Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.

Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for C1 biotransformation. Unfortunately, the lack of synthetic biology tools in P. pastoris limits its broad applications, particularly when multigene pathways should be manipulated. Here, the CRISPR/Cas9 system is established to efficiently integrate multiple heterologous genes to construct P. pastoris cell factories. In this protocol, with the 2,3-butanediol (BDO) biosynthetic pathway as a representative example, the procedures to construct P. pastoris cell factories are detailed using the established CRISPR-based multiplex genome integration toolkit, including donor plasmid construction, competent cell preparation and transformation, and transformant verification. The application of the CRISPR toolkit is demonstrated by the construction of engineered P. pastoris for converting methanol to BDO. This lays the foundation for the construction of P. pastoris cell factories harboring multi-gene biosynthetic pathways for the production of high-value compounds.

Phloem unloading in cultivated melon fruits follows an apoplasmic pathway during enlargement and ripening.

Melon (Cucumis melo L.) has a long history of cultivation worldwide. During cultivation, domestication, and selection breeding, the sugar content of mature melon fruits has been significantly increased. Compared with unsweet melon and wild melon, rapid sucrose accumulation can occur in the middle and late stages of sweet melon fruit development. The phloem unloading pathway during the evolution and development of melon fruit has not been identified and analyzed. In this study, the phloem unloading pathway and the function of related sugar transporters in cultivated and wild melon fruits were analyzed by CFDA [5(6)-carbofluorescein diacetate] and esculin tracing, cytological pathway observation, qRT-PCR, and gene function analysis, etc. Results show that the phloem unloading pathway of wild melon fruit is largely symplastic, whereas the phloem unloading pathway of cultivated melon fruit shifts from symplastic to apoplasmic during development. According to a fruit grafting experiment, the fruit sink accumulates sugars independently. Correlation analysis showed that the expression amounts of several sucrose transporter genes were positively correlated with the sucrose content of melon fruit. Furthermore, CmSWEET10 was proved to be a sucrose transporter located on the plasma membrane of the phloem and highly expressed in the premature stage of sweet melon fruits, which means it may be involved in phloem apoplast unloading and sucrose accumulation in sweet melon fruits. Finally, we summarize a functional model of related enzymes and sugar transporters involved in the apoplast unloading of sweet melon fruits during enlargement and sucrose accumulation.

Vacuolar sugar transporter EARLY RESPONSE TO DEHYDRATION6-LIKE4 affects fructose signaling and plant growth.

Regulation of intracellular sugar homeostasis is maintained by regulation of activities of sugar import and export proteins residing at the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 (ERDL4) protein, a member of the monosaccharide transporter family, resides in the vacuolar membrane in Arabidopsis (Arabidopsis thaliana). Gene expression and subcellular fractionation studies indicated that ERDL4 participates in fructose allocation across the tonoplast. Overexpression of ERDL4 increased total sugar levels in leaves due to a concomitantly induced stimulation of TONOPLAST SUGAR TRANSPORTER 2 (TST2) expression, coding for the major vacuolar sugar loader. This conclusion is supported by the finding that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. ERDL4 activity contributing to the coordination of cellular sugar homeostasis is also indicated by 2 further observations. First, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, and second, the ERDL4 gene is markedly expressed during cold acclimation, representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger rosettes and roots, a delayed flowering time, and increased total seed yield. Consistently, erdl4 knockout plants show impaired cold acclimation and freezing tolerance along with reduced plant biomass. In summary, we show that modification of cytosolic fructose levels influences plant organ development and stress tolerance.

Messenger RNA electroporated hepatitis B virus (HBV) antigen-specific T cell receptor (TCR) redirected T cell therapy is well-tolerated in patients with recurrent HBV-related hepatocellular carcinoma post-liver transplantation: results from a phase I trial.

