An Organism That Is Unicellular Biology Essay

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Introduction

A microorganism or microbe is an organism that is unicellular or lives in a colony of cellular organisms. The study of microorganisms is called microbiology, a subject that began with Anton van Leeuwenhoek's discovery of microorganisms in 1675, using amicroscope of his own design.

Microbes are also exploited by people in biotechnology, both in traditional food and beverage preparation, and in modern technologies based on genetic engineering. However, pathogenic microbes are harmful, since they invade and grow within other organisms, causing diseases that kill people, other animals and plants.

Microbial fermentation industries have undergone revolutionary development in recent years - development which has led to the large-scale production of various organic substances having nutritional, medicinal or other desirable characteristics. Some such uses of "industrial microorganisms".

The development of highly productive microbial strains is a prerequisite for efficient biotechnological processes. Up to the present time, strain improvement was mainly based on induced mutagenesis and only during the last few years has genetic recombination based on protoplast fusion become an additional practicable strategy.

In general, recombinant DNA techniques provide an enormous potential for strain improvement in industrial microorganisms. Basic cloning systems have now been developed for various microbial strains used in industrial processes. Not only bacteria and yeasts, but also many biotechnologically important species of filamentous fungi are now susceptible to rDNA techniques.

rDNA technology has already been successfully applied to the biotechnological production of polypeptides used pharmaceutically and originating from higher eukaryotes. For such ldquohigh price-low volumerdquoproducts, well-developed host systems such as E. coli are usually employed. The scale of production is usually small. Thus these processes are more or less comparable with laboratory operations. However, special problems have to be taken into account when genetically engineered strains are used in large-scale processes.

Process for strain development comprises of several stages-

Data collecting on species and cultivation parameters is required for further analysis of ways to handle the strain

Strain mutability evaluation includes period for probationary mutagenesis series when great variety of mutagen factor combinations affects the strain. All data for obtained mutants during this preliminary mutagenesis is analysed thoroughly for further stage

Screening and selection among great deal of mutant varieties is a very demanding stage: experts' deep knowledge in combination with many years of experience and scientific intuition makes it successful

Induced mutagenesis as the basis of Laboratory Know-How represents combination of classical methods for chemical and physical impact on the strain

Technology parameter optimisation

Process scale-up

Recovery Technology Development

Process for strain improvement comprises of several stages.

Data provided by Customer analysis: to plan further research

Strain and Technology parameters re-production

Strain mutability evaluation includes period for probationary mutagenesis series when great variety of mutagen factor combinations affects the strain. All data for obtained mutants during this preliminary mutagenesis is considered thoroughly for further stage

Screening and selection among great deal of mutant varieties is a very demanding stage: experts' deep knowledge in combination with many years of experience and scientific intuition makes it successful

Induced mutagenesis as the basis of Laboratory Know-How represents combination of classical methods for chemical and physical impact on the strain

Technology parameter optimisation

Process scale-up

Recovery Technology Improvement: under Customer request

Targets for industrial strain improvement

 Increase  product concentration:

(1) raise the gene dose;

(2) break down the gene regulation (e.g. catabolite derepression, metabolite resistance); or

(3) alter permeability to improve product export

Process improvement:

(1) decrease fermentation time;

(2) be able to metabolize inexpensive substrates; 

(3) do not produce undesirable by products e.g. pigments or substance chemically related to the main product;

(4) reduce oxygen needs;

(5) decrease foaming;

(6) tolerant to high concentrations of carbon or nitrogen sources;

(7) resistant to phage 

New product:

Changes in the genotype of microorganisms can lead to the biosynthesis of new metabolites.

Successful strain improvement

In a  balanced strain development program each method should complement the other. In the past, the successes are basically due to the extensive application of mutation and selection. Current data on the production levels of industrial high-performance mutants are rarely published. Today the penicillin yield is around 85,000 units /ml (approx. 50 g/l). Because of the low yield per weight of substrate used (weight of penicillin produced per weight of glucose used is around 0.12), continued increases can be expected in the future.

