Differential Protein Expression in Set5p-Mediated Acetic Acid Stress Response and Novel Targets for Engineering Yeast Stress Tolerance.

Acetic acid is a prevalent inhibitor in lignocellulosic hydrolysate, which represses microbial growth and bioproduction. Histone modification and chromatin remodeling have been revealed to be critical for regulating eukaryotic metabolism. However, related studies in chronic acetic acid stress responses remain unclear. Our previous studies revealed that overexpression of the histone H4 methyltransferase Set5p enhanced acetic acid stress tolerance of the budding yeast Saccharomyces cerevisiae. In this study, we examined the role of Set5p in acetic acid stress by analyzing global protein expression. Significant activation of intracellular protein expression under the stress was discovered, and the functions of the differential proteins were mainly involved in chromatin modification, signal transduction, and carbohydrate metabolism. Notably, a substantial increase of Set5p expression was observed in response to acetic acid stress. Functional studies demonstrated that the restriction of the telomere capping protein Rtc3p, as well as Ies3p and Taf14p, which are related to chromatin regulation, was critical for yeast stress response. This study enriches the understanding of the epigenetic regulatory mechanisms underlying yeast stress response mediated by histone-modifying enzymes. The results also benefit the development of robust yeast strains for lignocellulosic bioconversion.

AUXIN RESPONSE FACTORS 6 and 17 control the flag leaf angle in rice by regulating secondary cell wall biosynthesis of lamina joints.

Flag leaf angle impacts the photosynthetic capacity of densely grown plants and is thus an important agronomic breeding trait for crop architecture and yield. The hormone auxin plays a key role in regulating this trait, yet the underlying molecular and cellular mechanisms remain unclear. Here, we report that two rice (Oryza sativa) auxin response factors (ARFs), OsARF6 and OsARF17, which are highly expressed in lamina joint tissues, control flag leaf angle in response to auxin. Loss-of-function double osarf6 osarf17 mutants displayed reduced secondary cell wall levels of lamina joint sclerenchymatous cells (Scs), resulting in an exaggerated flag leaf angle and decreased grain yield under dense planting conditions. Mechanical measurements indicated that the mutant lamina joint tissues were too weak to support the weight of the flag leaf blade, resembling the phenotype of the rice increased leaf angle1 (ila1) mutant. We demonstrate that OsARF6 and OsARF17 directly bind to the ILA1 promoter independently and synergistically to activate its expression. In addition, auxin-induced ILA1 expression was dependent on OsARF6 and OsARF17. Collectively, our study reveals a mechanism that integrates auxin signaling with the secondary cell wall composition to determine flag leaf angle, providing breeding targets in rice, and potentially other cereals, for this key trait.

Transcriptome analysis of Kluyveromyces marxianus under succinic acid stress and development of robust strains.

Kluyveromyces marxianus has become an attractive non-conventional yeast cell factory due to its advantageous properties such as high thermal tolerance and rapid growth. Succinic acid (SA) is an important platform molecule that has been applied in various industries such as food, material, cosmetics, and pharmaceuticals. SA bioproduction may be compromised by its toxicity. Besides, metabolite-responsive promoters are known to be important for dynamic control of gene transcription. Therefore, studies on global gene transcription under various SA concentrations are of great importance. Here, comparative transcriptome changes of K. marxianus exposed to various concentrations of SA were analyzed. Enrichment and analysis of gene clusters revealed repression of the tricarboxylic acid cycle and glyoxylate cycle, also activation of the glycolysis pathway and genes related to ergosterol synthesis. Based on the analyses, potential SA-responsive promoters were investigated, among which the promoter strength of IMTCP2 and KLMA_50231 increased 43.4% and 154.7% in response to 15 g/L SA. In addition, overexpression of the transcription factors Gcr1, Upc2, and Ndt80 significantly increased growth under SA stress. Our results benefit understanding SA toxicity mechanisms and the development of robust yeast for organic acid production. KEY POINTS: • Global gene transcription of K. marxianus is changed by succinic acid (SA) • Promoter activities of IMTCP2 and KLMA_50123 are regulated by SA • Overexpression of Gcr1, Upc2, and Ndt80 enhanced SA tolerance.

