NANYANG TECHNOLOGICAL UNIVERSITY Biochemical Engineering Lecture 2 Notes PDF

CH3104 Biochemical Engineering PART2 Lecture 2: Metabolic Engineering Asst. Prof. CHEN Lin Quiz Time: 1st Nov. 2024 Duration: 1 h Venue: CBE-LT Format: MCQ and SAQ Content: Lecture 1 (Enzyme)-4 (Cell growth) Biochemical engineering is a process that uses living cells (such as microorganisms) or...

CH3104 Biochemical Engineering PART2 Lecture 2: Metabolic Engineering Asst. Prof. CHEN Lin Quiz Time: 1st Nov. 2024 Duration: 1 h Venue: CBE-LT Format: MCQ and SAQ Content: Lecture 1 (Enzyme)-4 (Cell growth) Biochemical engineering is a process that uses living cells (such as microorganisms) or biomolecules (such as enzymes) to carry out a chemical transformation leading to the production and ultimate recovery of valuable products. Cellular metabolism of yeast for biochemical engineering Metabolic pathways (Nielsen & Keasling, 2016) 4  Introduction Metabolic engineering, as first defined by Baily in 1981 , is “the improvement of cellular activities by manipulating enzymatic, regulatory, and transport functions of the cell with the use of recombinant DNA technology.” Metabolic engineering is a process for modulating the metabolism of the organisms so as to produce the required amounts of the desired metabolite through genetic manipulations. Metabolic engineering is not only widely applied in industrial fermentation for strain improvement and metabolite overproduction, but has also found many important applications in functional genomics, biological research (e.g., signal transduction),and medical research (e.g., drug discovery and gene therapy) 5  Metabolic engineering...... The use of term "engineering" implies that there is some precise understanding of the system, that is being modified Rate-limiting steps must be known A typical metabolic-engineering approach focuses on a particular metabolic intermediate or product such as starch, vitamin E, carotenoids, amino acids etc. 6  Methods to Investigate Cellular Metabolism DNA mRNA Enzyme Metabolites Genomics Transcriptomics Proteomics Metabolomics Metabolic fluxes (in vivo reaction rates): functional output of combined omics (Tang et al., 2008) 7 Analysis DNA analysis DNA sequencing Southern blotting RNA analysis Northern blotting qPCR (Real-Time PCR) Protein analysis Western blotting Eastern blotting Metabolite analysis Nuclear magnetic resonance Mass spectrometry 8  1. DNA analysis Chain-terminator method to determine the sequence of nucleotides along a DNA strand; Usually performed with dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators Figure 10-20 Essential Cell Biology (© Garland Science 2010) 9  Sanger DNA sequencing Step 1: Requires a DNA template, a DNA primer, a DNA polymerase, radioactively or fluorescently labeled ddNTPs that terminate DNA strand elongation. Step 2: The DNA sample is divided into four separate sequencing reactions, containing deoxynucleotides (dATP, dGTP, dCTP and dTTP) and DNA polymerase. To each reaction is added only one of the four dideoxynucleotides which are the chain-terminating nucleotides. 10 Southern blotting The analysis of DNA structure following its attachment to a solid support a. The DNA to be analyzed is first digested with a given restriction enzyme then the resultant DNA fragments are separated in an agarose gel. b. The gel is treated with NaOH to denature the DNA, then the NaOH is neutralized. c. The DNA is transferred from the gel to nitrocellulose or nylon filter paper by either capillary diffusion or under electric current. d. The DNA is fixed to the filter by baking or ultraviolet light treatment. e. The filter can then be probed for the presence of a given fragment of DNA by various radioactive or non-radioactive means. Essential Cell Biology (© Garland Science 2010) 11 Northern blotting Northern blotting involves the analysis of RNA following its attachment