Hereditary Congenital Methemoglobinemia Diagnosed at the Age of 79 Years: A Case Report.

Background: Cardiopulmonary disorders are the most common cause of central cyanosis, and methemoglobinemia is often overlooked in the differential diagnosis of patients with central cyanosis. In most cases, methemoglobinemia is acquired and hereditary congenital methemoglobinemia is rare. Only a few case reports of congenital methemoglobinemia can be found in PubMed. To date, only four cases of congenital methemoglobinemia diagnosed after the age of 50 years have been reported. Case Presentation: A 79-year-old Japanese woman presented at our hospital with the chief complaints of dyspnea and cyanosis. She exhibited cyanosis of the lips and extremities, and her SpO2 was 80%, with oxygen administration at 5 L/min. Blood gas analysis revealed a PaO2 of 325.4 mmHg and methemoglobin level of 36.9%. The SpO2 and PaO2 values were dissociated, and methemoglobin levels were markedly elevated. Genetic analysis revealed a nonsynonymous variant in the gene encoding nicotinamide adenine dinucleotide cytochrome (NADH) B5 reductase 3 (CYB5R3), and the patient was diagnosed with congenital methemoglobinemia. Conclusions: It is important to consider methemoglobinemia in the differential diagnosis of patients with central cyanosis. At 79 years of age, our patient represents the oldest patient with this diagnosis. This report indicates that it is crucial to consider the possibility of methemoglobinemia regardless of the patient's age.

Production of l-Theanine by Escherichia coli in the Absence of Supplemental Ethylamine.

l-Theanine is a nonproteinogenic amino acid present almost exclusively in tea plants and is beneficial for human health. For industrial production, l-theanine is enzymatically or chemically synthesized from glutamine/glutamate (or a glutamine/glutamate derivative) and ethylamine. Ethylamine is extremely flammable and toxic, which complicates and increases the cost of operational procedures. To solve these problems, we developed an artificial biosynthetic pathway to produce l-theanine in the absence of supplemental ethylamine. For this purpose, we identified and selected a novel transaminase (NCBI:protein accession number AAN70747) from Pseudomonas putida KT2440, which catalyzes the transamination of acetaldehyde to produce ethylamine, as well as γ-glutamylmethylamide synthetase (NCBI:protein accession number AAY37316) from Pseudomonas syringae pv. syringae B728a, which catalyzes the condensation of l-glutamate and ethylamine to produce l-theanine. Expressing these genes in Escherichia coli W3110S3GK and enhancing the production capacity of acetaldehyde and l-alanine achieved successful production of l-theanine without ethylamine supplementation. Furthermore, the deletion of ggt, which encodes γ-glutamyltranspeptidase (EC 2.3.2.2), achieved large-scale production of l-theanine by attenuating its decomposition. We show that an alanine decarboxylase-utilizing pathway represents a promising route for the fermentative production of l-theanine. Our study reports efficient methods to produce l-theanine in the absence of supplemental ethylamine.IMPORTANCE l-Theanine is widely used in food additives and dietary supplements. Industrial production of l-theanine uses the toxic and highly flammable precursor ethylamine, raising production costs. In this study, we used Escherichia coli to engineer two biosynthetic pathways that produce l-theanine from glucose and ammonia in the absence of supplemental ethylamine. This study establishes a foundation for safely and economically producing l-theanine.

Infective Endocarditis with No Underlying Disease for Which Bacterial Endophthalmitis Have Been the First Symptom.

Bacterial endophthalmitis is a rare complication of infective endocarditis (IE). We herein report a case of IE with no underlying disease for which endophthalmitis could have been the first symptom. A 58-year-old man was admitted to our hospital with a fever, vision disturbances, and pain in the left hand joint. His left eye was removed because fusion on the cornea progressed. Streptococcus agalactiae was detected in blood cultures, fluid cultures from his left hand joint, and the removed eye. Bacterial endophthalmitis may present as the first symptom of IE and develop without underlying disease due to S. agalactiae infection.

