Pseudomonas aeruginosa possesses three type VI secretion systems (T6SSs) that are involved in interspecies competition, internalization into epithelial cells, and virulence. Host-derived mucin glycans regulate the T6SSs through RetS, and attacks from other species activate the H1-T6SS. However, other environmental signals that control the T6SSs remain to be explored. Previously, we determined PitA to be a constitutive phosphate transporter, whose mutation reduces the intracellular phosphate concentration. Here, we demonstrate that mutation in the pitA gene increases the expression of the H2- and H3-T6SS genes and enhances bacterial uptake by A549 cells. We further found that mutation of pitA results in activation of the quorum sensing (QS) systems, which contributes to the upregulation of the H2- and H3-T6SS genes. Overexpression of the phosphate transporter complex genes pstSCAB or knockdown of the phosphate starvation response regulator gene phoB in the ΔpitA mutant reduces the expression of the QS genes and subsequently the H2- and H3-T6SS genes and bacterial internalization. Furthermore, growth of wild-type PA14 in a low-phosphate medium results in upregulation of the QS and H2- and H3-T6SS genes and bacterial internalization compared to those in cells grown in a high-phosphate medium. Deletion of the phoB gene abolished the differences in the expression of the QS and T6SS genes as well as bacterial internalization in the low- and high- phosphate media. Overall, our results elucidate the mechanism of PitA-mediated regulation on the QS system and H2- and H3-T6SSs and reveal a novel pathway that regulates the T6SSs in response to phosphate starvation. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogenic bacterium that causes acute and chronic infections in humans. The type VI secretion systems (T6SSs) have been shown to associate with chronic infections. Understanding the mechanism used by the bacteria to sense environmental signals and regulate virulence factors will provide clues for developing novel effective treatment strategies. Here, we demonstrate a relationship between a phosphate transporter and the T6SSs and reveal a novel regulatory pathway that senses phosphate limitation and controls bacterial virulence factors in P. aeruginosa.

类胡萝卜素具有丰富的颜色和强抗氧化活性,这赋予了该类产品较高的商业价值,加速了该类产品合成路径在异源底盘中的构建进程,使之成为发展非理性设计策略的有效研究对象。本文主要针对微生物合成类胡萝卜素细胞工厂构建中的瓶颈问题,从增强合成类胡萝卜素前体GGPP的供给利用以及缓解各类胡萝卜素产品对微生物底盘细胞的胁迫压力两个角度,系统综述非理性设计策略对元件优化、模块适配和多维调控等路径构建优化方法的丰富,以及对不同尺度的DNA变异和适应性进化等底盘进化手段的推动作用。并从扩展类胡萝卜素合成底盘物种的多样性和丰富非天然类胡萝卜素产品结构的可获得性的需求出发,对构建合成该类产品微生物细胞工厂自动化和智能化的发展趋势进行展望。

As an ancient and huge family of membrane proteins, ATP-binding cassette transporter (ABC transporter) plays an important physiological role in most organisms. Herein, we introduce the research progress in ABC transporters on the aspects of structural characteristic, transport mechanism and physiological functions. We also focus on the application of ABC transporters in the field of synthetic biology in recent years. Finally, we propose future research needs.

Aberrant activation of the cholesterol biosynthesis supports tumor cell growth. In recent years, significant progress has been made by targeting rate-limiting enzymes in cholesterol biosynthesis pathways to prevent carcinogenesis. However, precise mechanisms behind cholesterol degradation in cancer cells have not been comprehensively investigated. Here, we report that codon optimization of the orthologous cholesterol 7-desaturase, NVD-BM from Bombyx mori, significantly slowed melanoma cell proliferation and migration, and inhibited cancer cell engraftment in nude mice, by converting cholesterol to toxic 7-dehydrocholesterol. Based on these observations, we established a synthetic genetic circuit to induce melanoma cell regression by sensing tumor specific signals in melanoma cells. The dual-input signals, RELA proto-oncogene (RELA) and signal transducer and activator of transcription 1 (STAT1), activated NVD-BM expression and repressed melanoma cell proliferation and migration. Mechanically, we observed that NVD-BM decreased Akt1-ser473 phosphorylation and inhibited cytoplasmic RELA translocation. Taken together, NVD-BM was identified as a tumor suppressor in malignant melanoma, and we established a dual-input biosensor to promote cancer cell regression, via Akt1/NF-κB signaling. Our results demonstrate the potential therapeutic effects of cholesterol 7-desaturase in melanoma metabolism, and provides insights for genetic circuits targeting 7-dehydrocholesterol accumulation in tumors.

Geranyl diphosphate (GPP) is a direct precursor in the biosynthesis of monoterpenes. Previous studies focused on the manipulation of metabolic flux to improve GPP supply in yeast. However, if the subcellular distribution of GPP with monoterpene synthase is not coordinated, the usage of GPP becomes sub-optimal. Therefore, taking sabinene production in Saccharomyces cerevisiae as an exemplar, we confirmed the location of N-truncated sabinene synthase (t34SabS1) to be primarily in the cytosol. We also revealed the existence of GPP pools in the peroxisomes and mitochondria. Combined targeting of t34SabS1 into different combinations of subcellular locations demonstrated that the highest production of sabinene was obtained when expressed simultaneously in the cytosol and mitochondria. Further overexpression of mitochondria-related genes uncovered four novel molecular targets (FIS1, LSB3, MBA1, and AIM25) associated with sabinene output, with AIM25 resulting in the highest production. Eventually, integrating all of these engineered genes into the host chromosome achieved a sabinene production of 154.9 mg/L, an almost 60-fold increase from our original titer. This research highlights the strategy of organelle engineering to improve precursor utilization and to enhance compartmentalized pathways. It also provides a good reference for the synthesis of other valuable monoterpenes and their derivatives, in a eukaryotic host.