to a solid support. Similar to Southern blotting except: Southern blotting Northern blotting Separation of DNA RNA Denaturation NaOH treatment after gel Formaldehyde in the gel and RNA running samples Membrane Nitrocellulose filter Amino benzyloxymethyl filter membrane paper Hybridization DNA-DNA RNA-DNA 12  Sample Extraction , DNA fragmentation 1st generation sequencing and in vitro adapter ligation Sequence many identical molecules Sequencing in large gels or capilary tubing limits scale Clonal Amplification by Bridge PCR 2nd generation sequencing Sequence millions of clonally amplified molecules per run Using a reversible, stepwise sequencing chemistry Immobilized on a surface Sequencing-by-synthesis (Solexa Technology) 13 A novel isolecuine pathway is discovered. (Wu et al., 2010) 14 Application: Novel enzyme (pathway) for biofuel production Q: Why citrate malate can enhance butanol production? Citramalate Pathway for Bioproduction 5 steps (Feng et al., 2009) 15  2. RNA analysis: Gene expression studies Pattern of genes expressed in a cell is characteristic of its current state Virtually all differences in cell state or type are correlated with changes in mRNA levels of many genes Expression patterns of many previously uncharacterized genes may provide clues to their possible function by comparison Combine with metabolic schemas to understand how pathways are changed under varying conditions 16 Method 1: Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings. 17 18  Method 2: qPCR (Real-Time PCR) qPCR relies on plotting fluorescence against the number of cycles on a logarithmic scale. A threshold for detection of DNA-based fluorescence is set slightly above background. The number of cycles at which the fluorescence exceeds the threshold is called the cycle threshold, Ct. During the exponential amplification phase, the sequence of the DNA target threshold doubles every cycle. For example, a DNA sample whose Ct precedes that of another sample by 3 cycles contained 23 = 8 times more template. 19 Since the efficiency of amplification is often variable among primers and templates, the efficiency of a primer-template combination is assessed in a titration experiment with serial dilutions of DNA template to create a standard curve of the change in Ct with each dilution. The slope of the linear regression is then used to determine the efficiency of amplification. 20 Precisely Diagnose Coronavirus? Virus Sample RNA Reverse PCR Nonspecific Primer Transcription Detect the concentration Reverse Transcriptase Specific Primer of DNA DNA Polymerase Buffer Reagents Based on virus gene cDNA 21  Method 3: DNA microarray technology Detect expression of > 1000 genes simultaneously. Applications: Identification of complex genetic diseases ; Drug discovery and toxicology studies; Mutation/polymorphism detection (SNP’s); Pathogen analysis; Differing expression of genes over time, between tissues, and disease states Microarray technology evolved from Southern blotting, where fragmented DNA is attached to a substrate and then probed with a known gene or fragment. 22  DNA complementary to genes of interest is generated and laid out in microscopic quantities on solid surfaces at defined positions cDNA is eluted over the surface - complementary DNA binds Presence of bound DNA is detected by fluorescence following laser excitation Affymetrix chips Basic technology 23  The use of miniaturized microarrays DNA microarrays for gene expression profiling was first reported in 1995 and a complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997 (by Patrick Brown Group at Standford) DeRisi et al. (Science 278:680-686) used series of DNA microarrays to measure the relative expression of 6000 genes in yeast during the diauxic shift (from fermentation to respiration) by taking samples over a period of time and comparing them with the initial expression pattern. 