High-titer production of aromatic amines in metabolically engineered Escherichia coli

Aromatic amines are widely used in the pharmaceutical industry. Here, we reported the establishment of a bacterial platform for synthesizing three types of aromatic amines, namely, tyramine, dopamine, and phenylethylamine. Firstly, we expressed aromatic amino acid decarboxylase from Enterococcus faecium (pheDC) in an Escherichia coli strain with an increased shikimate (SHK) pathway flux toward L-tyrosine or L-phenylalanine synthesis. We found that glycerol served as a better carbon source than glucose, resulting in 940 mg/L tyramine from 4% glycerol. Next, the genes of lactate dehydrogenase (ldhA), formate acetyltransferase (pflB), phosphate acetyltransferase (pta), and alcohol dehydrogenase (adhE) were deleted to mitigate the fermentation byproduct formation. The tyramine level was further increased to 1.965 g/L in shake flasks, corresponding to 2.1 times improvement compared with that of the parental strain. By using a similar strategy, we also managed to produce 703 mg/L dopamine and 555 mg/L phenethylamine. In summary, we have demonstrated that the knockout of ldhA-pflB-pta-adhE is an effective strategy in improving aromatic amine productions, and achieved the highest aromatic amine titers in E. coli under shake flasks reported to date.

Is sleep bruxism related to the levels of enzymes involved in the serotonin synthesis pathway?

Objectives This exploratory research aimed to evaluate the levels of tryptophan hydroxylase 1 (TPH1) and aromatic l-amino acid decarboxylase (DDC), which play an important role in the serotonin synthesis pathway, in individuals with sleep bruxism (SB) diagnosed using audio–video polysomnography (vPSG) and compare them with that of individuals not presenting with SB. Materials and methods The study included adult patients hospitalized in the Department and Clinic of Internal Medicine, Occupational Diseases, Hypertension and Clinical Oncology at the Wroclaw Medical University. The participants underwent a single-night vPSG for the evaluation of the SB parameters. Peripheral blood samples were also collected from them for estimating the serum levels of TPH1 and DDC. Results A total of 105 patients (80 women and 25 men) were included in the study. All the patients were Caucasians and aged 18–63 years (mean age: 33.43 ± 10.8 years). Seventy-five patients (71.43%) presented with SB, of which 50 (47.62%) had severe SB, while the remaining 30 patients (28.57%) did not. No statistically significant differences in TPH1 and DDC levels were observed between the individuals with SB and without SB. A significant negative correlation was found between tonic SB episodes and DDC levels (p = 0.0012). Other correlations between the SB parameters and the levels of the studied enzymes were statistically insignificant (p > 0.05 for all comparisons). Conclusions The levels of the enzymes that are crucial for serotonin synthesis (TPH1 and DDC) did not seem to influence SB. Clinical relevance This study provides important insights for further research on the relationship between the serotonin pathway and SB, which should take into account not only the process of serotonin synthesis but also the effect of serotonin-dependent neurotransmission on SB.

Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS−1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS−1 (PPIase KS−1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS−1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS−1. The recombinant E. coli cells expressing the PPIase KS−1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS−1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS−1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.

Eliciting health state utilities for Aromatic L-amino Acid Decarboxylase (AADC) deficiency: a UK vignette study