BACKGROUND AND AIMS: Liver transplantation (LT) is the primary curative option for cirrhotic patients with early-stage hepatocellular carcinoma (HCC). However, tumor recurrence occurs in 15-20% of cases with unfavorable prognosis. We have developed a library of T cell receptors (TCRs) specific for different hepatitis B virus (HBV) antigens, restricted by different molecules of human leucocyte antigen (HLA)-class I, to redirect T cells against HBV antigens (Banu in Sci Rep 4:4166, 2014). We further demonstrated that these transiently functional T cells specific for HBV obtained through messenger RNA (mRNA) electroporation can eliminate HCC cells expressing HBV antigens in vitro and in vivo (Kah in J Clin Invest 127:3177-3188, 2017). A phase I clinical trial for patients with HCC recurrence post-liver transplant was conducted to assess the safety, tolerability, and anti-tumor efficacy of transiently functional HBV-TCR T cells. Here, we report the clinical findings with regard to the safety and anti-tumor efficacy of mRNA electroporated HBV-specific TCR-T cells. (ClinicalTrials.gov identifier: NCT02719782). PATIENTS AND METHODS: A total of six patients with HBV-positive recurrent HCC post-liver transplant and HLA-matched to TCR targeting hepatitis B surface antigen (HBsAg) or hepatitis B core antigen (HBcAg) (HLA-A*02:01/HBsAg, HLA-A*11:01/HBcAg, HLA-B*58:01/HBsAg or HLA-C*08:01/HBsAg) were enrolled in this study. The primary objective was to assess the safety of short-lived mRNA electroporated HBV-TCR T cells based on the incidence and severity of the adverse event (AE) graded per National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), Version 4.0. The secondary objective was to determine the effectiveness of HBV-TCR T cells as per RECIST 1.1 criteria. Patients were followed up for survival for 2 years post-end of treatment. RESULTS: The median age of the six patients was 35.5 years (range: 28-47). The median number of HBV-TCR T cell infusions administered was 6.5 (range: 4-12). The treatment-related AE included grade 1 pyrexia. This study reported no cytokine release syndrome nor neurotoxicity. One patient remained alive and five were deceased at the time of the data cutoff (30 April 2020). CONCLUSION: This study has demonstrated that multiple infusions of mRNA electroporated HBV-specific TCR T cells were well-tolerated in patients with HBV-positive recurrent HCC post-liver transplant.

Comprehensive investigation of long non-coding RNAs in an endophytic fungus Calcarisporium arbuscula NRRL 3705.

Long non-coding RNAs (lncRNAs) play an important role in eukaryotic cells. However, there is no report of lncRNAs in endophytic fungi Calcarisporium arbuscula. Here, in Calcarisporium arbuscula NRRL 3705, an endophytic fungus predominantly producing mycotoxins aurovertins, the genome-wide identification of lncRNAs was carried out based on RNA-Seq. Totally, 1332 lncRNAs were identified, including 1082 long intergenic noncoding RNAs, 64 long intronic noncoding RNAs and 186 long noncoding natural antisense transcripts. The average length of lncRNA and mRNA were 254 and 1102 bp, respectively. LncRNAs were shorter, with fewer exons and lower expression levels. Moreover, there were 39 up-regulated lncRNAs and 10 down-regulated lncRNAs in the ΔaurA mutant, which lacks the aurovertin biosynthetic enzyme AurA. Interestingly, expression of genes related to the metabolism of linoleic acid and methane were significantly down regulated in the ΔaurA mutant. This study enriches the endophytic fungal lncRNA database and provide a basis for further research.

Yolo-Pest: An Insect Pest Object Detection Algorithm via CAC3 Module.

Insect pests have always been one of the main hazards affecting crop yield and quality in traditional agriculture. An accurate and timely pest detection algorithm is essential for effective pest control; however, the existing approach suffers from a sharp performance drop when it comes to the pest detection task due to the lack of learning samples and models for small pest detection. In this paper, we explore and study the improvement methods of convolutional neural network (CNN) models on the Teddy Cup pest dataset and further propose a lightweight and effective agricultural pest detection method for small target pests, named Yolo-Pest, for the pest detection task in agriculture. Specifically, we tackle the problem of feature extraction in small sample learning with the proposed CAC3 module, which is built in a stacking residual structure based on the standard BottleNeck module. By applying a ConvNext module based on the vision transformer (ViT), the proposed method achieves effective feature extraction while keeping a lightweight network. Comparative experiments prove the effectiveness of our approach. Our proposal achieves 91.9% mAP0.5 on the Teddy Cup pest dataset, which outperforms the Yolov5s model by nearly 8% in mAP0.5. It also achieves great performance on public datasets, such as IP102, with a great reduction in the number of parameters.

