title: Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration.
abstract: Chromosomal integration of heterologous metabolic pathways is optimal for industrially relevant fermentation, as plasmid-based fermentation causes extra metabolic burden and genetic instabilities. In this work, chromosomal integration was adapted for the production of mevalonate, which can be readily converted into β-methyl-δ-valerolactone, a monomer for the production of mechanically tunable polyesters. The mevalonate pathway, driven by a constitutive promoter, was integrated into the chromosome of Escherichia coli to replace the native fermentation gene adhE or ldhA The engineered strains (CMEV-1 and CMEV-2) did not require inducer or antibiotic and showed slightly higher maximal productivities (0.38 to ∼0.43 g/liter/h) and yields (67.8 to ∼71.4% of the maximum theoretical yield) than those of the plasmid-based fermentation. Since the glycolysis pathway is the first module for mevalonate synthesis, atpFH deletion was employed to improve the glycolytic rate and the production rate of mevalonate. Shake flask fermentation results showed that the deletion of atpFH in CMEV-1 resulted in a 2.1-fold increase in the maximum productivity. Furthermore, enhancement of the downstream pathway by integrating two copies of the mevalonate pathway genes into the chromosome further improved the mevalonate yield. Finally, our fed-batch fermentation showed that, with deletion of the atpFH and sucA genes and integration of two copies of the mevalonate pathway genes into the chromosome, the engineered strain CMEV-7 exhibited both high maximal productivity (∼1.01 g/liter/h) and high yield (86.1% of the maximum theoretical yield, 30 g/liter mevalonate from 61 g/liter glucose after 48 h in a shake flask).
  
  
  
  Metabolic engineering has succeeded in producing various chemicals. However, few of these chemicals are commercially competitive with the conventional petroleum-derived materials. In this work, chromosomal integration of the heterologous pathway and subsequent optimization strategies ensure stable and efficient (i.e., high-titer, high-yield, and high-productivity) production of mevalonate, which demonstrates the potential for scale-up fermentation. Among the optimization strategies, we demonstrated that enhancement of the glycolytic flux significantly improved the productivity. This result provides an example of how to tune the carbon flux for the optimal production of exogenous chemicals.
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Jilong
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand.
authors.forename: Suthamat
authors.initials: S
authors.lastname: Niyompanich
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Yi-Shu
authors.initials: YS
authors.lastname: Tai
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Jingyu
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Wenqin
authors.initials: W
authors.lastname: Bai
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Prithviraj
authors.initials: P
authors.lastname: Mahida
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Tuo
authors.initials: T
authors.lastname: Gao
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota, USA kzhang@umn.edu.
authors.forename: Kechun
authors.initials: K
authors.lastname: Zhang
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering; D008798:Mevalonic Acid / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism
chemical_list: D005947:Glucose; D008798:Mevalonic Acid