EcCas6e-based antisense crRNA for gene repression and RNA editing in microorganisms.

Precise gene regulation and programmable RNA editing are vital RNA-level regulatory mechanisms. Gene repression tools grounded in small non-coding RNAs, microRNAs, and CRISPR-dCas proteins, along with RNA editing tools anchored in Adenosine Deaminases acting on RNA (ADARs), have found extensive application in molecular biology and cellular engineering. Here, we introduced a novel approach wherein we developed an EcCas6e mediated crRNA-mRNA annealing system for gene repression in Escherichia coli and RNA editing in Saccharomyces cerevisiae. We found that EcCas6e possesses inherent RNA annealing ability attributed to a secondary positively charged cleft, enhancing crRNA-mRNA hybridization and stability. Based on this, we demonstrated that EcCas6e, along with its cognate crRNA repeat containing a complementary region to the ribosome binding site of a target mRNA, effectively represses gene expression up to 25-fold. Furthermore, we demonstrated that multiple crRNAs can be easily assembled and can simultaneously target up to 13 genes. Lastly, the EcCas6e-crRNA system was developed as an RNA editing tool by fusing it with the ADAR2 deaminase domain. The EcCas6e-crRNA mediated gene repression and RNA editing tools hold broad applications for research and biotechnology.

Construction of an orthogonal transport system for Saccharomyces cerevisiae peroxisome to efficiently produce sesquiterpenes.

Subcellular compartmentalization is a crucial evolution characteristic of eukaryotic cells, providing inherent advantages for the construction of artificial biological systems to efficiently produce natural products. The establishment of an artificial protein transport system represents a pivotal initial step towards developing efficient artificial biological systems. Peroxisome has been demonstrated as a suitable subcellular compartment for the biosynthesis of terpenes in yeast. In this study, an artificial protein transporter ScPEX5* was firstly constructed by fusing the N-terminal sequence of PEX5 from S. cerevisiae and the C-terminal sequence of PEX5. Subsequently, an artificial protein transport system including the artificial signaling peptide YQSYY and its enhancing upstream 9 amino acid (9AA) residues along with ScPEX5* was demonstrated to exhibit orthogonality to the internal transport system of peroxisomes in S. cerevisiae. Furthermore, a library of 9AA residues was constructed and selected using high throughput pigment screening system to obtain an optimized signaling peptide (oPTS1*). Finally, the ScPEX5*-oPTS1* system was employed to construct yeast cell factories capable of producing the sesquiterpene α-humulene, resulting in an impressive α-humulene titer of 17.33 g/L and a productivity of 0.22 g/L/h achieved through fed-batch fermentation in a 5 L bioreactor. This research presents a valuable tool for the construction of artificial peroxisome cell factories and effective strategies for synthesizing other natural products in yeast.

Characterization of a pleiotropic regulator MtrA in Streptomyces avermitilis controlling avermectin production and morphological differentiation.

BACKGROUND: The macrolide antibiotic avermectin, a natural product derived from Streptomyces avermitilis, finds extensive applications in agriculture, animal husbandry and medicine. The mtrA (sav_5063) gene functions as a transcriptional regulator belonging to the OmpR family. As a pleiotropic regulator, mtrA not only influences the growth, development, and morphological differentiation of strains but also modulates genes associated with primary metabolism. However, the regulatory role of MtrA in avermectin biosynthesis remains to be elucidated. RESULTS: In this study, we demonstrated that MtrA, a novel OmpR-family transcriptional regulator in S. avermitilis, exerts global regulator effects by negatively regulating avermectin biosynthesis and cell growth while positively controlling morphological differentiation. The deletion of the mtrA gene resulted in an increase in avermectin production, accompanied by a reduction in biomass and a delay in the formation of aerial hyphae and spores. The Electrophoretic Mobility Shift Assay (EMSA) revealed that MtrA exhibited binding affinity towards the upstream region of aveR, the intergenic region between aveA1 and aveA2 genes, as well as the upstream region of aveBVIII in vitro. These findings suggest that MtrA exerts a negative regulatory effect on avermectin biosynthesis by modulating the expression of avermectin biosynthesis cluster genes. Transcriptome sequencing and fluorescence quantitative PCR analysis showed that mtrA deletion increased the transcript levels of the cluster genes aveR, aveA1, aveA2, aveC, aveE, aveA4 and orf-1, which explains the observed increase in avermectin production in the knockout strain. Furthermore, our findings demonstrate that MtrA positively regulates the cell division and differentiation genes bldM and ssgC, while exerting a negative regulatory effect on bldD, thereby modulating the primary metabolic processes associated with cell division, differentiation and growth in S. avermitilis, consequently impacting avermectin biosynthesis. CONCLUSIONS: In this study, we investigated the negative regulatory effect of the global regulator MtrA on avermectin biosynthesis and its effects on morphological differentiation and cell growth, and elucidated its transcriptional regulatory mechanism. Our findings indicate that MtrA plays crucial roles not only in the biosynthesis of avermectin but also in coordinating intricate physiological processes in S. avermitilis. These findings provide insights into the synthesis of avermectin and shed light on the primary and secondary metabolism of S. avermitilis mediated by OmpR-family regulators.