Genetic architecture of seed glycerolipids in Asian cultivated rice.

Glycerolipids are essential for rice development and grain quality but its genetic regulation remains unknown. Here we report its genetic base using metabolite-based genome-wide association study and metabolite-based quantitative traits locus (QTL) analyses based on lipidomic profiles of seeds from 587 Asian cultivated rice accessions and 103 chromosomal segment substitution lines, respectively. We found that two genes encoding phosphatidylcholine (PC):diacylglycerol cholinephosphotransferase (OsLP1) and granule-bound starch synthase I (Waxy) contribute to variations in saturated triacylglycerol (TAG) and lyso-PC contents, respectively. We demonstrated that allelic variation in OsLP1 sequence between indica and japonica results in different enzymatic preference for substrate PC-16:0/16:0 and different saturated TAG levels. Further evidence demonstrated that OsLP1 also affects heading date, and that co-selection of OsLP1 and a flooding-tolerant QTL in Aus results in the abundance of saturated TAGs associated with flooding tolerance. Moreover, we revealed that the sequence polymorphisms in Waxy has pleiotropic effects on lyso-PC and amylose content. We proposed that rice seed glycerolipids have been unintentionally shaped during natural and artificial selection for adaptive or import seed quality traits. Collectively, our findings provide valuable genetic resources for rice improvement and evolutionary insights into seed glycerolipid variations in rice.

Automatic Registration of Homogeneous and Cross-Source TomoSAR Point Clouds in Urban Areas.

Building reconstruction using high-resolution satellite-based synthetic SAR tomography (TomoSAR) is of great importance in urban planning and city modeling applications. However, since the imaging mode of SAR is side-by-side, the TomoSAR point cloud of a single orbit cannot achieve a complete observation of buildings. It is difficult for existing methods to extract the same features, as well as to use the overlap rate to achieve the alignment of the homologous TomoSAR point cloud and the cross-source TomoSAR point cloud. Therefore, this paper proposes a robust alignment method for TomoSAR point clouds in urban areas. First, noise points and outlier points are filtered by statistical filtering, and density of projection point (DoPP)-based projection is used to extract TomoSAR building point clouds and obtain the facade points for subsequent calculations based on density clustering. Subsequently, coarse alignment of source and target point clouds was performed using principal component analysis (PCA). Lastly, the rotation and translation coefficients were calculated using the angle of the normal vector of the opposite facade of the building and the distance of the outer end of the facade projection. The experimental results verify the feasibility and robustness of the proposed method. For the homologous TomoSAR point cloud, the experimental results show that the average rotation error of the proposed method was less than 0.1°, and the average translation error was less than 0.25 m. The alignment accuracy of the cross-source TomoSAR point cloud was evaluated for the defined angle and distance, whose values were less than 0.2° and 0.25 m.

Analysis of Interpolation Methods in the Validation of Backscattering Coefficient Products.

Validation is the basis of synthetic aperture radar (SAR) image quantification applications. Based on the point target of the field site, the radiation characteristics of the backscattering coefficient image can be used to optimize the SAR imaging, and the product production system can be more closely targeted, to ensure the image product accuracy in the actual quantification application. In this study, the validation of the backscattering coefficient image was examined using calibrators, and the radiometric properties of the image were evaluated by extracting the radar cross-section of each point target. Bilinear interpolation and fast Fourier transform (FFT) interpolation methods were introduced for the local area interpolation of point targets, and the two methods were compared from the perspective of response function imaging and validation accuracy. The results show that the FFT interpolation method is more favorable for validating the backscattering coefficient.

Two-color fluorescent proteins reporting survivin regulation in breast cancer cells for high throughput drug screening.

