Highly Efficient Production of Menaquinone-7 from Glucose by Metabolically Engineered Escherichia coli

Abstract

Menaquinone-7 (MK-7) possesses wide health and medical value, and the market demand for MK-7 has increased. Metabolic engineering for MK-7 production in Escherichia coli still remains challenging due to the characteristics of the competing quinone synthesis, and cells mainly synthesized menaquinones under anaerobic conditions. To increase the production of MK-7 in engineered E. coli strains under aerobic conditions, we divided the whole MK-7 biosynthetic pathway into three modules (MVA pathway, DHNA pathway, and MK-7 pathway) and systematically optimized each of them. First, by screening and enhancing Idi expression, the amounts of MK-7/DMK-7 increased significantly. Then, in the MK-7 pathway, by combinatorial overexpression of endogenous MenA and exogenous UbiE, and fine-tuning the expression of HepPPS, MenA, and UbiE, 70 μM MK-7 was achieved. Third, the DHNA synthetic pathway was enhanced, and 157 μM MK-7 was achieved. By the combinational metabolic engineering strategies and membrane engineering, an efficient metabolic engineered E. coli strain for MK-7 synthesis was developed, and 200 μM (129 mg/L) MK-7 was obtained in shake flask experiment, representing a 306-fold increase compared to the starting strain. In the scale-up fermentation, 2074 μM (1350 mg/L) MK-7 was achieved after 52 h fermentation with a productivity of 26 mg/L/h. This is the highest titer of MK-7 ever reported. This study offers an alternative method for MK-7 production from biorenewable feedstock (glucose) by engineered E. coli. The high titer of our process should make it a promising cost-effective resource for MK-7.

Recent advances in the metabolic pathways and microbial production of coenzyme Q

“…Nevertheless, a recent example of efficient menaquinone-7 (MK 7 ) production to a titer of 1350 mg/L has shown that quinone production by E. coli should not be underestimated. This was achieved by optimized heterologous expression of MVA pathway genes and screening several heterologous Idi enzymes to improve IPP supply, overexpression of endogenous and exogenous MK pathway genes and enhancing the flux from chorismate to 1,4-dihydroxy-2-naphthoate, the direct precursor for demethylmenaquinone (Gao et al 2021 ).…”

Production of Vitamin K by Wild-Type and Engineered Microorganisms

“…E. coli can be used to produce MK-7, even if E. coli mainly produces Q-8, MK-8, and demethylmenaquinone-8 (DMK-8) [ 116 ]. Recently, Gao et al (2021) produced MK-7 by E. coli through systematic metabolic engineering [ 100 ]. The authors categorized the MK-7 biosynthesis pathway of E. coli into three modules: the MVA pathway, the DHNA pathway, and the MK-7 pathway, and optimized each pathway to enhance MK-7 production.…”

“…They first overexpressed the isopentenyl diphosphate isomerase (Idi) enzyme from different species, Saccharomyces cerevisiae, Populus trichocarpa , and E. coli K12, which are involved in the supply of IPP/DMAPP precursor for the biosynthesis of MK-7. Next, they overexpressed the endogenous MenA and exogenous UbiE together with the heptaprenyl pyrophosphate synthase (HepPPS) from B. subtilis which enhanced MK-7 production up to 70 μM [ 100 ]. The HepPPS is an important key enzyme, allowing MK-7 production in E. coli under aerobic conditions.…”

“…Membrane engineering was further employed to increase MK-7, which led to 200 μM (129 mg/L) MK-7 in a shake flask experiment. The authors conducted a fed-batch fermentation in a minimal medium using glucose as a substrate and achieved the highest titer of MK-7 ever reported up to 1.35 g/L [ 100 ]. The engineered E. coli outperformed Bacillus and LAB by producing 1.35 g/L of MK-7 during 52 h fermentation with increased titer and productivity when compared to other strains [ 100 ].…”

“…The authors conducted a fed-batch fermentation in a minimal medium using glucose as a substrate and achieved the highest titer of MK-7 ever reported up to 1.35 g/L [ 100 ]. The engineered E. coli outperformed Bacillus and LAB by producing 1.35 g/L of MK-7 during 52 h fermentation with increased titer and productivity when compared to other strains [ 100 ]. This result suggested that the microbial cell factory platform can be engineered and used for overproducing vitamin K.…”