24  Method 3: RNA-seq "Whole Transcriptome Shotgun Sequencing”: sequence cDNA in order to get information about a sample's RNA content Step 1: ribosomal RNA represents over 90% of the RNA within a given cell, studies have shown that its removal increases the capacity to retrieve data from the remaining portion of the transcriptome. Step 2: reverse transcription. Once the cDNA is synthesized it can be further fragmented to reach the desired fragment. Step 3: high-throughput sequencing technologies generate millions of short reads from a library of nucleotide sequences, then integrate the information to obtain the expression level for each gene. 1. Microarrays 10-100 times cheaper than RNA-seq. 2. RNA-seq protocols still suffer from unknown biases. 3. Poor quantitative expression profiling: high abundance transcripts are responsible for the majority of the sequencing data. 5% of the genes give rise to 75% of the reads sequenced. As a consequence, it is hard to measure the abundances of the remaining genes reliably. 25 3) Proteomics analysis Proteomics analyzes protein profiles, particularly their structures and functions (e.g., detect novel enzymes). Separation Gas chromatography; High performance liquid NMR chromatography (HPLC); Capillary electrophoresis (CE). Detection methods 1. Mass spectrometry (detection based on LC-MS fragmentation patterns). 2. Nuclear magnetic resonance (NMR) spectroscopy. NMR is the only detection technique which does not rely on separation of the analytes, and the GC-MS sample can thus be recovered for further analyses. 26  Western blotting & Eastern blotting Western blotting: The Western blot detect specific proteins. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide or by the 3-D structure of the protein. The proteins are then transferred to a membrane, where they are probed using antibodies specific to the target protein. Eastern blotting: analyze protein post-translational modifications. It is most often used to detect carbohydrate epitopes. Thus, Eastern blotting is an extension of the biochemical technique of Western blotting. https://www.youtube.com/watch?v=Pt_NaNExry8 27  BLAST Search Protein sequences from this paper MSLVKLYDTTLRDGTQAEDISFLVEDKIRIAHKLDEIGIHYIEGGWPGSNPKDVAFFKDI KKEKLSQAKIAAFGSTRRAKVTPDKDHNLKTLIQAEPDVCTIFGKTWDFHVHEALRISLE ENLELIFDSLEYLKANVPEVFYDAEHFFDGYKANPDYAIKTLKAAQDAKADCIVLCDTNG GTMPFELVEIIREVRKHITAPLGIHTHNDSECAVANSLHAVSEGIVQVQGTINGFGERCG NANLCSIIPALKLKMKRECIGDDQLRKLRDLSRFVYELANLSPNKHQAYVGNSAFAHKGG VHVSAIQRHPETYEHLRPELVGNMTRVLVSDLSGRSNILAKAEEFNIKMDSKDPVTLEIL ENIKEMENRGYQFEGAEASFELLMKRALGTHRKFFSVIGFRVIDEKRHEDQKPLSEATIM VKVGGKIEHTAAEGNGPVNALDNALRKALEKFYPRLKEVKLLDYKVRVLPAGQGTASSIR VLIESGDKESRWGTVGVSENIVDASYQALLDSVEYKLHKSEEIEGSKK 28  CSI Miami Problem Dr. Smart identified trace protein sample in a drinking water sample. He did MS analysis of the protein and find the protein has the following amino acids sequences. >Unknown MKKCSYDYKLNNVNDPNFYKDIFPYEEVPKIVFNNIQLPMDLPDNIYITDTTFRDGQQSM PPYTSREIVRIFDYLHELDNNSGIIKQTEFFLYTKKDRKAAEVCMERGYEFPEVTSWIRA DKEDLKLVKDMGIKETGMLMSCSDYHIFKKLKMTRKETMDMYLDLAREALNNGIRPRCHL EDITRADFYGFVVPFVNELMKMSKEANIPIKIRACDTLGLGVPYNGVEIPRSVQGIIHGL RNICEVPSESIEWHGHNDFYGVVTNSSTAWLYGASSINTSFLGIGERTGNCPLEAMIFEY AQIKGNTKNMKLHVITELAQYFEKEIKYSVPVRTPFVGTDFNVTRAGIHADGILKDEEIY NIFDTDKILGRPVVVAVSQYSGRAGIAAWVNTYYRLKDEDKVNKNDSRIDQIKMWVDEQY RAGRTSVIGNNELELLVSKVMPEVIEKTEERAS Please use the NCBI Basic Local Alignment Search Tool (BLAST) to examine which bacterial species may contaminate the water system. https://www.ncbi.nlm.nih.gov/ 29  4. What is metabolomics? Metabolomics is the comprehensive, qualitative study and analysis of all the small molecules in an organism The rapidly emerging field of metabolomics combines strategies to identify and quantify cellular metabolites using sophisticated analytical technologies with the application of statistical and multivariant methods for information extraction and data interpretation. 