Reporter gene assay is widely used for high throughput drug screening and drug action mechanism evaluation. In this study, we developed a robust dual-fluorescent reporter assay to detect drugs repressing the transcription of survivin, a cancer biomarker from the inhibitor of apoptosis family, in breast cancer cells cultured in three-dimensional (3D) microbioreactors. Survivin is overexpressed in numerous malignancies but almost silent in normal tissue cells and is considered a lead target for cancer therapy. Breast cancer MCF-7 cells were engineered to express enhanced green fluorescent protein driven by a survivin promoter and red fluorescent protein driven by a cytomegalovirus promoter as internal control to detect changes in survivin expression in cells as affected by drugs. This 3D dual-fluorescent reporter assay was validated with YM155 and doxorubicin, which were known to downregulate survivin in cancer cells, and further evaluated with two widely used anticancer compounds, cisplatin, and epigallocatechin gallate, to evaluate their effects on survivin expression. The results showed that the 3D dual-fluorescent reporter assay was robust for high throughput screening of drugs targeting survivin in breast cancer cells.

Influences of dietary Eucommia ulmoides leaf extract on the hepatic lipid metabolism, inflammation response, intestinal antioxidant capacity, intestinal microbiota, and disease resistance of the channel catfish (Ictalurus punctatus).

The purpose of the study was to investigate the effects of Eucommia ulmoides leaf extract (ELE) on the common occurrence of liver steatosis, chronic inflammation, oxidative stress, disturbance of gut microbiota, and disease susceptibility in high-fat diet-fed channel catfish. Channel catfish fed three diets, including a high-fat diet (11% crude fat) and ELE-supplemented diets containing 1‰ or 2‰ ELE for 4 weeks. The results showed the contents of liver triacylglycerol of 1‰ and 2‰ ELE groups were reduced, and ELE treatments decreased the expression of lipogenesis related genes (srebp-1c, pparγ, and acc-1), and increased the expression of lipolysis related genes (pparα). In addition, the supplementation of ELE improved the inflammatory response of the liver and intestine. ELE could improve the destruction of intestinal morphology structure and increase the expression level of hif-1a and tight junction proteins (Occludin, Claudin2, Claudin15). 2‰ ELE significantly enhanced the antioxidant capacity of intestine by increasing the activity of SOD enzyme. Moreover, the supplement of ELE significantly increased the abundance of Cetobacterium and Romboutsia (p < 0.05). Compared with the control group, the expression of immune factor nf-κb had a significant decrease, and il-1ß showed a tendency to decrease in the ELE supplement groups after pathogenic bacteria challenge. In conclusion, the ELE alleviated fatty liver disease and inflammation response, improved the oxidative capacity and physiological structure of intestine, and improved the structure of intestinal microbiota and disease resistance in HFD-fed channel catfish.

A Lightweight YOLOv5-MNE Algorithm for SAR Ship Detection.

Unlike optical satellites, synthetic aperture radar (SAR) satellites can operate all day and in all weather conditions, so they have a broad range of applications in the field of ocean monitoring. The ship targets' contour information from SAR images is often unclear, and the background is complicated due to the influence of sea clutter and proximity to land, leading to the accuracy problem of ship monitoring. Compared with traditional methods, deep learning has powerful data processing ability and feature extraction ability, but its complex model and calculations lead to a certain degree of difficulty. To solve this problem, we propose a lightweight YOLOV5-MNE, which significantly improves the training speed and reduces the running memory and number of model parameters and maintains a certain accuracy on a lager dataset. By redesigning the MNEBlock module and using CBR standard convolution to reduce computation, we integrated the CA (coordinate attention) mechanism to ensure better detection performance. We achieved 94.7% precision, a 2.2 M model size, and a 0.91 M parameter quantity on the SSDD dataset.

Airborne SAR Radiometric Calibration Based on Improved Sliding Window Integral Method.

