Food Biotechnology CUBT402 Genetic Manipulation Of Microorganisms PDF

Food Biotechnology CUBT402 Genetic Manipulation Of Microorganisms Chinhoyi University of Technology - Department of Biotechnology  Objectives Describe how microorganisms can be genetically modified for use in food production  Contents Introduction Genetic m...

Food Biotechnology CUBT402 Genetic Manipulation Of Microorganisms Chinhoyi University of Technology - Department of Biotechnology  Objectives Describe how microorganisms can be genetically modified for use in food production  Contents Introduction Genetic manipulation of microorganisms Bacteria Filamentous fungi Yeasts  Introduction Microorganisms such as yeasts, filamentous fungi, bacteria and algae play a key role in many industrial processes ranging from the production of traditional fermented foods and beverages to recombinant proteins and other high-value molecules Most industries depend heavily on the model yeast S. cerevisiae for their processes S. cerevisiae strains are traditionally used to produce several products Rising demands for increased productivity, wider substrate range utilisation, production of non- conventional compounds and changing consumer preferences Great interest in further improving the current industrial strains and the selection or development of strains with novel properties - biotechnology  Cont.. Biotechnology in the food processing target to enhance process control, yields and efficiency, the quality, safety and consistency of bio-processed products (Steensels et al., 2014) Genetic modification/gene manipulation is a biological practice that alters the genetic material of all types of living organisms Use of recombinant DNA technology  Genetic manipulation of microorganisms Genetically modified microbes have the potential to withstand the fermentation conditions and enhance productivity E.g.cloning a gene that encodes for thermostability to the microbes is possible to survive high temperatures in bioreactors Creation of clones of a specific microorganism that is steady and could grow rapidly  Cont.. Strategies to improve of genetically and metabolically modified microorganisms Mallikarjuna andYellamma (2018)  Cont.. Traits considered for commercial food applications include: Sensory quality (flavour, aroma, visual appearance, texture and consistency) Virus (bacteriophage) resistance in dairy fermentations The ability to produce antimicrobial compounds (e.g. bacteriocins, hydrogen peroxide) for the inhibition of undesirable microorganisms In many developing countries, focus is on degradation or inactivation of natural toxins (e.g. cyanogenic glucosides in cassava), mycotoxins (in cereal fermentations) and anti- nutritional factors (e.g. phytates)  Genetic manipulation of bacteria Both traditional and molecular approaches are subject to improvement of bacteria, yeasts and moulds Traditional methods of genetic improvement such as classical mutagenesis, transformation, transduction and conjugation have been used in industrial starter culture development in bacteria Molecular methods include rDNA technology, genetic characterisation and genomics  a) Classical mutagenesis: Involves production of mutants by the exposure of microbial strains to mutagenic chemicals or UV rays to induce changes in their genomes – induced mutations Improved strains are then selected on the basis of specific properties such as improved flavour-producing ability or resistance to phage viruses Mutants show undesirable secondary mutations which can influence the behaviour of cultures during fermentation Spontaneous mutations can also occur Directed mutations – point mutations, deletions, insertions, substitutions, deletions  Mutagens Mutagen Mode of operation Genetic change Physical UV Crossing over; mitotic gene Frameshift conversion; pyrimidine dimers; mutations, base hydroxylated bases; cross-linking pair DNA strands; substitutions, reverse mutations transversions Ionising S- and d-strand breaks in DNA; Point mutations radiation deamination and dehydroxylated bases Chemical EMS Alkalylation GC-AT transitions MNNG Alkylation, acts close to replication Transitions, points transversions; clustered mutations EMS, ethyl methanesulfonate; MNNG, methylnitronitrosoguanidine  b) Conjugation: A natural process whereby genetic material is transferred among closely related microbial species as a result of physical contact between the donor and the recipient microbe Conjugation via sex pilus Only donor cells possess sex pili Horizontal gene transfer technique  c) Transduction: Foreign DNA is absorbed by host cell and integrates with its the genome Transformation occurs in ‘competent’ cells Competence can be artificially induced by treatment with a calcium salt and PEG Applied to enhance protease and amylase production in Bacillus spp  Genetic modifications: a) rDNA technology rDNA technology/cloning used for genetic modification of bacterial, yeast and mould strains to: promote expression of desirable genes hinder the expression of other undesirable genes alter specific genes or inactivate genes so as to block specific pathways Successful genetic modification for food bio-processing applications requires the development and use of food grade vectors i.e. plasmids which do not contain antibiotic resistance genes as markers and which consist of DNA sequences from microorganisms which are generally recognised as safe (GRAS)  b) Genetic characterisation: Genetic characterisation of microbial strains using