Lung histopathological pattern in a survivor with rapidly progressive interstitial lung disease and anti-melanoma differentiation-associated gene 5 antibody-positive clinically amyopathic dermatomyositis.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibodies are specific indicators of patients with dermatomyositis, particularly clinically amyopathic dermatomyositis (CADM). CADM is occasionally accompanied by fatal, treatment-resistant, rapidly-progressive interstitial lung disease (RP-ILD). All previous reports showed that histopathological findings in RP-ILD with anti-MDA5 antibody-positive CADM indicated diffuse alveolar damage (DAD). This is the first report describing a non-DAD pattern in RP-ILD with anti-MDA5 antibody-positive CADM, which was improved by immunosuppressive therapy. This case may be a milder clinical phenotype than a typical DAD pattern in RP-ILD with anti-MDA5 antibody-positive CADM.

Respiratory Collapse of the Inferior Vena Cava Reflects Volume Shift and Subsequent Fluid Refill in Acute Heart Failure Syndrome.

BACKGROUND: Fluid redistribution rather than fluid accumulation plays an important role in the development of acute heart failure (HF) syndrome. Patients with fluid redistribution develop acute HF without prominent volume overload. We investigated volume status by measuring the diameter of the inferior vena cava (IVC) and examining variations in hemoglobin and hematocrit. METHODS AND RESULTS: Seventy-four consecutive patients admitted for acute HF syndrome were analyzed. Blood tests and measurement of IVC diameter after stabilization of respiratory distress were performed on admission and were repeated after 24 h. IVC collapsibility index (IVC-CI) was calculated as (maximum IVC-minimum IVC)/maximum IVC. According to the initial IVC-CI, the patients were divided into the collapse group (IVC-CI ≥0.5: n=34) and the non-collapse group (IVC-CI <0.5: n=40). Initial blood pressure was higher in the collapse group (P<0.001). Although 24-h urine volume did not differ between the groups, hemoglobin (P<0.001) and hematocrit (P<0.001) decreased significantly in the collapse group but not in the non-collapse group after 24 h. Furthermore, IVC-CI significantly decreased in the collapse group after 24 h (P=0.003). CONCLUSIONS: In acute HF syndrome, IVC-CI ≥0.5 on admission suggests a volume shift from the central vein into the pulmonary vasculature. Fluid refill occurs within 24 h after admission. This observation could be helpful in selecting strategies for diuretic use. (Circ J 2016; 80: 1171-1177).

Metabolic engineering for L-glutamine overproduction by using DNA gyrase mutations in Escherichia coli.

An L-glutamine-overproducing mutant of an Escherichia coli K-12-derived strain was selected from randomly mutagenized cells in the course of L-alanyl-L-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism for L-glutamine overproduction in this mutant. Three mutations were identified that are related to the L-glutamine overproduction phenotype, namely, an intergenic mutation in the 5'-flanking region of yeiG and two nonsynonymous mutations in gyrA (Gly821Ser and Asp830Asn). Expression of yeiG, which encodes a putative esterase, was enhanced by the intergenic mutation. The nonsynonymous mutations in gyrA, a gene that encodes the DNA gyrase α subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (topA) and topoisomerase IV (parC and parE), an increase in L-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase in L-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed an L-glutamine-hyperproducing strain by attenuating cellular L-glutamine degradation activity. Although the reconstituted mutant (with yeiG together with gyrA) produced 200 mM L-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mM L-glutamine.

Identification of homophenylalanine biosynthetic genes from the cyanobacterium Nostoc punctiforme PCC73102 and application to its microbial production by Escherichia coli.

