Engineering Corynebacterium glutamicum for the production of 2,3-butanediol.
BACKGROUND
2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions.


RESULTS
A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol(-1) yield on glucose, an overall productivity of 0.2 g L(-1) h(-1) and a titer of 6.3 g L(-1).


CONCLUSIONS
We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República-EAN, 2780-157, Oeiras, Portugal. rados@itqb.unl.pt.
Dušica
D
Radoš
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República-EAN, 2780-157, Oeiras, Portugal. anac81@gmail.com.|Gut Health and Food Safety Programme, Institute of Food Research, Norwich Research Park, Norwich, UK. anac81@gmail.com.
Ana Lúcia
AL
Carvalho
Institute of Microbiology and Biotechnology, University of Ulm, 89069, Ulm, Germany. stefan.wieschalka@gmx.de.|Rentschler Biotechnologie GmbH, Erwin-Rentschler-Straße, 21, 88471, Laupheim, Germany. stefan.wieschalka@gmx.de.
Stefan
S
Wieschalka
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República-EAN, 2780-157, Oeiras, Portugal. arnevesrrr@gmail.com.|CED-Discovery, Chr Hansen A/S, 10-12 Bøge Alle, 2970, Hørsholm, Denmark. arnevesrrr@gmail.com.
Ana Rute
AR
Neves
Institute for Biochemical Engineering, University of Stuttgart, 70569, Stuttgart, Germany. blombach@ibvt.uni-stuttgart.de.
Bastian
B
Blombach
Institute of Microbiology and Biotechnology, University of Ulm, 89069, Ulm, Germany. bernhard.eikmanns@uni-ulm.de.
Bernhard J
BJ
Eikmanns
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República-EAN, 2780-157, Oeiras, Portugal. santos@itqb.unl.pt.|Lisbon Academy of Sciences, R. Academia das Ciências 19, 1249, Lisbon, Portugal. santos@itqb.unl.pt.
Helena
H
Santos
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors; D002072:Butylene Glycols / Q000737:chemistry / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000737:chemistry* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000172:deficiency / Q000235:genetics; D013294:Lactococcus lactis / Q000235:genetics; D060847:Metabolic Engineering; D005810:Multigene Family; D010100:Oxygen / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002072:Butylene Glycols; D011768:Pyruvate Dehydrogenase Complex; C026978:2,3-butylene glycol; D007770:L-Lactate Dehydrogenase; D005947:Glucose; D010100:Oxygen