SWATH-MS-based proteogenomic analysis reveals the involvement of alternative splicing in poplar upon lead stress.

Alternative splicing (AS) regulates gene expression and increases proteomic diversity for the fine tuning of stress responses in plants, but the exact mechanism through which AS functions in plant stress responses is not thoroughly understood. Here, we investigated how AS functions in poplar (Populus trichocarpa), a popular plant for bioremediation, in response to lead (Pb) stress. Using a proteogenomic analysis, we determine that Pb stress induced alterations in AS patterns that are characterized by an increased use of nonconventional splice sites and a higher abundance of Pb-responsive splicing factors (SFs) associated with Pb-responsive transcription factors. A strong Pb(II)-inducible chaperone protein, PtHSP70, that undergoes AS was further characterized. Overexpression of its two spliced isoforms, PtHSP70-AS1 and PtHSP70-AS2, in poplar and Arabidopsis significantly enhances the tolerance to Pb. Further characterization shows that both isoforms can directly bind to Pb(II), and PtHSP70-AS2 exhibits 10-fold higher binding capacities and a greater increase in expression under Pb stress, thereby reducing cellular toxicity through Pb(II) extrusion and conferring Pb tolerance. AS of PtHSP70 is found to be regulated by PtU1-70K, a Pb(II)-inducible core SF involved in 5'-splice site recognition. Because the same splicing pattern is also found in HSP70 orthologs in other plant species, AS of HSP70 may be a common regulatory mechanism to cope with Pb(II) toxicity. Overall, we have revealed a novel post-transcriptional machinery that mediates heavy metal tolerance in diverse plant species. Our findings offer new molecular targets and bioengineering strategies for phytoremediation and provide new insight for future directions in AS research.

Drying without dying: A genome database for desiccation-tolerant plants and evolution of desiccation tolerance.

Desiccation is typically fatal, but a small number of land plants have evolved vegetative desiccation tolerance (VDT), allowing them to dry without dying through a process called anhydrobiosis. Advances in sequencing technologies have enabled the investigation of genomes for desiccation-tolerant plants over the past decade. However, a dedicated and integrated database for these valuable genomic resources has been lacking. Our prolonged interest in VDT plant genomes motivated us to create the "Drying without Dying" database, which contains a total of 16 VDT-related plant genomes (including 10 mosses) and incorporates 10 genomes that are closely related to VDT plants. The database features bioinformatic tools, such as blast and homologous cluster search, sequence retrieval, Gene Ontology term and metabolic pathway enrichment statistics, expression profiling, co-expression network extraction, and JBrowser exploration for each genome. To demonstrate its utility, we conducted tailored PFAM family statistical analyses, and we discovered that the drought-responsive ABA transporter AWPM-19 family is significantly tandemly duplicated in all bryophytes but rarely so in tracheophytes. Transcriptomic investigations also revealed that response patterns following desiccation diverged between bryophytes and angiosperms. Combined, the analyses provided genomic and transcriptomic evidence supporting a possible divergence and lineage-specific evolution of VDT in plants. The database can be accessed at http://desiccation.novogene.com. We expect this initial release of the "Drying without Dying" plant genome database will facilitate future discovery of VDT genetic resources.

Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

The ScAPD1-like gene from the desert moss Syntrichia caninervis enhances resistance to Verticillium dahliae via phenylpropanoid gene regulation.

Soloist is a member of a distinct and small subfamily within the AP2/ERF transcriptional factor family that play important roles in plant biotic and abiotic stress responses. There are limited studies of Soloist genes and their functions are poorly understood. We characterized the abiotic and biotic stress tolerance function of the ScSoloist gene (designated as ScAPD1-like) from the desert moss Syntrichia caninervis. ScAPD1-like responded to multiple abiotic, biotic stresses and plant hormone treatments. ScAPD1-like protein located to the nucleus and bound to several DNA elements. Overexpression of ScAPD1-like in Arabidopsis did not alter abiotic stress resistance or inhibit Pseudomonas syringae pv. tomato (Pst) DC3000 infection. However, overexpression of ScAPD1-like significantly increased the resistance of transgenic Arabidopsis and S. caninervis to Verticillium dahliae infection, decreased reactive oxygen species accumulation and improved reactive oxygen species scavenging activity. ScAPD1-like overexpression plants altered the abundance of transcripts for lignin synthesis and promoted lignin accumulation in Arabidopsis. ScAPD1-like directly bind to RAV1, AC elements, and TATA-box in the promoters of AtPAL1 and AtC4H genes, respectively, in vitro. Chromatin immunoprecipitation-quantitative polymerase chain reaction assays demonstrated ScAPD1-like directly bound to PAL and C4H genes promoters in Arabidopsis and their homologs in S. caninervis. In S. caninervis, ScAPD1-like overexpression and RNAi directly regulated the abundance of ScPAL and ScC4H transcripts and modified the metabolites of phenylpropanoid pathway. We provide insight into the function of Soloist in plant defense mechanisms that likely occurs through activation of the phenylpropanoid biosynthesis pathway. ScAPD1-like is a promising candidate gene for breeding strategies to improve resistance to Verticillium wilt.