Background Monoterpenes and their derivatives play an important role as flavorings, perfume additives, pharmaceuticals and advanced biofuels. GPP (geranyl diphosphate) is the direct precursor to biosynthesize this class of products. The present study for monoterpenes synthesis in yeast focused on manipulation of metabolic flux to improve GPP supply. However, if the subcellular distribution of GPP and monoterpene synthase were not coincided with each other, it would be hard to well utilize GPP, leading to a waste of carbon sources.Results Herein, we took sabinene production in Saccharomyces cerevisiae as an instance, and confirmed the location of N-truncated sabinene synthase (t34SabS1) in yeast cytosol (C). We also revealed the existence of GPP pools in organelles [such as lipid monolayer membrane-bound peroxisomes (P) and bilayer membrane-bound mitochondria (M)] beside cytosol. In order to minimize the loss of GPP, an engineering strategy was proposed to coordinated compartmentalization of sabinene synthase. Initially, expression of t34SabS1 in an ERG20-downregulated host only obtained 19.4 mg/L sabinene. Combined targeting t34SabS1 into CM (cytosol and mitochondria) increased sabinene production to 64.6 mg/L. This titer was significantly higher than those generated by harnessing other combinations of subcellular locations ( i.e. CP, PM and even CPM). Further overexpression of the genes involved in mitochondria morphology uncovered four novel molecular targets ( i.e. FIS1 , LSB3 , MBA1 and AIM25 ) associating with sabinene output. Especially, overexpression of AIM25 enhanced the sabinene production to 90.4 mg/L. Eventually, integrating all above engineered genes into host chromosome achieved 154.9 mg/L of sabinene.Conclusions The engineering approaches of this study improve GPP utilization and boost sabinene accumulation almost 60-fold of the original titer. This research highlights the strategy of organelle engineering to improve precursor utilization and to enhance the compartmentalized pathway. It also sets a good reference to synthesize other valuable monoterpenes and their derivatives in eukaryotic hosts.

Melanoma is the most lethal cutaneous cancer with a high metastatic rate worldwide, causing ~55,500 deaths annually. Although the selective B-Raf oncogene serine/threonine-kinase (BRAF) inhibitors, dabrafenib and vemurafenib, have been approved for the treatment of BRAF-mutant metastatic melanoma, the 5-year survival rate remains unfavorable due to acquired therapy resistance. Therefore, it is of great importance to develop alternative therapeutic drugs and uncover their mechanisms for the treatment of melanoma. 7-dehydrocholesterol (7-DHC) has been demonstrated to inhibit melanoma, but the mechanism is unclear. Therefore, the present study aimed to elucidate the mechanisms of the inhibitory effect of 7-DHC in melanoma cells via analyzing the proliferation, migration, apoptosis, cell cycle and transcriptional sequencing of melanoma cells treated with 7-DHC, as well as constructing a gene signature according to public data of patients with melanoma. In the present study, 7-DHC, the precursor of vitamin D3, was able to induce apoptosis and inhibit cell proliferation and invasion of melanoma cells in a dose-dependent manner. RNA sequencing of melanoma cells treated with different concentrations of 7-DHC revealed that, compared with untreated melanoma cells, 65 genes were downregulated, and genes involved in the regulation of NF-ĸB import into the nucleus and NF-ĸB signaling were significantly repressed. Consistently, the Akt kinase family was one of most common somatic mutation hotspots in patients with melanoma according to The Cancer Genome Atlas enrichment analysis. Furthermore, 7-DHC decreased the phosphorylation of Akt1-Ser473 rather than that of MEK1, and the decreased phosphorylation of Akt1 subsequently inhibited the translocation of free RELA proto-oncogene NF-κB subunit to the nucleus. Finally, by intersecting downregulated genes by 7-DHC treatment and upregulated genes in patients with melanoma, a 7-DHC gene signature was identified, which was negatively associated with the prognosis. Overall, the present results demonstrated that 7-DHC suppressed melanoma cell proliferation and invasion via the Akt1/NF-ĸB signaling pathway, and 7-DHC key target genes were negatively associated with the prognosis. These findings highlight the potential application of 7-DHC for the treatment of melanoma in the future.

Although computational and reverse metabolic engineering approaches often lead to improved gene deletion mutants for cell factory engineering, the systems level effects of such gene deletions on the production phenotypes have not been extensively studied. Understanding the genetic and molecular function of such gene alterations on production strains will minimize the risk inherent in the development of large-scale fermentation processes, which is a daunting challenge in the field of industrial biotechnology. Therefore, we established a detailed experimental and systems biology approach to uncover the molecular mechanisms of YPL062W deletion in S. cerevisiae , which is shown to improve the production of all terpenoid classes. This study redefines the genetic function of YPL062W , demonstrates a strong correlation between YPL062W and terpenoid production, and provides a useful modification for the creation of terpenoid production platform strains. Further, this study underscores the benefits of detailed and systematic characterization of the metabolic effects of genetic alterations on engineered biosynthetic factories.