Development of sucrose-utilizing Escherichia coli K-12 strain by cloning β-fructofuranosidases and its application for L-threonine production.
Sucrose is one of the most promising carbon sources for industrial fermentation. To achieve sucrose catabolism, the sucrose utilization operons have been introduced into microorganisms that are not able to utilize sucrose. However, the rates of growth and sucrose uptake of these engineered strains were relatively low to be successfully employed for industrial applications. Here, we report a practical example of developing sucrose-utilizing microorganisms using Escherichia coli K-12 as a model system. The sucrose utilizing ability was acquired by introducing only β-fructofuranosidase from three different sucrose-utilizing organisms (Mannheimia succiniciproducens, E. coli W, and Bacillus subtilis). Among them, the M. succiniciproducens β-fructofuranosidase was found to be the most effective for sucrose utilization. Analyses of the underlying mechanism revealed that sucrose was hydrolyzed into glucose and fructose in the extracellular space and both liberated hexoses could be transported by their respective uptake systems in E. coli K-12. To prove that this system can also be applied for the production of useful metabolites, the M. succiniciproducens β-fructofuranosidase was introduced into the engineered L-threonine production strain of E. coli K-12. This recombinant strain was able to produce 51.1 g/L L-threonine by fed-batch culture, resulting in an overall yield of 0.284 g L-threonine per g sucrose. This simple approach to make E. coli K-12 to acquire sucrose-utilizing ability and its successful biotechnological application can be employed to develop sustainable bioprocesses using renewable biomass.
Lee
Jeong Wook
JW
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Program), BioProcess Engineering Research Center, Center for Systems and Synthetic Biotechnology, and Institute for BioCentury, KAIST, Daejeon 305-701, Republic of Korea.
Choi
Sol
S
Park
Jin Hwan
JH
Vickers
Claudia E
CE
Nielsen
Lars K
LK
Lee
Sang Yup
SY
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology*; D003470:Culture Media / Q000378:metabolism; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics; D005285:Fermentation; D007218:Industrial Microbiology; D031121:Mannheimia / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D015202:Protein Engineering; D013395:Sucrose / Q000378:metabolism*; D013912:Threonine / Q000096:biosynthesis*; D043324:beta-Fructofuranosidase / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D003470:Culture Media; D013912:Threonine; D013395:Sucrose; D043324:beta-Fructofuranosidase