Casuattimines A-N, fourteen new Lycopodium alkaloids from Lycopodiastrum casuarinoides with Cav3.1 channel inhibitory activity.

Two new dimeric Lycopodium alkaloids, casuattimines A and B (1 and 2), along with twelve previously undescribed Lycopodium alkaloids, casuattimines C-N (3-14), and eight known Lycopodium alkaloids, were isolated from Lycopodiastrum casuarinoides. Casuattimines A and B (1 and 2) are the first two ether-linked Lycopodium alkaloid dimers. Casuattimines C and D (3 and 4) are unique Lycopodium alkaloids characterized by a long fatty acid chain. Structural elucidation was achieved through HRESIMS, NMR, and electronic circular dichroism (ECD) calculations. In addition, the absolute configurations of compounds 7, 13, and 14 were determined by single crystal X-ray diffraction. Compounds 1, 2, and 4 demonstrated notable Cav3.1 channel inhibitory activities presenting IC50 values of 10.75 ± 1.02 µM, 9.33 ± 0.79 µM, and 7.14 ± 0.86 µM, respectively. The dynamics of compound 4 against the Cav3.1 channel and preliminary structure-activity relationships of these active Lycopodium alkaloids were also discussed.

CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii.

Komagataella phaffii, a nonconventional yeast, is increasingly attractive to researchers owing to its posttranslational modification ability, strict methanol regulatory mechanism, and lack of Crabtree effect. Although CRISPR-based gene editing systems have been established in K. phaffii, there are still some inadequacies compared to the model organism Saccharomyces cerevisiae. In this study, a redesigned gRNA plasmid carrying red and green fluorescent proteins facilitated plasmid construction and marker recycling, respectively, making marker recycling more convenient and reliable. Subsequently, based on the knockdown of Ku70 and DNA ligase IV, we experimented with integrating multiple DNA fragments at a single locus. A 26.5-kb-long DNA fragment divided into 11 expression cassettes for lycopene synthesis could be successfully integrated into a single locus at one time with a success rate of 57%. A 27-kb-long DNA fragment could also be precisely knocked out with a 50% positive rate in K. phaffii by introducing two DSBs simultaneously. Finally, to explore the feasibility of rapidly balancing the expression intensity of multiple genes in a metabolic pathway, a yeast combinatorial library was successfully constructed in K. phaffii using lycopene as an indicator, and an optimal combination of the metabolic pathway was identified by screening, with a yield titer of up to 182.73 mg/L in shake flask fermentation. KEY POINTS: • Rapid marker recycling based on the visualization of a green fluorescent protein • One-step multifragment integration and large fragment knockout in the genome • A random assembly of multiple DNA elements to create yeast libraries in K. phaffii.

Biosynthesis of eriodictyol in citrus waster by endowing P450BM3 activity of naringenin hydroxylation.

