US 9,926,580 B2  8  7  losporus  JCM5269,  Xanthophilomyces  dendrorhous  IFO10129,  Xanthobacter flavus  JCM1204,  Paecilomyces  lilacinus  ATCC10114,  Acremonium  chrysogenum  ATCC11550,  Hyphomonas hirschiana  ATCC33886,  Rhizo› bium meliloti  ATCC9930, and the like.  As the reduced coenzyme Q$_{10}$-producing microorgan› isms, not only the wild species of the above-mentioned  microorganisms but also microorganisms in which the tran› scription and translation activities of the genes relevant to  the biosynthesis of reduced coenzyme  Q$_{10 }$ in the above› mentioned microorganisms, or the enzyme activity of the  expressed protein are modified or improved can be used  preferably, for example.  As the means for modifying or improving the transcrip- tion and translation activities of the genes or the enzyme  activity of the expressed protein, there may be mentioned  gene recombination (including gene improvement, amplifi› cation and destruction by itself, external gene introduction,  and gene improvement and proliferation of thus-introduced  external genes) and mutagenesis by mutagens. In particular,  the mutagenesis by mutagens is preferred.  The more preferable microorganisms usable for the pres› ent invention are microorganisms containing reduced coen› zyme Q$_{10 }$at a ratio of not less than 70 mole %, preferably  not less than 75 mole %, more preferably not less than 80  mole t, still more preferably not less than 85 mole %, and  particularly preferably not less than 90 mole %, among the  entire coenzymes  Q$_{10 }$ in the case where the above-men› tioned modified or improved microorganisms, preferably  microorganisms mutated by mutagens, are evaluated by the  above-mentioned proliferation method and the measurement  method. In the fermentation production on the industrial  scale, it is preferable to use microorganisms having a  productivity of reduced coenzyme  Q$_{10 }$ per unit culture  medium of not less than 1 µg/mL,  preferably not less than 2  µg/mL, more preferably not less than 3 µg/mL, still more  preferably not less than 5 µg/mL,  particularly preferably not  less than 10 µg/mL, much more preferably not less than 15  µg/mL, and most preferably not less than 20 µg/mL.  The mutagenesis may be carried out by a single muta› genesis; however, mutagenesis is preferably carried out not  less than 2 times. That is because it was found that the  productivity of reduced coenzyme  Q$_{10 }$ can be improved in  the respective mutagenesis steps.  It  is needless to say that the  candidates of the microbial cells to be mutated are, gener› ally, those having a productivity of reduced coenzyme  Q$_{10 }$ as high as possible in the case where the evaluation is carried  out by the above-mentioned proliferation method and mea› surement method.  The mutagenesis can be carried out by using optional and  proper mutagens. The term "mutagen" encompasses, in a  board definition, not only chemical agents having mutagen› esis effects, for example, but also treatments such as UV  radiation having mutagenesis effects. As examples of proper  mutangens, there may be mentioned ethyl methanesulfonate,  UV  radiation,  N-methyl-N’-nitro-N-nitrosoguanidine,  nucleotide base analogues such as bromouracil, and acri› dines; however, they are not limited to these examples.  According to a conventional mutagenesis technique, suc› cessively to the mutagenesis, a proper selection of microbial  cells having high productivity of reduced coenzyme  Q$_{10 }$is  carried out. For that, the culture obtained from a single  colony should be evaluated, for example, by the above› mentioned proliferation method and measurement method.  Since a reduced coenzyme  Q$_{10 }$ crystal forms a white solid  layer or a colorless liquid phase, a productivity of reduced  To control the concentration of the carbon sources, a fed  batch culture method is preferably used. The carbon source  concentration in the broth can be controlled by adjusting the  supply of nutrient sources (especially carbon sources) based  on the culture control indexes such as pH, the dissolved  oxygen concentration (DO) or the remaining saccharide  concentration. Although it depends on the microorganism  species, the supply of the nutrient sources may be started  from the initial stage of the culture or during the culture. The  supply of the nutrient sources may be continuous or inter- In the fermentation production on the industrial scale,  although it depends on the microorganism species, the  concentration of the carbon sources (including the produced  alcohols) during the culture is preferably controlled to a  concentration that no adverse effects are substantially caused  on the productivity of reduced coenzyme Q$_{10}$. Accordingly,  it is preferable to control the culture so as to have the  concentration of the carbon sources that no adverse effects  are substantially caused on the productivity of reduced  coenzyme Q$_{10}$, that is, generally to not more than 20 g/L,  preferably not more than 5 g/L, and more preferably not  more than 2 g/L in the broth.  In general, the culture is carried out at a pH range of 4 to  9, preferably 5 to 8.  If  the pH is not more than 3 or not less  than 10, proliferation of microorganisms tends to be easily  inhibited.  The culture is generally carried out at a temperature range  of 15 to 45°  C., preferably 20 to 37°  C.  If  it is below 15°  C.,  the proliferation speed of microorganisms tends to be too  slow to allow the industrial production and at high tempera- tures exceeding 45° C., the viability of microorganisms  tends to be easily hindered.  The culture is carried out, in general, in a culture medium  containing major nutrients and micronutrients suited for  microorganism proliferation. As the above-mentioned nutri› ents, there may be mentioned, for example, carbon sources  ( e.g. hydrocarbons such as glucose, sucrose, maltose, starch,  corn syrup and molasses; alcohols such as methanol and  ethanol), nitrogen sources ( e.g. com steep liquor, ammo› nium sulfate, animonium phosphate, animonium hydroxide,  urea and peptone ), phosphorus sources ( e.g. animonium  phosphate and phosphoric acid) and micronutrients ( e.g.  minerals such as magnesium, potassium, zinc, copper, iron,  manganese, molybdenum, sulfuric acid and hydrochloric  acid; vitamins such as biotin, desthiobiotin and vitamin Bl;  amino acids such as alanine and histidine; and natural raw  materials containing vitamins such as yeast extract and malt  extract); however, these are not !imitative ones, and com› monly used ones may be used. Incidentally, in natural  components of a culture medium, such as yeast extract,  phosphorus sources such as phosphates are contained. The  above-mentioned nutrients can be appropriately used in  combination.  In the processes of the present invention, high productiv- ity ofreduced coenzyme Q$_{10 }$in the fermentation production  on the industrial scale can be achieved partially by using the  microbial cells containing reduced coenzyme Q$_{10 }$at a ratio  of not less than 70 mole % among the entire coenzymes Q$_{10 }$ and, partially, by using the suitable conditions of culture  (fermentation) for increasing a productivity of reduced coen› zyme  Q$_{10 }$per unit culture medium as described below.  It  is  particularly preferable to combinedly use suitable microbial  cells described above and the suitable conditions of culture  (fermentation) as described below.  coenzyme Q$_{10 }$ can be suitably evaluated by the above› mentioned measurement method at the time of selection of  the colony. 