Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on ß-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

Effect of resveratrol on herpesvirus encephalitis: Evidences for its mechanisms of action.

BACKGROUND: Herpes simplex virus type 1 (HSV-1)-induced herpes simplex encephalitis (HSE) has a high mortality rate in clinically immunocompromised patients, while recovered patients often experience neurological sequelae due to neuroinflammation. Nucleoside drugs and nucleoside analogues such as acyclovir and ganciclovir are mainly used in clinical treatment, and the emergence of resistant viral strains makes the development of new anti-herpesvirus encephalitis drugs urgent. Resveratrol is a multifunctional, plant-derived bioactive compound and its antiviral potential is attracting much attention. PURPOSE: This study aimed to investigate the anti-HSV-1 mechanism of resveratrol in microglial cells and in the HSE mouse model. METHODS: The antiviral effect of resveratrol on HSV-1 infection was investigated by plaque assay, virus titer, immunofluorescence, Western blot and time-of-addition assay. The influence of resveratrol on stimulator of interferon gene (STING)/Nuclear Factor kappa B (NF-κB) signaling pathway-mediated neuroinflammation was examined by Western blot, RT-qPCR and ELISA. The interaction between resveratrol and STING/heat shock protein 90 beta (HSP90ß) was evaluated by molecular modeling, co-immunoprecipitation, and drug affinity responsive target stability assay. The therapeutic effect of resveratrol on HSE was evaluated in the HSE mouse model by analyzing weight loss, neurodegenerative symptoms and histopathological scores. RESULTS: Resveratrol inhibited the early process of HSV-1 infection, and interfered with the STING/NF-κB signaling pathway to attenuate HSV-1-induced neuroinflammation and microglial M1 polarization, independent of its classical target Sirtuin1. Mechanistically, resveratrol completely bound to Glu515 and Lys491 of HSP90ß, thus disrupting the HSP90ß-STING interaction and promoting STING degradation. Resveratrol also significantly alleviated viral encephalitis and neuroinflammation caused by HSV-1 in the HSE mouse model. CONCLUSION: Resveratrol acted as a non-classical HSP90ß inhibitor, binding to the STING-HSP90ß interaction site to promote STING degradation and attenuate HSV-1-induced encephalitis and neuroinflammation. These findings suggest the alternative strategy of targeting HSP90ß and resveratrol-mediated inhibition of HSP90ß as a potential antiviral approach.

MiR-339-5p inhibits replication of porcine reproductive and respiratory syndrome virus by targeting viral gene regions.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a variable virus, whose spread cannot be totally stopped by vaccination. PRRSV infection results in abortion and respiratory symptoms in pregnant pigs. One crucial component of the anti-viral infection strategy is microRNA (miRNA), a class of multifunctional small molecules. It is unknown whether miR-339-5p can specifically target the PRRSV gene and prevent the virus from replicating, despite the fact that miR-339-5p is markedly up-regulated during the PRRSV infection. In this pursuit, the present study revealed that the two PRRSV areas targeted by miR-339-5p were PRRSV nsp2-3378 to 3403 and PRRSV nsp2-3112 to 3133 using the miRanda program. Dual luciferase reporter assays showed that the miR-339-5p target region of the PRRSV gene sequence exhibited 100% homology and was highly conserved. Furthermore, the ability of miR-339-5p to target PRRSV gene areas was verified. It was found that the overexpression of miR-339-5p markedly reduced the PRRSV replication through PRRSV infection trials. The precursor sequence of ssc-miR-339-5p was amplified using the DNA of pig lung tissue as a template in order to create a fragment of 402 bp of porcine-derived miR-339-5p precursor sequence, which was then used to produce the eukaryotic expression plasmid of miR-339-5p. In conclusion, miR-339-5p can target the specific PRRSV gene areas and prevent PRRSV replication, offering fresh perspectives for the creation of medications that combat the PRRSV infection.