To verify the performance of the high-resolution fully polarimetric synthetic aperture radar (SAR) sensor carried by the Xinzhou 60 remote-sensing aircraft, we used corner reflectors to calibrate the acquired data. The target mechanism in high-resolution SAR images is more complex than it is in low-resolution SAR images, the impact of the point target pointing error on the calibration results is more obvious, and the target echo signal of high-resolution images is more easily affected by speckle noise; thus, more accurate extraction of the point target position and the response energy is required. To solve this problem, this paper introduces image context information and proposes a method to precisely determine the integration region of the corner reflector using sliding windows based on the integral method. The validation indicates that the fully polarimetric SAR sensor on the Xinzhou 60 remote-sensing aircraft can accurately reflect the radiometric characteristics of the ground features and that the integral method can obtain more stable results than the peak method. The sliding window allows the position of the point target to be determined more accurately, and the response energy extracted from the image via the integral method is closer to the theoretical value, which means that the high-resolution SAR system can achieve a higher radiometric calibration accuracy. Additionally, cross-validation reveals that the airborne SAR images have similar quality levels to Sentinel-1A and Gaofen-3 images.

Two rice MYB transcription factors maintain male fertility in response to photoperiod by modulating sugar partitioning.

Photoperiod-dependent male fertility is a critical enabler of modern hybrid breeding. A MYB transcription factor, CSA, is a key regulator of sugar partitioning in rice anthers, disruption of which causes photoperiod-sensitive male sterility. However, little is known about the molecular mechanisms governing plant fertility in response to photoperiod. Here, we have obtained another rice photoperiod-sensitive male sterile mutant, csa2, which exhibits semi-sterility under long-day (LD) conditions, with normal fertility under short-day (SD) conditions. CSA2 specifically expressed in anthers, and here is shown to be indispensable for sugar partitioning to anthers under LD conditions. The CSA2 protein can restore the fertility of csa mutants under SD conditions when expressed in a CSA-specific pattern, indicating that the two proteins share common downstream regulatory targets. Transcriptomic analyses also reveal discrete regulatory targets in anthers. Furthermore, the regulatory role of CSA2 in sugar transport was influenced by the photoperiod conditions during floral initiation, not simply during anther development. Collectively, we propose that rice evolved at least two MYB proteins, CSA2 and CSA, that regulate sugar transport in anthers under LD and SD conditions, respectively. This finding provides insight into the molecular mechanisms that regulate male fertility in response to photoperiod.

Retracted: Anticancer Activity of Metformin, an Antidiabetic Drug, Against Ovarian Cancer Cells Involves Inhibition of Cysteine-Rich 61 (Cyr61)/Akt/Mammalian Target of Rapamycin (mTOR) Signaling Pathway.

An editorial decision has been made to retract this manuscript due to breach of publishing guidelines, following the identification of non-original and manipulated figures. Reference: Fengli Zhang, Huixiao Chen, Jing Du, Bin Wang, Lixiao Yang: Anticancer Activity of Metformin, an Antidiabetic Drug, Against Ovarian Cancer Cells Involves Inhibition of Cysteine-Rich 61 (Cyr61)/Akt/Mammalian Target of Rapamycin (mTOR) Signaling Pathway. Med Sci Monit 2018; 24: 6093-6101. 10.12659/MSM.909745.

OsFH3 Encodes a Type II Formin Required for Rice Morphogenesis.

