Glutamicum S9114 Research Articles

Cellulosic hydrocarbons production by engineering dual synthesis pathways in Corynebacterium glutamicum

BackgroundLignocellulose provides the only practical carbohydrates feedstock for sustainable bioproduction of hydrocarbons as future alternative of fossil fuels. Production of hydrocarbons from lignocellulose is achieved by a biorefinery process chain including pretreatment to breakdown the crystalline structure for cellulase-catalyzed hydrolysis, detoxification of inhibitory compounds generated during pretreatment, enzymatic hydrolysis to fermentable monosaccharide sugars, and fermentation to hydrocarbon products. The major barriers on fermentative production of hydrocarbons from lignocellulose include two aspects: one is the inherent stress of pretreatment-derived inhibitors on microbial cells, the other is the toxicity of hydrocarbons to cell membranes. The microbial cell factory should be tolerant to both inhibitor stress and hydrocarbons toxicity.ResultsCorynebacterium glutamicum was selected as the starting strain of hydrocarbons synthesis since it is well adapted to lignocellulose hydrolysate environment. The dual hydrocarbon synthesis pathways were constructed in an industrial C. glutamicum S9114 strain. The first pathway was the regular one in microalgae composed of fatty acyl-acyl carrier protein (fatty acyl-ACP) reductase (AAR) and aldehyde deformylating oxygenase (ADO) with fatty acyl-ACP as precursor. The second pathway was the direct decarboxylation of free fatty acid by fatty acid decarboxylase (OleT) using the rich fatty acids from the disruption of the transcriptional regulator fasR gene. The transmembrane transportation of hydrocarbon products was avoided by secretively expressing the fatty acid decarboxylase (OleT) to the extracellular space. The hydrocarbons generation from glucose reached 29.2 mg/L, in which the direct decarboxylation pathway contributed more than 70% of the total hydrocarbons generation, and the AAR–ADO pathway contributed the rest 30%.ConclusionThe dual hydrocarbon synthesis pathways (OleT and AAR–ADO pathways) were constructed in the inhibitors tolerant C. glutamicum S9114 strain for hydrocarbon production using lignocellulose feedstock as the starting feedstock. When corn stover was used for hydrocarbons production after dry acid pretreatment and biodetoxification, the hydrocarbons generation reached 16.0 mg/L. This study provided a new strategy for hydrocarbons synthesis using microbial cell factory suitable for lignocellulose feedstock.

Synergistic improvement of N-acetylglucosamine production by engineering transcription factors and balancing redox cofactors

The regulation of single gene transcription level in the metabolic pathway is often failed to significantly improve the titer of the target product, and even leads to the imbalance of carbon/nitrogen metabolic network and cofactor network. Global transcription machinery engineering (gTME) can activate or inhibit the synergistic expression of multiple genes in specific metabolic pathways, so transcription factors with specific functions can be expressed according to different metabolic regulation requirements, thus effectively increasing the synthesis of target metabolites. In addition, maintaining intracellular redox balance through cofactor engineering can realize the self-balance of cofactors and promote the efficient synthesis of target products. In this study, we rebalanced the central carbon/nitrogen metabolism and redox metabolism of Corynebacterium glutamicum S9114 by gTME and redox cofactors engineering to promote the production of the nutraceutical N-acetylglucosamine (GlcNAc). Firstly, it was found that the overexpression of the transcription factor RamA can promote GlcNAc synthesis, and the titer was further improved to 16 g/L in shake flask by using a mutant RamA (RamAM). Secondly, a CRISPR interference (CRISPRi) system based on dCpf1 was developed and used to inhibit the expression of global negative transcriptional regulators of GlcNAc synthesis, which promoted the GlcNAc titer to 27.5 g/L. Thirdly, the cofactor specificity of the key enzymes in GlcNAc synthesis pathway was changed by rational protein engineering, and the titer of GlcNAc in shake flask was increased to 36.9 g/L. Finally, the production of GlcNAc was scaled up in a 50-L fermentor, and the titer reached 117.1 ± 1.9 g/L, which was 6.62 times that of the control group (17.7 ± 0.4 g/L), and the yield was increased from 0.19 g/g to 0.31 g/g glucose. The results obtained here highlight the importance of engineering the global regulation of central carbon/nitrogen metabolism and redox metabolism to improve the production performance of microbial cell factories.