Methods used in strain improvement

In  vivo mutagenesis:

Spontaneous mutation- A mutation occurring in the absence of mutagens, usually due to errors in the normal functioning of cellular enzymes.

radiation (short-wavelength uv, 200-300nm, opt. 254nm / ionizing radiation, e.g. X-rays, -rays, and -rays);

chemical agents (mutagens which affect nonreplicating DNA, e.g. nitrous acid (HNO2), hydroxylamine (NH2OH),  alkylating agents / base analogs or frameshift mutagens, which are incorporated into or enter into replicating DNA;

transposon insertion- An insertion sequence (also known as an IS, an insertion sequence element, or an IS element) is a short DNA sequence that acts as a simple transposable element. Insertion sequences have two major characteristics: they are small relative to other transposable elements (generally around 700 to 2500 bp in length) and only code for proteins implicated in the transposition activity (they are thus different from other transposons, which also carry accessory genes such as antibiotic resistance genes).

suicide vector and recombination.

In  vitro mutagenesis:

Random mutagenesis- Low mutation rate (single amino acid-changes).

Methods for random mutagenesis--

• E.coli XL1red

• UV irradiation

• chemical methods

- deamination

- alkylation

- Base-Analog Mutagens

• PCR methods

- DNA shuffling

•site directed random mutagenesis

Directed mutagenesis- also known as directed mutation, is a hypothesis proposing that organisms can respond to environmental stresses through directing mutations to certaingenes or areas of the genome.

Recombination:

Genetic recombination is a process by which a molecule of nucleic acid (usually DNA, but can also be RNA) is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method of DNA repair in both bacteria and eukaryotes. In eukaryotes, recombination also occurs in meiosis, where it facilitates chromosomal crossover. 

Recombination in fungi: sexual and parasexual cycles;

Parasexual recombination is a valuable tool in the laboratory, particularly for asexual fungi, and a number of developments in methodology are outlined. In biotechnology, the parasexual cycle has proved less useful than at one time predicted, but it retains a function in analysis of the products of genetic manipulation, and as a convenient detection system for environmental chemicals that may disturb mitosis. In nature, recent evidence suggests that parasexual recombination is rare, in part at least because of the prevalence of heterokaryon incompatibility of many wild fungi.

Recombination in bacteria: transformation, transduction and conjugation;

Transformation is the genetic alteration of a cell resulting from the uptake, incorporation and expression of exogenous genetic material (DNA) that is taken up through the cell wall(s). Transformation occurs most commonly in bacteria and in some species occurs naturally.

Transduction is the process by which DNA is transferred from one bacterium to another by a virus.  It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells.

in vivo rearrangements by transposable genetic elements

protoplast fusion- Protoplasts are the cells of which cell walls are removed and cytoplasmic membrane is the outermost layer in such cells.Protoplast can be obtained by specific lytic enzymes to remove cell wall. Protoplast fusion an important tools in strain improvement for bringing genetic recombinations and developing hybrid strains in filamentous fungi. Protoplast fusion has been used to combine genes from different organisms to create strains with desired properties.These are the powerful techniques for engineering of microbial strains for desirable industrial properties.

in vitro recombinant DNA techniques

Spontaneous mutations  

Mutation rate  depends on growth conditions: 10-10 to 10-5 per generation and per gene; usually 10-7 to 10-6. All mutant types are found, although deletions are relatively frequent.

Spontaneous mutations on the molecular level can be caused by:

Tautomerism - A base is changed by the repositioning of a hydrogen atom, altering the hydrogen bonding pattern of that base resulting in incorrect base pairing during replication.

Depurination - Loss of a purine base (A or G) to form an apurinic site (AP site).

Deamination - Hydrolysis changes a normal base to an atypical base containing a keto group in place of the original amine group. Examples include C → U and A → HX (hypoxanthine), which can be corrected by DNA repair mechanisms; and 5MeC (5-methylcytosine) → T, which is less likely to be detected as a mutation because thymine is a normal DNA base.

Slipped strand mispairing - Denaturation of the new strand from the template during replication, followed by renaturation in a different spot ("slipping"). This can lead to insertions or deletions.

The mutation frequency (proportion of mutants in the population) can be significantly increased by using mutagenic agents (mutagens): may increase to 10-5-10-3 for the isolation of improved secondary metabolite producers or even up to 10-2-10-1 for the isolation of auxotrophic mutants.

Mutagenesis through:-

Radiation by short-wavelength ultraviolet (UV)

Effective  wavelengths: 200-300 nm (opt. at 254 nm).