30  Targeted metabolomics Untargeted metabolomics Known metabolites for specific pathways Are global in scale. are targeted. Have the goal of simultaneously measuring as many metabolites as possible. Approach to specific biochemical questions in pharmokinetic studies of drug It includes biological samples without bias in order to generate a metabolic profile of a metabolism. whole sample. Measuring the influence of therapeutics or genetic modifications on a specific enzyme. 31 Targeted vs. Untargeted (Patti et al., 2012)  Metabolomics Steps in metabolomic techniques Sample preparation Sample extraction Chromatography Data analysis Detection 33 Techniques Separation Techniques Gas Chromatography (GC) Capillary Electrophoresis (CE) High Performance Liquid Chromatography (HPLC) Ultra Performance Liquid Chromatography (UPLC) Detection Techniques Nuclear Magnetic Resonance Spectroscopy (NMR) Mass Spectrometry (MS) Combination of Techniques GC-MS HPLC-MS 34 Chromatography “Color writing” Separation technique. The separation of components in a mixture that involves passing the mixture dissolved in a "mobile phase" through a stationary phase. Separation based on differential partitioning between the mobile and stationary phases 35  Gas Chromatography Involves a sample being vaporized to a gas and injected into a column Samples are usually derivatized with trimethylsilyl (TMS) to make them volatile Sample is transported through the column by an inert gas mobile phase Column has a liquid or polymer stationary phase that is adsorbed to the surface of a metal tube Columns are 1.5-10 m in length and 2-4 mm in internal diameter 36 High Pressure (Performance) Liquid Chromatography - HPLC Developed in 1970’s Uses high pressures (6000 psi) and smaller (5 um), pressure-stable particles Allows compounds to be detected at ppt (parts per trillion) level Allows separation of many types of polar and nonpolar compounds 37 HPLC Modalities Reversed phase – for separation of non-polar molecules (non-polar stationary phase, polar mobile phase) Normal phase – for separation of polar molecules (polar stationary phase, non-polar/organic mobile phase) HILIC – hydrophilic interaction liquid chromatography for separation of polar molecules (polar stationary phase, mixed polar/nonpolar mobile phase) 38 Detection: Nuclear Magnetic Resonance Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical technique used to observe the magnetic properties of certain atomic nuclei. It provides detailed information about the structure, dynamics, and chemical environment of molecules. A current generates a strong magnetic field polarizes the nuclei in sample material Generated nuclear quantum “dance” Quantum signals is recorded The signals Fourier transform (FT) shows "lines" for different nuclei in different electronic environments. 39 Detection: Mass Spectrometry Mass spectrometry is a technique to measure the mass of ions (m/z) All mass spectrometers perform three main tasks: 1. Ionize molecules. 2. Acceleration: Use electric and magnetic fields to accelerate ions and manipulate their flight. 3. Detect ions (convert to electronic signal) 40  NMR excels in non-destructive analysis and quantification of metabolites, while MS offers superior sensitivity and broader applicability across diverse metabolite classes. Nuclear Magnetic Resonance (NMR) Mass Spectrometry (MS) Measures the magnetic properties of atomic nuclei in Measures mass-to-charge ratios of ionized Principle a magnetic field, providing structural information molecules to identify and quantify metabolites. about metabolites. Highly sensitive; can detect metabolites at Less sensitive; detects metabolites at concentrations Sensitivity concentrations as low as 10-100 nM, often >1 μM, typically identifying 50-200 metabolites. identifying over 1000 metabolites. Requires sample ionization and