Peroxisome-based metabolic engineering for biomanufacturing and agriculture.

Subcellular compartmentalization of metabolic pathways plays a crucial role in metabolic engineering. The peroxisome has emerged as a highly valuable and promising compartment for organelle engineering, particularly in the fields of biological manufacturing and agriculture. In this review, we summarize the remarkable achievements in peroxisome engineering in yeast, the industrially popular biomanufacturing chassis host, to produce various biocompounds. We also review progress in plant peroxisome engineering, a field that has already exhibited high potential in both biomanufacturing and agriculture. Moreover, we outline various experimentally validated strategies to improve the efficiency of engineered pathways in peroxisomes, as well as prospects of peroxisome engineering.

Acute toxicological evaluation of AT-533 and AT-533 gel in Sprague-Dawley rats.

BACKGROUND: AT-533 is a novel heat shock protein 90 inhibitor that exerting anti-inflammatory, antiviral, and antitumor efficacy. Furthermore, the gel made of AT-533 as raw material named AT-533 gel has the function of repairing keratitis and dermatitis caused by herpes virus infection. However, the acute safety evaluation of AT-533 and AT-533 gel has not been conducted. METHODS AND RESULTS: Herein, we performed acute toxicological studies of AT-533 and AT-533 gel in Sprague-Dawley rats. Fifteen-day acute toxicity study of AT-533 was conducted in both male and female Sprague-Dawley rats at doses of 5, 50, 250 and 500 mg/kg and AT-533 gel at 5 g/kg in the study. During experiment, food consumption and mortality were observed and body weight, hematology, serum biochemistry and histopathological assessment of rats were carried out. No abnormal changes were observed in rats percutaneously treated with AT-533 at 5 mg/kg and 50 mg/kg and AT-533 gel. However, loss of appetite and body weight, adverse reactions, toxicologically relevant alterations in hematology and biochemistry were found in rats percutaneously treated with AT-533 at 250 mg/kg and 500 mg/kg during 15-day acute dermic toxicity study. CONCLUSIONS: The aforementioned results suggested that the LD50 of AT-533 is 228.382 mg/kg and the LD50 of AT-533 gel is greater than 5 g/kg. These findings indicated that AT-533 is non-toxic in rats when the dose less than 50 mg/kg and AT-533 gel can be considered a gel with no toxicity at doses less than 5 g/kg.

Activity-guided isolation and identification of antiherpesvirus and antineuroinflammatory active terpenoids from Artemisia vulgaris L. based on the LC-MS/MS molecular network.

Seven undescribed terpenoids, comprising two guaiane-type sesquiterpene lactones (1-2), one eucalyptol-type sesquiterpene (3), one monolactone (4), and three triterpenoids (5-7), along with 35 known analogues, were isolated from the leaves of Artemisia vulgaris L. Their structures and configurations were analysed by extensive spectroscopy. Compounds 1, 2, 8-10, 13, 17, 19, and 28 showed antineuroinflammatory activity, and compounds 1 and 2 revealed remarkable antineuroinflammatory effects, with an IC50 value of 2.2 ± 0.1 and 1.6 ± 0.1 µM, more potent than the positive control drug dexamethasone. Furthermore, compounds 1 and 2 could inhibit the expression of BV-2 inflammatory genes (IL-6, TNF-α, IL-1ß) induced by LPS, downregulate the critical inflammatory protein production of iNOS and COX-2. The anti-HSV-1 activity screening revealed that compounds 28, 29 and 38 exhibited inhibitory activity against HSV-1 proliferation. Particularly, compound 28 exhibited a significant anti-HSV-1 effect, inhibiting the proliferation of HSV-1 and acyclovir-resistant strains of HSV-1/153 and HSV-1/Blue. Our research identified compounds 1, 2, and 28 from A. vulgaris., which could potentially serve as lead compounds for antineuroinflammatory and anti-HSV-1 activities.

