Terpenoids and Their Biosynthesis in Cyanobacteria

Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP) pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids.

[1]  Chhandak Basu,et al.  The production of the sesquiterpene β-caryophyllene in a transgenic strain of the cyanobacterium Synechocystis. , 2011, Journal of plant physiology.

[2]  H. Moghimi,et al.  Stereoselective Permeation of Tretinoin and Isotretinoin through Enhancer-Treated Rat Skin. I. Effect of Ethanol and Sodium Dodecyl Sulfate , 2003 .

[3]  E. Gantt,et al.  Evidence of a Role for LytB in the Nonmevalonate Pathway of Isoprenoid Biosynthesis , 2000, Journal of bacteriology.

[4]  C. Cockell,et al.  Cyanobacterial bacteriohopanepolyol signatures from cultures and natural environmental settings , 2008 .

[5]  O. Sticher,et al.  A novel extracellular diterpenoid with antibacterial activity from the cyanobacterium Nostoc commune. , 1999, Journal of natural products.

[6]  T. Asaeda,et al.  Culture temperature affects gene expression and metabolic pathways in the 2-methylisoborneol-producing cyanobacterium Pseudanabaena galeata. , 2014, Journal of plant physiology.

[7]  G. Asghari,et al.  Biotransformation of Aromatic Aldehydes by Cell Cultures of Peganum harmala L. and Silybum marianum (L.) Gaertn , 2004 .

[8]  N. Kubota,et al.  Asymmetric reduction of enones with Synechococcus sp. PCC 7942 , 2004 .

[9]  Victoria H. Work,et al.  Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002 , 2014, Front. Bioeng. Biotechnol..

[10]  Robert E. Jinkerson,et al.  Toward a photosynthetic microbial platform for terpenoid engineering , 2015, Photosynthesis Research.

[11]  M. Hancianu,et al.  Linalool: a review on a key odorant molecule with valuable biological properties , 2014 .

[12]  P. Wright,et al.  Exploiting cyanobacterial P450 pathways. , 2010, Current opinion in microbiology.

[13]  E. Gantt,et al.  Isoprenoid Biosynthesis in Synechocystis sp. Strain PCC6803 Is Stimulated by Compounds of the Pentose Phosphate Cycle but Not by Pyruvate or Deoxyxylulose-5-Phosphate , 2002, Journal of bacteriology.

[14]  Xiang-Qun Xie,et al.  Beta-caryophyllene is a dietary cannabinoid , 2008, Proceedings of the National Academy of Sciences.

[15]  Z. Gombos,et al.  Carotenoids, versatile components of oxygenic photosynthesis. , 2013, Progress in lipid research.

[16]  H. Sahm,et al.  Content and composition of hopanoids in Zymomonas mobilis under various growth conditions , 1991, Journal of bacteriology.

[17]  G. Daum,et al.  Squalene – biochemistry, molecular biology, process biotechnology, and applications , 2011 .

[18]  Elizabeth J Johnson The role of carotenoids in human health. , 2002, Nutrition in clinical care : an official publication of Tufts University.

[19]  J. Bergquist,et al.  Production of Squalene in Synechocystis sp. PCC 6803 , 2014, PloS one.

[20]  C. Poulter,et al.  Identification of an Archaeal Type II Isopentenyl Diphosphate Isomerase in Methanothermobacter thermautotrophicus , 2004, Journal of bacteriology.

[21]  H. Lokstein,et al.  Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803. , 2005, Biochimica et biophysica acta.

[22]  E. Gantt,et al.  Inactivation of sll1556 in Synechocystis Strain PCC 6803 Impairs Isoprenoid Biosynthesis from Pentose Phosphate Cycle Substrates In Vitro , 2004, Journal of bacteriology.

[23]  E. Gantt,et al.  Impaired Isoprenoid Biosynthesis: A Competitive Disadvantage Under Light Stress in Synechocystis PCC 6803 , 2008 .

[24]  C. Ghelardini,et al.  Local anaesthetic activity of β-caryophyllene ☆ , 2001 .

[25]  H. Sahm,et al.  Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. , 1993, The Biochemical journal.

[26]  J. Heilmann,et al.  New antibacterial metabolites from the cyanobacterium Nostoc commune(EAWAG 122b). , 2000, Journal of natural products.

[27]  G. Riccioni Carotenoids and cardiovascular disease , 2009, Current atherosclerosis reports.

[28]  R. Hill,et al.  The Biogenetic Anatomy of Vitamin B6 , 1996, The Journal of Biological Chemistry.

