An oleaginous yeast platform for renewable 1-butanol synthesis based on a heterologous CoA-dependent pathway and an endogenous pathway.

BACKGROUND: Microbial biofuel production provides a promising sustainable alternative to fossil fuels. 1-Butanol is recognized as an advanced biofuel and is gaining attention as an ideal green replacement for gasoline. In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was first engineered with a heterologous CoA-dependent pathway and an endogenous pathway, respectively. RESULTS: The co-overexpression of two heterologous genes ETR1 and EutE resulted in the production of 1-butanol at a concentration of 65 µg/L. Through the overexpression of multiple 1-butanol pathway genes, the titer was increased to 92 µg/L. Cofactor engineering through endogenous overexpression of a glyceraldehyde-3-phosphate dehydrogenase and a malate dehydrogenase further led to titer improvements to 121 µg/L and 110 µg/L, respectively. In addition, the presence of an endogenous 1-butanol production pathway and a gene involved in the regulation of 1-butanol production was successfully identified in Y. lipolytica. The highest titer of 123.0 mg/L was obtained through this endogenous route by combining a pathway gene overexpression strategy. CONCLUSIONS: This study represents the first report on 1-butanol biosynthesis in Y. lipolytica. The results obtained in this work lay the foundation for future engineering of the pathways to optimize 1-butanol production in Y. lipolytica.

Efficient production of 22(R)-hydroxycholesterol via combination optimization of Saccharomyces cerevisiae.

22(R)-hydroxycholesterol (22(R)-HCHO) is a crucial precursor of steroids biosynthesis with various biological functions. However, the production of 22(R)-HCHO is expensive and unsustainable due to chemical synthesis and extraction from plants or animals. This study aimed to construct a microbial cell factory to efficiently produce 22(R)-HCHO through systems metabolic engineering. First, we tested 7-dehydrocholesterol reductase (Dhcr7s) and cholesterol C22-hydroxylases from different sources in Saccharomyces cerevisiae, and the titer of 22(R)-HCHO reached 128.30 mg L-1 in the engineered strain expressing Dhcr7 from Columba livia (ClDhcr7) and cholesterol 22-hydroxylase from Veratrum californicum (VcCyp90b27). Subsequently, the 22(R)-HCHO titer was significantly increased to 427.78 mg L-1 by optimizing the critical genes involved in 22(R)-HCHO biosynthesis. Furthermore, hybrid diploids were constructed to balance cell growth and 22(R)-HCHO production and to improve stress tolerance. Finally, the engineered strain produced 2.03 g L-1 of 22(R)-HCHO in a 5-L fermenter, representing the highest 22(R)-HCHO titer reported to date in engineered microbial cell factories. The results of this study provide a foundation for further applications of 22(R)-HCHO in various industrially valuable steroids.

Application of multiple strategies to enhance oleanolic acid biosynthesis by engineered Saccharomyces cerevisiae.

Oleanolic acid and its derivatives are widely used in the pharmaceutical, agricultural, cosmetic and food industries. Previous studies have shown that oleanolic acid production levels in engineered cell factories are low, which is why oleanolic acid is still widely extracted from traditional medicinal plants. To construct a highly efficient oleanolic acid production strain, rate-limiting steps were regulated by inducible promoters and the expression of key genes in the oleanolic acid synthetic pathway was enhanced. Subsequently, precursor pool expansion, pathway refactoring and diploid construction were considered to harmonize cell growth and oleanolic acid production. The multi-strategy combination promoted oleanolic acid production of up to 4.07 g/L in a 100 L bioreactor, which was the highest level reported.

Isotoxic dose escalated radiotherapy for glioblastoma based on diffusion-weighted MRI and tumor control probability-an in-silico study.