sometimes Sample Minimal preparation; non-destructive and allows for chemical derivatization, which can be Preparation analysis of intact samples. destructive. Can quantify metabolites but may require Provides quantitative information with high Quantification calibration curves and is subject to ion reproducibility; suitable for qNMR (quantitative NMR). suppression effects. Ideal for studying metabolic flux, in vivo analysis, and Widely used for targeted analysis, structural Applications complex mixtures; excellent for polar compounds like characterization, and high-throughput screening sugars and organic acids. of diverse metabolites. 41 Data analysis Trimethylsilylpropanoic acid (TSP) A typical 1H NMR and 1H-13C nuclear magnetic resonance (NMR) spectra of mung beans Metabolites Assignments 1H chemical shifts (ppm) 13C chemical shifts (ppm) 1 Isoleucine αCH; βCH; γCH2; γ'CH3; δCH3 3.61 (d); 1.95 (m); 1.33, 1.00 (m), 0.94 (d); 0.84 (t) 59.6; 36.3; 24.6; 17.9; 13.1 2 Leucine αCH; βCH2; γCH; δCH3; δ'CH3 3.59 (m); 1.61(m); 1.62 (m); 0.99 (d); 0.87 (d) 54.1; 40.4; 24.3; 24.1; 21.6 3 Valine αCH; βCH; γCH3; γ'CH3 3.61 (d); 2.26 (m); 0.95 (d); 1.04 (d) 62.2; 29.3; 17.9; 20.4 4 Ethanol αCH2; βCH3 3.61 (m); 1.17 (t) 60.1; 17.0 5 Threonine αCH; βCH; γCH3 3.48 (d); 4.25 (m); 1.25 (d) 62.7; 68.3; 22.0 6 Lactic acid αCH; βCH3 4.18 (q); 1.26 (d) 71.7; 22.0 7 Alanine αCH; βCH3 3.73 (q); 1.32 (d) 52.5; 19.7 8 Lysine αCH; βCH2; γCH2; δCH2; εCH2 3.61 (t); 1.90 (m); 1.34 (m); 1.61 (m); 2.94 (t) 56.8; 32.6; 22.3; 28.8; 38.9 9 Arginine αCH; βCH2; γCH2; δCH2 3.71 (t); 1.89 (dd); 1.59 (m); 3.16 (t) 56.4; 28.0; 25.1; 40.4 10 Acetic acid CH3 1.78 (s) 24.4 11 γ-Aminobutyric acid αCH2; βCH2; γCH2 2.24 (t); 1.87 (m); 3.00 (t) 34.9; 23.3; 42.0 12 Glutamine αCH; βCH2; γCH2 3.70 (m); 2.11 (m); 2.33 (m) 56.4; 27.1; 33.6 13 Succinic acid CH2 2.35 (s) 33.7 14 Ketoglutaric acid αCH2; βCH2 3.00 (t); 2.45 (t) 36.9; 31.1 15 Aspartic acid αCH; βCH2 3.81 (dd); 2.66, 2.71 (dd) 54.9; 38.2, 38.3 16 Asparagine αCH; βCH2 3.82 (q); 2.84, 2.92 (dd) 52.1; 36.1 17 Tyrosine C1H; C2H2; C4H; C5H 3.90 (dd); 2.94, 3.19 (dd); 7.13 (d); 6.82 (m) 58.1; 36.0; 130.1; 115.4 18 Phenylalanine C1H; C2H2; C4H; C5H; C6H 3.89 (dd); 2.97, 3.03 (dd); 7.32 (q); 7.32 (t); 7.33 (m) 58.0; 36.9; 129.1; 128.6; 127.1 19 Histidine αCH; βCH2; NCHC; NCHN 3.88 (dd); 3.06, 3.21 (dd); 7.04 (d); 7.83 (d) 55.4; 28.1; 118.7; 138.9 (Chen et al., 2019) 42  Gas Chromatography analysis Target metabolites are identified by exact retention times and their corresponding mass spectra (B) as shown for the co- eluting peaks of malate, gamma-amino butyric acid (GABA), and an unidentified compound. m/z, Ratio of mass to charge. 43  Once metabolic composition is determined, data reduction techniques can elucidate patterns and connections. Principal component analysis (PCA) can efficiently reduce the dimensions of a dataset to a few which explain the greatest variation. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) is a statistical method used for analyzing complex datasets, particularly in metabolomics. OPLS-DA is particularly useful in filtering out noise and irrelevant data, thereby improving the reliability of results in distinguishing between different biological conditions or treatments. (23.1%) Score plot (71.2%) Loading plot (Chen et al., 2019) 44 Metabolic pathways databases PFBP http://www.ebi.ac.uk/research/pfmp KEGG http://www.genome.ad.jp/kegg/ BRENDA http://srs6.ebi.ac.uk/ PathDB http://www.ncgr.org/research/pathdb/ SRS http://srs6.ebi.ac.uk/ EcoCyc http://ecocyc.panbio.com/ecocyc/ MPW-EMP http://wit.mcs.anl.gov/WIT2/ CSNbd http://geo.nihs.go.jp/csndb/ Transfac http://transfac.gbf.de/ RegulonDB http://www.cifn.unam.mx/Computational_Biology/regulondb/ DPinteract http://arep.med.harvard.edu/dpinteract/ ooTFD http://www.isbi.net/ JGI http://www.jgi.doe.gov/ MicrobesOnline http://www.microbesonline.org/ 45 Sucrose