[29]  Keith E. J. Tyo,et al.  Terpenoids: opportunities for biosynthesis of natural product drugs using engineered microorganisms. , 2008, Molecular pharmaceutics.

[30]  R. Summons,et al.  2‐Methylhopanoids are maximally produced in akinetes of Nostoc punctiforme: geobiological implications , 2009, Geobiology.

[31]  H. Sahm,et al.  Glyceraldehyde 3-Phosphate and Pyruvate as Precursors of Isoprenic Units in an Alternative Non-mevalonate Pathway for Terpenoid Biosynthesis , 1996 .

[32]  Yi Tang,et al.  Synthetic biological approaches to natural product biosynthesis. , 2012, Current opinion in biotechnology.

[33]  William N. Hunter,et al.  The Non-mevalonate Pathway of Isoprenoid Precursor Biosynthesis* , 2007, Journal of Biological Chemistry.

[34]  H. Sahm,et al.  Effect of azasqualene on hopanoid biosynthesis and ethanol tolerance of Zymomonas mobilis , 1991 .

[35]  R. Bos,et al.  Mono- and sesqui-terpene hydrocarbons of the essential oil of Cannabis sativa. , 1975 .

[36]  K. Timmis Handbook of hydrocarbon and lipid microbiology , 2010 .

[37]  T. Heuser,et al.  A Synechococcus leopoliensis SAUG 1402‐1 operon harboring the 1‐deoxyxylulose 5‐phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis , 1999, FEBS letters.

[38]  P. León,et al.  1-Deoxy-d-xylulose-5-phosphate Synthase, a Limiting Enzyme for Plastidic Isoprenoid Biosynthesis in Plants* , 2001, The Journal of Biological Chemistry.

[39]  J. Sacchettini,et al.  Creating Isoprenoid Diversity , 1997, Science.

[40]  D. Newman,et al.  Identification and characterization of Rhodopseudomonas palustris TIE‐1 hopanoid biosynthesis mutants , 2012, Geobiology.

[41]  A. Melis,et al.  Regulation of β-phellandrene synthase gene expression, recombinant protein accumulation, and monoterpene hydrocarbons production in Synechocystis transformants , 2014, Planta.

[42]  Cordelia J. Hwang,et al.  Geosmin and 2-Methylisoborneol from Cyanobacteria in Three Water Supply Systems , 1982, Applied and environmental microbiology.

[43]  Using Synthetic Biology to Retarget Biosynthetic Pathways to the Chloroplast for Direct Access to the Products of Photosynthesis , 2013 .

[44]  M. Rohmer,et al.  Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[45]  T. Sharkey,et al.  Isoprene Increases Thermotolerance of Isoprene-Emitting Species , 1997, Plant physiology.

[46]  S. Takaichi Carotenoids in Algae: Distributions, Biosyntheses and Functions , 2011, Marine drugs.

[47]  M. Takagi,et al.  An unusual isopentenyl diphosphate isomerase found in the mevalonate pathway gene cluster from Streptomyces sp. strain CL190. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[48]  C. Fox Squalene Emulsions for Parenteral Vaccine and Drug Delivery , 2009, Molecules.

[49]  M. Valvano,et al.  Hopanoid Production Is Required for Low-pH Tolerance, Antimicrobial Resistance, and Motility in Burkholderia cenocepacia , 2011, Journal of bacteriology.

[50]  A. Melis,et al.  Paradigm of Monoterpene (β-phellandrene) Hydrocarbons Production via Photosynthesis in Cyanobacteria , 2013, BioEnergy Research.

[51]  A. Melis,et al.  Diffusion‐based process for carbon dioxide uptake and isoprene emission in gaseous/aqueous two‐phase photobioreactors by photosynthetic microorganisms , 2012, Biotechnology and bioengineering.

[52]  Jonathan Williams,et al.  Isoprene emission from phytoplankton monocultures: the relationship with chlorophyll-a, cell volume and carbon content , 2010 .

[53]  P. Su,et al.  Research progress relating to the role of cytochrome P450 in the biosynthesis of terpenoids in medicinal plants , 2014, Applied Microbiology and Biotechnology.

[54]  Hung‐wen Liu,et al.  Current development in isoprenoid precursor biosynthesis and regulation. , 2013, Current opinion in chemical biology.

[55]  D. Werck-Reichhart,et al.  Cytochromes P450: a success story , 2000, Genome Biology.

