Metabolic engineering of Pichia pastoris for overproduction of cis-trans nepetalactol.

Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical application is limited due to challenges in plant extraction. Microbial production has emerged as a promising strategy to meet the clinical demands for MIAs. The biosynthetic pathway of cis-trans nepetalactol, which serves as the universal iridoid scaffold for all MIAs, has been successfully identified and reconstituted. However, bottlenecks and challenges remain to construct a high-yielding platform strain for cis-trans nepetalactol production, which is vital for subsequent MIAs biosynthesis. In the present study, we focused on engineering of Pichia pastoris cell factories to enhance the production of geraniol, 8-hydroxygeraniol, and cis-trans nepetalactol. By targeting the biosynthetic pathway from acetyl-CoA to geraniol in both peroxisomes and cytoplasm, we achieved comparable geraniol titers in both compartments. Through protein engineering, we found that either G8H or CPR truncation increased the production of 8-hydroxygeraniol, with a 47.8-fold and 14.0-fold increase in the peroxisomal and cytosolic pathway strain, respectively. Furthermore, through a combination of dynamical control of ERG20, precursor and cofactor supply engineering, diploid engineering, and dual subcellular compartmentalization engineering, we achieved the highest ever reported production of cis-trans nepetalactol, with a titer of 4429.4 mg/L using fed-batch fermentation in a 5-L bioreactor. We anticipate our systematic metabolic engineering strategies to facilitate the development of P. pastoris cell factories for sustainable production of MIAs and other plant natural products.

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.

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.

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.

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.

RETRACTED: Ginsenoside Rh2 inhibits proliferation but promotes apoptosis and autophagy by down-regulating microRNA-638 in human retinoblastoma cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. The journal was initially contacted by the corresponding author to report that the article was submitted without the consent of an author. Given the comments of Dr Elisabeth Bik regarding this article "… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated ", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Overexpression of the tonoplast sugar transporter CmTST2 in melon fruit increases sugar accumulation.

Fruits are an important part of the human diet and sugar content is a major criterion used to evaluate fruit quality. Melon fruit accumulate high sugar concentrations during their development; however, the mechanism through which these sugars are transported into the vacuoles of fruit cells for storage remains unclear. In this study, three tonoplast sugar transporters (TSTs), CmTST1, CmTST2, and CmTST3, were isolated from melon plants. Analysis of subcellular localization revealed that all these proteins were targeted to the tonoplast, and evaluation of spatial expression and promoter-GUS activity indicated that they had different expression patterns in the plant. RT-PCR and qRT-PCR results indicated that CmTST2 exhibited the highest expression level among the TST isoforms during melon fruit development. Histochemical and immunohistochemistry localization experiments were performed to identify the tissue- and cell-type localization of CmTST2 in the fruit, and the results indicated that both its transcription and translation were in the mesocarp and vascular cells. Overexpressing the CmTST2 gene in strawberry fruit and cucumber plants by transient expression and stable transformation, respectively, both increased sucrose, fructose, and glucose accumulation in the fruit. The results indicate that CmTST2 plays an important role in sugar accumulation in melon fruit.

Pumpkin CmHKT1;1 Controls Shoot Na⁺ Accumulation via Limiting Na⁺ Transport from Rootstock to Scion in Grafted Cucumber.

Soil salinity adversely affects the growth and yield of crops, including cucumber, one of the most important vegetables in the world. Grafting with salt-tolerant pumpkin as the rootstock effectively improves the growth of cucumber under different salt conditions by limiting Na⁺ transport from the pumpkin rootstock to the cucumber scion. High-affinity potassium transporters (HKTs) are crucial for the long distance transport of Na⁺ in plants, but the function of pumpkin HKTs in this process of grafted cucumber plants remains unclear. In this work, we have characterized CmHKT1;1 as a member of the HKT gene family in Cucurbita moschata and observed an obvious upregulation of CmHKT1;1 in roots under NaCl stress conditions. Heterologous expression analyses in yeast mutants indicated that CmHKT1;1 is a Na⁺-selective transporter. The transient expression in tobacco epidermal cells and in situ hybridization showed CmHKT1;1 localization at plasma membrane, and preferential expression in root stele. Moreover, ectopic expression of CmHKT1;1 in cucumber decreased the Na⁺ accumulation in the plants shoots. Finally, the CmHKT1;1 transgenic line as the rootstock decreased the Na⁺ content in the wild type shoots. These findings suggest that CmHKT1;1 plays a key role in the salt tolerance of grafted cucumber by limiting Na⁺ transport from the rootstock to the scion and can further be useful for engineering salt tolerance in cucurbit crops.

Comprehensive characterization of a theophylline riboswitch reveals two pivotal features of Shine-Dalgarno influencing activated translation property.