Important products: dimers between adjacent pyri-midines (T-T, T-C, and C-C) or between pyrimidines of complementary strands (results in crosslinks). Mainly induces transitions of GC→AT, transversions, frameshift and deletions are also found. To prevent error-free mechanisms of photoreactiva-tion and excision repair: carrying out all manipulation under long-wavelength visible light ( > 600 nm) and/or the use of caffeine or similar inhibitors of repair. The SOS repair system is primarily responsible for the production of mutations.

Alkylating agents

Most potent mutagen for practical application, except UV. Compounds frequently used are ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), diethylsulfate (DES), diepoxybutane (DEB), N-methyl-N'-nitro-N-nitrosoguanidine (NTG, the most effective chemical mutagens, but its use in a mutation program is difficult because of its carcinogenic effects), N-methyl-N-nitroso-urea and mustard gas. Formation of alkylated bases in DNA, along with phosphotriester, purine-free sites and single-strand breaks / 7-alkylguanine is the most common product but it does not result in mutations / O6-alkyl-guanine and O4-alkylthymine are the most important premutational lesions, and as a result of pairing errors, mainly AT→GC transitions are elicited (direct mutagenesis). Transitions, transversions, deletions and frameshift mutations. A second process results in mutation is the induction of error-prone SOS repair when relatively high doses of mutagen are used.

NTG

A large proportion of mutants under optimal conditions with a low killing rate. 8-10% of  the survival Streptomy-ces coelicolor are auxotrophs, ~ 50% of the survival E. coli population consists of mutants. 90% mutations are GC→AT transitions; also small extent deletions and frameshift after the deletion of GC pairs.Easily decomposed in vivo; formed nitrous acid in acidic solutions (not effective as mutagen in pH 6-9 where NTG is active; diazomethane (a strongly methylating agent) is formed under alkaline conditions. Alkylation of nonreplicating DNA and the main point of action of at the replication point of DNA, through a change in DNA polymerase III during DNA replication (there is incorrect duplication in a short segment of the DNA until the defective polymerase is replaced by an intact molecule). This explains that NTG mutations frequently occur in gene clusters.

IS-Elements  

DNA sequencesof variable length can incorporated in different sites of the genome and released again ( recA-independent  recombination). They destroy the function of the gene at the site of their integration. IS2 bears a promotor, which, when incorporated in the appropriate orientation, results in the constitutive expression of genes located downstream. In particular IS1 causes deletions, whereas IS2 causes duplications.

Transposons

Genetic elements containing flanking IS-elements in inverse orientation, often with antibiotic-resistance genes. Available for a wide variety of purposes in gene technology: Tn5 contains an aminoglycoside antibiotic-resistance gene which can be expressed in a wide variety of both procaryotes and eucaryotes. Several transposons have been integrated into plasmids (e.g. Tn1 and Tn3), others in either plasmids or chromosomes (e.g. Tn5). Transposon mutagenesis offers a wide variety of advantages: (1) can obtained a mutant phenotype with a very low reversion rate, (2) relatively easy to isolate insertion mutations, (3) the site at which the transposon has been integrated can be readily determined.

Selection of improved  mutants

Random  selection

Directed selection

Rational selection

 Selection of improved  mutants - 1 

Random  selection: 

(1) 5-10 best strains are repeatedly mutated and selected;

(2) Hundreds to thousands of isolates per mutation cycle must be tested (to screen a small number of survivors, about 20-50, after many different mutagen treatments and to continue mutating strains having small yield increases as quickly as possible is more economical);

(3) With nonautomatic methods the number of isolates that can be tested per unit times is usually limited to 1000-2000 per week;

(4) Two stages of screening (one fermentation sample per isolation in the first stage when the test error is smaller than the yield increase expected; 10-30% best isolates are tested in a second stage and the number of replicates are chosen statistically).

Selection of improved  mutants - 2 

Directed  selection

Selection of improved  mutants - 3 

Rational  selection: 

(1) Antibiotic-resistant mutants: genetic marker / increased cell permeability / protein synthesis with a higher turnover;

(2) Antimetabolite-resistant mutants: analog-resistant mutants can form an excess of metabolites, in some cases through changed regulatory mechanisms (elimination of allosteric inhibition, constitutive product formation);

(3) Auxotrophs: products formed via branching biosynthetic pathways such as amino acids and nucleotides.