The flavonoid naringenin is abundantly present in pomelo peels, and the unprocessed naringenin in wastes is not friendly for the environment once discarded directly. Fortunately, the hydroxylated product of eriodictyol from naringenin exhibits remarkable antioxidant and anticancer properties. The P450s was suggested promising for the bioconversion of the flavonoids, but less naturally existed P450s show hydroxylation activity to C3' of the naringenin. By well analyzing the catalytic mechanism and the conformations of the naringenin in P450, we proposed that the intermediate Cmpd I ((porphyrin)Fe = O) is more reasonable as key conformation for the hydrolyzation, and the distance between C3'/C5' of naringenin to the O atom of CmpdI determines the hydroxylating activity for the naringenin. Thus, the "flying kite model" that gradually drags the C-H bond of the substrate to the O atom of CmpdI was put forward for rational design. With ab initio design, we successfully endowed the self-sufficient P450-BM3 hydroxylic activity to naringenin and obtained mutant M5-5, with kcat, Km, and kcat/Km values of 230.45 min-1, 310.48 µM, and 0.742 min-1 µM-1, respectively. Furthermore, the mutant M4186 was screened with kcat/Km of 4.28-fold highly improved than the reported M13. The M4186 also exhibited 62.57% yield of eriodictyol, more suitable for the industrial application. This study provided a theoretical guide for the rational design of P450s to the nonnative compounds. KEY POINTS: •The compound I is proposed as the starting point for the rational design of the P450BM3 •"Flying kite model" is proposed based on the distance between O of Cmpd I and C3'/C5' of naringenin •Mutant M15-5 with 1.6-fold of activity than M13 was obtained by ab initio modification.

Aldicarb disturbed bile acid, steroid hormone and oxylipin homeostasis in C57BL/6 J mice.

Mounting evidence has shown that the gut microbiota plays a key role in human health. The homeostasis of the gut microbiota could be affected by many factors, including environmental chemicals. Aldicarb is a carbamate insecticide used to control a variety of insects and nematode pests in agriculture. Aldicarb is highly toxic and its wide existence has become a global public health concern. In our previous study, we have demonstrated that aldicarb disturbed the gut microbial community structure and composition. However, the impacts of aldicarb on gut microbiota-derived metabolites, bile acids, remain elusive. In present study, we performed targeted metabolomics analysis to explore the effects of aldicarb exposure on bile acids, as well as steroid hormones and oxylipins in the serum, feces and liver of C57BL/6 J mice. Our results showed that aldicarb exposure disturbed the level of various bile acids, steroid hormones and oxylipins in the serum and feces of C57BL/6 J mice. In the liver, the level of cortisol was decreased, meanwhile 15,16-dihydroxyoctadeca-9,12-dienoic acid was increased in aldicarb-treated mice. Metagenomic sequencing analysis showed that the relative abundance of a bile salt hydrolase, choloylglycine hydrolase (EC:3.5.1.24) and a sulfatase enzyme involved in steroid hormone metabolism, arylsulfatase, was significantly increased by aldicarb exposure. Furthermore, correlations were found between gut microbiota and various serum metabolites. The results from this study are helpful to improve the understanding of the impact of carbamate insecticides on host and microbial metabolism.

Lycopodium Alkaloids from Huperzia goebelii.

One new fawcettimine-type Lycopodium alkaloid, hupertimine F (1), together with five known (2-6) Lycopodium alkaloids were isolated from Huperzia goebelii. The structure of 1 was elucidated by 1D and 2D NMR spectra, HRESIMS, and X-ray diffraction. Structurally, 1 represents the fourth example of Lycopodium alkaloids characterized by a 5/5/5/5/6 pentacyclic ring system with a 1-aza-7-oxabicyclo[2.2.1]heptane moiety. These known compounds 2, 3, 5, and 6 were isolated from H. goebelii for the first time. Compounds 1-6 were evaluated for acetylcholinesterase, butyrylcholinesterase and monoamine oxidase B inhibitory activities in vitro.

Lycocasine A, a Lycopodium Alkaloid from Lycopodiastrum casuarinoides and Its Acid-Sensing Ion Channel 1a Inhibitory Activity.

A novel Lycopodium alkaloid, lycocasine A (1), and seven known Lycopodium alkaloids (2-8), were isolated from Lycopodiastrum casuarinoides. Their structures were determined through NMR, HRESIMS, and X-ray diffraction analysis. Compound 1 features an unprecedented 5/6/6 tricyclic skeleton, highlighted by a 5-aza-tricyclic[6,3,1,02,6]dodecane motif. In bioactivity assays, compound 1 demonstrated weak inhibitory activity against acid-sensing ion channel 1a.

Metabolomic Analysis of the Desert Moss Syntrichia caninervis Provides Insights into Plant Dehydration and Rehydration Response.