[56]  T. Eggelte,et al.  Artemisinin drugs in the treatment of malaria: from medicinal herb to registered medication. , 1999, Trends in pharmacological sciences.

[57]  E. Gantt,et al.  Interactions of Isoprenoid Pathway Enzymes and Indirect Stimulation of Isoprenoid Biosynthesis by Pentose Phosphate Cycle Substrates in Synechocystis PCC 6803 , 2012 .

[58]  J. Schopf,et al.  Early Archean (3.3-billion to 3.5-billion-year-old) microfossils from Warrawoona Group, Australia. , 1987, Science.

[59]  R. V. Solís,et al.  Two new antibacterial norabietane diterpenoids from cyanobacteria, Microcoleous lacustris , 2008, Journal of Natural Medicines.

[60]  Y. Xue,et al.  Functional expression of an Arabidopsis p450 enzyme, p-coumarate-3-hydroxylase, in the cyanobacterium Synechocystis PCC 6803 for the biosynthesis of caffeic acid , 2014, Journal of Applied Phycology.

[61]  A. Melis,et al.  Photosynthesis-to-fuels: from sunlight to hydrogen, isoprene, and botryococcene production , 2012 .

[62]  C. Schmidt-Dannert,et al.  Identification of Sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. Strain PCC 7120 , 2008, Journal of bacteriology.

[63]  E. Gantt,et al.  Isopentenyl diphosphate isomerase deficiency in Synechocystis sp. strain PCC6803 , 2000, FEBS letters.

[64]  E. Pichersky,et al.  Structure and evolution of linalool synthase. , 1998, Molecular biology and evolution.

[65]  J. Keasling,et al.  Engineering a mevalonate pathway in Escherichia coli for production of terpenoids , 2003, Nature Biotechnology.

[66]  T. Hase,et al.  Cyanobacterial Non-mevalonate Pathway , 2005, Journal of Biological Chemistry.

[67]  M. Rohmer The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. , 1999, Natural product reports.

[68]  S. Tabata,et al.  Complete genome structure of the unicellular cyanobacterium Synechocystis sp. PCC6803. , 1997, Plant & cell physiology.

[69]  F. Arnold,et al.  Evolution of the C30 Carotenoid Synthase CrtM for Function in a C40 Pathway , 2002, Journal of bacteriology.

[70]  F. Jüttner,et al.  Sesquiterpenes of the Geosmin-Producing Cyanobacterium Calothrix PCC 7507 and their Toxicity to Invertebrates , 2009, Zeitschrift für Naturforschung C - A Journal of Biosciences.

[71]  M. Rodríguez-Concepcíon,et al.  Elucidation of the Methylerythritol Phosphate Pathway for Isoprenoid Biosynthesis in Bacteria and Plastids. A Metabolic Milestone Achieved through Genomics1 , 2002, Plant Physiology.

[72]  Joshua S. Yuan,et al.  Ecological functions of terpenoids in changing climates , 2013 .

[73]  Mari L. Salmi,et al.  Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. , 2009, Journal of experimental botany.

[74]  W. Janzen,et al.  Anti-AIDS agents, 11. Betulinic acid and platanic acid as anti-HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally related triterpenoids. , 1994, Journal of natural products.

[75]  T. Sharkey,et al.  ISOPRENE EMISSION FROM PLANTS. , 2003, Annual review of plant physiology and plant molecular biology.

[76]  Jia-You Fang,et al.  Biological and Pharmacological Activities of Squalene and Related Compounds: Potential Uses in Cosmetic Dermatology , 2009, Molecules.

[77]  J. Keasling,et al.  Microbial engineering for the production of advanced biofuels , 2012, Nature.

[78]  P. Proteau,et al.  Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp. PCC6803. , 2003, Biochimica et biophysica acta.

[79]  K. Poralla,et al.  Hopanoid Biosynthesis and Function in Bacteria , 1999, Naturwissenschaften.

[80]  G. McFadden,et al.  Rewiring and regulation of cross-compartmentalized metabolism in protists , 2010, Philosophical Transactions of the Royal Society B: Biological Sciences.

[81]  M. Valvano,et al.  Elucidation of the Burkholderia cenocepacia hopanoid biosynthesis pathway uncovers functions for conserved proteins in hopanoid-producing bacteria. , 2015, Environmental microbiology.

[82]  T. Sharkey,et al.  Isoprene emission from plants: why and how. , 2007, Annals of botany.

[83]  D. Cane,et al.  Isolation and characterization of the gene associated with geosmin production in cyanobacteria. , 2008, Environmental science & technology.