molecular diagnostics contributes to understanding of fermentation processes - rely mostly on PCR Provides outstanding tools for the detection, identification and characterisation of microbial strains for bio-processing and for improvement of fermentation processes Application of molecular diagnostics and related-techniques, along with molecular markers for bacterial strains facilitates understanding of the ecological interactions of microbial strains, their roles, succession, competition and prevalence in food fermentations Also allows the correlation of these features to desirable quality attributes of the final product  c) Genomics: Functional genomics aims to determine patterns of gene expression and interaction in the genome based on the knowledge of extensive or complete genomic sequence of an organism Provides knowledge on how microbes respond to environmental factors at genetic level Therefore allow adaptation of conditions to improve technological processes Macroarrays were used to analyse expression of E. coli genes in a glucose-limited medium Functional genomics shed light on common genetic mechanisms which allow microbes to utilise certain sugars during fermentation and how strains perform better than others It also holds potential for defining and modifying metabolic mechanisms used by microbes At protein level, proteomics offers potential for improving fermentation technologies Proteomics aims to identify and characterise complete sets of protein, and protein-protein interactions in a given species  Genetic manipulation of filamentous fungi Filamentous fungi and moulds grow and quickly on simple and cheap media They are preferred cell factories for due to their outstanding capacity in expression and secretion of heterologous proteins with posttranslational modification Applied in manufacturing of a wide range of food products, organic acids and commercial enzymes The advent of genetic transformation techniques is a breakthrough to genetically modify fungal strains  Cont.. Foreign DNA is transformed into filamentous fungi to obtain desired strains by many different ways: Protoplast-mediated transformation (PMT) Agrobacterium-mediated transformation (AMT) Electroporation method Biolistic transformation Shock-wave-mediated transformation (SWMT)  1. Protoplast-mediated transformation (PMT): PMT is the most widely used fungal transformation method It was first applied to S. cerevisiae PMT relies on a large number of competent fungal protoplasts Basic principle:  Commercial enzymes are used to degrade fungal complex cell wall components for generation protoplasts  Chemical agents e.g. PEG are used to promote the fusion of exogenous nucleic acids and protoplasts  Non-enzymatic methods e.g. physical methods such as grinding and sonication tried to remove protoplasts  These methods have low yield of protoplasts  Cont.. Basic steps for PMT Li et al. (2017)  Cont.. Advantages of PMT: Simple and effective No use of expensive equipment Disadvantages of PMT: Several steps and critical reagents involved Requires experienced personnel Careful monitoring of growth status of fungi under transformation  2. Agrobacterium-mediated transformation (AMT): Agrobacterium tumefaciens Gram-negative bacterium commonly found in soil The bacterium carries a tumour-inducing plasmid (Ti plasmid) commnly referred to as transfer-DNA (T-DNA) A. tumefaciens infect injured plants conjugatively and integrate part of the Ti plasmid into the genome of infected pant cells This results in a condition called Crown gall disease A. tumefaciens now used as a vehicle to integrate target gene into fungal genome to improve its properties  Cont.. Basic steps of AMT Li et al. (2017)  Cont.. Advantages of AMT: Diversified transformation recipients including protoplasts, hyphae and spores Ability to integrate exogenous genes into the genome to form stable transformants High transformation efficiency Disadvantages of AMT: Requires binary vectors which are tedious to prepare Multiple factors must be considered in optimisation of the transformation  3. Electroporation transformation: Electric charges are stored in a capacitor to build a high voltage The sample is then struck by impulse voltage and the exogenous nucleic acid is transferred instantly into cells Transformation of fungi make use of square waves or exponential- decay waves Exponential-decay pulses are generated by simply charging and discharging the capacitor Square wave is non-sinusoidal periodic waveform in which amplitude alternates between fixed minimum and maximum values  Cont.. Advantages of electroporation: Simple Rapid Efficient transformation of filamentous fungi  4. Biolistic method: Biolistic transformation is also known as particle bombardment Foreign is adsorbed on the surface of tungsten or gold particles The particles are injected into host cells under the push of high pressure The method can realised both stable and transient transformation Particle bombardment is the most powerful amongst all the genetic transformation methods Has been successfully utilised to transform A. nidulans and T. reesei  Cont.. Advantages of biolistic method: It is not affected by limitations of cell type of host or species Sufficiently efficient for fungi that are difficult to culture or from which protoplasts are difficult to prepare Easy and convenient to operate Disadvantages of biolistic method: Instruments and consumables are expensive Applicable in cases where other method have failed  5. Shock-wave-mediated