Desiccation-tolerant (DT) plants can survive extreme dehydration and tolerate the loss of up to 95% of their water content, making them ideal systems to determine the mechanism behind extreme drought stress and identify potential approaches for developing drought-tolerant crops. The desert moss Syntrichia caninervis is an emerging model for extreme desiccation tolerance that has benefited from high-throughput sequencing analyses, allowing identification of stress-tolerant genes; however, its metabolic response to desiccation is unknown. A liquid chromatography-mass spectrometry analysis of S. caninervis at six dehydration-rehydration stages revealed 912 differentially abundant compounds, belonging to 93 metabolic classes. Many (256) metabolites accumulated during rehydration in S. caninervis, whereas only 71 accumulated during the dehydration period, in contrast to the pattern observed in vascular DT plants. During dehydration, nitrogenous amino acids (l-glutamic acid and cysteinylglycine), alkaloids (vinleurosine) and steroids (physalin D) accumulated, whereas glucose 6-phosphate decreased. During rehydration, γ-aminobutyric acid, glucose 6-phosphate and flavonoids (karanjin and aromadendrin) accumulated, as did the plant hormones 12-oxo phytodienoic acid (12-OPDA) and trans-zeatin riboside. The contents ofl-arginine, maltose, turanose, lactulose and sucrose remained high throughout dehydration-rehydration. Syntrichia caninervis thus accumulates antioxidants to scavenge reactive oxygen species, accumulating nitrogenous amino acids and cytoprotective metabolites and decreasing energy metabolism to enter a protective state from dehydration-induced damage. During subsequent rehydration, many metabolites rapidly accumulated to prevent oxidative stress and restore physiological activities while repairing cells, representing a more elaborate rehydration repair mechanism than vascular DT plants, with a faster and greater accumulation of metabolites. This metabolic kinetics analysis in S. caninervis deepens our understanding of its dehydration mechanisms and provides new insights into the different strategies of plant responses to dehydration and rehydration.

Inhibition of astroglial hemichannels ameliorates infrasonic noise induced short-term learning and memory impairment.

As a kind of environmental noise, infrasonic noise has negative effects on various human organs. To date, research has shown that infrasound impairs cognitive function, especially the ability for learning and memory. Previously, we demonstrated that impaired learning and memory induced by infrasound was closely related with glia activation; however, the underlying mechanisms remain unclear. Connexin 43 hemichannels (Cx43 HCs), which are mainly expressed in hippocampal astrocytes, are activated under pathological conditions, lending support to the hypothesis that Cx43 HCs might function in the impaired learning and memory induced by infrasound. This study revealed that that blocking hippocampal Cx43 HCs or downregulating hippocampal Cx43 expression significantly alleviated impaired learning and memory induced by infrasound. We also observed that infrasound exposure led to the abundant release of glutamate and ATP through Cx43 HCs. In addition, the abundant release of glutamate and ATP depended on proinflammatory cytokines. Our finds suggested that the enhanced release of ATP and glutamate by astroglial Cx43 HCs may be involved in the learning and memory deficits caused by infrasound exposure.

Dose-Response Relationship between Red Blood Cell Distribution Width and In-Hospital Mortality in Oldest Old Patients with Acute Ischemic Stroke.

INTRODUCTION: It is crucial to identify predictors of mortality in the early stage of acute ischemic stroke for the oldest old (aged ≥80 years) because of their poor overall survival outcomes. However, limited data are available as the oldest old have often been excluded from previous clinical studies. Hence, we aimed to assess the predictive effect of red blood cell distribution width on in-hospital mortality and the dose-response relationship between the red blood cell distribution width and in-hospital mortality in oldest old with acute ischemic stroke. METHODS: A retrospective cohort study was performed in two tertiary hospitals. Patients aged ≥80 years admitted due to acute ischemic stroke from January 1, 2014, to January 31, 2020, were included in the study. We divided the eligible patients into 3 groups with tertiles of red blood cell distribution width. Restrictive cubic spline and robust locally weighted regression analysis were performed to test the dose-response relationship between red blood cell distribution width and the in-hospital mortality risk. All-cause in-hospital mortality was the main study outcome. RESULTS: Overall, 606 patients were included in the final analysis. Red blood cell distribution width was categorized into 3 groups (T1: <13.7%, T2: 13.8-15.7%, and T3: >15.7%). The rationality of this categorization was then validated with restricted cubic spline and robust locally regression smoothing scatterplot, respectively. After adjusting for demographic and clinical features, a higher red blood cell distribution width was independently associated with in-hospital mortality and the hazard ratio (HR) was 3.31 (95% CI 2.47-4.45, p < 0.001). There was a positive dose-response relationship between red blood cell distribution width and mortality risk. Sensitivity analysis identified no conspicuous change in the HR. CONCLUSIONS: Red blood cell distribution width may be a valuable and simple measure for predicting in-hospital mortality in oldest old patients with acute ischemic stroke.

