Lysine production from the sugar alcohol mannitol: Design of the cell factory Corynebacterium glutamicum SEA-3 through integrated analysis and engineering of metabolic pathway fluxes

Abstract

The amino acid lysine is among the world's most important biotechnological products, and enabling its manufacture from the most attractive new materials is an ever-present challenge. In this study, we describe a cell factory of Corynebacterium glutamicum, which produces lysine from mannitol. A preliminary mutant C. glutamicum SEA-1 obtained by the deletion of the mannitol repressor MtlR in the glucose-based, lysine-producing strain C. glutamicum LYS-12 produced only small amounts of lysine. This limitation was due to a significant accumulation of fructose and a limited NADPH supply, which caused a low flux of only 6% into the oxidative pentose phosphate (PP) pathway. Subsequent expression of fructokinase slightly increased production but failed to substantially redirect the flux from the Emden-Meyerhof-Parnas (EMP) pathway to the PP pathway. This suggested the design of C. glutamicum SEA-3, which overexpressed the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase GapN from Streptococcus mutans and coupled the EMP pathway flux to NADPH formation. When grown on mannitol, the SEA-3 strain had a lysine yield of 0.24 mol mol and a specific productivity of 1.3 mmol g h, approximately 60% and 75% higher, respectively, than those of the basic producer SEA-1. A computational pathway analysis revealed that this design would potentially enable a lysine yield of 0.9 mol mol, providing room for further development. Our findings open new avenues for lysine production from marine macroalgae, which is farmed globally as an attractive third-generation renewable resource. Mannitol is a major constituent of these algae (up to 30% and higher) and can be easily extracted from their biomass with hot water.

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

“…As a proof of concept demonstration, base editing was used to remove feedback inhibition of aspartokinase encoded by lysC ( cgl0251 ) by introducing an amino acid transition T311I (C932T or C932T&C933T base editing), which cannot be achieved by our previous MACBETH method (Y. Wang et al, ). This modification was reported to deregulate lysine biosynthesis pathway and cause overproduction of lysine, one of the world's most important biotechnological products (Hoffmann et al, ) (Figure a). nxCas9 3.7(D10A)‐AID was selected for its higher editing efficiency at this genomic locus.…”

“…The average of three biological replicates is plotted. [Color figure can be viewed at wileyonlinelibrary.com] lysine, one of the world's most important biotechnological products (Hoffmann et al, 2018) (Figure 7a). nxCas9 3.7(D10A)-AID was selected for its higher editing efficiency at this genomic locus.…”

Metabolic engineering of carbohydrate metabolism systems in Corynebacterium glutamicum for improving the efficiency of l-lysine production from mixed sugar

“…However, there is no fructokinase (ScrK) in C. glutamicum [36], resulting in that the intracellular fructose should be firstly excreted into extracellular and then reassimilated via fructose-PTS (PTS Fru ). Previous results indicated that hetero-expression of ScrK is beneficial to increase the production of NADPH-dependent products with sucrose or mixed sugar as carbon source [7,[37][38][39]. Therefore, hetero-expression of ScrK from C. acetobutylicum at pfkB gene loci was set as a potential strategy for enhancing l-lysine production on mixed sugar in this study.…”

Production of Value-Added Chemicals by Bacillus methanolicus Strains Cultivated on Mannitol and Extracts of Seaweed Saccharina latissima at 50°C

“…E. coli can naturally assimilate both mannitol and glucose and has been engineered to produce 17.4 g/l of succinic acid from a hydrolyzate made from brown seaweed Saccharina japonica (Bai et al, 2015). Recently, the lysine producing strain C. glutamicum LYS-12 was engineered to assimilate mannitol and efficiently produce 2 g/l L-lysine from this substrate in shake flask experiments (Hoffmann et al, 2018). However, the high salt content of seaweed-based media represents a typical challenge in microbial cultivations and therefore strains with high salt tolerance are required.…”