Comparative analysis of sugar, acid, and volatile compounds in CPPU-treated and honeybee-pollinated melon fruits during different developmental stages.

Plant growth regulator N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) is widely used in fruit production. However, the mechanism in which CPPU affects melon fruit quality, especially aroma compound, remains unclear. Here, gas chromatography-mass spectrometry was performed to detect the sugar, citric acid, and aroma content in CPPU-treated and pollinated melon fruit. Results showed that the application of CPPU decreased the sugar and aroma content in melon fruit. The relative content of several important esters, including isobutyl acetate, ethyl acetate, 2-methylbutyl acetate, methyl acetate, benzyl acetate, and phenethyl acetate, in CPPU-treated fruits was significantly lower than that in honeybee-pollinated fruits. The content of many amino acids (isoleucine, leucine, valine, methionine, and l-phenylalanine), which could be metabolized into aroma compounds, in CPPU-treated fruits was significantly higher than that in honeybee-pollinated fruits. In conclusion, CPPU application interferes with amino-acid metabolism and affects the production of aromatic esters in melon fruit.

Development of a Pichia pastoris cell factory for efficient production of germacrene A: a precursor of ß-elemene.

ß-Elemene, an active ingredient found in medicinal plants like turmeric and zedoary, is a sesquiterpene compound with antitumor activity against various cancers. However, its current mode of production through plant extraction suffers from low efficiency and limited natural resources. Recently, there has been an increased interest in establishing microbial cell factories to produce germacrene A, which can be converted to ß-elemene by a one-step reaction in vitro. In this study, we constructed an engineered Pichia pastoris cell factory for producing germacrene A. We rerouted the fluxes towards germacrene A biosynthesis through the optimization of the linker sequences between germacrene A synthase (GAS) and farnesyl pyrophosphate synthase (ERG20), overexpression of important pathway genes (i.e., IDI1, tHMG1, and ACS), and multi-copy integration of related expression cassettes. In combination with medium optimization and bioprocess engineering, the final titer of germacrene A in a 1 L fermenter reached 1.9 g/L through fed-batch fermentation. This represents the first report on the production of germacrene A in P. pastoris and demonstrates its advantage in producing terpenoids and other value-added natural products.

Improving the immunomodulatory function of mesenchymal stem cells by defined chemical approach.

Mesenchymal stem cell (MSC) is a potential therapeutic material that has self-renewal, multilineage differentiation, and immunomodulation properties. However, the biological function of MSCs may decline due to the influence of donor differences and the in vitro expansion environment, which hinders the advancement of MSC-based clinical therapy. Here, we investigated a method for improving the immunomodulatory function of MSCs with the help of small-molecule compounds, A-83-01, CHIR99021, and Y27632 (ACY). The results showed that small-molecule induced MSCs (SM-MSCs) could enhance their immunosuppressive effects on T cells and macrophages. In vivo studies showed that, in contrast to control MSCs (Ctrl-MSCs), SM-MSCs could inhibit the inflammatory response in mouse models of delayed hypersensitivity and acute peritonitis more effectively. In addition, SM-MSCs showed the stronger ability to inhibit the infiltration of pro-inflammatory T cells and macrophages. Thus, small-molecule compounds ACY could better promote the immunomodulatory effect of MSCs, indicating it could be a potential improving method in MSC culture.

Comparative analysis of pumpkin rootstocks mediated impact on melon sensory fruit quality through integration of non-targeted metabolomics and sensory evaluation.