Tuneable gene expression controlled by synthetic biological elements is of great importance to biotechnology and synthetic biology. The synthetic riboswitch is a pivotal type of elements that can easily control the heterologous gene expression in diverse bacteria. In this study, the theophylline-dependent synthetic riboswitch and the corresponding variants with varied spacings between Shine-Dalgarno (SD) sequence and start codon were employed to comprehensively characterize the induction and regulation properties through combining a strong promoter aprE in Bacillus subtilis. Amongst the sets of newly constructed expression elements, the expression element with 9-bp spacing exhibited the higher expression level, a superior induction fold performance, and a considerably lower leaky expression than those with longer or shorter spacings. The riboswitch expression element with 9-bp spacing showed an approximately linear dose dependence from 0 to 8 mM of theophylline. Modification of the SD sequence through the insertion of a single A base prior to the native sequence enables the increase of the expression level post induction while decreasing the induction fold as a result of the elevated leaky level. The riboswitch elements with the engineered SD and the optimal 9-bp spacing exhibit an altered dose dependency in which the approximately linear range shifts to 0-4 mM, although it has a similar profile to the induction process. These results not only provide comprehensive data for the induced expression by a theophylline riboswitch combined with a strong native promoter from B. subtilis but also provide the two pivotal features of SD essential to the modular design of other synthetic riboswitches.

Down-Regulating CsHT1, a Cucumber Pollen-Specific Hexose Transporter, Inhibits Pollen Germination, Tube Growth, and Seed Development.

Efficient sugar transport is needed to support the high metabolic activity of pollen tubes as they grow through the pistil. Failure of transport results in male sterility. Although sucrose transporters have been shown to play a role in pollen tube development, the role of hexoses and hexose transporters is not as well established. The pollen of some species can grow in vitro on hexose as well as on sucrose, but knockouts of individual hexose transporters have not been shown to impair fertilization, possibly due to transporter redundancy. Here, the functions of CsHT1, a hexose transporter from cucumber (Cucumis sativus), are studied using a combination of heterologous expression in yeast (Saccharomyces cerevisiae), histochemical and immunohistochemical localization, and reverse genetics. The results indicate that CsHT1 is a plasma membrane-localized hexose transporter with high affinity for glucose, exclusively transcribed in pollen development and expressed both at the levels of transcription and translation during pollen grain germination and pollen tube growth. Overexpression of CsHT1 in cucumber pollen results in a higher pollen germination ratio and longer pollen tube growth than wild-type pollen in glucose- or galactose-containing medium. By contrast, antisense suppression of CsHT1 leads to inhibition of pollen germination and pollen tube elongation in the same medium and results in a decrease of seed number per fruit and seed size when antisense transgenic pollen is used to fertilize wild-type or transgenic cucumber plants. The important role of CsHT1 in pollen germination, pollen tube growth, and seed development is discussed.

Enhancement of a high efficient autoinducible expression system in Bacillus subtilis by promoter engineering.

Quorum-sensing related promoter srfA (PsrfA) was used to construct autoinducible expression system for production of recombinant proteins in Bacillus subtilis. PsrfA was prominent in the unique property of inducer-free activity that is closely correlated with cell density. Here, using green fluorescent protein (GFP) as the reporter protein, PsrfA was optimized by shortening its sequences and changing the nucleotides at the conserved regions of -35 -15 and -10 regions, obtaining a library of PsrfA derivatives varied in the strength of GFP production. Among all the promoter mutants, the strongest promoter P10 was selected and the strength in GFP expression was 150% higher than that of PsrfA. Heterologous protein of aminopeptidase and nattokinase could be overexpressed by P10, the activities of which were 360% and 50% higher than that of PsrfA, respectively. These results suggested that the enhanced promoter P10 could be used to develop autoinducible expression system for overexpression of heterologous proteins in B. subtilis.

Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis.