The actin cytoskeleton is crucial for plant morphogenesis, and organization of actin filaments (AF) is dynamically regulated by actin-binding proteins. However, the roles of actin-binding proteins, particularly type II formins, in this process remain poorly understood in plants. Here, we report that a type II formin in rice, Oryza sativa formin homolog 3 (OsFH3), acts as a major player to modulate AF dynamics and contributes to rice morphogenesis. osfh3 mutants were semi-dwarf with reduced size of seeds and unchanged responses to light or gravity compared with mutants of osfh5, another type II formin in rice. osfh3 osfh5 mutants were dwarf with more severe developmental defectiveness. Recombinant OsFH3 could nucleate actin, promote AF bundling, and cap the barbed end of AF to prevent elongation and depolymerization, but in the absence of profilin, OsFH3 could inhibit AF elongation. Different from other reported type II formins, OsFH3 could bind, but not bundle, microtubules directly. Furthermore, its N-terminal phosphatase and tensin homolog domain played a key role in modulating OsFH3 localization at intersections of AF and punctate structures of microtubules, which differed from other reported plant formins. Our results, thus, provide insights into the biological function of type II formins in modulating plant morphology by acting on AF dynamics.

EDDAMAP: efficient data-dependent approach for monitoring asymptomatic patient.

BACKGROUND: A pandemic affects healthcare delivery and consequently leads to socioeconomic complications. During a pandemic, a community where there lives an asymptomatic patient (AP) becomes a potential endemic zone. Assuming we want to monitor the travel and/or activity of an AP in a community where there is a pandemic. Presently, most monitoring algorithms are relatively less efficient to find a suitable solution as they overlook the continuous mobility instances and activities of the AP over time. Conversely, this paper proposes an EDDAMAP as a compelling data-dependent technique and/or algorithm towards efficient continuous monitoring of the travel and/or activity of an AP. METHODS: In this paper, it is assumed that an AP is infected with a contagious disease in which the EDDAMAP technique exploits a GPS-enabled mobile device by tagging it to the AP along with its travel within a community. The technique further examines the Spatio-temporal trajectory of the AP to infer its spatial time-bounded activity. The technique aims to learn the travels of the AP and correlates them to its activities to derive some classes of point of interests (POIs) in a location. Further, the technique explores the natural occurring POIs via modelling to identify some regular stay places (SP) and present them as endemic zones. The technique adopts concurrent object feature localization and recognition, branch and bound formalism and graph theory to cater for the worst error-guaranteed approximation to obtain a valid and efficient query solution and also experiments with a real-world GeoLife dataset to confirm its performance. RESULTS: The EDDAMAP technique proofs a compelling technique towards efficient monitoring of an AP in case of a pandemic. CONCLUSIONS: The EDDAMAP technique will promote the discovery of endemic zones and hence some public healthcare facilities can rely on it to facilitate the design of patient monitoring system applications to curtail a global pandemic.

Analysis of Combining SAR and Optical Optimal Parameters to Classify Typhoon-Invasion Lodged Rice: A Case Study Using the Random Forest Method.

Lodging, a commonly occurring rice crop disaster, seriously reduces rice quality and production. Monitoring rice lodging after a typhoon event is essential for evaluating yield loss and formulating suitable remedial policies. The availability of Sentinel-1 and Sentinel-2 open-access remote sensing data provides large-scale information with a short revisit time to be freely accessed. Data from these sources have been previously shown to identify lodged crops. In this study, therefore, Sentinel-1 and Sentinel-2 data after a typhoon event were combined to enable monitoring of lodging rice to be quickly undertaken. In this context, the sensitivity of synthetic aperture radar (SAR) features (SF) and spectral indices (SI) extracted from Sentinel-1 and Sentinel-2 to lodged rice were analyzed, and a model was constructed for selecting optimal sensitive parameters for lodging rice (OSPL). OSPL has high sensitivity to lodged rice and strong ability to distinguish lodged rice from healthy rice. After screening, Band 11(SWIR-1) and Band 12(SWIR-2) were identified as optimal spectral indices (OSI), and VV, VV + VH and Shannon Entropy were optimal SAR features (OSF). Three classification results of lodging rice were acquired using the Random Forest classification (RFC) method based on OSI, OSF and integrated OSI-OSF stack images, respectively. Results indicate that an overall level of accuracy of 91.29% was achieved with the combination of SAR and optical optimal parameters. The result was 2.91% and 6.05% better than solely using optical or SAR processes, respectively.