OBJECTIVES: Glioblastoma (GBM) is the most common malignant primary brain tumor with local recurrence after radiotherapy (RT), the most common mode of failure. Standard RT practice applies the prescription dose uniformly across tumor volume disregarding radiological tumor heterogeneity. We present a novel strategy using diffusion-weighted (DW-) MRI to calculate the cellular density within the gross tumor volume (GTV) in order to facilitate dose escalation to a biological target volume (BTV) to improve tumor control probability (TCP). METHODS: The pre-treatment apparent diffusion coefficient (ADC) maps derived from DW-MRI of ten GBM patients treated with radical chemoradiotherapy were used to calculate the local cellular density based on published data. Then, a TCP model was used to calculate TCP maps from the derived cell density values. The dose was escalated using a simultaneous integrated boost (SIB) to the BTV, defined as the voxels for which the expected pre-boost TCP was in the lowest quartile of the TCP range for each patient. The SIB dose was chosen so that the TCP in the BTV increased to match the average TCP of the whole tumor. RESULTS: By applying a SIB of between 3.60 Gy and 16.80 Gy isotoxically to the BTV, the cohort's calculated TCP increased by a mean of 8.44% (ranging from 7.19 to 16.84%). The radiation dose to organ at risk is still under their tolerance. CONCLUSIONS: Our findings indicate that TCPs of GBM patients could be increased by escalating radiation doses to intratumoral locations guided by the patient's biology (i.e., cellularity), moreover offering the possibility for personalized RT GBM treatments. ADVANCES IN KNOWLEDGE: A personalized and voxel level SIB radiotherapy method for GBM is proposed using DW-MRI, which can increase the tumor control probability and maintain organ at risk dose constraints.

[Oxidosqualene cyclases in triterpenoids biosynthesis: a review].

Triterpenoids are one of the most diverse compounds in plant metabolites, and they have a wide variety of physiological activities and are of important economic value. Oxidosqualene cyclases catalyze the cyclization of 2, 3-oxidosqualene to generate different types of sterols and plant triterpenoids, which is of great significance to the structural diversity of natural products. However, the mechanism of the diversified cyclization of 2, 3-oxidosqualene catalyzed by oxidosqualene cyclases remains unclear. This review summarized the research progress of oxidosqualene cyclases from the aspects of catalytic function, molecular evolutionary relationship between genes and proteins, protein structure, molecular simulation and molecular calculations, which may provide a reference for protein engineering and metabolic engineering of triterpene cyclase.

Proteomic Response of Bacillus subtilis Spores under High Pressure Combined with Moderate Temperature and Random Peptide Mixture LK Treatment.

In this study, a method of Bacillus subtilis spore inactivation under high pressure (P, 200 MPa) combined with moderate temperature (T, 80 °C) and the addition of antimicrobial peptide LK (102 µg/mL) was investigated. Spores presented cortex hydrolysis and inner membrane (IM) damage with an 8.16 log reduction in response to treatment with PT-LK, as observed by phase-contrast and inverted fluorescence microscopy and flow cytometry (FCM) analysis. Furthermore, a tandem mass tag (TMT) quantitative proteomics approach was utilized because Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis data were used. After treatment with PT-LK, 17,017 polypeptides and 3166 proteins were detected from B. subtilis spores. Among them, 78 proteins showed significant differences in abundance between the PT-LK-treated and control groups, with 49 proteins being upregulated and 29 proteins being downregulated in the PT-LK-treated group. Genetic information processing, metabolism, cellular process, and environmental information processing were the main mechanisms of PT-LK-mediated spore inactivation.

Pre-treatment analysis of non-rigid variations can assist robust intensity-modulated proton therapy plan selection for head and neck patients.

