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Biosynthesis
The complete biosynthesis of Gentamicin is not entirely elucidated. The genes controlling the biosynthesis of Gentamicin are of particular interest due to the difficulty in obtaining the antibiotic after production. Since Gentamicin is collected at the cell surface and the cell surface must be perforated some how to obtain the antibiotic. Many propose the amount of Gentamicin collected after production could increase if the genes are identified and re-directed to secrete the antibiotic instead of collecting Gentamicin at the cell surface. Literature also agrees with the Gentamicin biosynthesis pathway starting with D-Glucose-6-phosphate being dephopsphorylated, transaminated, dehydrogenated and finally glycosylated with D-glucosamine to generate paromamine inside Micromonospora echinospora. The addition of D-xylose leads to the first intermediate of the Gentamicin C complex pathway, Gentamicin A2. Gentamicin A2 is C-methylated and epimerized into Gentamicin X2, the first branch point of this biosynthesis pathway

When X2 is acted on by the cobalamin-dependent radical S-adenosyl-L-methionine enzyme GenK, the carbon position 6' is methylated to form the pharmacologically active intermediate G418 G418 then undergoes dehydrogenation and amination at the C6' position  by the dehydrogenase gene, GenQ, to generate the pharmacologically active JI-20B, although another intermediate, 6'-dehydro-6'oxo-G418 (6'DOG) is porposed to be in-between this step and for which the gene GenB1 is proposed as the aminating gene. JI-20B is dehydroxylated and epimerized to first component of the Gentamicin C complex, Gentamicin C2a which then undergoes an epimerization by GenB2 and then a N-methylation by an unconfirmed gene to form the final product in this branch point, Gentamicin C1.

When X2 bypasses GenK and is directly dehydrogenated and aminated by the GenQ enzyme, the other pharmacologically relevant intermediate JI-20A is formed. . Although, there has been identification of an intermediate for this step, 6'-dehydro-6'-oxo-gentamicin X2 (6'-DOX), for which the enzyme GenB1 is purposed as the aminating enzyme. JI-20A is then dehydroxylated into the first component of the Gentamicin C complex for this branch, Gentamicin C1a via a catalytic reaction with GenB4. C1a then undergoes an N-methylation by an unconfirmed enzyme to form the final component, Gentamicin C2b.

Fermentation

Gentamicin is only synthesized via submerged fermentation and inorganic sources of nutrients have been found to reduce production. Traditional fermentation used yeast beef broth, but there has been research into optimizing the growth medium for producing Gentamicin C complex due to the C complex currently being the only pharmaceutically relevant component. The main components of the growth medium are carbon sources, mainly sugars, but several studies found increased Gentamicin production by adding vegetable and fish oils and decreased Gentamicin production with the addition of Glucose, Xylose and several carboxylic acids. Tryptone and various forms of yeast and yeast derivatives are traditionally used as the nitrogen source in the growth medium, but several amino acids], [[soybean meal, Corn steep liquor, ammonium sulfate, and ammonium chloride have proven to be beneficial additives. Phosphate ions, metal ions (cobalt and a few others at low concentration), various vitamins (mostly B vitamins), purine and pyrimidine bases are also supplemented into the growth medium to increase Gentamicin production, but the margin of increase is dependent on the species of Micromonospora and the other components in the growth medium. With all of these aforementioned additives, pH and aeration are key determining factors for the amount of Gentamicin produced. A range of pH from 6.8-7.5 is used for Gentamicin biosynthesis and the aeration is determined by independent experimentation reliant on type of growth medium and species of Micromonospora.  FixedC1primeStereospecific Gentamicin IUPAC Numbered copy.png Structure

Since Gentamicin is derived from the species Micromonospora, the backbone for this antibiotic is the aminocyclitol 2-deoxystreptamine. . This six carbon ring is substituted at the carbon positions 4 and 6 by the amino sugar molecules cyclic purpurosamine and garosamine, respectively. The Gentamicin complex, is differentiated into five major components (C1, C1a, C2, C2a, C2b) and multiple minor components by substitution at the 6' carbon of the purpurosamine unit indicated in the image to the right by R1 and R2. The R1 and R2 can have the follow substitutions for some of the species in the Gentamicin complex.