Purpose The aim of this study was to generate health state utilities for aromatic L-amino acid decarboxylase (AADC) deficiency, a rare genetic, lifelong neurogenerative condition predominantly manifesting in young infants. Methods Participants were presented with health state vignettes. These had been previously developed based on published literature, clinician input, interviews with parents of AADC deficiency patients and expert opinion. A total of 5 health state vignettes were presented: bedridden, head control, sitting unsupported, standing with assistance and walking with assistance. Health state utilities (HSU) were elicited using time-trade off (TTO; 10-year time horizon) and the standard gamble (SG). The vignettes were completed online by panel participants drawn from a representative sample of the United Kingdom residential population. Results A total of 1598 participants completed the vignettes. Around 21% had incongruent responses (higher utilities for the bedridden compared to walking health states). Incongruent responses were associated with shorter task completion times, gender and parental status. These responses were removed from the analysis. Health state utilities (HSU) increased correspondingly as health states improved for both the TTO and SG. The mean HSU (standard deviation) for the TTO task were: bedridden state 0.49 (0.34); head control 0.54 (0.33), sitting unsupported 0.63 (0.31); standing with assistance 0.68 (0.31); and walking with assistance 0.73 (0.31). For the SG, mean health state utilities were: 0.56 (0.28), 0.57 (0.27), 0.67 (0.24), 0.70 (0.24), and 0.75 (0.25), respectively. Conclusion Health state utilities were derived for AADC deficiency through a vignette study. These will be used for a cost-effectiveness model of an AADC deficiency treatment.

Technological and Safety Characterization of Kocuria rhizophila Isolates From Traditional Ethnic Dry-Cured Ham of Nuodeng, Southwest China

Nuodeng ham is known for its unique processing techniques and flavor. In the present study, proteolytic microorganisms from cured artisanal Nuodeng ham were investigated in order to identify and select potential starter cultures for its faster and safer fermentation. Eight isolates, accounting for 57% of proteolytic microorganisms, were found to be related to Kocuria rhizophila. Relevant properties of K. rhizophila as potential starter culture were evaluated in vitro for the first time. Intra-species diversities were found in phylogenetic and physiological properties of K. rhizophila isolates. Nevertheless, desirable attributes, such as halo-tolerance, nitrate reductase and protease activity, as well as the absence of antimicrobial resistance and amino acid decarboxylase activity, were observed in selected isolates. Moreover, genome analysis of isolates K24 and K45 confirmed their lack of typical genes for virulence, antimicrobial resistance and amino acid decarboxylase. K. rhizophila may thus represent a novel starter candidate of coagulase-negative cocci group and contribute to color and flavor development of fermented meats.

Safety of AADC Gene Therapy for Moderately Advanced Parkinson Disease: Three-Year Outcomes From the PD-1101 Trial

Objective:To report final, 36-month safety and clinical outcomes from the PD-1101 trial of NBIb-1817 (VY-AADC01) in participants with moderately advanced Parkinson’s disease (PD) and motor fluctuations.Methods:PD-1101 was a phase 1b, open-label, dose escalation trial of VY-AADC01, an experimental AAV2 gene therapy encoding the human aromatic L-amino acid decarboxylase (AADC) enzyme. VY-AADC01 was delivered via bilateral, intraoperative MRI-guided putaminal infusions to 3 cohorts (n = 5 participants per cohort): cohort 1, ≤7.5x1011 vector genomes (vg); cohort 2, ≤1.5x1012 vg; cohort 3, ≤4.7x1012 vg.Results:No serious adverse events (SAEs) attributed to VY-AADC01 were reported. All 4 non-vector–related SAEs (atrial fibrillation and pulmonary embolism in 1 participant and 2 events of small bowel obstruction in another participant) resolved. Requirements for PD medications were reduced by 21-30% in the 2 highest dose cohorts at 36 months. Standard measures of motor function (PD diary, UPDRS III off-medication and on-medication scores), global impressions of improvement (CGI-I, PGI-I), and quality of life (PDQ-39) were stable or improved compared with baseline at 12, 24, and 36 months following VY-AADC01 administration across cohorts.Conclusions:VY-AADC01 and the surgical administration procedure were well-tolerated and resulted in stable or improved motor function and quality of life across cohorts, as well as reduced PD medication requirements in cohorts 2 and 3 over 3 years.Clinicaltrials.gov identifier: NCT01973543Classification of evidence:This study provides Class IV evidence that, in patients with moderately advanced PD and motor fluctuations, putaminal infusion of VY-AADC01 is well tolerated and may improve motor function.