PURPOSE: To incorporate small non-rigid variations of head and neck patients into the robust evaluation of intensity-modulated proton therapy (IMPT) for the selection of robust treatment plans. METHODS: A cohort of 20 nasopharynx cancer patients with weekly kilovoltage CT (kVCT) and 15 oropharynx cancer patients with weekly cone-beam CT (CBCT) were retrospectively included. Anatomical variations between week 0/week 1 of treatment were acquired using deformable image registration (DIR) for all 35 patients and then applied to the planning CT of four patients who have kVCT scanned each week to simulate potential small non-rigid variations (sNRVs). The robust evaluations were conducted on IMPT plans with: (1) different number of beam fields from 3-field to 5-field; (2) different beam angles. The robust evaluation before treatment, including the sNRVs and setup uncertainty, referred to as sNRV+R evaluation was compared with the conventional evaluation (without sNRVs) in terms of robustness consistency with the gold standard evaluation based on weekly CT. RESULTS: Among four patients (490 scenarios), we observed a maximum difference in the sNRV+R evaluation to the nominal dose of: 9.37% dose degradation on D95 of clinical target volumes (CTVs), increase in mean dose (D mean $_{\text{mean}}$ ) of parotid 11.87 Gy, increase in max dose (D max $_{\text{max}}$ ) of brainstem 20.82 Gy. In contrast, in conventional evaluation, we observed a maximum difference to the nominal dose of: 7.58% dose degradation on D95 of the CTVs, increase in parotid D mean $_{\text{mean}}$ by 4.88 Gy, increase in brainstem D max $_{\text{max}}$ by 13.5 Gy. In the measurement of the robustness ranking consistency with the gold standard evaluation, the sNRV+R evaluation was better or equal to the conventional evaluation in 77% of cases, particularly, better on spinal cord, parotid glands, and low-risk CTV. CONCLUSION: This study demonstrated the additional dose discrepancy that sNRVs can make. The inclusion of sNRVs can be beneficial to robust evaluation, providing information on clinical uncertainties additional to the conventional rigid isocenter shift.

Medical Imaging Biomarker Discovery and Integration Towards AI-Based Personalized Radiotherapy.

Intensity-modulated radiation therapy (IMRT) has been used for high-accurate physical dose distribution sculpture and employed to modulate different dose levels into Gross Tumor Volume (GTV), Clinical Target Volume (CTV) and Planning Target Volume (PTV). GTV, CTV and PTV can be prescribed at different dose levels, however, there is an emphasis that their dose distributions need to be uniform, despite the fact that most types of tumour are heterogeneous. With traditional radiomics and artificial intelligence (AI) techniques, we can identify biological target volume from functional images against conventional GTV derived from anatomical imaging. Functional imaging, such as multi parameter MRI and PET can be used to implement dose painting, which allows us to achieve dose escalation by increasing doses in certain areas that are therapy-resistant in the GTV and reducing doses in less aggressive areas. In this review, we firstly discuss several quantitative functional imaging techniques including PET-CT and multi-parameter MRI. Furthermore, theoretical and experimental comparisons for dose painting by contours (DPBC) and dose painting by numbers (DPBN), along with outcome analysis after dose painting are provided. The state-of-the-art AI-based biomarker diagnosis techniques is reviewed. Finally, we conclude major challenges and future directions in AI-based biomarkers to improve cancer diagnosis and radiotherapy treatment.

Separation and purification of plant terpenoids from biotransformation.

The production of plant terpenoids through biotransformation has undoubtedly become one of the research hotspots, and the continuous upgrading of the corresponding downstream technology is also particularly important. Downstream technology is the indispensable technical channel for the industrialization of plant terpenoids. How to efficiently separate high-purity products from complex microbial fermentation broths or enzyme-catalyzed reactions to achieve high separation rates, high returns and environmental friendliness has become the focus of research in recent years. This review mainly introduces the common separation methods of plant terpenoids based on biotransformation from the perspectives of engineering strain construction, unit separation technology, product properties and added value. Then, further attention was paid to the application prospects of intelligent cell factories and control in the separation of plant terpenoids. Finally, some current challenges and prospects are proposed, which provide possible directions and guidance for the separation and purification of terpenoids and even industrialization.

