Enhanced succinic acid production by Mannheimia employing optimal malate dehydrogenase

Abstract

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 μM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L −1 of SA with the maximum productivity of 21.3 g L −1 h −1 , demonstrating the importance of enzyme optimization in strain development.

Uncoupling growth and succinic acid production in an industrial Saccharomyces cerevisiae strain

“…Researchers from KAIST applied metabolic engineering to improve succinic acid production of the capnophilic rumen bacterium Mannheimia succiniciproducens (Lee et al, 2002). They obtained succinic acid yields up to 1.64 mol/mol glucose equivalent (Ahn et al, 2016; Lee et al, 2016) and titers up to 134 g/L (Ahn et al, 2020) by accomplishing almost homo‐succinic acid production. Myriant applied an engineered Escherichia coli strain for large‐scale SA production (Ahn et al, 2016).…”

Insights on the Advancements of In Silico Metabolic Studies of Succinic Acid Producing Microorganisms: A Review with Emphasis on Actinobacillus succinogenes

“…The power of in silico metabolic modeling has also been seen in a recent SA production study using M. succiniciproducens as a biocatalyst. In this research, GEM [75] was analyzed to characterize malate dehydrogenase (MDH) and finally the overexpression of genes encoding MDH led to the production of the highest overall SA reported to date [99]. This is one of the latest tangible pieces of evidence of applications of model-guided in silico studies in the journey of developing industrially compatible SA-producing strains.…”

“…Integration of genome-scale metabolic model with dynamic modeling and genetic algorithm to provide simpified gene deletion strategies for the complex evolutionary goals containing multiple targets 2020 [98] M. succiniciproducens Flux variability scanning using genome-scale metabolic model to identify amplification target genes for improved SA production 2020 [99] Likewise, other potential microbes were also evaluated for their metabolic capability of SA production. The fundamental difference in these in silico studies, besides the algorithms employed, is the scope of metabolic network coverage.…”

Label-free three-dimensional analyses of live cells with deep-learning-based segmentation exploiting refractive index distributions

“…Quantitative phase imaging (QPI) is a powerful method to observe the morphology of a live specimen without any perturbation; this includes dye staining or fluorescence protein expression 6 . Recently developed three dimensional (3D) QPI techniques provide the 3D refractive index (RI) distributions, containing quantitative information on the concentration of a material, and have been exploited in various applications including biomolecular condensates 7 , biotechnology 8 , microbiology 9 , and cell biology 10 . Although the 3D QPI image can provide the physical properties corresponding to each voxel, a universal and versatile segmentation method is required to simultaneously monitor quantitative dynamics in a whole cell and its organelles.…”