Highly efficient biosynthesis of l-ornithine from mannitol by using recombinant Corynebacterium glutamicum

Mannitol is a promising six-carbon sugar alcohol that is widely found in macroalgae. The potential of mannitol as a renewable raw material is of interest due to the advantages of ocean farms. Herein, the biobased production of l-ornithine from mannitol was resoundingly demonstrated for the first time in engineered Corynebacterium glutamicum S9114 through the deletion of the mannitol repressor MtlR. By modulating the expression of mtlD and reinforcing the fructose metabolic pathway, we generated the strain MTL13 that produced 54.56 g/L of l-ornithine with a yield of 0.47 g/g on mannitol. These results illustrate the robust conversion from mannitol to l-ornithine using engineered Corynebacterium glutamicum, providing a reference for the biobased production of additional chemicals from mannitol.

Development of a DNA double-strand break-free base editing tool in Corynebacterium glutamicum for genome editing and metabolic engineering

As a traditional amino acid producing bacterium, Corynebacterium glutamicum is a platform strain for production of various fine chemicals. Based on the CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system, gene editing tools that enable base conversion in the genome of C. glutamicum have been developed. However, some problems such as genomic instability caused by DNA double-strand break (DSB) and off-target effects need to be solved. In this study, a DSB-free single nucleotide genome editing system was developed by construction of a bi-directional base conversion tool TadA-dCas9-AID. This system includes cytosine base editors (CBEs): activation-induced cytidine deaminase (AID) and adenine deaminase (ABEs): tRNA adenosine deaminase (TadA), which can specifically target the gene through a 20-nt single guide RNA (sgRNA) and achieve the base conversion of C-T, C-G and A-G in the 28-bp editing window upstream of protospacer adjacent motif. Finally, as a proof-of-concept demonstration, the system was used to construct a mutant library of zwf gene in C. glutamicum S9114 genome to improve the production of a typical nutraceutical N-acetylglucosamine (GlcNAc). The GlcNAc titer of the mutant strain K293R was increased by 31.9% to 9.1 ​g/L in shake flask. Here, the developed bases conversion tool TadA-dCas9-AID does not need DNA double-strand break and homologous template, and is effective for genome editing and metabolic engineering in C. glutamicum.

Proteome analysis guided genetic engineering of Corynebacterium glutamicum S9114 for tween 40-triggered improvement in l-ornithine production

Backgroundl-ornithine is a valuable amino acid with a wide range of applications in the pharmaceutical and food industries. However, the production of l-ornithine by fermentation cannot compete with other methods, because of the low titers produced with this technique. Development of fermentation techniques that result in a high yield of l-ornithine and efficient strategies for improving l-ornithine production are essential.ResultsThis study demonstrates that tween 40, a surfactant promoter of the production of glutamate and arginine, improves l-ornithine production titers in engineered C. glutamicum S9114. The intracellular metabolism under tween 40 triggered fermentation conditions was explored using a quantitative proteomic approach, identifying 48 up-regulated and 132 down-regulated proteins when compared with the control. Numerous proteins were identified as membrane proteins or functional proteins involved in the biosynthesis of the cell wall. Modulation of those genes revealed that the overexpression of CgS9114_09558 and the deletion of CgS9114_13845, CgS9114_02593, and CgS9114_02058 improved the production of l-ornithine in the engineered strain of C. glutamicum Orn8. The final strain with all the exploratory metabolic engineering manipulations produced 25.46 g/L of l-ornithine, and a yield of 0.303 g l-ornithine per g glucose, which was 30.6% higher than that produced by the original strain (19.5 g/L).ConclusionThese results clearly demonstrate the positive effect of tween 40 addition on l-ornithine accumulation. Proteome analysis was performed to examine the impact of tween 40 addition on the physiological changes in C. glutamicum Orn8 and the results showed several promising modulation targets for developing l-ornithine-producing strains.

