Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

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

BACKGROUND: L-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. RESULTS: This 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). CONCLUSION: These 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.

Thermostable arginase from Sulfobacillus acidophilus with neutral pH optimum applied for high-efficiency L-ornithine production.

This study aims to use neutral pH optimum arginase as the catalyst for high-efficiency L-ornithine production. Sulfobacillus acidophilus arginase was firstly cloned and overexpressed in Escherichia coli. The purified enzyme was obtained, and the molecular mass determination showed that this arginase was a hexamer. S. acidophilus arginase possessed similarities with the other arginases such as the conserved sequences, purification behavior, and the necessity for Mn2+ as a cofactor. The maximum enzyme activity was obtained at pH 7.5 and 70 °C. Thermostability and pH stability analysis showed that the arginase was stable at 30-60 °C and pH 7.0-8.5, respectively. The kinetic parameters suggested that S. acidophilus arginase could efficiently hydrolyze L-arginine. Bioconversion with this neutral pH optimum arginase had the advantages of avoiding producing by-product, high molar yield, and high-level production of L-ornithine. When the bioconversion was performed with a fed-batch strategy and a coupled-enzyme system involving S. acidophilus arginase and Jack bean urease, the final production of 2.87 mol/L was obtained with only 1.72 mmol/L L-arginine residue, and the molar yield was 99.9%. The highest production record suggests that S. acidophilus arginase has a great prospect in industrial L-ornithine production.

Development of a System of High Ornithine and Citrulline Production by a Plant-Derived Lactic Acid Bacterium, Weissella confusa K-28.

As a bacterium used in industry for production of several amino acids, an endotoxin-free Corynebacterium (C.) glutamicum is well known. However, it is also true that the endotoxin-producing other Corynebacterium species is present. An aim of this study is to obtain a lactic acid bacterium (LAB) that produces ornithine and citrulline at high levels. We successfully isolated a strain, designated K-28, and identified it as Weissella (W.) confusa. The production of ornithine and citrulline by K-28 was 18 ± 1 and 10 ± 2 g/L, respectively, with a 100 ± 9% conversion rate when arginine was continuously fed into a jar fermenter. Although the ornithine high production using C. glutamicum is industrially present, the strains have been genetically modified. In that connection, the wild-type of C. glutamicum produces only 0.5 g/L ornithine, indicating that W. confusa K-28 is superior to C. glutamicum to use a probiotic microorganism. We confirmed that W. confusa K-28 harbors an arginine deiminase (ADI) gene cluster, wkaABDCR. The production of ornithine and the expression of these genes significantly decreased under the aerobic condition rather than anaerobic one. The expression level of the five genes did not differ with or without arginine, suggesting that the production of amino acids in the K-28 strain was not induced by exogenous arginine.

Bacterial ornithine lipid, a surrogate membrane lipid under phosphate-limiting conditions, plays important roles in bacterial persistence and interaction with host.

Ornithine lipids (OLs) are bacteria-specific lipids that are found in the outer membrane of Gram (-) bacteria and increase as surrogates of phospholipids under phosphate-limited environmental conditions. We investigated the effects of OL increase in bacterial membranes on pathogen virulence and the host immune response. In Pseudomonas aeruginosa, we increased OL levels in membranes by overexpressing the OL-synthesizing operon (olsBA). These increases changed the bacterial surface charge and hydrophobicity, which reduced bacterial susceptibility to antibiotics and antimicrobial peptides (AMPs), interfered with the binding of macrophages to bacterial cells and enhanced bacterial biofilm formation. When grown under low phosphate conditions, P. aeruginosa became more persistent in the treatment of antibiotics and AMPs in an olsBA-dependent manner. While OLs increased persistence, they attenuated P. aeruginosa virulence; in host cells, they reduced the production of inflammatory factors (iNOS, COX-2, PGE2 and nitric oxide) and increased intracellular Ca2+ release. Exogenously added OL had similar effects on P. aeruginosa and host cells. Our results suggest that bacterial OL plays important roles in bacteria-host interaction in a way that enhances bacterial persistence and develops chronic adaptation to infection.

Metabolic evolution and a comparative omics analysis of Corynebacterium glutamicum for putrescine production.