Effects of the pleiotropic regulator DasR on lincomycin production in Streptomyces lincolnensis.

The lincoamide antibiotic lincomycin, derived from Streptomyces lincolnensis, is widely used for the treatment of infections caused by gram-positive bacteria. As a common global regulatory factor of GntR family, DasR usually exists as a regulatory factor that negatively regulates antibiotic synthesis in Streptomyces. However, the regulatory effect of DasR on lincomycin biosynthesis in S. lincolnensis has not been thoroughly investigated. The present study demonstrates that DasR functions as a positive regulator of lincomycin biosynthesis in S. lincolnensis, and its overexpression strain OdasR exhibits a remarkable 7.97-fold increase in lincomycin production compared to the wild-type strain. The effects of DasR overexpression could be attenuated by the addition of GlcNAc in the medium in S. lincolnensis. Combined with transcriptome sequencing and RT-qPCR results, it was found that most structural genes in GlcNAc metabolism and central carbon metabolism were up-regulated, but the lincomycin biosynthetic gene cluster (lmb) were down-regulated after dasR knock-out. However, DasR binding were detected with the DasR responsive elements (dre) of genes involved in GlcNAc metabolism pathway through electrophoretic mobility shift assay, while they were not observed in the lmb. These findings will provide novel insights for the genetic manipulation of S. lincolnensis to enhance lincomycin production. KEY POINTS: • DasR is a positive regulator that promotes lincomycin synthesis and does not affect spore production • DasR promotes lincomycin production through indirect regulation • DasR correlates with nutrient perception in S. lincolnensis.

BER for a space uplink laser communication system under an actual optical distortion with a cake-cutting method.

A numerical calculating model is proposed for characterizing the BER performance of the detector working among the 2-D asymmetric distorted spot on the effects of atmospheric turbulence under weak turbulent conditions. Based on the isotropy of the beam wander effect, we introduce a beam wander vector to describe the behavior of the detector drifting among the receiving plane. Furthermore, using the cake-cutting method, the overall PDF of the light intensity is approximated by the average PDF of light intensity intercepted by the detector drifting in all different directions. The results demonstrate that the model obtains the overall PDF of the light intensity received by the detector and analyzes the BER performance of the communication system efficiently. Being an extension of the traditional 1-D calculation, our proposed model has important implications for designing the space uplink optical communication system.

Characterization and designing of an SAM riboswitch to establish a high-throughput screening platform for SAM overproduction in Saccharomyces cerevisiae.