Tolerance and transcriptional analysis of Corynebacterium glutamicum on biotransformation of toxic furaldehyde and benzaldehyde inhibitory compounds.

Furaldehydes and benzaldehydes are among the most toxic inhibitors from lignocellulose pretreatment on microbial growth and metabolism. The bioconversion of aldehyde inhibitors into less toxic alcohols or acids (biotransformation) is the prerequisite condition for efficient biorefinery fermentations. This study found that Corynebacterium glutamicum S9114 demonstrated excellent tolerance and biotransformation capacity to five typical aldehyde inhibitors including two furaldehydes: 2-furaldehyde (furfural), 5-(hydroxymethyl)-2-furaldehyde, and three benzaldehydes: 4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde (vanillin), and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde). Transcription levels of 93 genes hypothesized to be responsible for five aldehydes biotransformation were examined by qRT-PCR. Multiple genes showed significantly up-regulated expression against furaldehydes or benzaldehydes. Overexpression of CGS9114_RS01115 in C. glutamicum resulted in the increased conversion of all five aldehyde inhibitors. The significant oxidoreductase genes responsible for each or multiple inhibitors biotransformation identified in this study will serve as a component of key gene device library for robust biorefinery fermentation strains development in the future biorefinery applications.

Metabolic engineering of Corynebacterium glutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis

Glucosamine (GlcN) and its acetylated derivative N-acetylglucosamine (GlcNAc) are widely used in the pharmaceutical industries. Here, we attempted to achieve efficient production of GlcNAc via genomic engineering of Corynebacterium glutamicum. Specifically, we ligated the GNA1 gene, which converts GlcN-6-phosphate to GlcNAc-6-phosphate by transferring the acetyl group in Acetyl-CoA to the amino group of GlcN-6-phosphate, into the plasmid pJYW4 and then transformed this recombinant vector into the C. glutamicum ATCC 13032, ATCC 13869, ATCC 14067, and S9114 strains, and we assessed the GlcNAc titers at 0.5 g/L, 1.2 g/L, 0.8 g/L, and 3.1 g/L from each strain, respectively. This suggested that there were likely to be significant differences among the key genes in the glutamate and GlcNAc synthesis pathways of these C. glutamicum strains. Therefore, we performed whole genome sequencing of the S9114 strain, which has not been previously published, and found that there are many differences among the genes in the glutamate and GlcNAc synthesis pathways among the four strains tested. Next, nagA (encoding GlcNAc-6-phosphate deacetylase) and gamA (encoding GlcN-6-phosphate deaminase) were deleted in C. glutamicum S9114 to block the catabolism of intracellular GlcNAc, leading to a 54.8% increase in GlcNAc production (from 3.1 to 4.8 g/L) when grown in a shaker flask. In addition, lactate synthesis was blocked by knockout of ldh (encoding lactate dehydrogenase); thus, further increasing the GlcNAc titer to 5.4 g/L. Finally, we added a key gene of the GlcN synthetic pathway, glmS, from different sources into the expression vector pJYW-4-ceN, and the resulting recombinant strain CGGN2-GNA1-CgglmS produced the GlcNAc titer of 6.9 g/L. This is the first report concerning the metabolic engineering of C. glutamicum, and the results of this study provide a good starting point for further metabolic engineering to achieve industrial-scale production of GlcNAc.

Engineering Corynebacterium glutamicum triggers glutamic acid accumulation in biotin-rich corn stover hydrolysate