Strain improvement in antibiotic-producing microorganisms

In the context of commercial strain development for improved antibiotic production it is important to stress the efficiency and practicability of the procedures used. Typical constraints are the novelty of the microorganism, time and manpower. Established and more novel techniques applied to strain improvement can be divided into the induction of beneficial alterations and the screen for and recognition of such altered mutants. Recent advances have contributed mainly to the former of these two divisions. The empiricism of mutagenesis can be removed as more is known about mutation induction with respect to repair phenomena. Mutagens must be assessed for efficiency with rapid test systems for forward and reverse mutation. Enhanced mutation frequencies can be achieved with repair-deficient mutants or chemical inhibitors of repair processes. It is widely accepted that recombinational methods are an important part of strain development although published examples of its successful application are rare. Parasexuality in asexual fungi and protoplast fusion in both bacteria and fungi facilitate recombination and therefore the construction of improved strains. As a result of these advances it has become necessary to deviate from the classical, linear mutation and selection programmes. The application of recombinant DNA technology to strain development is undoubtedly promising. In the field of antibiotic research this approach may necessitate extensive basic research before its potential can be realised. Major constraints may be poor knowledge of both the biosynthesis of the antibiotic and the genetic systems in the producing microorganism. It will also be necessary to develop suitable vector systems. This approach clearly consumes time and resources which are important considerations in commercial terms. The ability to recognise and isolate improved mutants is just as important as their production and again time and efficiency is paramount. Shake-flask screening has been central to most programmes although its format may be changing to accommodate multiline screens to capitalise on recombination techniques. Its major limitation as a primary screen is in the work involved in testing a comparatively small number of strains. To circumvent this problem many laboratories are miniaturising screens or moving towards 'plate-based' screens in which colony productivity is assessed on agar. Plate screens also permit less empirical screening procedures such as reversion of mutants impaired in antibiotic production or the use of chemical inhibitors of antibiotic biosynthesis. This approach has the advantage of allowing large numbers to be screened as well as providing information of a biosynthetic nature if the mutants and inhibitors are subject to investigation in their own right.

Industrial strain improvement: Mutagenesis and random screening procedures

Abstract

Industrial strain improvement plays a central role in the commercial development of microbial fermentation processes. In recent years new procedures such as rational screening and genetic engineering have begun to make a significant contribution to this activity but mutagenesis and selection - so-called 'random screening' - is still a cost-effective procedure, and for reliable short-term strain development is frequently the method of choice. The current practice of strain improvement by mutagenesis and selection is a highly developed technique drawing on the latest advances from a wide range of scientific and technical disciplines. Mutagenic procedures can be optimized in terms of type of mutagen and dose, mutagen specificity effects can be taken into account and mutagenesis itself can be enhanced or directed in order to obtain the maximum frequency of desirable mutant types among the isolates to be screened. Screens can be designed to allow maximum expression of the desirable mutant types and the application of statistically-based screening procedures will maximize the probability of detecting them. Automated procedures can be developed using robotics and microprocessors to increase the numbers of isolates that can be processed through a screen per unit time. The relationship between screening and production conditions can be organized so as to minimize the probability of improved isolates selected by the screen failing to scale up.

Strain improvement of Rhizopus oryzae for over-production of Image (+)-lactic acid and metabolic flux analysis of mutants

Abstract

Strain improvement has been conventionally achieved through mutation and selection. However, the genetic and metabolic profiles of mutant strains were poorly characterized and mutagenesis remained a random process. Metabolic flux analysis (MFA) of mutants will enable us to better understand the change of flux profile in vivo and the mechanism of mutagenesis. A Image (+)-lactic acid over-producing mutant, Rhizopus oryzae R1021, was isolated by mutagenizing the parent strain (R. oryzae R3017) with UV, diethyl sulfate (DES) and 60Co. Starting with a concentration of 120 g/l corn starch, mutant R1021 produced 79.4 g/l Image (+)-lactic acid after 60 h in flasks, 52% higher than that produced by the parent strain. The Image (+)-lactic acid purity was 99.05% by weight based on the amount of total lactic acid. The mutant R1021 was also morphologically different from the parent strain. The results of carbon flux analysis of the parent strain and mutants showed that the pyruvate node in the metabolic model was the principle and flexible node. The results show that the key steps of the pathways in parent strain where most carbon is lost from lactic acid formation are the reactions form pyruvate to acetyl-CoA, oxaloacetate and ethanol. Although the fractions of the carbon from pyruvate to ethanol and acetyl-CoA were reduced in mutants, these two pathways are still the steps that are most likely to be further targeted to reroute the pyruvate metabolism to improve lactic acid production.

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