Heavy metal pollution and health risk assessment of agricultural soil near a smelter in an industrial city in China.

To assess heavy metal pollution and human health risk, a total of 28 topsoil samples were collected during four seasons from seven agricultural soil sites near a famous smelter in Jiyuan, China. The maximum concentrations of Cd, Pb, Hg, As, Zn, Cu, Ni, and Cr were 26.00, 2601.00, 3.29, 65.00, 410.00, 156.30, 54.80, and 73.60 mg kg-1, respectively. Compared with the sampling site nearest to the smelter, the concentrations of six metals at the farthest site were decreased significantly (P < 0.05). All sites were heavily contaminated, with Nemerow index (P) >3.0, and all sites had very high ecological risks related to Cd and Hg. The non-carcinogenic risk for children (based on combined exposure to the eight metals) was above the safety level. The carcinogenic risk of As for adults (8.98 × 10-6) and children (1.49 × 10-5) exceeded the acceptable level (1 × 10-6). Results suggest a serious health risk in the polluted areas, particularly for children.Abbreviation Cd: Cadmium; Pb: Lead; Hg: Mercury; As: Arsenic; Zn: Zinc; Cu: Copper; Ni: Nickel; Cr: Chromium; P: Nemerow index; RI: Potential ecological risk index; Ei: Monomial potential ecological risk of a specific heavy metal; HI: non-carcinogenic hazard index; CR: Carcinogenic risk; TN: Total nitrogen; TP: Total phosphorus; OM: Organic matter; MC: Moisture content; ADD: Average daily dose.

Treatment effect of apatinib combined chemotherapy as second-line or above therapy in patients with advanced gastric cancer or adenocarcinoma of the gastroesophageal junction.

This study aimed to compare the therapeutic effects between apatinib combined chemotherapy and chemotherapy alone as second-line or above therapy in advanced gastric cancer (GC) or adenocarcinoma of the gastroesophageal junction (AGEJ). The clinical data of advanced GC or AGEJ patients, including sex, age, Eastern Cooperative Oncology Group (ECOG) grading, chemotherapy regimen, pathological grading, location of primary lesion, previous gastrectomy, metastases, previous chemotherapy or radiotherapy were retrospectively collected, and the progression-free survival (PFS) was recorded. 127 patients underwent apatinib combined chemotherapy and 60 patients underwent chemotherapy regimen alone. Disease control rate (DCR) of patients with apatinib combined chemotherapy was higher than that of chemotherapy alone (P=0.033). A Kaplan-Meier (KM) plot showed that PFS was significantly longer in patients receiving apatinib combined chemotherapy than those treated by chemotherapy alone (P = 0.002). The PFS of patients with a number of metastatic lesions ≤ 2 was obviously longer than that of patients with a number of metastatic lesions > 2 (P < 0.001). Cox regression analysis revealed that PFS was independently associated with the number of metastatic lesions >2 (HR=2.129, 95% CI: 1.256-3.608, P=0.005) and treatment methods (chemotherapy alone or apatinib combined chemotherapy) (HR=1.427, 95% CI: 1.055-1.930, P=0.021). Compared with chemotherapy alone, apatinib combined chemotherapy could significantly improve DCR and prolong the PFS in advanced GC or AGEJ cases who had failed in at least first-line chemotherapy with acceptable tolerance.

An engineered mouse embryonic stem cell model with survivin as a molecular marker and EGFP as the reporter for high throughput screening of embryotoxic chemicals in vitro.