[84]  Derick R. Peterson,et al.  The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells. , 2005, Blood.

[85]  J. Schrader,et al.  Biooxidation of monoterpenes with bacterial monooxygenases , 2011 .

[86]  R. Moreau,et al.  Hopanoid lipids compose the Frankia vesicle envelope, presumptive barrier of oxygen diffusion to nitrogenase. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[87]  C. Olsen,et al.  Anchoring a Plant Cytochrome P450 via PsaM to the Thylakoids in Synechococcus sp. PCC 7002: Evidence for Light-Driven Biosynthesis , 2014, PloS one.

[88]  D. Tholl Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. , 2006, Current opinion in plant biology.

[89]  C. C. Giri,et al.  Biotransformations using plant cells, organ cultures and enzyme systems: current trends and future prospects. , 2001, Biotechnology advances.

[90]  B. Witholt,et al.  Biotransformation of limonene by bacteria, fungi, yeasts, and plants , 2003, Applied Microbiology and Biotechnology.

[91]  D. Speert,et al.  Identification of Hopanoid Biosynthesis Genes Involved in Polymyxin Resistance in Burkholderia multivorans , 2011, Antimicrobial Agents and Chemotherapy.

[92]  Tsair-Fuh Lin,et al.  Seasonal change and correlation with environmental parameters for 2-MIB in Feng-Shen Reservoir, Taiwan , 2008, Environmental monitoring and assessment.

[93]  J. Gutterman,et al.  Avicins: Triterpenoid saponins from Acacia victoriae (Bentham) induce apoptosis by mitochondrial perturbation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[94]  A. Melis,et al.  Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism. , 2010, Metabolic engineering.

[95]  B. M. Lange,et al.  Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[96]  S. Lochyński,et al.  Biotransformations of monoterpenes by photoautotrophic micro‐organisms , 2014, Journal of applied microbiology.

[97]  D. Newman,et al.  Probing the Subcellular Localization of Hopanoid Lipids in Bacteria Using NanoSIMS , 2014, PloS one.

[98]  M. Rohmer,et al.  Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: Direct thylakoid/ferredoxin‐dependent photoreduction of GcpE/IspG , 2006, FEBS letters.

[99]  J. Nielsen,et al.  Advanced biofuel production by the yeast Saccharomyces cerevisiae. , 2013, Current opinion in chemical biology.

[100]  R. Zhou,et al.  Engineering cyanobacteria for the production of a cyclic hydrocarbon fuel from CO2 and H2O , 2014 .

[101]  D. Millie,et al.  EVALUATING THE RELATIONSHIP BETWEEN PHOTOPIGMENT SYNTHESIS AND 2‐METHYLISOBORNEOL ACCUMULATION IN CYANOBACTERIA , 1999 .

[102]  M. Komatsu,et al.  Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol , 2008, Proceedings of the National Academy of Sciences.

[103]  Firoz Mohammad,et al.  Role of secondary metabolites in defense mechanisms of plants , 2011 .

[104]  C. Poulter,et al.  Cloning, Solubilization, and Characterization of Squalene Synthase from Thermosynechococcus elongatus BP-1 , 2008, Journal of bacteriology.

[105]  M. Rohmer Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosynthesis. Elucidation and distribution , 2003 .

[106]  U. Jürgens,et al.  Localization and distribution of hopanoids in membrane systems of the cyanobacterium Synechocystis PCC 6714. , 1992, FEMS microbiology letters.

[107]  B. L. Wylie,et al.  Tolypodiol, an antiinflammatory diterpenoid from the cyanobacterium Tolypothrix nodosa. , 1996, Journal of natural products.

[108]  Kaoru Nakamura,et al.  Reduction of (+)- and (−)-camphorquinones by cyanobacteria , 2004 .

[109]  Seon-Won Kim,et al.  An update on microbial carotenoid production: application of recent metabolic engineering tools , 2007, Applied Microbiology and Biotechnology.

[110]  K. Shadan,et al.  Available online: , 2012 .

[111]  D. Newman,et al.  Hopanoids Play a Role in Membrane Integrity and pH Homeostasis in Rhodopseudomonas palustris TIE-1 , 2009, Journal of bacteriology.

[112]  Min Yang,et al.  Earthy odor compounds production and loss in three cyanobacterial cultures. , 2012, Water research.

[113]  J. Schwender,et al.  Distribution of the mevalonate and glyceraldehyde phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714. , 1998, The Biochemical journal.