S-adenosyl- l-methionine (SAM) is a high-value compound widely used in the treatment of various diseases. SAM can be produced through fermentation, but further enhancing the microbial production of SAM requires novel high-throughput screening methods for rapid detection and screening of mutant libraries. In this work, an SAM-OFF riboswitch capable of responding to the SAM concentration was obtained and a high-throughput platform for screening SAM overproducers was established. SAM synthase was engineered by semirational design and directed evolution, which resulted in the SAM2S203F,W164R,T251S,Y285F,S365R mutant with almost twice higher catalytic activity than the parental enzyme. The best mutant was then introduced into Saccharomyces cerevisiae BY4741, and the resulting strain BSM8 produced a sevenfold higher SAM titer in shake-flask fermentation, reaching 1.25 g L-1 . This work provides a reference for designing biosensors to dynamically detect metabolite concentrations for high-throughput screening and the construction of effective microbial cell factories.

Construction and optimization of Saccharomyces cerevisiae for synthesizing forskolin.

Forskolin, one of the primary active metabolites of labdane-type diterpenoids, exhibits significant medicinal value, such as anticancer, antiasthmatic, and antihypertensive activities. In this study, we constructed a Saccharomyces cerevisiae cell factory that efficiently produced forskolin. First, a chassis strain that can accumulate 145.8 mg/L 13R-manoyl oxide (13R-MO), the critical precursor of forskolin, was constructed. Then, forskolin was produced by integrating CfCYP76AH15, CfCYP76AH11, CfCYP76AH16, ATR1, and CfACT1-8 into the 13R-MO chassis with a titer of 76.25 µg/L. We confirmed that cytochrome P450 enzymes (P450s) are the rate-limiting step by detecting intermediate metabolite accumulation. Forskolin production reached 759.42 µg/L by optimizing the adaptations between CfCYP76AHs, t66CfCPR, and t30AaCYB5. Moreover, multiple metabolic engineering strategies, including regulation of the target genes' copy numbers, amplification of the endoplasmic reticulum (ER) area, and cofactor metabolism enhancement, were implemented to enhance the metabolic flow to forskolin from 13R-MO, resulting in a final forskolin yield of 21.47 mg/L in shake flasks and 79.33 mg/L in a 5 L bioreactor. These promising results provide guidance for the synthesis of other natural terpenoids in S. cerevisiae, especially for those containing multiple P450s in their synthetic pathways. KEY POINTS: • The forskolin biosynthesis pathway was optimized from the perspective of system metabolism for the first time in S. cerevisiae. • The adaptation and optimization of CYP76AHs, t66CfCPR, and t30AaCYB5 promote forskolin accumulation, which can provide a reference for diterpenoids containing complex pathways, especially multiple P450s pathways. • The forskolin titer of 79.33 mg/L is the highest production currently reported and was achieved by fed-batch fermentation in a 5 L bioreactor.

Biosynthesis of valerenic acid by engineered Saccharomyces cerevisiae.

OBJECTIVE: To produce valerenic acid (VA) in Saccharomyces cerevisiae by engineering a heterologous synthetic pathway. RESULT: Valerena-4,7(11)-diene synthase (VDS) derived from Valeriana officinalis (valerian) was expressed in S. cerevisiae to generate valerena-4,7(11)-diene as the precursor of VA. By overexpressing the key genes of the mevalonate pathway ERG8, ERG12 and ERG19, and integrating 4 copies of MBP (maltose-binding protein)-VDS-ERG20 gene expression caskets into the genome, the production of valerena-4,7(11)-diene was improved to 75 mg/L. On this basis, the cytochrome P450 monooxygenase LsGAO2 derived from Lactuca sativa was expressed to oxidize valerena-4,7(11)-diene to produce VA, and the most effective VA production strain was used for fermentation. The yield of VA reached 2.8 mg/L in the flask and 6.8 mg/L in a 5-L bioreactor fed glucose. CONCLUSIONS: An S. cerevisiae strain was constructed and optimized to produce VA, but the valerena-4,7(11)-diene oxidation by LsGAO2 is still the rate-limiting step for VA synthesis that needs to be further optimized in future studies.

Bioinformatics analysis of the prognostic value of NEK8 and its effects on immune cell infiltration in glioma.