Biosynthetic Pathway and the Potential Role of Melatonin at Different Abiotic Stressors and Developmental Stages in Tolypocladium guangdongense

Melatonin, a bioactive compound and an important signaling molecule produced in plants and animals, is involved in many biological processes. However, its function and synthetic pathways in fungi are poorly understood. Here, the samples from Tolypocladium guangdongense, a highly valued edible fungus with functional food properties, were collected under different experimental conditions to quantify the levels of melatonin and its intermediates. The results showed that the intracellular melatonin content was markedly improved by Congo red (CR), cold, and heat stresses; the levels of intracellular melatonin and its intermediates increased at the primordial (P) and fruiting body (FB) stages. However, the levels of most intermediates exhibited a notable decrease under CR stress. Several genes related to melatonin synthesis, excluding AADC (aromatic-L-amino-acid decarboxylase), were markedly upregulated at an early stage of CR stress but downregulated later. Compared to the mycelial stage, those genes were significantly upregulated at the P and FB stages. Additionally, exogenous melatonin promoted resistance to several abiotic stressors and P formation in T. guangdongense. This study is the first to report melatonin biosynthesis pathway in macro-fungi. Our results should help in studying the diversity of melatonin function and melatonin-synthesis pathways and provide a new viewpoint for melatonin applications in the edible-medicinal fungus.

Are levodopa preparationas with benserazide and carbidopa clinically equivalent?

Parkinson’s disease is the only neurodegenerative disease that can be effectively treated symptomatically. Treatment of motor symptoms is based primarily on the use of drugs that increase the activity of the nigrostriatal dopaminergic system and compensate for dopamine deficiency. Levodopa remains the gold standard of dopaminergic therapy. It is the most effective and best tolerated anti-parkinsonian drug, it causes the fewest side effects, also in the elderly patients. Oral preparations of levodopa additionally contain one of the aromatic L-amino acid decarboxylase inhibitors: benserazide or carbidopa. Inhibitors have a beneficial effect on the bioavailability of levodopa in the central nervous system, its clinical efficacy and tolerability. In practice, according to common opinion, the preparations of levodopa with carbidopa and levodopa with benserazide are clinically equivalent and can be used interchangeably. The case of a 69-year-old patient treated for 6 years for Parkinson’s disease is presented. The patient presented motor symptoms of advanced Parkinson’s disease: wearing-off motor fluctuations and peak dose dyskinesia. The was treated with levodopa in a dose of 5 × 200 mg (preparation of levodopa with benserazide) as a monotherapy. Due to the worsening availability of the drug used so far in pharmacies, it was changed to a preparation containing levodopa and carbidopa, while maintaining the same dose of levodopa. During the next visit, the patient reported that the change of the formulation had a beneficial effect in the form of a slight but significant reduction in the incidence and severity of peak dose dyskinesia. Pharmacokinetic studies showed that the mean maximum concentration of levodopa after administration of levodopa + benserazide was significantly higher than after administration of levodopa + carbidopa. The preparation containing benserazide caused a rapid increase and then a rapid decrease of the lewodopa plasma concentration. When levodopa was combined with carbidopa, the concentration of levodopa increased and decreased slowly. The results of these pharmacokinetic studies may explain the patient’s observation of the amelioration of peak dose dyskinesia after switching from a levodopa + benserazide formulation to a levodopa + carbidopa combination.

Reduced Immunogenicity of Intraparenchymal Delivery of Adeno-Associated Virus Serotype 2 Vectors: Brief Overview

: Preexisting immunity to adeno-associated virus (AAV) poses a concern in AAV vector–mediated gene therapy. Localized administration of low doses of carefully chosen AAV serotypes can mitigate the risk of an immune response. This article will illustrate the low risk of immune response to AAV serotype 2 vector–mediated gene therapy to the brain with support from clinical trial data in aromatic L-amino acid decarboxylase deficiency and Parkinson disease.