[114]  D. V. Banthorpe,et al.  The biosynthesis of monoterpenes. , 1972, Sogo kango. Comprehensive nursing, quarterly.

[115]  M. Ikeuchi,et al.  Engineering of cyanobacteria for the photosynthetic production of limonene from CO2. , 2014, Journal of biotechnology.

[116]  R. Zhou,et al.  Genetically engineering cyanobacteria to convert CO2, water, and light into the long-chain hydrocarbon farnesene , 2014, Applied Microbiology and Biotechnology.

[117]  A. Glieder,et al.  Engineering primary metabolic pathways of industrial micro-organisms. , 2007, Journal of biotechnology.

[118]  Jonathan Williams,et al.  Evidence for marine production of monoterpenes , 2008 .

[119]  R. Kurihara,et al.  Stereoselective biotransformation of limonene and limonene oxide by cyanobacterium, Synechococcus sp. PCC 7942. , 2003, Journal of bioscience and bioengineering.

[120]  Hung‐wen Liu,et al.  Methylerythritol phosphate pathway of isoprenoid biosynthesis. , 2013, Annual review of biochemistry.

[121]  Edgard Gnansounou,et al.  Cyanobacteria and microalgae: a positive prospect for biofuels. , 2011, Bioresource technology.

[122]  Y. Ghasemi,et al.  Biotransformation of monoterpenes by immobilized microalgae , 2011, Journal of Applied Phycology.

[123]  M. Havaux Carotenoids as membrane stabilizers in chloroplasts , 1998 .

[124]  Zhongjie Wang,et al.  Genes Associated with 2-Methylisoborneol Biosynthesis in Cyanobacteria: Isolation, Characterization, and Expression in Response to Light , 2011, PloS one.

[125]  D. Jendrossek,et al.  Squalene-Hopene Cyclases , 2011, Applied and Environmental Microbiology.

[126]  A. Melis,et al.  Heterologous expression of the mevalonic acid pathway in cyanobacteria enhances endogenous carbon partitioning to isoprene. , 2014, Molecular plant.

[127]  D. V. Von Hoff,et al.  Taxol: a new and effective anti-cancer drug. , 1991, Anti-cancer drugs.

[128]  R. Chizzola Regular Monoterpenes and Sesquiterpenes (Essential Oils) , 2013 .

[129]  F. Güneş Medical use of squalene as a natural antioxidant , 2013 .

[130]  J. Komenda,et al.  Involvement of carotenoids in the synthesis and assembly of protein subunits of photosynthetic reaction centers of Synechocystis sp. PCC 6803. , 2010, Plant & cell physiology.

[131]  K. Berthelot,et al.  Isopentenyl diphosphate isomerase: A checkpoint to isoprenoid biosynthesis. , 2012, Biochimie.

[132]  D. Cane,et al.  Biosynthesis of 2-methylisoborneol in cyanobacteria. , 2011, Environmental science & technology.

[133]  Jeffrey C Way,et al.  Engineering cyanobacteria to generate high-value products. , 2011, Trends in biotechnology.

[134]  T. Sharkey,et al.  Methylerythritol 4-phosphate (MEP) pathway metabolic regulation. , 2014, Natural product reports.

[135]  M. Behmanesh,et al.  Biosynthesis, regulation and properties of plant monoterpenoids , 2014 .

[136]  R. Prinn,et al.  Isoprene production by Prochlorococcus, a marine cyanobacterium, and other phytoplankton , 2003 .

[137]  M. Ihara,et al.  Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis. , 2014, Journal of bioscience and bioengineering.

[138]  J. Sinisterra,et al.  Biotransformation of Terpenoids: A Green Alternative for Producing Molecules with Pharmacological Activity , 2009 .

[139]  A. Orav,et al.  Effect of storage on the essential oil composition of Piper nigrum L. fruits of different ripening states. , 2004, Journal of agricultural and food chemistry.

[140]  C. Poulter,et al.  Type II Isopentenyl Diphosphate Isomerase from Synechocystis sp. Strain PCC 6803 , 2004, Journal of bacteriology.

[141]  N. Meskhidze,et al.  Production and Emissions of Marine Isoprene and Monoterpenes: A Review , 2010 .

[142]  Seon-Won Kim,et al.  Synthetic Biology and Metabolic Engineering for Marine Carotenoids: New Opportunities and Future Prospects , 2014, Marine drugs.

[143]  Mo Xian,et al.  Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway , 2011, Applied Microbiology and Biotechnology.