BackgroundLignocellulose biomass contains high amount of biotin and resulted in an excessive biotin condition for cellulosic glutamic acid accumulation by Corynebacterium glutamicum. Penicillin or ethambutol triggers cellulosic glutamic acid accumulation, but they are not suitable for practical use due to the fermentation instability and environmental concerns. Efficient glutamic acid production from lignocellulose feedstocks should be achieved without any chemical inductions.ResultsAn industrial strain C. glutamicum S9114 was metabolically engineered to achieve efficient glutamic acid accumulation in biotin-excessive corn stover hydrolysate. Among the multiple metabolic engineering efforts, two pathway regulations effectively triggered the glutamic acid accumulation in lignocellulose hydrolysate. The C-terminal truncation of glutamate secretion channel MscCG (ΔC110) led to the successful glutamic acid secretion in corn stover hydrolysate without inductions. Then the α-oxoglutarate dehydrogenase complex (ODHC) activity was attenuated by regulating odhA RBS sequence, and glutamic acid accumulation was further elevated for more than fivefolds. The obtained C. glutamicum XW6 strain reached a record-high titer of 65.2 g/L with the overall yield of 0.63 g/g glucose using corn stover as the starting feedstock without any chemical induction.ConclusionsMetabolic engineering method was successfully applied to achieve efficient glutamic acid in biotin-rich lignocellulose hydrolysate for the first time. This study demonstrated the high potential of glutamic acid production from lignocellulose feedstock.

Metabolic engineering of Corynebacterium glutamicum S9114 to enhance the production of l-ornithine driven by glucose and xylose

l-ornithine, an important amino acid, is widely used in food and medicine industries. l-ornithine production mainly relies on microbial fermentation, which may not meet the industrial requirement owing to the poor fermentation ability of available strains. Herein, mscCG2 deletion, CgS9114_12202 (gdh2) overexpression and rational modulation in tricarboxylic acid cycle was firstly demonstrated to increase l-ornithine production in engineered Corynebacterium glutamicum S9114. By further modulate glucose utility result in strain SO26 that produced 38.5 g/L or 43.6 g/L of l-ornithine in shake flask and fed-batch fermentation, respectively. This was 25% higher than that of the original strain (30.8 g/L) and exhibits highest titer reported in shake-flask. Moreover, the incorporation of xylose pathway in the engineered strain resulted in the highest l-ornithine production titer (18.9 g/L) and yield (0.40 g/g xylose) with xylose substrate. These results illustrate the tremendous potential of the engineered strain C. glutamicum S9114 in l-ornithine production.

Optimization of \u029f-ornithine production in recombinant Corynebacterium glutamicum S9114 by cg3035 overexpression and manipulating the central metabolic pathway

Backgroundʟ-Ornithine is an important amino acid with broad applications in pharmaceutical and food industries. Despite lagging ʟ-ornithine productivity and cost reduction, microbial fermentation is a promising route for sustainable ʟ-ornithine production and thus development of robust microbial strains with high stability and productivity is essential.ResultsPreviously, we systematically developed a new strain, SO1 originate from Corynebacterium glutamicum S9114, for ʟ-ornithine production. In this work, overexpression of cg3035 encoding N-acetylglutamate synthase (NAGS) using a plasmid or by inserting a strong Ptac promoter into the chromosome was found to increase ʟ-ornithine production in the engineered C. glutamicum SO1. The genome-based cg3035 modulated strain was further engineered by attenuating the expression of pta and cat, inserting a strong Peftu promoter in the upstream region of glycolytic enzymes such as pfkA, gap, and pyk, and redirecting carbon flux to the pentose phosphate pathway. The final strain with all the exploratory metabolic engineering manipulations produced 32.3 g/L of ʟ-ornithine, a yield of 0.395 g ornithine per g glucose, which was 35.7% higher than that produced by the original strain (23.8 g/L).ConclusionThese results clearly demonstrated that enhancing the expression of NAGS promoted ʟ-ornithine production and provide a promising alternative systematic blueprint for developing ʟ-ornithine-producing C. glutamicum strains.

Pathway engineering in Corynebacterium glutamicum S9114 for 5-aminolevulinic acid production.