Size-dependent effects of polystyrene microplastics on gut metagenome and antibiotic resistance in C57BL/6 mice.

Microplastic pollution is an emerging threat for marine and terrestrial ecosystems, which has raised global concerns about its implications for human health. Mounting evidence has shown that the gut microbiota plays a key role in human health and diseases. The gut bacteria could be disturbed by many environmental factors, including the microplastic particles. However, the size effect of polystyrene microplastics on mycobiome, as well as gut functional metagenome has not been well studied. In this study, we performed ITS sequencing to explore the size effect of polystyrene microplastics on the fungal composition, in combination with the shotgun metagenomics sequencing to reveal the size effects of polystyrene on the functional metagenome. We found that polystyrene microplastic particles with 0.05-0.1 µm diameter showed greater impact on the bacterial and fungal composition of gut microbiota as well as the metabolic pathways than the polystyrene microplastic particles with 9-10 µm diameter. Our results suggested that size-depended effects should not be ignored in the health risk assessment of microplastics.

Three New Selaginellin Derivatives from Selaginella pulvinata and Their α-Glucosidase Inhibitory Activity.

Three new selaginellin derivatives, selaginpulvilins V-X (1-3), together with seven known analogs (4-10) were isolated from whole plants of Selaginella pulvinata. Their structures were determined by extensive spectroscopic methods including 1D and 2D NMR, HR-ESI-MS and chemical derivatization method. Compound 1 represents a rare example of naturally occurring selaginellin with an alkynylphenol-trimmed skeleton. Biological evaluation showed that compounds 2, 6 and 8 displayed moderate inhibition against α-glucosidase with IC50 values of 3.71, 2.04 and 4.00 µM, respectively.

Alternative Splicing: From Abiotic Stress Tolerance to Evolutionary Genomics.

The post-transcriptional regulation of gene expression, in particular alternative splicing (AS) events, substantially contributes to the complexity of eukaryotic transcriptomes and proteomes [...].

Association of serum uric acid with risk of stroke in US adults: A cross-sectional study from NHANES 1999-2020.

BACKGROUND AND AIMS: The validity of high uric acid levels as an independent cause of stroke remains controversial, and the association between its low concentration and stroke is unclear. This study determines how different serum uric acid (SUA) levels are associated with stroke risk. METHODS: This cross-sectional study used continuous National Health and Nutrition Examination Survey data in the United States during 1999-2020. The SUA levels of 6.0, 6.8, and 9.0 mg/dL were all considered as cut-off points. Restricted cubic spline interpolation and logistic regression models were used to evaluate the different associations. Subgroup analyses and sensitivity analyses were conducted to evaluate the influence of multiple factors on the outcomes. RESULTS: The study included 23,413 participants aged ≥ 20 years. A J-shaped curve existed between SUA and stroke risk, and the risk of stroke was positively correlated with SUA levels in the overall population. Subgroup analysis of all adults in the SUA 6.8-9.0 mg/dL group showed that stroke risk for non-Hispanic white, obese, ex-smoker, and heavy drinking groups was increased, but for the other Hispanic group was reduced. In the SUA < 6.0 mg/dL group, stroke risk for ex-smoker, heavy drinkers, and no chronic kidney disease groups was increased. CONCLUSION: Our findings indicate a J-shaped relationship between SUA levels and stroke risk. Low and high SUA levels increased stroke risk for different populations, except in the other Hispanic population. Early SUA management is highly significant for stroke prevention in high-risk populations.

To dry perchance to live: Insights from the genome of the desiccation-tolerant biocrust moss Syntrichia caninervis.