Putrescine is widely used in the industrial production of bioplastics, pharmaceuticals, agrochemicals, and surfactants. Because the highest titer of putrescine is much lower than that of its precursor L-ornithine reported in microorganisms to date, further work is needed to increase putrescine production in Corynebacterium glutamicum. We first compared 7 ornithine decarboxylase genes and found that the Enterobacter cloacae ornithine decarboxylase gene speC1 was most suitable for putrescine production in C. glutamicum. Increasing NADPH availability and blocking putrescine oxidation and acetylation were chosen as targets for metabolic engineering. The putrescine producer C. glutamicum PUT4 was first constructed by deleting puo, butA and snaA genes, and replacing the fabG gene with E. cloacae speC1. After adaptive evolution with C. glutamicum PUT4, the evolved strain C. glutamicum PUT-ALE, which produced an 96% higher amount of putrescine compared to the parent strain, was obtained. The whole genome resequencing indicates that the SNPs located in the odhA coding region may be associated with putrescine production. The comparative proteomic analysis reveals that the pentose phosphate and anaplerotic pathway, the glyoxylate cycle, and the ornithine biosynthetic pathway were upregulated in the evolved strain C. glutamicum PUT-ALE. The aspartate family, aromatic, and branched chain amino acid and fatty acid biosynthetic pathways were also observed to be downregulated in C. glutamicum PUT-ALE. Reducing OdhA activity by replacing the odhA native start codon GTG with TTG and overexpression of cgmA or pyc458 further improved putrescine production. Repressing the carB, ilvH, ilvB and aroE expression via CRISPRi also increased putrescine production by 5, 9, 16 and 19%, respectively.

Improved L-ornithine production in Corynebacterium crenatum by introducing an artificial linear transacetylation pathway.

L-Ornithine is a non-protein amino acid with extensive applications in the food and pharmaceutical industries. In this study, we performed metabolic pathway engineering of an L-arginine hyper-producing strain of Corynebacterium crenatum for L-ornithine production. First, we amplified the L-ornithine biosynthetic pathway flux by blocking the competing branch of the pathway. To enhance L-ornithine synthesis, we performed site-directed mutagenesis of the ornithine-binding sites to solve the problem of L-ornithine feedback inhibition for ornithine acetyltransferase. Alternatively, the genes argA from Escherichia coli and argE from Serratia marcescens, encoding the enzymes N-acetyl glutamate synthase and N-acetyl-L-ornithine deacetylase, respectively, were introduced into Corynebacterium crenatum to mimic the linear pathway of L-ornithine biosynthesis. Fermentation of the resulting strain in a 5-L bioreactor allowed a dramatically increased production of L-ornithine, 40.4 g/L, with an overall productivity of 0.673 g/L/h over 60 h. This demonstrates that an increased level of transacetylation is beneficial for L-ornithine biosynthesis.

The Arabidopsis N-Acetylornithine Deacetylase Controls Ornithine Biosynthesis via a Linear Pathway with Downstream Effects on Polyamine Levels.

Arabidopsis thaliana At4g17830 codes for a protein showing sequence similarity with the Escherichia coli N-acetylornithine deacetylase (EcArgE), an enzyme implicated in the linear ornithine (Orn) biosynthetic pathway. In plants, N-acetylornithine deacetylase (NAOD) activity has yet to be demonstrated; however, At4g17830-silenced and mutant (atnaod) plants display an impaired reproductive phenotype and altered foliar levels of Orn and polyamines (PAs). Here, we showed the direct connection between At4g17830 function and Orn biosynthesis, demonstrating biochemically that At4g17830 codes for a NAOD. These results are the first experimental proof that Orn can be produced in Arabidopsis via a linear pathway. In this study, to identify the role of AtNAOD in reproductive organs, we carried out a transcriptomic analysis on atnaod mutant and wild-type flowers. In the atnaod mutant, the most relevant effects were the reduced expression of cysteine-rich peptide-coding genes, known to regulate male-female cross-talk during reproduction, and variation in the expression of genes involved in nitrogen:carbon (N:C) status. The atnaod mutant also exhibited increased levels of sucrose and altered sensitivity to glucose. We hypothesize that AtNAOD participates in Orn and PA homeostasis, contributing to maintain an optimal N:C balance during reproductive development.

Systematic pathway engineering of Corynebacterium glutamicum S9114 for L-ornithine production.