Fructose Linolenic acid Isoleucine Valine Oleic acid Linoleic acid Glutamine Proline beta-D-Glucose alpha-D-glucose Ethanol Serine Dopamine leucine Pathway Name Match Status p -log(p) Holm p FDR Impact Details Glycolate Glyoxylate and Malic acid dicarboxylate 6/32 9.4519E-6 5.0245 7.5615E-4 7.5615E-4 0.22752 KEGG Acetic acid metabolism maltose Alanine, aspartate Glycine KEGG SMP SMP S and glutamate 5/28 7.5414E-5 4.1225 0.0059577 0.0030166 0.42388 sterols MP metabolism Choline gamma-Aminobutyric acid Valine, leucine and isoleucine 3/8 2.3793E-4 3.6235 0.018559 0.0063449 0.0 KEGG SMP Pyridoxamine biosynthesis asparagine Glycolysis / Aspartic Acid 4/26 7.9263E-4 3.1009 0.061033 0.015853 0.03796 KEGG SMP SMP Gluconeogenesis Alanine Gallic acid Starch and tyrosine sucrose 3/18 0.0030909 2.5099 0.2349 0.049454 0.12786 KEGG SMP metabolism https://www.metaboanalyst.ca/ 46 Integrated metabolomics and transcriptomics reveal the adaptive responsesof Salmonella enterica serovar Typhimurium (Chen et al., 2022) What is Metabolic Engineering doing? Engineer microbes to behave as expected 1. Feedstocks degradation 2. Valued chemicals production 3. Easy manipulation and wide choice of expression system 48 Ways to engineer metabolic pathways: Directly by manipulating the genes encoding the enzymes catalyzing the reactions in the pathways Indirectly by altering the regulatory pathways affecting gene expression and enzyme activity 49 Fundamental requirements for metabolic engineering 1.The biosynthetic pathway of the chemical to be produced. 2.Genes encoding the related enzymes. 3.Regulation of such enzymes, with ability to transfer and express or suppress the required genes in the host organism. 4.Mutate the gene in vivo and in vitro to be able to alter properties of the encoded enzyme. 5.Assemble an array of genes for their expression inside the host cell. 50  Different approaches of metabolic engineering 1. One of the most obvious approaches is over expressing the gene encoding the rate-limiting enzyme of the biosynthetic pathway of the desired end-product. Example: the vitamin E content of Arabidopsis (a model plant system) has been increased by over expression of a gene encoding the enzyme γ tocopherol methyltransferase. 2. Another way is to inhibit the competing metabolic reactions which involve the same substrate. In this way, the substrate is metabolically channeled specifically towards the desired chemical. Example: the production of 1,2-propanediol is increased, (which is mainly used for production of biodegradable polymers) by inhibiting the lactate dehydrogenase and glyoxylase genes which encode for the competing enzymes. 51 3. The third strategy is, the production of the desired biochemical can be carried out in the non-native organism, i.e., heterologous host. In this method, a gene can be isolated from the organism which naturally produces the desired biochemical and can be expressed in another organism which might be easier to cultivate than the host organism. In such a case, an important factor is the availability of the substrate of the desired pathway. Thus, multiple genes encoding an array of the enzymes of a pathway can be expressed in the non-native host. Example: the production of biofuel molecules – fatty acid ethyl esters – in E. coli by expressing the genes encoding the successive enzymes of the pathway which are obtained from various sources such as plants and bacteria. 