Melon fruits are popular because of sweet taste and pleasant aroma. Grafting has been extensively used for melons to alleviate abiotic stresses and control soil borne diseases. However, use of grafting for vegetable fruit quality improvement is less studies. In modern age fruit quality particularly sensory quality characteristics have key importance from consumer eye lens. We performed liquid chromatography-mass spectrometry and metabonomic analysis to examine sensory fruit quality of melon grafted onto ten different pumpkin rootstocks. Bases on the result of our study, 478 metabolites were detected and 184 metabolites consisting of lipids, amino acids and organic oxygen compounds were differentially expressed in grafted melon fruits. The results from metabolomic, physiochemical and sensory analysis explain the differences in melon fruit flavor from two contrasting rootstocks. In conclusion the fruits from Tianzhen No. 1 rootstock exhibited better organoleptic characteristics and higher soluble sugars content [glucose (19.87 mg/g), fructose (19.68 mg/g) and sucrose (169.45 mg/g)] compared with other rootstocks used in this study. Moreover, the contents of bitterness causing amino acids such as L-arginine, L-asparagine, Histidinyl-histidine and Acetyl-DL-valine were found lower in Tianzhen No. 1-grafted melon fruits compared with Sizhuang No. 12-grafted melon fruits. These fruit quality characteristics made Tianzhen No. 1 rootstock suitable for commercial cultivation of Yuniang melon.

A Small-Molecule Cocktails-Based Strategy in Culture of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have a variety of unique properties, such as stem cell multipotency and immune regulation, making them attractive for use in cell therapy. Before infusion therapy, MSCs are required to undergo tissue separation, purification, and expansion in vitro for a certain duration. During the process of in vitro expansion of MSCs, the influence of culture time and environment can lead to cell senescence, increased heterogeneity, and function attenuation, which limits their clinical applications. We used a cocktail of three small-molecule compounds, ACY (A-83-01, CHIR99021, and Y-27632), to increase the proliferation activity of MSCs in vitro and reduce cell senescence. ACY inhibited the increase in heterogeneity of MSCs and conserved their differentiation potential. Additionally, ACY maintained the phenotype of MSCs and upregulated the expression of immunomodulatory factors. These results suggest that ACY can effectively improve the quantity and quality of MSCs.

Contributions of sugar transporters to crop yield and fruit quality.

The flux, distribution, and storage of soluble sugars regulate crop yield in terms of starch, oil, protein, and total carbohydrates, and affect the quality of many horticultural products. Sugar transporters contribute to phloem loading and unloading. The mechanisms of phloem loading have been studied in detail, but the complex and diverse mechanisms of phloem unloading and sugar storage in sink organs are less explored. Unloading and subsequent transport mechanisms for carbohydrates vary in different sink organs. Analyzing the transport and storage mechanisms of carbohydrates in important storage organs, such as cereal seeds, fruits, or stems of sugarcane, will provide information for genetic improvements to increase crop yield and fruit quality. This review discusses current research progress on sugar transporters involved in carbohydrate unloading and storage in sink organs. The roles of sugar transporters in crop yield and the accumulation of sugars are also discussed to highlight their contribution to efficient breeding.

A target and efficient synthetic strategy for structural and bioactivity optimization of a fungal natural product.

Drugs have been largely inspired from natural products, while enzymes underlying their biosynthesis have enabled complex structures and diverse bioactivities. Nevertheless, the high enzyme specificity and limited in vivo precursor types have restricted the natural product reservoir, but Nature has imprinted natural products with active sites, which can be readily modified by chemosynthesis with various functional groups for more favorable druggability. Here in the less exploited fungal natural products, we introduced CtvA, a polyketide synthase for a mycotoxin citreoviridin biosynthesis in Aspergillus, into an endophytic fungus Calcarisporium arbuscula to expand tetrahydrofuran (THF) into a dioxabicyclo-octane (DBO) ring moiety based on versatility and promiscuity of the aurovertin biosynthetic enzyme. Alternative acylations on the hydroxyl groups essential for cell toxicity by chemosynthesis produced compounds with improved anti-tumor activities and pharmacokinetics. Thus, we showed an effective strategic way to optimize the fungal natural product efficiently for more promising drug development.

Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms.

There is a growing interest in establishing the methylotrophic yeast Pichia pastoris as microbial cell factories for producing fuels, chemicals, and natural products, particularly with methanol as the feedstock. Although CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technology has been established for the integration of multigene biosynthetic pathways, long (500-1000 bp) homology arms are generally required, probably due to low homologous recombination (HR) efficiency in P. pastoris. To achieve efficient genome integration of heterologous genes with short homology arms, we aimed to enhance HR efficiency by introducing the recombination machinery from Saccharomyces cerevisiae. First, we overexpressed HR related genes, including RAD52, RAD59, MRE11, and SAE2, and evaluated their effects on genome integration efficiency. Then, we constructed HR efficiency enhanced P. pastoris, which enabled single-, two-, and three-loci integration of heterologous gene expression cassettes with ∼40 bp homology arms with efficiencies as high as 100%, ∼98%, and ∼81%, respectively. Finally, we demonstrated the construction of ß-carotene producing strain and the optimization of betaxanthin producing strain in a single step. The HR efficiency enhanced P. pastoris strains can be used for the construction of robust cell factories, and our machinery engineering strategy can be employed for the modification of other nonconventional yeasts.

Discovery of a Potential Liver Fibrosis Inhibitor from a Mushroom Endophytic Fungus by Genome Mining of a Silent Biosynthetic Gene Cluster.

Liver fibrosis has accounted for liver diseases and overall mortality, but no relevant drug has been developed. Filamentous fungi are important resources of natural products for pharmaceutical development. Calcarisporium arbuscula is a mushroom endophytic fungus, which primarily produces aurovertins. Here, in an aurovertin null-production mutant, one silent gene cluster (mca17) was activated by overexpression of a pathway-specific zinc finger transcriptional regulator, and a tetramic acid-type compound (1, MCA17-1) was identified. Along with detailed structural characterization, its biosynthesis was proposed to be produced from the core PKS-NRPS hybrid enzyme. Moreover, 1 suppressed the activation of LX-2 upon transforming growth factor-ß (TGF-ß) challenge and had stronger bioactivity than the positive control obeticholic acid (OCA) against liver fibrosis. Our work suggested that this engineered fungus could be a producer of 1 for promising pharmaceutical development, and alternatively, it would be developed as a mushroom ingredient in dietary therapy to prevent liver fibrosis.

A Cell Factory of a Fungicolous Fungus Calcarisporiumarbuscula for Efficient Production of Natural Products.

Fungal natural products are rich sources of clinical drugs. Particularly, the fungicolous fungi have a large number of biosynthetic gene clusters (BGCs) to produce numerous bioactive natural products, but most BGCs are silent in the laboratory. We have shown that a fungicolous fungus Calcarisporiumarbuscula NRRL 3705 predominantly produces the highly reduced polyketide-type mycotoxins aurovertins. Here after evaluation of the aurovertin-null mutant ΔaurA as an efficient host, we further screened two strong promoters aurBp and A07068p based on RNA-Seq, and successfully activated an endogenous gene cluster from C. arbuscula as well as three additional exogenous BGCs from other fungi to produce polyketide-type natural products. Thus, we showed an efficient expression system from the fungicolous fungus C. arbuscula, which will be highly beneficial and complementary to the conventional Aspergillus and Penicillium fungal cell factories, and provides a useful toolkit for genome-wide mining of bioactive natural products from fungicolous fungi.

Genomic and transcriptomic survey of an endophytic fungus Calcarisporium arbuscula NRRL 3705 and potential overview of its secondary metabolites.