BACKGROUND: L-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. RESULTS: In 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. CONCLUSION: These results demonstrate the potential of Corynebacterium glutamicum S9114 for efficient L-ornithine production and provide new targets for strain development.

Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis.

Staphylococcus aureus assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5'-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress.

Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli.

Mycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacterium Cylindrospermum stagnale PCC 7417 revealed a new gene cluster with homology to MAA synthase from Nostoc punctiforme This newly identified gene cluster is unusual because it has five biosynthesis genes (mylA to mylE), compared to the four found in other MAA gene clusters. Heterologous expression of mylA to mylE in Escherichia coli resulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced in E. coli and structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods. IMPORTANCE: Mycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs in E. coli is also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens.

Arginase 1 is a negative regulator of osteoclast differentiation.

Arginase 1 (Arg1) limits the availability of l-arginine for producing nitric oxide (NO) and ornithine, a substrate for polyamine synthesis. Anti-osteoclastogenic activities of NO and polyamines, and the involvement of Arg1 on the dendritic cell differentiation of dendritic cells have been reported, but the relevance of Arg1 to osteoclast differentiation has not been investigated. Here, we observed Arg1 down-regulation during the RANKL-induced differentiation of bone marrow-derived macrophages into osteoclasts. Arg1 overexpression significantly inhibited osteoclast differentiation with low NO production, while Arg1 knockdown enhanced osteoclast differentiation with high NO production. These results suggest that Arg1 and NO have reciprocal roles as negative and positive regulators, respectively, of osteoclast differentiation. We conclude that Arg1 is down-regulated during osteoclast differentiation and may negatively regulate osteoclast differentiation by regulating NO production.

Cloning, expression, and characterization of a thermostable l-arginase from Geobacillus thermodenitrificans NG80-2 for l-ornithine production.

Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) can efficiently catalyze conversion of arginine to ornithine. Therefore, this enzyme can be used to produce l-ornithine from l-arginine. In this article, the l-arginase gene encoding the Geobacillus thermodenitrificans NG80-2 was cloned and overexpressed in Escherichia coli. The specific activity of the purified enzyme was 138.3 U/mg. The molecular mass of the l-arginase was approximately 33.0 kDa as estimated by SDS-PAGE and 192.0 kDa as determined by gel-filtration chromatography. Manganese ions were the optimum metal cofactor for activity, whereas the enzyme was slightly inhibited by Mg(2+) , Cu(2+) , Ba(2+) , Ca(2+) , and Zn(2+) . Activity was optimal at pH 9.0 and 80 °C, and the protein was stable at 40 and 50 °C. The recombinant enzyme was a uricotelic arginase. Using arginine as the substrate, the Michaelis-Menten constant (Km ) and catalytic efficiency (kcat /Km ) were measured to be 171.9 mM and 3.8 mM(-1)  s(-1) , respectively. Trp and His residues were directly involved in the l-arginase activity evaluated by inactivation agents. The biosynthesis yield of l-ornithine by the purified enzyme was 36.9 g/L, and the molar yield was 97.2%.

Discovery of a bifunctional acyltransferase responsible for ornithine lipid synthesis in Serratia proteamaculans.

Ornithine lipids (OLs) are phosphorus-free membrane lipids that can be formed by many bacteria but that are absent from archaea and eukaryotes. A function for OLs in stress conditions and in host-bacteria interactions has been shown in some bacteria. Some bacterial species have been described that can form OLs, but lack the known genes (olsBA) involved in its biosynthesis, which implied the existence of a second pathway. Here we describe the bifunctional protein OlsF from Serratia proteamaculans involved in OL formation. Expression of OlsF and its homologue from Flavobacterium johnsoniae in Escherichia coli causes OL formation. Deletion of OlsF in S. proteamaculans caused the absence of OL formation. Homologues of OlsF are widely distributed among γ-, δ- and ε-Proteobacteria and in the Cytophaga-Flavobacterium-Bacteroidetes group of bacteria, including several well-studied pathogens for which the presence of OLs has not been suspected, such as for example Vibrio cholerae and Klebsiella pneumonia. Using genomic data, we predict that about 50% of bacterial species can form OLs.

High-level expression of human arginase I in Pichia pastoris and its immobilization on chitosan to produce L-ornithine.