5-Aminolevulinic acid (ALA) is a non-protein amino acid with a significant potential for cancer treatment and plant stress resistance. Microbial fermentation has gradually replaced the traditional chemical-based method for ALA production, thus increasing the need for high-ALA-producing strains. In this study, we engineered the glutamate producing strain, Corynebacterium glutamicum S9114, for ALA production. To efficiently convert l-glutamate to ALA, hemA and hemL from Salmonella typhimurium and Escherichia coli were tandemly overexpressed. In addition, ncgl1221 encoding a glutamate transporter was deleted to block glutamate secretion and thus improve ALA production. Furthermore, the intrinsic ribosome-binding site (RBS) of hemB was replaced by a relatively weak RBS to reduce the conversion of ALA to porphyrin. Transcriptional and fermentation data confirmed that inactivation of lysE and putP reduced the conversion of glutamate to arginine and proline, which also contribute to ALA production. The final SA14 strain produced 895mg/L concentration of ALA after 72h incubation in a shake flask. This amount was 58-fold higher than that obtained by the parent strain C. glutamicum S9114. The results demonstrate the potential of C. glutamicum S9114 for efficient ALA production and provide new targets for the development of ALA-producing strains.

Improvement of l-ornithine production by attenuation of argF in engineered Corynebacterium glutamicum S9114

l-Ornithine, a non-essential amino acid, has enormous industrial applications in food, pharmaceutical, and chemical industries. Currently, l-ornithine production is focused on microorganism fermentation using Escherichia coli or Corynebacterium glutamicum. In C. glutamicum, development of high l-ornithine producing C. glutamicum was achieved by deletion of argF, but was accompanied by growth deficiency and arginine auxotrophy. l-Arginine has been routinely added to solve this problem; however, this increases production cost and causes feedback inhibition of N-acetyl-l-glutamate kinase activity. To avoid the drawbacks of growth disturbance due to disruption of ArgF, strategies were adopted to attenuate its expression. Firstly, ribosome binding site substitution and start codon replacement were introduced to construct recombinant C. glutamiucm strains, which resulted in an undesirable l-ornithine production titer. Then, we inserted a terminator (rrnB) between argD and argF, which significantly improved l-ornithine production and relieved growth disturbance. Transcription analysis confirmed that a terminator can be used to downregulate expression of argF and simultaneously improve the transcriptional level of genes in front of argF. Using disparate terminators to attenuate expression of argF, an optimal strain (CO-9) with a T4 terminator produced 6.1 g/L of l-ornithine, which is 42.8% higher than that produced by strain CO-1, and is 11.2-fold higher than that of the parent CO strain. Insertion of terminators with gradient termination intensity can be a stable and powerful method to exert precise control of the expression level of argF in the development of l-ornithine producing strains, with potential applications in metabolic engineering and synthetic biology.

Tolerance improvement of Corynebacterium glutamicum on lignocellulose derived inhibitors by adaptive evolution.

Robustness of fermenting strains to lignocellulose derived inhibitors is critical for efficient biofuel and biochemical productions. In this study, the industrial fermenting strain Corynebacterium glutamicum S9114 was evolved for improved inhibitor tolerance using long-term adaptive evolution by continuously transferring into the inhibitors containing corn stover hydrolysate every 24h, and finally a stably evolved C. glutamicum was obtained after 128days of serial transfers. The evolved strain exhibited the highly increased conversion rate to the typical lignocellulose derived inhibitors including furfural, 5-hydroxymethylfurfural, vanillin, syringaldehyde, 4-hydroxybenzaldehyde, and acetic acid. Glucose consumption was obviously accelerated, and 22.4g/L of glutamic acid was achieved in the corn stover hydrolysate, approximately 68.4% greater than that by the original strain. Whole genome re-sequencing revealed various mutations with the potential connection to the improved performance of the evolved strain. Transcriptional analysis further demonstrated that the glucose-PTS transport and the pentose phosphate pathway were significantly upregulated in the evolved strain, which very likely contributed to the accelerated glucose consumption, as well as sufficient NAD(P)H supply for aldehyde inhibitors reduction conversion and thus enhanced the inhibitor tolerance. This study provided important experimental evidences and valuable genetic information for robust strain construction and modification in lignocellulose biorefining processes.