Glioma is the most common malignancy of the nervous system with high rates of recurrence and mortality, even after surgery. The 5-year survival rate is only about 5%. NEK8 is involved in multiple biological processes in a variety of cancers; however, its role in glioma is still not clear. In the current study, we evaluated the prognostic value of NEK8, as well as its role in the pathogenesis of glioma. Using a bioinformatics approach and RNA-seq data from public databases, we found that NEK8 expression is elevated in glioma tissues; we further verified this result by RT-PCR, Western blotting and immunochemistry using clinical samples. Functional enrichment analyses of genes with correlated expression indicated that elevated NEK8 expression is associated with increased immune cell infiltration in glioma and may affect the tumour microenvironment via the regulation of DNA damage/repair. Survival analyses revealed that high levels of NEK8 are associated with a poorer prognosis; higher WHO grade, IDH status, 1p/19q codeletion, age and NEK8 were identified as an independent prognostic factor. These findings support the crucial role of NEK8 in the progression of glioma via effects on immune cell infiltration and suggest that it is a new prognostic biomarker.

Progress in heterologous biosynthesis of forskolin.

Forskolin, a class of labdane-type diterpenoid, has significant medicinal value in anticancer, antiasthmatic, antihypertensive, and heart-strengthening treatments. The main source of natural forskolin is its extraction from the cork tissue of the root of Coleus forskohlii. However, conventional modes of extraction pose several challenges. In recent years, the construction of microbial cell factories to produce medicinal natural products via synthetic biological methods has effectively solved the current problems and is a research hotspot in this field. This review summarizes the recent progress in the heterologous synthesis of forskolin via synthetic biological technology, analyzes the current challenges, and proposes corresponding strategies.

Uncovering fungal community composition in natural habitat of Ophiocordyceps sinensis using high-throughput sequencing and culture-dependent approaches.

BACKGROUND: The fungal communities inhabiting natural Ophiocordyceps sinensis play critical ecological roles in alpine meadow ecosystem, contribute to infect host insect, influence the occurrence of O. sinensis, and are repertoire of potential novel metabolites discovery. However, a comprehensive understanding of fungal communities of O. sinensis remain elusive. Therefore, the present study aimed to unravel fungal communities of natural O. sinensis using combination of high-throughput sequencing and culture-dependent approaches. RESULTS: A total of 280,519 high-quality sequences, belonging to 5 fungal phyla, 15 classes, 41 orders, 79 families, 112 genera, and 352 putative operational taxonomic units (OTUs) were obtained from natural O. sinensis using high-throughput sequencing. Among of which, 43 genera were identified in external mycelial cortices, Ophiocordyceps, Sebacinia and Archaeorhizomyces were predominant genera with the abundance of 95.86, 1.14, 0.85%, respectively. A total of 66 genera were identified from soil microhabitat, Inocybe, Archaeorhizomyces, unclassified Thelephoraceae, Tomentella, Thelephora, Sebacina, unclassified Ascomycota and unclassified fungi were predominant genera with an average abundance of 53.32, 8.69, 8.12, 8.12, 7.21, 4.6, 3.08 and 3.05%, respectively. The fungal communities in external mycelial cortices were significantly distinct from soil microhabitat. Meanwhile, seven types of culture media were used to isolate culturable fungi at 16 °C, resulted in 77 fungal strains identified by rDNA ITS sequence analysis, belonging to 33 genera, including Ophiocordyceps, Trichoderma, Cytospora, Truncatella, Dactylonectria, Isaria, Cephalosporium, Fusarium, Cosmospora and Paecilomyces, etc.. Among all culturable fungi, Mortierella and Trichoderma were predominant genera. CONCLUSIONS: The significantly differences and overlap in fungal community structure between two approaches highlight that the integration of high-throughput sequencing and culture-dependent approaches would generate more information. Our result reveal a comprehensive understanding of fungal community structure of natural O. sinensis, provide new insight into O. sinensis associated fungi, and support that microbiota of natural O. sinensis is an untapped source for novel bioactive metabolites discovery.

Promotion of compound K production in Saccharomyces cerevisiae by glycerol.