BACKGROUND: L-ornithine (L-Orn), is an intermediate metabolite in the urea cycle that plays a significant role in humans. L-Orn can be obtained from the catalysis of L-arginine (L-Arg) by arginase. The Pichia pastoris expression system offers the possibility of generating a large amount of recombinant protein. The immobilized enzyme technology can overcome the difficulties in recovery, recycling and long-term stability that result from the use of free enzyme. METHODS: The recombinant human arginase I (ARG I) was obtained using an optimized method with the Pichia pastoris GS115 as the host strain. Chitosan paticles were cross-linked with glutaraldehyde and rinsed exhaustively. Then the expressed ARG I was immobilized on the crosslinked chitosan particles, and the enzymatic properties of both the free and immobilized enzymes were evaluated. At last, the immobilized ARG I was employed to catalyze L-Arg to L-Orn. RESULTS: The results indicated that these two states both exhibited optimal activity under the same condition of pH10 at 40 °C. However, the immobilized ARG I exhibited the remarkable thermal and long-term stability as well as broad adaptability to pH, suggesting its potential for wide application in future industry. After a careful analysis of its catalytic conditions, immobilized ARG I was employed to catalyze the conversion of L-Arg to L-Orn under optimal condition of 1 % glutaraldehyde, 1 mM Mn(2+), 40 °C, pH10 and an L-arginine (L-Arg) concentration of 200 g/L, achieving a highly converted content of 149.g/L L-Orn. CONCLUSIONS: In this work, ARG Ι was abundantly expressed, and an efficient, facile and repeatable method was developed to synthesize high-quality L-Orn. This method not only solved the problem of obtaining a large amount of arginase, but also provided a promising alternative for the future industrial production of L-Orn.

Construction of a highly efficient Bacillus subtilis 168 whole-cell biocatalyst and its application in the production of L-ornithine.

L-Ornithine, a non-protein amino acid, is usually extracted from hydrolyzed protein as well as produced by microbial fermentation. Here, we focus on a highly efficient whole-cell biocatalyst for the production of L-ornithine. The gene argI, encoding arginase, which catalyzes the hydrolysis of L-arginine to L-ornithine and urea, was cloned from Bacillus amyloliquefaciens B10-127 and expressed in GRAS strain Bacillus subtilis 168. The recombinant strain exhibited an arginase activity of 21.9 U/mg, which is 26.7 times that of wild B. subtilis 168. The optimal pH and temperature of the purified recombinant arginase were 10.0 and 40 °C, respectively. In addition, the recombinant arginase exhibited a strong Mn(2+) preference. When using whole-cell biocatalyst-based bioconversion, a hyper L-ornithine production of 356.9 g/L was achieved with a fed-batch strategy in a 5-L reactor within 12 h. This whole-cell bioconversion study demonstrates an environmentally friendly strategy for L-ornithine production in industry.

Biosynthesis and defensive function of Nδ-acetylornithine, a jasmonate-induced Arabidopsis metabolite.

Since research on plant interactions with herbivores and pathogens is often constrained by the analysis of already known compounds, there is a need to identify new defense-related plant metabolites. The uncommon nonprotein amino acid N(δ)-acetylornithine was discovered in a targeted search for Arabidopsis thaliana metabolites that are strongly induced by the phytohormone methyl jasmonate (MeJA). Stable isotope labeling experiments show that, after MeJA elicitation, Arg, Pro, and Glu are converted to Orn, which is acetylated by NATA1 to produce N(δ)-acetylornithine. MeJA-induced N(δ)-acetylornithine accumulation occurs in all tested Arabidopsis accessions, other Arabidopsis species, Capsella rubella, and Boechera stricta, but not in less closely related Brassicaceae. Both insect feeding and Pseudomonas syringae infection increase NATA1 expression and N(δ)-acetylornithine accumulation. NATA1 transient expression in Nicotiana tabacum and the addition of N(δ)-acetylornithine to an artificial diet both decrease Myzus persicae (green peach aphid) reproduction, suggesting a direct toxic or deterrent effect. However, since broad metabolic changes that are induced by MeJA in wild-type Arabidopsis are attenuated in a nata1 mutant strain, there may also be indirect effects on herbivores and pathogens. In the case of P. syringae, growth on a nata1 mutant is reduced compared with wild-type Arabidopsis, but growth in vitro is unaffected by N(δ)-acetylornithine addition.