N-Doping Coupled with Co-Vacancies Activating Sulfur Atoms and Narrowing Bandgap for CoS Toward Synergistically Accelerating Hydrogen Evolution.

The combination of hetero-elemental doping and vacancy engineering will be developed as one of the most efficient strategies to design excellent electrocatalysts for hydrogen evolution reaction (HER). Herein, a novel strategy for N-doping coupled with Co-vacancies is demonstrated to precisely activate inert S atoms adjacent to Co-vacancies and significantly improve charge transfer for CoS toward accelerating HER. In this strategy, N-doping favors the presence of Co-vacancies, due to greatly decreasing their formation energy. The as-developed strategy realizes the upshift of S 3p orbitals followed by more overlapping between S 3py and H 1s orbitals, which results in the favorable hydrogen atom adsorption free energy change (ΔGH ) to activate inert S atoms as newborn catalytical sites. Besides, this strategy synergistically decreases the bandgap of CoS, thereby achieving satisfactory electrical conductivity and low charge-transfer resistance for the as-obtained electrocatalysts. With an excellent HER activity of -89.0 mV at 10.0 mA cm-2 in alkaline environments, this work provides a new approach to unlocking inert sites and significantly improving charge transfer toward cobalt-based materials for highly efficient HER.

Metabolic Engineering of Pichia pastoris for the Production of Triacetic Acid Lactone.

Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds.

GDF11 inhibits adipogenesis of human adipose-derived stromal cells through ALK5/KLF15/ß-catenin/PPARγ cascade.

Obesity is a metabolic disease characterized by excessive fat storage, and the adipogenic differentiation of adipose-derived stromal cells (ADSCs) is closely linked to its occurrence. Growth differentiation factor 11 (GDF11), a well-known molecule in the field of anti-aging, also has great potential in regulating stem cell differentiation. In this study, we found that GDF11 inhibited adipogenic differentiation of human ADSCs in vitro by activating the WNT/ß-catenin and SMAD2/3 pathways while inhibiting the AKT pathway. Moreover, the transcription factor Kruppel-like factor 15 (KLF15) was discovered to be an important downstream factor for GDF11 in inhibiting adipogenesis via the WNT/ß-catenin pathway. Furthermore, AlphaFold2 structure prediction and inhibitor-blocking experiments revealed that ALK5 is a functional receptor of GDF11. Collectively, we demonstrated that GDF11 is a potential target for inhibiting adipogenic differentiation and combating obesity.

Ketogenic diet restrains herpes simplex encephalitis via gut microbes.

Herpes simplex virus type 1 (HSV-1) infection-associated herpes simplex encephalitis (HSE) is an occasionally but severe neuronal disease that causes behavioral disorder and impairs cognition. Herein, we demonstrate that the consumption of ketogenic diet (KD), a low-carbohydrate high-fat diet, restricts the neurotropic infection of HSV-1 and HSE progression in mice. KD reduced weight loss, neurodegenerative symptoms, virus production and neuroinflammation, resulting in the enhanced survival rate of HSE mice. Notably, depletion of gut microbes by antibiotics attenuated the protective function of KD on HSV-1-related neuroinflammation and HSE development. Therefore, KD represents as an alternative therapeutic strategy to alleviate or prevent HSE via gut microbiota.

Engineering Rich Active Sites and Efficient Water Dissociation for Ni-Doped MoS2/CoS2 Hierarchical Structures toward Excellent Alkaline Hydrogen Evolution.