Sustainable production of FAEE biodiesel using the oleaginous yeast Yarrowia lipolytica.

Fatty acid ethyl esters (FAEEs) are fatty acid-derived molecules and serve as an important form of biodiesel. The oleaginous yeast Yarrowia lipolytica is considered an ideal host platform for the production of fatty acid-derived products due to its excellent lipid accumulation capacity. In this proof-of-principle study, several metabolic engineering strategies were applied for the overproduction of FAEE biodiesel in Y. lipolytica. Here, chromosome-based co-overexpression of two heterologous genes, namely, PDC1 (encoding pyruvate decarboxylase) and ADH1 (encoding alcohol dehydrogenase) from Saccharomyces cerevisiae, and the endogenous GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) gene of Y. lipolytica resulted in successful biosynthesis of ethanol at 70.8 mg/L in Y. lipolytica. The engineered Y. lipolytica strain expressing the ethanol synthetic pathway together with a heterologous wax ester synthase (MhWS) exhibited the highest FAEE titer of 360.8 mg/L, which is 3.8-fold higher than that of the control strain when 2% exogenous ethanol was added to the culture medium of Y. lipolytica. Furthermore, a synthetic microbial consortium comprising an engineered Y. lipolytica strain that heterologously expressed MhWS and a S. cerevisiae strain that could provide ethanol as a substrate for the production of the final product in the final engineered Y. lipolytica strain was created in this study. Finally, this synthetic consortium produced FAEE biodiesel at a titer of 4.8 mg/L under the optimum coculture conditions.

Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil.

BACKGROUND: Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. RESULTS: In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. CONCLUSIONS: This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

[Advances in metabolic engineering for the microbial production of naturally occurring terpenes-limonene and bisabolene: a mini review].

Limonene (C10H16) and bisabolene (C15H24) are both naturally occurring terpenes in plants. Depending on the number of C5 units, limonene and bisabolene are recognized as representative monoterpenes and sesquiterpenes, respectively. Limonene and bisabolene are important pharmaceutical and nutraceutical products used in the prevention and treatment of cancer and many other diseases. In addition, they can be used as starting materials to produce a range of commercially valuable products, such as pharmaceuticals, nutraceuticals, cosmetics, and biofuels. The low abundance or yield of limonene and bisabolene in plants renders their isolation from plant sources non-economically viable. Isolation of limonene and bisabolene from plants also suffers from low efficiency and often requires harsh reaction conditions, prolonged reaction times, and expensive equipment cost. Recently, the rapid developments in metabolic engineering of microbes provide a promising alternative route for producing these plant natural products. Therefore, producing limonene and bisabolene by engineering microbial cells into microbial factories is becoming an attractive alternative approach that can overcome the bottlenecks, making it more sustainable, environmentally friendly and economically competitive. Here, we reviewed the status of metabolic engineering of microbes that produce limonene and bisabolene including microbial hosts, key enzymes, metabolic pathways and engineering of limonene/bisabolene biosynthesis. Furthermore, key challenges and future perspectives were discussed.

Photogenerated carriers transfer in dye-graphene-SnO2 composites for highly efficient visible-light photocatalysis.

The visible-light-driven photocatalytic activities of graphene-semiconductor catalysts have recently been demonstrated, however, the transfer pathway of photogenerated carriers especially where the role of graphene still remains controversial. Here we report graphene-SnO2 aerosol nanocomposites that exhibit more superior dye adsorption capacity and photocatalytic efficiency compared with pure SnO2 quantum dots, P25 TiO2, and pure graphene aerosol under the visible light. This study examines the origin of the visible-light-driven photocatalysis, which for the first time links to the synergistic effect of the cophotosensitization of the dye and graphene to SnO2. We hope this concept and corresponding mechanism of cophotosensitization could provide an original understanding for the photocatalytic reaction process at the level of carrier transfer pathway as well as a brand new approach to design novel and versatile graphene-based composites for solar energy conversion.