With global climate change, water scarcity threatens whole agro/ecosystems. The desert moss Syntrichia caninervis, an extremophile, offers novel insights into surviving desiccation and heat. The sequenced S. caninervis genome consists of 13 chromosomes containing 16 545 protein-coding genes and 2666 unplaced scaffolds. Syntenic relationships within the S. caninervis and Physcomitrella patens genomes indicate the S. caninervis genome has undergone a single whole genome duplication event (compared to two for P. patens) and evidence suggests chromosomal or segmental losses in the evolutionary history of S. caninervis. The genome contains a large sex chromosome composed primarily of repetitive sequences with a large number of Copia and Gypsy elements. Orthogroup analyses revealed an expansion of ELIP genes encoding proteins important in photoprotection. The transcriptomic response to desiccation identified four structural clusters of novel genes. The genomic resources established for this extremophile offer new perspectives for understanding the evolution of desiccation tolerance in plants.

Perfluorooctanoic Acid-Disturbed Serum and Liver Lipidome in C57BL/6 Mice.

Perfluorooctanoic acid is a manufactured material extensively utilized in industrial and consumer products. As a persistent organic pollutant, perfluorooctanoic acid has raised increasing public health concerns recently. Although perfluorooctanoic acid is known to induce lipid accumulation in the liver, the impact of perfluorooctanoic acid on different lipid classes has not been fully evaluated. In this study, we performed untargeted lipidomics analysis to investigate the impact of perfluorooctanoic acid on the lipid homeostasis in C57BL/6 male mice. Perfluorooctanoic acid disturbed the lipid profiles in serum and liver, with a variety of lipid classes significantly altered. Greater impacts were observed in the liver lipidome than the serum lipidome. In particular, some lipid clusters in the liver were altered by both high- and low-dose perfluorooctanoic acid exposure, including the increase of unsaturated triglycerides and the decrease of sphingomyelins, saturated phosphatidylcholines, saturated lysophosphatidylcholines, and phospholipid ethers. In parallel with an increase in the liver, a decrease of saturated phosphatidylcholines was found in the serum of high-dose perfluorooctanoic acid-treated mice. The findings from this study are helpful to improve the understanding of perfluorooctanoic acid-induced dysregulation of lipid metabolism and perfluorooctanoic acid-associated health effects in liver.

One-pot biosynthesis of 7ß-hydroxyandrost-4-ene-3,17-dione from phytosterols by cofactor regeneration system in engineered mycolicibacterium neoaurum.

BACKGROUND: 7ß-hydroxylated steroids (7ß-OHSt) possess significant activities in anti-inflammatory and neuroprotection, and some of them have been widely used in clinics. However, the production of 7ß-OHSt is still a challenge due to the lack of cheap 7ß-hydroxy precursor and the difficulty in regio- and stereo-selectively hydroxylation at the inert C7 site of steroids in industry. The conversion of phytosterols by Mycolicibacterium species to the commercial precursor, androst-4-ene-3,17-dione (AD), is one of the basic ways to produce different steroids. This study presents a way to produce a basic 7ß-hydroxy precursor, 7ß-hydroxyandrost-4-ene-3,17-dione (7ß-OH-AD) in Mycolicibacterium, for 7ß-OHSt synthesis. RESULTS: A mutant of P450-BM3, mP450-BM3, was mutated and engineered into an AD producing strain for the efficient production of 7ß-OH-AD. The enzyme activity of mP450-BM3 was then increased by 1.38 times through protein engineering and the yield of 7ß-OH-AD was increased from 34.24 mg L- 1 to 66.25 mg L- 1. To further enhance the performance of 7ß-OH-AD producing strain, the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) for the activity of mP450-BM3-0 was optimized by introducing an NAD kinase (NADK) and a glucose-6-phosphate dehydrogenase (G6PDH). Finally, the engineered strain could produce 164.52 mg L- 1 7ß-OH-AD in the cofactor recycling and regeneration system. CONCLUSIONS: This was the first report on the one-pot biosynthesis of 7ß-OH-AD from the conversion of cheap phytosterols by an engineered microorganism, and the yield was significantly increased through the mutation of mP450-BM3 combined with overexpression of NADK and G6PDH. The present strategy may be developed as a basic industrial pathway for the commercial production of high value products from cheap raw materials.