Besides improving charge transfer, there are two key factors, such as increasing active sites and promoting water dissociation, to be deeply investigated to realize high-performance MoS2-based electrocatalysts in alkaline hydrogen evolution reaction (HER). Herein, we have demonstrated the synergistic engineering to realize rich unsaturated sulfur atoms and activated O-H bonds toward the water for Ni-doped MoS2/CoS2 hierarchical structures by an approach to Ni doping coupled with in situ sulfurizing for excellent alkaline HER. In this work, the Ni-doped atoms are evolved into Ni(OH)2 during alkaline HER. Interestingly, the extra unsaturated sulfur atoms will be modulated into MoS2 nanosheets by breaking Ni-S bonds during the formation of Ni(OH)2. On the other hand, the higher the mass of the Ni precursor (mNi) for the fabrication of our samples, the more Ni(OH)2 is evolved, indicating a stronger ability for water dissociation of our samples during alkaline HER. Our results further reveal that regulating mNi is crucial to the HER activity of the as-synthesized samples. By regulating mNi to 0.300 g, a balance between increasing active sites and promoting water dissociation is achieved for the Ni-doped MoS2/CoS2 samples to boost alkaline HER. Consequently, the optimal samples present the highest HER activity among all counterparts, accompanied by reliable long-term stability. This work will promise important applications in the field of electrocatalytic hydrogen evolution in alkaline environments.

Nicotinamide n-Oxide Attenuates HSV-1-Induced Microglial Inflammation through Sirtuin-1/NF-κB Signaling.

HSV-1 is a typical neurotropic virus that infects the brain and causes keratitis, cold sores, and occasionally, acute herpes simplex encephalitis (HSE). The large amount of proinflammatory cytokines induced by HSV-1 infection is an important cause of neurotoxicity in the central nervous system (CNS). Microglia, as resident macrophages in CNS, are the first line of defense against neurotropic virus infection. Inhibiting the excessive production of inflammatory cytokines in overactivated microglia is a crucial strategy for the treatment of HSE. In the present study, we investigated the effect of nicotinamide n-oxide (NAMO), a metabolite mainly produced by gut microbe, on HSV-1-induced microglial inflammation and HSE. We found that NAMO significantly inhibits the production of cytokines induced by HSV-1 infection of microglia, such as IL-1ß, IL-6, and TNF-α. In addition, NAMO promotes the transition of microglia from the pro-inflammatory M1 type to the anti-inflammatory M2 type. More detailed studies revealed that NAMO enhances the expression of Sirtuin-1 and its deacetylase enzymatic activity, which in turn deacetylates the p65 subunit to inhibit NF-κB signaling, resulting in reduced inflammatory response and ameliorated HSE pathology. Therefore, Sirtuin-1/NF-κB axis may be promising therapeutic targets against HSV-1 infection-related diseases including HSE.

Hypoxia-induced HMGB1 promotes glioma stem cells self-renewal and tumorigenicity via RAGE.

Glioma stem cells (GSCs) in the hypoxic niches contribute to tumor initiation, progression, and recurrence in glioblastoma (GBM). Hypoxia induces release of high-mobility group box 1 (HMGB1) from tumor cells, promoting the development of tumor. Here, we report that HMGB1 is overexpressed in human GBM specimens. Hypoxia promotes the expression and secretion of HMGB1 in GSCs. Furthermore, silencing HMGB1 results in the loss of stem cell markers and a reduction in self-renewal ability of GSCs. Additionally, HMGB1 knockdown inhibits the activation of RAGE-dependent ERK1/2 signaling pathway and arrests the cell cycle in GSCs. Consistently, FPS-ZM1, an inhibitor of RAGE, downregulates HMGB1 expression and the phosphorylation of ERK1/2, leading to a reduction in the proliferation of GSCs. In xenograft mice of GBM, HMGB1 knockdown inhibits tumor growth and promotes mouse survival. Collectively, these findings uncover a vital function for HMGB1 in regulating GSC self-renewal potential and tumorigenicity.

Boosting Highly Active Exposed Mo Atoms by Fine-Tuning S-Vacancies of MoS2-Based Materials for Efficient Hydrogen Evolution.