52 4. The fourth approach is to engineer an enzyme which is not found in nature. This mode relies on creating mutations in the related gene so that the amino acid composition of the enzyme is altered. This might result in alteration in the substrate and product specificities of the enzymes, as they are dependent on the amino acid composition and sequence. Example: Such a method has been used for the production of a nonnatural amino acid L-homoalanine, (which is an important precursor of the many drugs), by creating rational mutations in glutamate dehydrogenase enzyme in E. coli. 53  APPLICATIONS ❖ The application of metabolic engineering to industrial biotechnology has mainly focused on improving cell metabolism to increase productivity – higher product yield, production rate, and cell growth efficiency (energy efficiency), and to eliminate or reduce undesirable byproducts. ❖ It has also been applied to eliminate or reduce feedback inhibition. By recruiting heterologous activities, a partial pathway in an organism can be completed for the production of the desirable product. ❖ Also, hybrid or new metabolic pathways can be developed to produce new products, extend substrate range, or enhance processing characteristics. 54  Amino acids Amino acids are the basic building blocks of proteins and have many important applications in food and biomedical industries. The essential amino acids, such as lysine, methionine, threonine, and phenylalanine are important nutritional supplement in animal feed. MSG (monosodium glutamate) is used as a flavoring agent, and tryptophan is an important pharmaceutical ingredient. Since the discovery of glutamate-overproducing Corynebacterium glutamicum in 1957, traditional strain development and, more recently, metabolic engineering have been widely studied and applied to enhance the production of various L-amino acids from sugars by fermentation. Today, except for methionine and a few others, many important amino acids are commercially produced from sugars by microbial fermentation in large quantities with high product titers and yields. 55 56 ❖To overproduce amino acids in the aspartate group, a change in the metabolic flux resulting from mutations affecting the repression or feedback inhibition of the key biosynthetic enzymes is usually required. For example, lysine synthesis in C. glutamicum is controlled by aspartate kinase (AsK), which is allosterically inhibited by lysine and threonine; this feedback control is eliminated by mutations in the β-subunit of the kinase. ❖Threonine synthesis in corynebacteria is regulated by the activity of HDH, which is strongly inhibited by threonine. In addition, the genes coding for homoserine dehydrogenase (hom) and homoserine kinase (thrB) are on the same operon, which is repressed by methionine. 57  Antibiotics β-lactam antibiotics such as penicillins are among the oldest and largest industrial products produced by fermentation. Recombinant DNA technology can provide direct and more efficient approaches to strain improvement, and has recently been extensively studied and used to further improve the production of β-lactam antibiotics, including penicillins, cephalosporins, and cephamycins. For example, penicillin production in P. chrysogenum was increased significantly when additional copies of the pcbC (Isopenicillin N synthase) and penDE genes (Acyl- coenzyme A:6-aminopenicillanic-acid-acyltransferase PenDE )were introduced. Cephalosporin C production in A. chrysogenum was increased 2 to 3-fold by overexpressing the cefG gene alone. 58  Metabolic engineering in plants PLANTS: THE NATURAL FACTORIES Man has also known how to grow, harvest, and process plant tissues since the beginning of modern agricultural practice. Production of fine chemicals, pharmaceuticals, and industrial products. Plants contain an amazing diversity of genes encoding enzymes for thousands of distinct biochemical reactions. Plant cells are highly compartmentalized, facilitating. 