numbers of mutants for the rare combination of traits sequentially obtained that lead to overproduction, and (ii) the possibility that the vigour of the producing strain may be substantially weakened following several rounds of mutagenesis 83 Fig.5.12: Overproduction of primary metabolites by decreasing the concentration of a repressing or inhibiting end product (Stanbury et al., 1994).. 5.3.3 RECOMBINANT DNA TECHNOLOGY More recent approaches employ recombinant DNA technology to develop strains that are capable of overproducing primary metabolites. This rationale for strain construction relies largely on the same principles of regulation discussed previously, but aims at assembling the appropriate characteristics by means of in vitro recombinant DNA techniques. This is particularly valuable in organisms with complex regulatory systems, where deregulation would involve many genetic alterations. Metabolic engineering of rational strain development is therefore a targeted manipulation of the producer organism. Production of a particular primary metabolite by deregulated organisms may inevitably be limited by the inherent capacity of a particular organism to make the appropriate enzymes, i.e., even in the absence of repressive mechanisms, there may not be enough of the enzyme made to obtain high productivity. One way to overcome this is to increase the number of copies of structural genes coding for these enzymes by genetic engineering. Another way often used in combination with this strategy is to increase the frequency of transcription, which is related to the frequency of 84 binding of RNA polymerase to the promoter region. The former can be achieved by cloning the synthetic genes in vitro into a plasmid that, when introduced into the cell through transformation, will replicate into multiple copies. Increasing the frequency of transcription involves the construction of a recombinant plasmid in vitro that contains the structural genes of the biosynthetic enzymes but lacks the regulatory sequences (promoter and operator) normally associated with them. Instead the structural genes are cloned downstream of an efficient promoter, thus facilitating a higher level of expression. The ideal plasmid for metabolite synthesis would contain a regulatory region with a constitutive phenotype, preferably not subject to nutritional repression. Novel genetic technologies such as "genome-based strain reconstruction" achieve the construction of a superior strain that contains only mutations crucial to hyperproduction, but not other unknown mutations that accumulate by brute-force mutagenesis and screening. This approach has successfully been used to improve lysine production. The directed improvement of product formation or cellular properties via modification of specific biochemical reactions or introduction of new ones with the use of recombinant DNA technology is known as "metabolic engineering." Analytical methods are combined to quantify fluxes and to control them with molecular biological techniques in order to implement suggested genetic modifications. Different means of analyzing flux are (i) kinetic-based models, (ii) control theories, (iii) tracer experiments, (iv) magnetization transfer, (v) metabolite balancing, (vi) enzyme analysis, and (vii) genetic analysis. The overall flux through metabolic pathway depends on several steps, not just a single rate-limiting reaction. A genome-wide transcript expression analysis called “massive parallel signature sequencing" has been successfully used to discover new targets for further improvement of riboflavin production by the fungus Ashbya gossypii. The development and combined application of the above technologies will help to develop "inverse metabolic engineering” which in turn will be used to construct certain phenotypes that are ideal for commercial purposes. Molecular breeding techniques such as "DNA shuffling" come closer to mimicking natural recombination by allowing in vitro homologous recombination. These techniques not only recombine DNA fragments but also introduce point mutations at a very low but controlled rate. Unlike site directed mutagenesis, this method of pooling and recombining parts of similar genes from different species or strains has yielded remarkable improvements in enzymes in a very short amount of time. “Whole genome shuffling" is a novel technique for strain improvement combining the advantage of multi-parental crossing allowed by DNA shuffling with the recombination of entire genomes.