L-Homophenylalanine (L-Hph) is a useful chiral building block for synthesis of several drugs, including angiotensin-converting enzyme inhibitors and the novel proteasome inhibitor carfilzomib. While the chemoenzymatic route of synthesis is fully developed, we investigated microbial production of L-Hph to explore the possibility of a more efficient and sustainable approach to L-Hph production. We hypothesized that L-Hph is synthesized from L-Phe via a mechanism homologous to 3-methyl-2-oxobutanoic acid conversion to 4-methyl-2-oxopentanoic acid during leucine biosynthesis. Based on bioinformatics analysis, we found three putative homophenylalanine biosynthesis genes, hphA (Npun_F2464), hphB (Npun_F2457), and hphCD (Npun_F2458), in the cyanobacterium Nostoc punctiforme PCC73102, located around the gene cluster responsible for anabaenopeptin biosynthesis. We constructed Escherichia coli strains harboring hphABCD-expressing plasmids and achieved the fermentative production of L-Hph from L-Phe. To our knowledge, this is the first identification of the genes responsible for homophenylalanine synthesis in any organism. Furthermore, to improve the low conversion efficiency of the initial strain, we optimized the expression of hphA, hphB, and hphCD, which increased the yield to ∼630 mg/liter. The L-Hph biosynthesis and L-Leu biosynthesis genes from E. coli were also compared. This analysis revealed that HphB has comparatively relaxed substrate specificity and can perform the function of LeuB, but HphA and HphCD show tight substrate specificity and cannot complement the LeuA and LeuC/LeuD functions, and vice versa. Finally, the range of substrate tolerance of the L-Hph-producing strain was examined, which showed that m-fluorophenylalanine, o-fluorophenylalanine, and L-tyrosine were accepted as substrates and that the corresponding homoamino acids were generated.

Identification of novel L-amino acid alpha-ligases through Hidden Markov Model-based profile analysis.

L-Amino acid alpha-ligase (Lal), catalyzing the formation of alpha-dipeptides from unprotected L-amino acids in an ATP-dependent manner, is used in cost-effective fermentative production of dipeptides. We searched for novel Lals by in silico screening using Hidden Markov Model-based profile analysis, and identified five novel Lals that showed low similarity and different substrate specificity from known Lals.

Effect of multidrug-efflux transporter genes on dipeptide resistance and overproduction in Escherichia coli.

L-Alanyl-L-glutamine (Ala-Gln) is a clinically and nutritionally important dipeptide. We have already shown a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing L-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids. In the course of Ala-Gln-producing strain development, it was revealed that Lal expression caused growth inhibition. We also found that the addition of some dipeptides, including Ala-Gln, inhibited the growth of a multiple peptidase-deficient strain. To further increase the productivity by overcoming the inhibitory effect of dipeptides, we focused on dipeptide transport systems. The four genes (bcr, norE, ydeE and yeeO) were selected from 34 genes encoding a multidrug-efflux transporter of E. coli as those conferring resistance to growth inhibitory dipeptides. Intracellular concentration of Ala-Gln was reduced by overexpressing these genes in a multiple peptidase-deficient strain. Furthermore, overexpression of each gene in the dipeptide-producing strains resulted in the increase of Ala-Gln and L-alanyl-L-branched chain amino acids titers. These results indicate that some multidrug-efflux transporters of E. coli can transport dipeptides and that enhancement of their activities is effective for fermentative production of dipeptides.

Fermentative production of L-alanyl-L-glutamine by a metabolically engineered Escherichia coli strain expressing L-amino acid alpha-ligase.

In spite of its clinical and nutritional importance, l-alanyl-l-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing l-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of the dpp and pep genes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologous l-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lal and ald expressed under the control of the uspA promoter) or pPE177 (lal and ald expressed under the control of the rpoH promoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containing d-amino acids were formed.

ywfE in Bacillus subtilis codes for a novel enzyme, L-amino acid ligase.

The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as d-alanyl-d-alanine, polyglutamate, and gamma-peptide, but, curiously, no enzyme synthesizing alpha-dipeptides of l-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming l-alanyl-l-glutamine from l-alanine and l-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of l-amino acids. Among 231 combinations of l-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of alpha-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the d-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, l-amino acid ligase.

Production of mevalonate by a metabolically-engineered Escherichia coli.

Mevalonate is biosynthesized from acetyl-CoA and metabolized to isoprenoid compounds in a wide variety of organisms although certain types of prokaryotes employ another route for isoprenoid biosynthesis (the non-mevalonate pathway). To establish a fermentative process for mevalonate production, enzymes for mevalonate synthesis from Enterococcus faecalis were expressed in Escherichia coli, a non-mevalonate pathway bacterium. Mevalonate was accumulated, indicating a redirection of acetate metabolism by the expressed enzyme. The recombinant E. coli produced 47 g mevalonate l(-1) in 50 h of fed-batch cultivation in a 2 l jar fermenter; this is the highest titer ever reported demonstrating the superiority of E. coli in its ability of acetyl-CoA supply and its inability is degrade mevalonate.