59  PLANT METABOLIC ENGINEERING The inhibition methods of plant genes: Knocking out gene function Interfering with protein function using specific inhibitors or antibodies. The expression of genes in plants or plant cells is dependent on several factors that include: Method to introduce genes into the plant. Promoters Source for the gene encoding the enzyme of interest. Choice of plant system for metabolic engineering Whole Plants Plant Cells in Culture Cell Using Plant Genes in Microorganisms 60 Strategies for increasing flux of existing pathways Manipulating the Activity of ‘‘Rate Limiting’’ Steps Metabolic Flux Analysis and Modelling: Metabolic flux is the rate at which the material is processed through a specific pathway. Expression of Multiple Genes in Plants : Progress and Limitations As exemplified by the attempts to increase the levels of monoterpenoid alkaloid biosynthesis by coexpressing tryptophan decarboxylase and strictosamide synthase. Targeting Entire Pathways with Transcription Factors Anthocyanin accumulation is regulated in many plant species by the concerted action of transcription factors corresponding to the MYB and bHLH families. 61 MAKING NEW COMPOUNDS IN PLANTS Plants as a Source of Industrial Materials Polyhydroxyalkanoates (PHA) in plants. These polyesters of 3-hydroxyacids have unique biodegradable and elastomeric properties. Starch Plants as Pharmaceutical Factories Plants as Nutraceutical Factories Levels of provitamin A carotenoids in food staples such as corn, wheat, and rice by metabolic engineering. A similar success was recently reported in the manipulation of folates (including tetrahydrofolate) in tomato fruits The next generation of engineering plant metabolic pathways Additionally, plants can be gene-edited with constructs that are composed exclusively of DNA sequence derived from the same or similar plant species. These so-called cisgenic plants, which could in principle be obtained through standard breeding practices, may be more readily accepted by the public and regulatory bodies. 62 Engineering plant pathways to create better biofuels 63 ❖ A key enzyme in lignin biosynthesis, cinammoyl-CoA reductase(CCR), which catalyzes the conversion of hydroxycinnamoyl-CoA esters to the corresponding aldehydes. Field trials on poplar plants have shown that biomass from transgenic plants with downregulation of CCR is more easily processed to production of bioethanol ❖ In another attempt enzyme monolignol ferulate transferase (MFT) was targeted. The introduction of MTF into transgenic poplar plants alters the pool of monolignols, with an increase of monolignol ferulate conjugates. Since the ferulate conjugates are capable of introducing readily cleavable ester bonds into the lignin backbone without affecting the plant development lignification process. ❖ Another strategy to improve access to biofuels is to increase the content of triacylglycerols (TAGs) in plant vegetative tissues. A variety of genes that enhance TAG accumulation levels have been identified: the transcription factor WRIN-KLED1, the TAG biosynthetic gene diacylglycerol acyl-transferase1-2 (DGAT1-2) and a gene encoding a structural protein oleosin1 (OLE1) that impacts oil body formation. 64 Golden Rice: Metabolically engineered plants Nutrient content of a crop plant can be improved by metabolic engineering. Rice, maize, wheat, tomatoes and other vegetables are breaded to enhance the level of certain nutrients. Addition of β-carotene pathway to rice to yield rice having higher levels of vitamin A. 65 Engineering new traits into crops by engineering plant metabolism 1. Phenylpropanoids are plant metabolites that act as antioxidant agents, and therefore have essential health promoting properties. Phenylpropanoid production was substantially upregulated in tomato fruits by introducing fruit-specific expression of the A. thaliana transcription factor AtMYB12. 2. AtMYB12 increases phenylpropanoid levels by transcriptionally activating the biosynthetic genes of these pathways. 3. This transcription factor also appears to direct carbon flux towards aromatic amino acid biosynthesis, which in turn increases the supply of substrate for phenylpropanoid metabolism. 66 Key Takeaways Metabolic pathway: omics Metabolic engineering strategies 67