Construction of microbial platform for an energy-requiring bioprocess: practical 2'-deoxyribonucleoside production involving a C-C coupling reaction with high energy substrates.
BACKGROUND
Reproduction and sustainability are important for future society, and bioprocesses are one technology that can be used to realize these concepts. However, there is still limited variation in bioprocesses and there are several challenges, especially in the operation of energy-requiring bioprocesses. As an example of a microbial platform for an energy-requiring bioprocess, we established a process that efficiently and enzymatically synthesizes 2'-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase. This method consists of the coupling reactions of the reversible nucleoside degradation pathway and energy generation through the yeast glycolytic pathway.


RESULTS
Using E. coli that co-express deoxyriboaldolase and phosphopentomutase, a high amount of 2'-deoxyribonucleoside was produced with efficient energy transfer under phosphate-limiting reaction conditions. Keeping the nucleobase concentration low and the mixture at a low reaction temperature increased the yield of 2'-deoxyribonucleoside relative to the amount of added nucleobase, indicating that energy was efficiently generated from glucose via the yeast glycolytic pathway under these reaction conditions. Using a one-pot reaction in which small amounts of adenine, adenosine, and acetone-dried yeast were fed into the reaction, 75 mM of 2'-deoxyinosine, the deaminated product of 2'-deoxyadenosine, was produced from glucose (600 mM), acetaldehyde (250 mM), adenine (70 mM), and adenosine (20 mM) with a high yield relative to the total base moiety input (83%). Moreover, a variety of natural dNSs were further synthesized by introducing a base-exchange reaction into the process.


CONCLUSIONS
A critical common issue in energy-requiring bioprocess is fine control of phosphate concentration. We tried to resolve this problem, and provide the convenient recipe for establishment of energy-requiring bioprocesses. It is anticipated that the commercial demand for dNSs, which are primary metabolites that accumulate at very low levels in the metabolic pool, will grow. The development of an efficient production method for these compounds will have a great impact in both fields of applied microbiology and industry and will also serve as a good example of a microbial platform for energy-requiring bioprocesses.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
Nobuyuki
N
Horinouchi
Takafumi
T
Sakai
Takako
T
Kawano
Seiichiro
S
Matsumoto
Mie
M
Sasaki
Makoto
M
Hibi
Jun
J
Shima
Sakayu
S
Shimizu
Jun
J
Ogawa
D000079:Acetaldehyde / Q000378:metabolism; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism*; D004734:Energy Metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods*; D015394:Molecular Structure; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D003839:Deoxyadenosines; D000079:Acetaldehyde; D005947:Glucose; C058118:2'-deoxyadenosine