Reconstruction of the Diaminopimelic Acid Pathway to Promote L-lysine Production in Corynebacterium glutamicum

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.

Starch extracted from pineapple (Ananas comosus) plant stem as a source for amino acids production

Background Pineapple plant (Ananas comosus) is one of the largest productions in Asia and its increasing production has generated a huge amount of pineapple wastes. Pineapple plant stem is made up of high concentration of starch which can potentially be converted into value-added products, including amino acids. Due to the increasing demand in animal feed grade amino acids, especially for methionine and lysine, the utilisation of cheap and renewable source is deemed to be an essential approach. This study aimed to produce amino acids from pineapple plant stem hydrolysates through microbial fermentation by Pediococcus acidilactici Kp10. Dextrozyme was used for hydrolysis of starch and Celluclast 1.5 L for saccharification of cellulosic materials in pineapple plant stem. Results The hydrolysates obtained were used in the fermentation to produce methionine and lysine. Pineapple plant stem showed high starch content of 77.78%. Lignocellulosic composition of pineapple plant stem consisted of 46.15% hemicellulose, 31.86% cellulose, and 18.60% lignin. Saccharification of alkaline-treated pineapple plant stem gave lower reducing sugars of 13.28 g/L as compared to untreated, where 18.56 g/L reducing sugars obtained. Therefore, the untreated pineapple plant stem was selected for further process. Starch hydrolysis produced 57.57 g/L reducing sugar (100% hydrolysis yield) and saccharification of cellulosic materials produced 24.67 g/L reducing sugars (56.93% hydrolysis yield). The starch-based and cellulosic-based of pineapple plant stem were subjected as carbon source in methionine and lysine production by P. acidilactici Kp10. Conclusions In conclusion, higher methionine and lysine production were produced from starch-based hydrolysis (40.25 mg/L and 0.97 g/L, respectively) as compared to cellulosic-based saccharification (37.31 mg/L and 0.84 g/L, respectively) of pineapple plant stem. Graphical Abstract

Evolving a New Efficient Mode of Fructose Utilization for Improved Bioproduction in Corynebacterium glutamicum

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.

Reconstruction of the Diaminopimelic Acid Pathway to Promote L-lysine Production in Corynebacterium Glutamicum

Background: The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and great potentials to increase the production of L-lysine. Results: The aim of this work is to enhance the carbon flux in dehydrogenase pathway to promote L-lysine production. Firstly, the effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3±4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189±8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35±0.05 g/(g·h) in a 5-L jar fermenter. Conclusions: The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.

Genomic and Transcriptomic Investigation of the Physiological Response of the Methylotroph Bacillus methanolicus to 5-Aminovalerate

The methylotrophic thermophile Bacillus methanolicus can utilize the non-food substrate methanol as its sole carbon and energy source. Metabolism of L-lysine, in particular its biosynthesis, has been studied to some detail, and methanol-based L-lysine production has been achieved. However, little is known about L-lysine degradation, which may proceed via 5-aminovalerate (5AVA), a non-proteinogenic ω-amino acid with applications in bioplastics. The physiological role of 5AVA and related compounds in the native methylotroph was unknown. Here, we showed that B. methanolicus exhibits low tolerance to 5AVA, but not to related short-chain (C4–C6) amino acids, diamines, and dicarboxylic acids. In order to gain insight into the physiological response of B. methanolicus to 5AVA, transcriptomic analyses by differential RNA-Seq in the presence and absence of 5AVA were performed. Besides genes of the general stress response, RNA levels of genes of histidine biosynthesis, and iron acquisition were increased in the presence of 5AVA, while an Rrf2 family transcriptional regulator gene showed reduced RNA levels. In order to test if mutations can overcome growth inhibition by 5AVA, adaptive laboratory evolution (ALE) was performed and two mutants—AVA6 and AVA10—with higher tolerance to 5AVA were selected. Genome sequencing revealed mutations in genes related to iron homeostasis, including the gene for an iron siderophore-binding protein. Overexpression of this mutant gene in the wild-type (WT) strain MGA3 improved 5AVA tolerance significantly at high Fe2+ supplementation. The combined ALE, omics, and genetics approach helped elucidate the physiological response of thermophilic B. methanolicus to 5AVA and will guide future strain development for 5AVA production from methanol.

Evolving a new efficient mode of fructose utilization for improved bioproduction in Corynebacterium glutamicum

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (~10 %) compared to glucose (~60 %). Consequently, the biosynthesis of NADPH demanding products, e.g. L-lysine, by C. glutamicum is largely decreased, when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and consequently NADPH availability. Here, we generated deletion strains either lacking in the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose specific PTS, as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased biomass formation, reduced side-product accumulation, and increased L-lysine production by 50 %.