BACKGROUND: Ginsenoside compound K (CK), one of the primary active metabolites of protopanaxadiol-type ginsenosides, is produced by the intestinal flora that degrade ginseng saponins and exhibits diverse biological properties such as anticancer, anti-inflammatory, and anti-allergic properties. However, it is less abundant in plants. Therefore, enabling its commercialization by construction of a Saccharomyces cerevisiae cell factory is of considerable significance. RESULTS: We induced overexpression of PGM2, UGP1, and UGT1 genes in WLT-MVA5, and obtained a strain that produces ginsenoside CK. The production of CK at 96 h was 263.94 ± 2.36 mg/L, and the conversion rate from protopanaxadiol (PPD) to ginsenoside CK was 64.23 ± 0.41%. Additionally, it was observed that the addition of glycerol was beneficial to the synthesis of CK. When 20% glucose (C mol) in the YPD medium was replaced by the same C mol glycerol, CK production increased to 384.52 ± 15.23 mg/L, which was 45.68% higher than that in YPD medium, and the PPD conversion rate increased to 77.37 ± 3.37% as well. As we previously observed that ethanol is beneficial to the production of PPD, ethanol and glycerol were fed simultaneously in the 5-L bioreactor fed fermentation, and the CK levels reached 1.70 ± 0.16 g/L. CONCLUSIONS: In this study, we constructed an S. cerevisiae cell factory that efficiently produced ginsenoside CK. Glycerol effectively increased the glycosylation efficiency of PPD to ginsenoside CK, guiding higher carbon flow to the synthesis of ginsenosides and effectively improving CK production. CK production attained in a 5-L bioreactor was 1.7 g/L after simultaneous feeding of glycerol and ethanol.

Alpha-Terpineol production from an engineered Saccharomyces cerevisiae cell factory.

BACKGROUND: Alpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction. RESULTS: α-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L. CONCLUSIONS: An efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.

High-titer production of 13R-manoyl oxide in metabolically engineered Saccharomyces cerevisiae.

BACKGROUND: Diterpenoids are a large class of natural products with complex structures and broad commercial applications as food additives, important medicines, and fragrances. However, their low abundance in plants and high structural complexity limit their applications. Therefore, it is important to create an efficient diterpenoid-producing yeast cell factory of the production of various high-value diterpenoid compounds in a cost-effective manner RESULTS: In this study, 13R-manoyl oxide (13R-MO; 2.31 mg/L) was produced by expressing CfTPS2 and CfTPS3 from Coleus forskohlii in Saccharomyces cerevisiae. The 13R-MO titer was increased by 142-fold to 328.15 mg/L via the stepwise metabolic engineering of the original strain, including the overexpression of the rate-limiting genes (tHMG1 and ERG20) of the mevalonate pathway, transcription and protein level regulation of ERG9, Bts1p and Erg20F96Cp fusion, and the overexpression of tCfTPS2 and tCfTPS3 (excision of the N-terminal plastid transit peptide sequences of CfTPS2 and CfTPS3). The final titer of 13R-MO reached up to 3 g/L by fed-batch fermentation in a 5 L bioreactor. CONCLUSIONS: In this study, an efficient 13R-MO yeast cell factory was constructed, which achieved the de novo production of 3 g/L of 13R-MO from glucose. To the best of our knowledge, this is the highest 13R-MO titer reported to date. Furthermore, the metabolic engineering strategies presented here could be used to produce other valuable diterpenoid compounds in yeast.

Yarrowia lipolytica construction for heterologous synthesis of α-santalene and fermentation optimization.