BACKGROUND: Secondary metabolites as natural products from endophytic fungi are important sources of pharmaceuticals. However, there is currently little understanding of endophytic fungi at the omics levels about their potential in secondary metabolites. Calcarisporium arbuscula, an endophytic fungus from the fruit bodies of Russulaceae, produces a variety of secondary metabolites with anti-cancer, anti-nematode and antibiotic activities. A comprehensive survey of the genome and transcriptome of this endophytic fungus will help to understand its capacity to biosynthesize secondary metabolites and will lay the foundation for the development of this precious resource. RESULTS: In this study, we reported the high-quality genome sequence of C. arbuscula NRRL 3705 based on Single Molecule Real-Time sequencing technology. The genome of this fungus is over 45 Mb in size, larger than other typical filamentous fungi, and comprises 10,001 predicted genes, encoding at least 762 secretory-proteins, 386 carbohydrate-active enzymes and 177 P450 enzymes. 398 virulence factors and 228 genes related to pathogen-host interactions were also predicted in this fungus. Moreover, 65 secondary metabolite biosynthetic gene clusters were revealed, including the gene cluster for the mycotoxin aurovertins. In addition, several gene clusters were predicted to produce mycotoxins, including aflatoxin, alternariol, destruxin, citrinin and isoflavipucine. Notably, two independent gene clusters were shown that are potentially involved in the biosynthesis of alternariol. Furthermore, RNA-Seq assays showed that only expression of the aurovertin gene cluster is much stronger than expression of the housekeeping genes under laboratory conditions, consistent with the observation that aurovertins are the predominant metabolites. Gene expression of the remaining 64 gene clusters for compound backbone biosynthesis was all lower than expression of the housekeeping genes, which partially explained poor production of other secondary metabolites in this fungus. CONCLUSIONS: Our omics data, along with bioinformatics analysis, indicated that C. arbuscula NRRL 3705 contains a large number of biosynthetic gene clusters and has a huge potential to produce a profound number of secondary metabolites. This work also provides the basis for development of endophytic fungi as a new resource of natural products with promising biological activities.

Overexpression of Melon Tonoplast Sugar Transporter CmTST1 Improved Root Growth under High Sugar Content.

Sugar allocation is based on the source-to-sink and intracellular transport between different organelles, and sugar transporters are usually involved in these processes. Tonoplast sugar transporters (TST) are responsible for transporting sugar into vacuoles; however, the role of TSTs in root growth and the response to abiotic stress is poorly studied. Here, RNA analysis and promoter-ß-glucuronidase staining revealed that a melon TST1 gene (CmTST1) is highly expressed in the roots. The sugar feeding experiment results showed that the expression of CmTST1 in the roots was induced by a relatively high level of sucrose (6%), glucose (3%), and fructose (3%). The ectopic overexpression of CmTST1 in Arabidopsis improved the root and shoot growth of seedlings under high exogenous sugar stress. Furthermore, the ectopic expression of CmTST1 promoted the expression of plasma membrane-located sugar transporters. We proposed that CmTST1 plays a key role in importing sugar transport into the vacuoles of roots in response to metabolic demands to maintain cytosolic sugar homeostasis.

Realization of Robust and Precise Regulation of Gene Expression by Multiple Sigma Recognizable Artificial Promoters.

Precise regulation of gene expression is fundamental for tailor-made gene circuit design in synthetic biology. Current strategies for this type of development are mainly based on directed evolution beginning with a native promoter template. The performances of engineered promoters are usually limited by the growth phase because only one promoter is recognized by one type of sigma factor (σ). Here, we constructed multiple-σ recognizable artificial hybrid promoters (AHPs) composed of tandems of dual and triple natural minimal promoters (NMPs). These NMPs, which use σA, σH and σW, had stable functions in different growth phases. The functions of these NMPs resulted from an effect called transcription compensation, in which AHPs sequentially use one type of σ in the corresponding growth phase. The strength of the AHPs was influenced by the combinatorial order of each NMP and the length of the spacers between the NMPs. More importantly, the output of the precise regulation was achieved by equipping AHPs with synthetic ribosome binding sites and by redesigning them for induced systems. This strategy might offer promising applications to rationally design robust synthetic promoters in diverse chassis to spur the construction of more complex gene circuits, which will further the development of synthetic biology.