Embryonic stem cell test (EST) is the only generally accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of EST for regulatory embryotoxicity screening are impeded by its technical complexity, long testing period, and limited endpoint data. In this study, a high throughput embryotoxicity screening based on mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by a human survivin promoter and a human cytomegalovirus promoter, respectively, was developed. These EGFP expressing mESCs were cultured in three-dimensional (3D) fibrous scaffolds in microbioreactors on a multiwell plate with EGFP fluorescence signals as cell responses to chemicals monitored noninvasively in a high throughput manner. Nine chemicals with known developmental toxicity were used to validate the survivin-based embryotoxicity assay, which showed that strongly embryotoxic compounds such as 5-fluorouracil, retinoic acid, and methotrexate downregulated survivin expression by more than 50% in 3 days, while weakly embryotoxic compounds such as boric acid, methoxyacetic acid, and tetracyclin showed modest downregulation effect and nonembryotoxic saccharin, penicillin G, and acrylamide had negligible downregulation effect on survivin expression, confirming that survivin can be used as a molecular endpoint for high throughput screening of embryotoxicants. The potential developmental toxicity of three Chinese herbal medicines were also evaluated using this assay, demonstrating its application in in vitro developmental toxicity test for drug safety assessment.

Symbiochlorum hainanensis gen. et sp. nov. (Ulvophyceae, Chlorophyta) isolated from bleached corals living in the South China Sea.

Light/scanning electron/transmission microscopy-based morphological analyses and multiple nucleotide sequences-based molecular phylogenetic analyses are used to identify and assess the phylogenetic position of a new unidentified green alga isolated from bleached corals living in the South China Sea. This new unidentified green alga is a unicellular marine alga and has uninucleate vegetative cells and multiple chloroplasts with a pyrenoid. It can form aplanosporangium covered by cell walls and reproduces by releasing autospore. These features differ substantially from those of the two genera Ignatius and Pseudocharacium. Those two genera have been accommodated in the Ignatius clade. Nucleotide sequences of the nuclear small subunit ribosomal RNA gene (18S rRNA), internal transcribed spacer 2 of ribosomal RNA gene (ITS2) and ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit gene (rbcL, partial) are obtained and compared with published green algal sequences. The results from the morphology, ultrastructure, and multiple nucleotide sequences data support the placement of the new unidentified green alga in Ulvophyceae. This new unidentified isolate is described as Symbiochlorum hainanensis gen. et sp. nov., a new sister lineage to the Ignatius clade, Ulvophyceae, Chlorophyta.

Comprehensive optimization of precursor-directed production of BC194 by Streptomyces rochei MB037 derived from the marine sponge Dysidea arenaria.

BC194, a derivative of borrelidin (BN) that features a lower cytotoxicity than that of BN due to an altered starter unit, trans-1,2-cyclobutanedicarboxylic acid (trans-1,2-CBDA), is a potent inhibitor of angiogenesis. However, BC194 production has only been reported to occur via mutasynthesis, which requires tedious, multistep genetic manipulation. In this study, we surveyed several factors contributing to the precursor-directed biosynthesis of BC194 and provided an alternative method for the production of BC194 that is directly applicable to other BN-producing strains. First, the precursor-directed biosynthesis of BC194 by a BN-producing strain, Streptomyces rochei MB037 derived from sponge Dysidea arenaria, was carried out in modified Radix astragali (RA) medium with 5 mM trans-1,2-CBDA. Next, possible inhibitors of BN starter unit trans-1,2-cyclopentanedicarboxylic acid (trans-1,2-CPDA) biosynthesis were investigated. It was found that potassium ferricyanide was a possible inhibitor of 3,4-dihydroxyphenylacetate 2,3-dioxygenase (DHPAO) and capable of suppressing the yield of BN and increasing the BC194 yield by 112.5% (from 5.2 ± 0.76 to 11.9 ± 0.59 mg/L). BC194 yield was further enhanced in the presence of 50 mM trans-1,2-CBDA, reaching 20.2 ± 0.62 mg/L. Furthermore, 3% macroporous adsorbent DA-201 resin was added to the fermentation broth, enabling a further 36.6% increase in BC194 production and reaching 27.59 ± 1.15 mg/L. Moreover, an efficient separation of BC194 with approximately 95% purity was developed by employing high-speed counter-current chromatography (HSCCC), achieving an improved recovery (approximately 93%).