BACKGROUND: Bacillus subtilis, a Gram-positive organism, has been developed to be an attractive expression platform to produce both secreted and cytoplasmic proteins owing to its prominent biological characteristics. We previously developed an auto-inducible expression system containing the srfA promoter (PsrfA) which was activated by the signal molecules acting in the quorum-sensing pathway for competence. The P srfA promoter exhibited the unique property of inducer-free activity that is closely correlated with cell density. RESULTS: To improve the PsrfA-mediated expression system to the high-cell-density fermentation for industrial production in the B. subtilis mutant strain that is unable to sporulate, a spore mutant strain BSG1682 was developed, and the PsrfA promoter was enhanced by promoter engineering. Using green fluorescent protein (GFP) as the reporter, higher fluorescent intensity was observed in BSG1682 with expression from either plasmid or chromosome than that of the wild type B. subtilis 168. Thereafter, the PsrfA was engineered, yielding a library of PsrfA derivatives varied in the strength of GFP expression. The P23 promoter exhibited the best performance, almost twofold stronger than that of P srfA. Two heterologous proteins, aminopeptidase (AP) and nattokinase (NK), were successfully overproduced under the control of P23 in BSG1682. Finally, the capacity of the expression system was demonstrated in batch fermentation in a 5-L fermenter. CONCLUSIONS: The expression system demonstrates prominence in the activity of the auto-inducible promoter. Desired proteins could be highly and stably produced by integrating the corresponding genes downstream of the promoter on the plasmid or the chromosome in strain BSG1682. The expression system is conducive to the industrial production of pharmaceuticals and heterologous proteins in high-cell-density fermentation in BSG1682.

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch.

BACKGROUND: Synthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches. RESULTS: A novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43'-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43'-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed ß-glucuronidase at equal levels, and induction ratio was similar to that of GFP. CONCLUSION: The constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.

Construction and development of an auto-regulatory gene expression system in Bacillus subtilis.

BACKGROUND: Bacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA). RESULTS: The promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the -10 and -35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level. CONCLUSION: The auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression system that has an expression pattern that is split between cell-growth and over-expression, leading to an increase in cell density and elevating the overall expression levels of heterologously expressed proteins. The broad applicability of this system and inducer-free expression property in B. subtilis facilitate the industrial scale-up and medical applications for the over-production of a variety of desired proteins.

MiR-124 protects human hepatic L02 cells from H2O2-induced apoptosis by targeting Rab38 gene.

BACKGROUND: Hepatic ischemia reperfusion injury (IRI) is an inevitable clinical problem for liver surgeons. Because microRNAs (miRNAs) participate in various hepatic pathophysiological processes, this study aimed to explore the role and potential mechanism of miR-124 in hepatic IRI. METHODS: A liver IRI model was established in rats. The differential expression of miRNAs was detected using microarrays, and the expression of miR-124 was measured by qRT-PCR. A hydrogen peroxide (H2O2)-induced oxidative stress apoptosis model was also established. Cell apoptosis was detected by flow cytometry, and viability was detected by CCK8. The expression of Rab38 was detected by Western blotting and qRT-PCR, and a luciferase reporter assay was used to verify the expression of the miR-124 target gene. RESULTS: The miRNA spectrum changes dramatically after hepatic IRI in rats, and miR-124 is significantly down-regulated after liver IRI. MiR-124 decreases the H2O2-induced apoptosis of human hepatic L02 cells by up-regulating the activation of the AKT pathway. Rab38 is a target gene of miR-124 and is involved in H2O2-induced apoptosis. Interference with the expression of the Rab38 gene can protect hepatic L02 from H2O2-induced apoptosis by increasing the phosphorylation of AKT. These protective effects of miR-124 are attenuated by over-expression of Rab38. CONCLUSIONS: Many miRNAs are involved in hepatic IRI in rats, and miR-124 is significantly decreased in this model. MiR-124 significantly decreases the H2O2-induced apoptosis of human hepatic L02 cells by targeting the Rab38 gene and activating the AKT pathway.

Antisense suppression of cucumber (Cucumis sativus†L.) sucrose synthase 3 (CsSUS3) reduces hypoxic stress tolerance.

Sucrose synthase (SUS; EC 2.4.1.13) plays important roles in sugar metabolism and abiotic stress response. But the genes encoding SUS in cucumber (Cucumis sativus L.) have not been well studied. Here, we isolated four cucumber sucrose synthase genes (CsSUS). Among them, CsSUS3, which highly expressed in the roots, was chosen for further study. Immunolocalization and subcellular localization analysis indicated that CsSUS3 localized in the cytosol and the plasma membrane, and mainly existed in the companion cells of phloem in the roots. When suffering hypoxia stress from flooding, CsSUS3 expression and SUS activity in roots increased, especially in the lateral roots; moreover, the soluble SUS activity increased clearly, but the membrane fraction hardly changed. Compared with the wild-type cucumbers, the transgenic lines with antisense expression of CsSUS3 were more sensitive to flooding. After 6 d of flooding, the SUS activity, soluble sugar and uridine 5'-diphosphate glucose (UDPG) content and the ratio of ATP/ADP in the roots of transgenic plants were significantly lower than that in wild-type plants. Moreover, the transgenic lines grew more slowly with more yellow necrosis in the leaves. These findings suggested CsSUS3 participated in resisting hypoxic stress. Furthermore, the mechanism of CsSUS3 in resisting hypoxic stress was also discussed.

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.