Sandalwood oil is a valuable resource derived from Santalum album. It has antibacterial, cosmetic, and sedative effects. α-Santalene is the precursor of α-santalol, the main component of sandalwood oil. Yarrowia lipolytica is an oleaginous yeast, which has been metabolically engineered to produce valuable compounds such as terpenoids and biofuel. This study presents a method for the heterologous synthesis of α-santalene by Y. lipolytica. Using Y. lipolytica ATCC 201249, a codon-optimized plant-origin α-santalene synthase (STS) was integrated into the genome, and a yield of 5.19 mg/L α-santalene was obtained after fermentation. Upstream genes in the MVA pathway (ERG8, ERG10, ERG12, ERG13, ERG19, ERG20, HMG1, and tHMG1) were overexpressed, and we found that the key genes ERG8, HMG1, and tHMG1 can increase the supply of FPP and the yield of α-santalene. ERG8 and HMG1 were overexpressed in multiple-copy formats, and the plasmid pERG8HMG1 and ERG8-HMG1 expression modules were optimized as single-copy and multiple-copy formats, respectively. The overexpression of single-copy plasmid pERG8HMG1 led to α-santalene yield of 13.31 mg/L. The optimal feeding strategy was determined by initial carbon source concentration optimizations and five feeding methods. Using 50 g/L glucose as the initial carbon source, maintaining the carbon source concentration at 5-20 g/L during the feeding process is most conducive to increased production. These results were verified in a 5-L fermenter by batch and fed-batch fermentation. The OD of fed-batch fermentation broth reached 79.09, and the production of α-santalene reached 27.92 mg/L; 5.38 times of the initial yield, without by-products farnesol and trans-α-bergamotene.

Serum metabolic profiling using small molecular water-soluble metabolites in individuals with schizophrenia: A longitudinal study using a pre-post-treatment design.

AIM: We sought to compare alterations in serum bioenergetic markers within a well-characterized sample of adults with schizophrenia at baseline and after 8 weeks of pharmacological treatment with the hypothesis that treatment would be associated with significant changes in bioenergetic markers given the role of bioenergetic dysfunction in schizophrenia. METHODS: We recruited adults with schizophrenia (n = 122) who had not received pharmacological treatment for at least 1 month prior to enrollment, including drug-naïve (i.e., first-episode) participants and treatment non-adherent participants. Pre- and post-treatment serum samples were analyzed using liquid chromatography-tandem mass spectrometry. RESULTS: Metabolites with the greatest change, when comparing pre- and post-treatment levels, were identified revealing 14 water-soluble metabolites of interest. The composition of these metabolites was: amino acids (n = 6), carnitines (n = 4), polar lipids (n = 3), and organic acid (n = 1). All amino acids and lysophosphatidylcholines (LysoPC) were increased, while the four carnitines - oleoylcarnitine, L-palmitoylcarnitine, linoleyl carnitine, and L-acetylcarnitine - were decreased post-treatment. Of these metabolite biomarkers, six - oleoylcarnitine, linoleyl carnitine, L-acetylcarnitine, LysoPC(15:0), D-glutamic acid, and L-arginine - were identified as having most consistently and predictably changed after 8 weeks of treatment. CONCLUSION: The current study identified several bioenergetic markers that consistently change with pharmacological treatment. These bioenergetic changes may provide further insights into the pathophysiology of schizophrenia along with furthering our understanding of the mechanisms subserving both the effects (e.g., antipsychotic effects) and side-effects (e.g., metabolic syndrome) of antipsychotics.

Production of sesquiterpenoid zerumbone from metabolic engineered Saccharomyces cerevisiae.

Zerumbone, the predominant sesquiterpenoid component of Zingiber zerumbet, exhibits diverse pharmacological properties. In this study, de novo production of zerumbone was achieved in a metabolically engineered yeast cell factory by introducing α-humulene synthase (ZSS1), α-humulene 8-hydroxylase (CYP71BA1) and zerumbone synthase variant (ZSD1S114A) from Z. zerumbet, together with AtCPR1 from Arabidopsis thaliana into the yeast strain. Multistep metabolic engineering strategies were applied, including the over-expression of the mevalonate (MVA) pathway rate-limiting enzymes tHMG1 and ERG20, regulation of ERG9 by an inducible promoter and competitive pathway deletion to redirect metabolic flux toward the desired product. In the engineered strain, α-humulene production increased by 18-fold, to 92 mg/L compared to that in the original strain. Five cytochrome P450 reductases (CPRs) from different sources were selected for CYP71BA1 adaptability tests, and AtCPR1 from A. thaliana was found to be the optimal, producing 113.16 µg/L of 8-hydroxy-α-humulene. Multicopy integration of CYP71BA1, AtCPR1, ZSS1 and ICE2 (type III membrane protein) genes resulting in strain LW14 increased the production of 8-hydroxy-α-humulene by 134-fold to 15.2 mg/L. Expressing ZSD1S114A in the ura3 site of strain LW14 resulted in the production of 7 mg/L zerumbone. Multicopy integration of ZSD1S114A increased the production of zerumbone to 20.6 mg/L. The high zerumbone-producing strain was used for batch and fed-batch fermentation in a 5-L bioreactor and zerumbone degradation by yeast was observed; the production of zerumbone finally reached 40 mg/L by fed-batch fermentation in a 5-L bioreactor.

