Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl‐CoA supply

Abstract

Microbial production of mevalonate from renewable feedstock is a promising and sustainable approach for the production of value‐added chemicals. We describe the metabolic engineering of Escherichia coli to enhance mevalonate production from glucose and cellobiose. First, the mevalonate‐producing pathway was introduced into E. coli and the expression of the gene atoB, which encodes the gene for acetoacetyl‐CoA synthetase, was increased. Then, the deletion of the pgi gene, which encodes phosphoglucose isomerase, increased the NADPH/NADP+ ratio in the cells but did not improve mevalonate production. Alternatively, to reduce flux toward the tricarboxylic acid cycle, gltA, which encodes citrate synthetase, was disrupted. The resultant strain, MGΔgltA‐MV, increased levels of intracellular acetyl‐CoA up to sevenfold higher than the wild‐type strain. This strain produced 8.0 g/L of mevalonate from 20 g/L of glucose. We also engineered the sugar supply by displaying β‐glucosidase (BGL) on the cell surface. When cellobiose was used as carbon source, the strain lacking gnd displaying BGL efficiently consumed cellobiose and produced mevalonate at 5.7 g/L. The yield of mevalonate was 0.25 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing mevalonate from cellobiose or cellooligosaccharides using an engineered E. coli strain.

Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineered E. coli

“…More broadly, improving NADPH availability or flux, useful in the synthesis of numerous metabolites as well as cell based bioconversions, has been a long standing challenge in metabolic engineering. [13][14][15][16][17] We sought to apply two-stage dynamic metabolic control (DMC) to improve NADPH flux and xylitol production using xylose as a sole feedstock. 18 Dynamic control over metabolism has become a popular approach in metabolic engineering, and has been used for the production of various products from 3-hydroxypropionic acid to myo-inositol and many others.…”

Microbial Upgrading of Acetate into Value-Added Products—Examining Microbial Diversity, Bioenergetic Constraints and Metabolic Engineering Approaches

“…Another example is the pathway for mevalonate synthesis which also demands NADPH. In a recent study, the importance of NADPH regeneration for the production of mevalonate was revealed [171]. However, the strategy presented would need to be thoroughly revised for acetate-based processes since it was established for glucose as a carbon source and so far, the implementation for acetate-based production is lacking.…”

Recent Progress on Chemical Production From Non-food Renewable Feedstocks Using Corynebacterium glutamicum

“…Due to the absence of a cellobiose transporter and low permeability of cellobiose, expression of βglucosidase in microorganisms invariably requires secretion or surface display in order for the enzyme to contact the substrate. Traditionally, secreted expression or surface display of βglucosidase has been applied for biorefinery chemical production in a multitude of industrial strains, including Clostridium thermocellum (Dash et al, 2019), S. cerevisiae (Yun et al, 2018), Pseudomonas putida (Dvoøák and de Lorenzo, 2018), E. coli (Satowa et al, 2020), and C. glutamicum (Adachi et al, 2013). C. glutamicum displays high β-glucosidase activity derived from Saccharophagus degradans, successfully displaying β-glucosidase fused with the C-terminus of the anchor protein PorC.…”