Enhancement of L-ornithine production by disruption of three genes encoding putative oxidoreductases in Corynebacterium glutamicum.

Recently, Corynebacterium glutamicum has been shown to exhibit gluconate bypass activity, with two key enzymes, glucose dehydrogenase (GDH) and gluconate kinase, that provides an alternate route to 6-phosphogluconate formation. In this study, gene disruption analysis was used to examine possible metabolic functions of three proteins encoded by open reading frames having significant sequence similarity to GDH of Bacillus subtilis. Chromosomal in-frame deletion of three genes (NCgl0281, NCgl2582, and NCgl2053) encoding putative NADP⁺-dependent oxidoreductases led to the absence of GDH activity and correlated with increased specific glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. This finding suggested that enhanced carbon flux from glucose was directed toward the oxidative pentose phosphate (PP) pathway, when the mutant was cultivated with 6 % glucose. Consequently, the mutant showed 72.4 % increased intracellular NADPH and 66.3 % increased extracellular L-ornithine production. The enhanced activities of the oxidative PP pathway in the mutant explain both the increased intracellular NADPH and the high extracellular concentration of L-ornithine. Thus, the observed metabolic changes in this work corroborate the importance of NADPH in L-ornithine production from C. glutamicum.

[Efficient biosynthesis of guanidoacetic acid by a recombinant strain of Bacillus subtilis].

Guanidinoacetic acid, as an energetic substance, has a wide range of applications in the food, pharmaceutical, and feed industries. However, the biosynthesis of guanidinoacetic acid has not been applied in industrial production. In this study, we designed the synthetic route of guanidinoacetic acid in a food-grade strain of Bacillus subtilis. By regulating the expression of key enzymes, lifting feedback inhibition, and increasing membrane permeability, we achieved the efficient synthesis of guanidinoacetic acid by whole-cell catalysis. Firstly, the optimal L-arginine:glycine amidinotransferase was screened based on the phylogenetic tree, and the expression of the key enzyme was enhanced by a strategy combining strong promoter and genome integration. Secondly, the ornithine cycle for L-arginine synthesis in Corynebacterium glutamicum was introduced to alleviate the feedback inhibition of the enzyme by the byproduct L-ornithine, and the L-arginine degradation pathway was knocked down to enhance substrate regeneration. Thirdly, the expression of N-acetylmuramoyl-L-alanine amidase (LytC) was up-regulated to increase the cell membrane permeability. Finally, after optimization of whole-cell production conditions, strain Bs-13 achieved guanidinoacetic acid production at a titer of 13.1 g/L after 24 h, with a proudction rate of 0.54 g/(L·h) and a glycine conversion rate of 92.7%. The above strategy improved the production of guanidinoacetic acid and provided a reference for the biosynthesis of guanidinoacetic acid.

Metabolic evolution of Corynebacterium glutamicum for increased production of L-ornithine.

BACKGROUND: L-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. The commercial applications mean that efficient biotechnological production of L-ornithine has become increasingly necessary. Adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. The evolved strains can be further optimised by metabolic engineering. Thus, metabolic evolution strategy was used for engineering Corynebacterium glutamicum to enhance L-ornithine production. RESULTS: A C. glutamicum strain was engineered by using a combination of gene deletions and adaptive evolution with 70 passages of growth-based selection. The metabolically evolved C. glutamicum strain, named ΔAPE6937R42, produced 24.1 g/L of L-ornithine in a 5-L bioreactor. The mechanism used by C. glutamicum ΔAPE6937R42 to produce L-ornithine was investigated by analysing transcriptional levels of select genes and NADPH contents. The upregulation of the transcription levels of genes involved in the upstream pathway of glutamate biosynthesis and the elevated NADPH concentration caused by the upregulation of the transcriptional level of the ppnK gene promoted L-ornithine production in C. glutamicum ΔAPE6937R42. CONCLUSIONS: The availability of NADPH plays an important role in L-ornithine production in C. glutamicum. Our results demonstrated that the combination of growth-coupled evolution with analysis of transcript abundances provides a strategy to engineer microbial strains for improving production of target compounds.