Heterologous production of levopimaric acid in Saccharomyces cerevisiae.

BACKGROUND: Levopimaric acid (LA), a type of diterpene resin acid produced by plants, is a significant industrial intermediate that is mainly produced via phytoextraction. This work aimed to apply synthetic biology to produce LA in yeast strains from a simple carbon source. RESULTS: Levopimaradiene (LP), the precursor of LA, was produced via LP synthase (LPS) expression in yeast. LPS was then modified by N-terminal truncating and site-directed mutagenesis. The strain containing t79LPSMM (79 N-terminal amino acid truncating and M593I/Y700F mutation) produced 6.92 mg/L of LP, which were 23-fold higher than the strain containing LPS. Next, t79LPSMM was expressed in a new metabolically engineered chassis, and the final LP production increased 164-folds to 49.21 mg/L. Three cytochrome P450 reductases (CPRs) were co-expressed with CYP720B1 (the enzyme responsible for LA production from LP) in yeast to evaluate their LA producing abilities, and the CPR from Taxus cuspidata (TcCPR) was found to be the best (achieved 23.13 mg/L of LA production). CYP720B1 and TcCPR genes overexpression in the multi-copy site of the S.cerevisiae genome led to a 1.9-fold increase in LA production to 45.24 mg/L in a shake-flask culture. Finally, LA production was improved to 400.31 mg/L via fed-batch fermentation in a 5-L bioreactor. CONCLUSIONS: This is the first report to produce LA in a yeast cell factory and the highest titer of LA is achieved.

Enhanced Electron Collection in Perovskite Solar Cells Employing Thermoelectric NaCo2 O4 /TiO2 Coaxial Nanofibers.

Thermoelectric NaCo2 O4 /TiO2 coaxial nanofibers are prepared and distributed in the perovskite solar cells. Under illumination, p-type NaCo2 O4 can convert unwanted heat to thermal voltage, and thus promote the electron extraction and transport with the action of electrostatic force. These advantages collectively contribute to an overall power conversion efficiency improvement of ≈20% (15.14% vs 12.65%).

[Diversity and antimicrobial activities of cultivable bacteria isolated from Jiaozhou Bay].

Objective: Marine microorganisms have a great potential in producing biologically active secondary metabolites. In order to study the diversity and antimicrobial activity, we explored 9 sediment samples in different observation sites of Jiaozhou bay. Methods: We used YPD and Z2216E culture medium to isolate bacteria from the sediments; 16S rRNA was sequenced for classification and identification of the isolates. Then, we used Oxford cup method to detect antimicrobial activities of the isolated bacteria against 7 test strains. Lastly, we selected 16 representatives to detect secondary-metabolite biosynthesis genes:PKSI, NRPS, CYP, PhzE, dTGD by PCR specific amplification. Results: A total of 76 bacterial strains were isolated from Jiaozhou bay; according to the 16S rRNA gene sequence analysis. These strains could be sorted into 11 genera belonging to 8 different families:Aneurinibacillus, Brevibacillus, Microbacterium, Oceanisphae, Bacillus, Marinomonas, Staphylococcus, Kocuria, Arthrobacters, Micrococcus and Pseudoalteromonas. Of them 34 strains showed antimicrobial activity against at least one of the tested strains. All 16 strains had at least one function genes, 5 strains possessed more than three function genes. Conclusion: Jiaozhou bay area is rich in microbial resources with potential in providing useful secondary metabolites.