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 plasmidbased 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 ␤-methyl-␦-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 ϳ0.43 g/liter/h) and yields (67.8 to ϳ71.4% 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 fedbatch 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 (ϳ1.01 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). IMPORTANCEMetabolic 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. Mevalonate pathways, important in the production of isoprenoids, are broadly present in eukaryotes, archaea, and some prokaryotes (1-3). Besides a precursor for the production of isoprenoids (2, 3), mevalonate has been used in cosmetics and as a building block for the production of sustainable polymers (4). Recently, it has been demonstrated that mevalonate could be transformed, by simple chemical steps, into a branched lactone ␤-methyl-␦-valerolactone (␤M␦VL), and its copolymerization with lactides results in a new class of polyesters with tunable mechanical properties (5). These potential applications motivate the development of ...

Optogeneticlacoperon to control chemical and protein production inEscherichia coliwith light

“…The favorable performance of our OptoLAC circuits to control GFP expression compared to IPTG induction encouraged us to test them in light-controlled bacterial fermentations for chemical production. We first tested our optogenetic circuits to control the biosynthesis of mevalonate, an important terpenoid precursor previously produced in E. coli (Figure 3a) 24–26 . We adapted to our optogenetic platform a previously described IPTG-inducible plasmid 24 containing the three genes to produce mevalonate from acetyl-CoA ( atoB , HMGS , and tHMGR ), resulting in plasmid pMAL487 (see Methods).…”

Effect of precise control of flux ratio between the glycolytic pathways on mevalonate production in Escherichia coli

“…Each single colony was inoculated into 5 ml Luria–Bertani medium and incubated for 12 hr at 37°C. In preculture, the starter was inoculated to an initial optical density at 600 nm (OD 600 ) of 0.05 into 50 ml M9 medium with 4 g/L glucose as the sole carbon source (Wada et al, ), and incubated for 24 hr. In main cultures, the preculture was inoculated to an OD 600 of 0.05 into 50 ml M9 medium with 4 g/L [1– 13 C] glucose (Cambridge Isotope Laboratories, Andover, MA) as the sole carbon source.…”

“…Therefore, extensive efforts have been made to enhance the NADPH supply by pathway engineering. Mevalonate is one of these useful compounds which consume NADPH for their synthesis, and is used in cosmetic products (Z. Q. Beck, Eliot, Peres, & Vaviline, ; Wang et al, ). In addition, because this compound is a key intermediate metabolite for the synthesis of isoprenoids which are used as advanced fuels and pharmaceuticals (Martin, Pitera, Withers, Newman, & Keasling, ; Xu, Xie, Zhao, Xian, & Liu, ), it is highly desirable to enhance the productivity in bioprocess using microorganisms, such as Escherichia coli ( E.coli ).…”

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

“…Even though the endogenous MEP pathway is less efficient than expressing the heterologous MVA pathway (Morrone et al, 2010), quite some work has been devoted to optimize its performance and further understand its wiring (Kim and Keasling, 2001;Zhou et al, 2012;Bongers et al, 2015). The emerging picture indicates that further optimization is needed when the MVA pathway is used to release metabolic bottlenecks and reduce toxicity of some metabolic intermediates therein (Martin et al, 2003;Ajikumar et al, 2010;Ma et al, 2011;Wang et al, 2016a). The adoption of alternative bacterial chassis could certainly be a way forward to overcome some of these limitations.…”