Biosensor for branched-chain amino acid metabolism in yeast and applications in isobutanol and isopentanol production

Branched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.

Easily Misdiagnosed cblC Deficiency in Adolescents: the Clinical and Metabolic Studies

Purpose: Adolescents are easily attacked by potential inherited metabolic disorders. cblC deficiency is the most common type of methylmalonic aciduria in China. The late-onset patients present with varied non-specific symptoms and usually being misdiagnosed. The purpose of this study is to investigate the clinical features of patients with adolescence-onset cblC deficiency and explore the prevention and control strategies. Methods: Fifty-seven patients (34 males and 23 females) with adolescence-onset cblC deficiency were admitted in our clinic from 2002 to September 2021. The diagnosis was confirmed by metabolic and genetic tests. The clinical and biochemical features, disease triggers, outcome and genotypes-phenotypes correlation were examined.Results: The onset ages ranged from 10 to 25 years old (median age was 12 years). 16 cases (28.0%) presented with symptoms after infection or sports training. 46 patients (80.7%) had neuropsychiatric diseases. 14 patients (24.6%) displayed cardiovascular diseases. Five cases (8.9%) showed pulmonary hypertension. Renal damage was observed in seven cases (12.3%). 23 mutations were identified from the MMACHC gene of 57 patients. 37 patients demonstrated c.482G>A (64.9%) and 16 cases had c.609G>A (26.3%). Among 13 patients that exhibited spastic paraplegia as a main manifestation, 10 patients had c.482G>A (76.9%). Five patients presented with psychotic disorders and spastic paraplegia with c.482G>A. All patients improved after metabolic treatment with cobalamin, L-carnitine, and betaine. 30 school-aged patients returned to school. Two patients were married and had healthy babies.Conclusion: Patients with adolescence-onset cblC deficiency presented with varied neuropsychiatric symptoms or multiple organ damage. Metabolic studies and individualized treatment are keys to improve the outcome of the patients.

Determination of the Absolute Configuration of Bioactive Indole-Containing Pyrazino[2,1-b]quinazoline-3,6-Diones and Study of Their In Vitro Metabolic Profile

In recent decades, fungi-derived naturally occurring quinazolines have emerged as potential drug candidates. Nevertheless, most studies are conducted for bioactivity assays, and little is known about their absorption, distribution, metabolism, and elimination (ADME) properties. To perform metabolic studies, the synthesis of the naturally occurring quinazolinone, fiscalin B (1), and its chloro derivative, 4-((1H-indol-3-yl)methyl)-8,10-dichloro-1-isobutyl-1,2-dihydro-6H-pyrazino[2,1-b]quinazoline-3,6(4H)-dione (2), disclosed as an antibacterial agent, was performed in a gram scale using a microwave-assisted polycondensation reaction with 22% and 17% yields, respectively. The structure of the non-natural (+)-fiscalin B was established, for the first time, by X-ray crystallography as (1R,4S)-1, and the absolute configuration of the naturally occurring fiscalin B (-)-1 was confirmed by comparison of its calculated and experimental electronic circular dichroism (ECD) spectra as (1S,4R)-1. In vitro metabolic studies were monitored for this class of natural products for the first time by ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). The metabolic characteristics of 1 and 2 in human liver microsomes indicated hydration and hydroxylation mass changes introduced to the parent drugs.

IMA genome - F14

Draft genomes of Penicillium roqueforti, Fusarium sororula, Chalaropsis populi, and Chrysoporthe puriensis are presented. Penicillium roqueforti is a model fungus for genetics, physiological and metabolic studies, as well as for biotechnological applications. Fusarium sororula and Chrysoporthe puriensis are important tree pathogens, and Chalaropsis populi is a soil-borne root-pathogen. The genome sequences presented here thus contribute towards a better understanding of both the pathogenicity and biotechnological potential of these species.