transformation (SWMT): SWMT utilise the principle of energy transformation and transmission to generate transient pressure disturance and twisting force across cells to form transient cavitation effect The method changes the permeability of cell membranes through acoustic cavitation resulting in uptake of exogenous nucleic acid into the cells Successful application in fungi has been demonstrated in A. niger, Fusarium oxysporum and Phanerochaete chrysosporium SWMT has been applied in medical treatment such as orthopedics and crushing kidney stones  Cont.. Advantages of SWMT: Directly act on spores not protoplasts Physical parameters are easily controllable Efficient transformation Disadvantages of SWMT: Large of amount of DNA is damaged in shock wave treatment Expensive and inconvenient to generate of plasmid in laboratory for SWMT Requires expensive shock wave sources and equipment  Genetic manipulation of yeasts S. cerevisiae is most widely used industrial yeast Traditionally used in the food industry for production of alcoholic beverages (beer, wine, and sake) and for bread fermentation Non-conventional yeasts, such as Scheffersomyces stipitis, Yarrowia lipolytica,Kluyveromyces lactis, and Dekkera bruxellensis are also important in industrial fermentations Industrial processes are rarely using the most suited or best- performing yeast strain Need for improvement of yeast strains to provide specific yeast to meet suitable industrial goals  Cont.. Multiple strategies to improve yeast industrial strains include: Mutagenesis Protoplast fusion Breeding Genome shuffling Modification strategies to alter traits in a more targeted way Global transcription machinery engineering’(gTME), etc  Breeding/sexual hybridisation: a) Direct mating/sexual hybridisation  The most spontaneous way of breeding organisms  Similar to selective breeding in agriculture  Involves mating two selected parents with an interesting phenotype  An effective way to obtain hybrids but time-consuming  In yeast strains: cell-to-cell, spore to-cell, and spore-to-spore mating  Spore-to-spore and spore-to-cell mating are used to create novel interspecific hybrids for the fermentation industry  Applicability depends on the sexual cycle of the parental strains  Applied to generate wine yeasts with improved cryotolerance or introduce flocculation in yeast strains for production of sparkling wines  Cont.. b) Rare mating/asexual hyridisation Solve challenges of natural and industrial yeasts with low sporulation efficiencies and low spore viability to obtain hybrids Cells in diploid condition can become homozygous for the mating-type locus and can be ‘force-mated’ with a cell of the opposite mating type Applied to study inter-specific hybridisation and improvement of multiple yeast traits S. cerevisiae and S. cerevisiae (var. diastaticus) crossed to produce yeast to ferment dextrins to produce low-calorie beers Also used to construct cryotolerant wine yeasts, dextrin-fermenting and high, ethanol-producing yeasts and yeasts with higher leavening ability in dough fermentations  Cytoduction: Selectively transfer non-Mendelian traits from a donor to a recipient strain without disrupting the nuclear integrity of the recipient strain Non-Mendelian traits include factors not embedded in nuclear DNA but found in mitochondrial DNA (e.g. respiration-related genes) or present in cytoplasm (e.g. killer plasmids) Donor strain contains a cytoplasmically transferable factor that carries a dysfunctional KAR1 gene A KAR1 mutant is defective in karyogamy (=nuclear fusion) after hybridisation  Cont.. Mating or protoplast fusion results in a zygote-like transient heterokaryon Mitotic divisions of the zygote bud off haploid heteroplasmons Heteroplasmons contains only one genome but mixed cytoplasmic factors One or a few chromosomes of the second parent can be transferred to the other nucleus - single-chromosome transfer Results in ‘exceptional’ cytoductants that can be used to examine individual chromosomes of industrial yeast strains  Directed evolution: Also as known as adaptive or experimental evolution Relies on natural or induced genetic variation and selection Yeast cells grown under continuous selection for many generations Random mutants will evolve in this population Combine the use of mutagens and sexual hybridisation in evolving population(s) to increase the genetic and phenotypic variability After specific mutation(s), the variant will be selected and enriched in the population  Mass mating/genome reshuffling: Involves cultivating many individual cells and generation of many crosses quickly and execute consecutive rounds of crossing Large numbers of haploid yeast cells from different parents are mixed and allowed to mate randomly Useful for homothallic strains, strains with low mating efficiency, or for the creation of inter-specific hybrids E.g. mass mating and selection of two of bakery strains: (1) high osmotolerance strains used in sweet dough and good maltose utilisation, and strains used in un-sugared dough  Genetic modification: GM yeasts for brewing and baking have been approved for use (e.g. approval was granted in the UK for use of a GM yeast (S. cerevisiae) in beer production) None of GM yeasts are used in industries  Genomics: Recently, genome sequences of many food-related microbes have been completed S. cerevisiae is the first eucaryote to have its genome sequenced in 1996 Huge numbers of microbial genome sequencing projects are also in progress --END--