Enhanced l-ornithine production by systematic manipulation of l-ornithine metabolism in engineered Corynebacterium glutamicum S9114

l-Ornithine is a non-protein amino acid with extensive applications in medicine and the food industry. Currently, l-ornithine is produced by microbial fermentation; however, this process needs to be further improved in terms of l-ornithine productivity and cost reduction. In this study, overexpression of LysE was observed to increase l-ornithine production in engineered Corynebacterium glutamicum S9114. To overcome the drawbacks of using a plasmid to express LysE, Ptac, a strong promoter, was inserted in the upstream region of lysE on the chromosome. This strain was further engineered by attenuating the expression of ncgl2228 and proB, and enhancing the expression of gdh and argCJBD. Combination of those targets resulted in l-ornithine production at a titer of 25 g/L, which was 63.4% higher than that produced by the original strain (15.3 g/L). These results demonstrated the positive effects of overexpressing LysE on l-ornithine production and provided novel targets for developing l-ornithine-producing C. glutamicum strains.

Systematic pathway engineering of Corynebacterium glutamicum S9114 for l-ornithine production

Backgroundl-Ornithine is a non-protein amino acid with extensive applications in medicine and the food industry. Currently, l-ornithine production is based on microbial fermentation, and few microbes are used for producing l-ornithine owing to unsatisfactory production titer.ResultsIn this study, Corynebacterium glutamicum S9114, a high glutamate-producing strain, was developed for l-ornithine production by pathway engineering. First, argF was deleted to block l-ornithine to citrulline conversion. To improve l-ornithine production, ncgl1221 encoding glutamate transporter, argR encoding arginine repressor, and putP encoding proline transporter were disrupted. This base strain was further engineered by attenuating oxoglutarate dehydrogenase to increase l-ornithine production. Plasmid-based overexpression of argCJBD operon and lysine/arginine transport protein LysE was tested to strengthen l-ornithine synthesis and transportation. This resulted in efficient l-ornithine production at a titer of 18.4 g/L.ConclusionThese results demonstrate the potential of Corynebacterium glutamicum S9114 for efficient l-ornithine production and provide new targets for strain development.

Reconstruction and analysis of a genome-scale metabolic network of Corynebacterium glutamicum S9114

Corynebacterium glutamicum S9114 is commonly used for industrial glutamate production. Therefore, a comprehensive understanding of the physiological and metabolic characteristics of C. glutamicum is important for developing its potential for industrial production. A genome-scale metabolic model, iJM658, was reconstructed based on genome annotation and literature mining. The model consists of 658 genes, 984 metabolites and 1065 reactions. The model quantitatively predicted C. glutamicum growth on different carbon and nitrogen sources and determined 129 genes to be essential for cell growth. The iJM658 model predicted that C. glutamicum had two glutamate biosynthesis pathways and lacked eight key genes in biotin synthesis. Robustness analysis indicated a relative low oxygen level (1.21mmol/gDW/h) would improve glutamate production rate. Potential metabolic engineering targets for improving γ-aminobutyrate and isoleucine production rate were predicted by in silico deletion or overexpression of some genes. The iJM658 model is a useful tool for understanding and optimizing the metabolism of C. glutamicum and a valuable resource for future metabolic and physiological research.

Genome Sequence of Corynebacterium glutamicum S9114, a Strain for Industrial Production of Glutamate

Here we report the genome sequence of Corynebacterium glutamicum S9114, an industrial producer widely used in production of glutamate in China. Preliminary comparison with the sequences of the Corynebacterium glutamicum strains ATCC 13032 and R revealed some notable mutagenesis that might be related to the high yield of glutamate.

On-line prediction of products concentrations in glutamate fermentation using metabolic network model and linear programming

The experimental data showed that, in glutamate fermentation with strain Corynebacterium glutamicum S9114, glutamate produced in different ways with different patterns of OUR, CER and lactate accumulation at different DO levels. A metabolic network model combined with the linear programming optimization was thus developed to on-line predict glutamate production under different DO levels, with only OUR and CER be on-line measured. The results indicated the advantages of the metabolic network model over the traditional unstructured models, in terms of easy parameter identification, prediction accuracy, and the universal abilities. The proposed model potentially supplies an alternative way for on-line control and optimization of fermentation processes.