Guided by the theoretical calculation, achieving an efficient hydrogen evolution reaction (HER) by S-vacancy engineering toward MoS2-based materials is quite challenging due to the contradictory relationship between the adsorption free energy of hydrogen atoms (ΔGH) of the exposed Mo atoms (EMAs) and the number of EMAs per unit area (NEMAs). Herein, we demonstrate a novel one-pot incorporating-assisted compositing strategy to realize fine-tuning the concentration of S-vacancies (CS-vacancies) of MoS2-based materials to boost highly active EMAs for efficient HER. In our strategy, S-vacancies are modulated into basal planes of MoS2 via decreasing the formation energy of S-vacancies by oxygen incorporation; moreover, CS-vacancies of the basal planes is precisely regulated by simply controlling the molar amount of the Co precursor based on the electron injection effect. At low or excessively high CS-vacancies, the as-synthesized electrocatalysts lack "highly active EMAs" in quantity or nature. The balance between the intrinsic activity of EMAs and NEMAs is realized for boosting EMAs with high catalytic performance. The optimal electrocatalysts exhibit excellent activity and stability at fine-tuning CS-vacancies to 9.61%. Our results will pave a novel strategy for unlocking the potential of an inert basal plane in MoS2 for high-performance HER.

Synergistic Regulation of S-Vacancy of MoS2-Based Materials for Highly Efficient Electrocatalytic Hydrogen Evolution.

Low or excessively high concentration of S-vacancy (C S-vacancy) is disadvantageous for the hydrogen evolution reaction (HER) activity of MoS2-based materials. Additionally, alkaline water electrolysis is most likely to be utilized in the industry. Consequently, it is of great importance for fine-tuning C S-vacancy to significantly improve alkaline hydrogen evolution. Herein, we have developed a one-step Ru doping coupled to compositing with CoS2 strategy to precisely regulate C S-vacancy of MoS2-based materials for highly efficient HER. In our strategy, Ru doping favors the heterogeneous nucleation and growth of CoS2, which leads to a high crystallinity of Ru-doped CoS2 (Ru-CoS2) and rich heterogeneous interfaces between Ru-CoS2 and Ru-doped MoS2-x (Ru-MoS2-x). This facilitates the electron transfer from Ru-CoS2 to Ru-MoS2-x, thereby increasing C S-vacancy of MoS2-based materials. Additionally, the electron injection effect increases gradually with an increase in the mass of Co precursor (m Co), which implies more S2- leaching from MoS2 at higher m Co. Subsequently, C S-vacancy of the as-synthesized samples is precisely regulated by the synergistic engineering of Ru doping and compositing with CoS2. At C S-vacancy = 17.1%, a balance between the intrinsic activity and the number of exposed Mo atoms (EMAs) to boost highly active EMAs should be realized. Therefore, the typical samples demonstrate excellent alkaline HER activity, such as a low overpotential of 170 mV at 100 mA cm-2 and a TOF of 4.29 s-1 at -0.2 V. Our results show promise for important applications in the fields of electrocatalysis or energy conversion.

Establishing Komagataella phaffii as a Cell Factory for Efficient Production of Sesquiterpenoid α-Santalene.

Santalene, a major component of the sandalwood essential oil, is a typical representative of sesquiterpenes and has important applications in medicine, food, flavors, and other fields. Due to the limited supply of natural sandalwood resources, there is a growing interest in engineering microbial cell factories for the mass production of santalene. In the present study, Komagataella phaffii (also known as Pichia pastoris) was established as a cell factory for high-level production of α-santalene for the first time. The metabolic fluxes were rewired toward α-santalene biosynthesis through the optimization of promoters to drive the expression of the α-santalene synthase (SAS) gene, overexpression of the key mevalonate pathway genes (i.e., tHMG1, IDI1, and ERG20), and multi-copy integration of the SAS expression cassette. In combination with medium optimization and bioprocess engineering, the optimal strain (STE-9) was able to produce α-santalene with a titer as high as 829.8 ± 70.6 mg/L, 4.4 ± 0.3 g/L, and 21.5 ± 1.6 g/L in a shake flask, batch fermenter, and fed-batch fermenter, respectively. These represented the highest production of α-santalene ever reported, highlighting the advantages of K. phaffii cell factories for the production of terpenoids and other natural products.

The Effects of AT-533 and AT-533 gel on Liver Cytochrome P450 Enzymes in Rats.

BACKGROUND AND OBJECTIVES: AT-533 is a novel heat shock protein 90 inhibitor, which exhibits various biological activities in vitro and in vivo. Cytochrome P450 (CYP) enzymes in the liver are involved in the biotransformation of drugs and considered to be essential indicators of liver toxicity. The aim of this study was to assess the effect of AT-533, either as active pharmaceutical ingredient or in gel form, on liver CYP enzymes. METHODS: The effect of AT-533 or AT-533 gel on rat liver cytochrome P450 enzymes, including CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, was analyzed using LC-MS/MS. RESULTS: AT-533 and AT-533 gel did not significantly increase or reduce the enzymatic activity of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 at any treatment dose. CONCLUSIONS: AT-533 and AT-533 gel did not have any effect on CYP activity and may be considered safe for external use in gel form, as an alternative to conventional treatment.

Synthetic Biology Toolkit for Marker-Less Integration of Multigene Pathways into Pichia pastoris via CRISPR/Cas9.

Pichia pastoris, an important methylotrophic yeast, is currently mainly used for the expression of recombinant proteins and has great potential applications in the production of value-added compounds (e.g., chemical and natural products). However, the construction of P. pastoris cell factories is largely hindered by the lack of genetic tools for the manipulation of multigene biosynthetic pathways. Therefore, the present study aimed to establish a CRISPR-based synthetic biology toolkit for the integration and assembly of multigene biosynthetic pathways into the chromosome of P. pastoris. First, 23 intergenic regions were selected and characterized as potential integration sites, with a focus on the integration efficiency and heterologous gene expression levels. In addition, a panel of constitutive and methanol-inducible promoters with different strengths (weak, medium, and strong promoters) were characterized to control the expression of biosynthetic pathway genes to the desirable levels. With a series of gRNA plasmids (for single-locus, two-loci, and three-loci integration) and donor plasmids (containing homology arms for integration and promoters and terminators for driving heterologous gene expression) as major components, a CRISPR-based synthetic biology toolkit was established, which enabled the integration of one locus, two loci, and three loci with efficiencies as high as ∼100, ∼93, and ∼75%, respectively, in P. pastoris GS115 strain. Finally, the application of the toolkit was demonstrated by the construction of a series of P. pastoris cell factories, which could produce 2,3-butanediol, ß-carotene, zeaxanthin, and astaxanthin with methanol as the sole carbon and energy source. The P. pastoris synthetic biology toolkit is highly standardized and can be employed to construct P. pastoris cell factories with high efficiency.

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.

De Novo Biosynthesis of Vindoline and Catharanthine in Saccharomyces cerevisiae.

Vinblastine has been used clinically as one of the most potent therapeutics for the treatment of several types of cancer. However, the traditional plant extraction method suffers from unreliable supply, low abundance, and extremely high cost. Here, we use synthetic biology approach to engineer Saccharomyces cerevisiae for de novo biosynthesis of vindoline and catharanthine, which can be coupled chemically or biologically to vinblastine. On the basis of a platform strain with sufficient supply of precursors and cofactors for biosynthesis, we reconstituted, debottlenecked, and optimized the biosynthetic pathways for the production of vindoline and catharanthine. The vindoline biosynthetic pathway represents one of the most complicated pathways ever reconstituted in microbial cell factories. Using shake flask fermentation, our engineered yeast strains were able to produce catharanthine and vindoline at a titer of 527.1 and 305.1 µg·liter-1, respectively, without accumulating detectable amount of pathway intermediates. This study establishes a representative example for the production of valuable plant natural products in yeast.