Coupled strategy based on regulator manipulation and medium optimization empowers the biosynthetic overproduction of lincomycin.

The biosynthesis of bioactive secondary metabolites, specifically antibiotics, is of great scientific and economic importance. The control of antibiotic production typically involves different processes and molecular mechanism. Despite numerous efforts to improve antibiotic yields, joint engineering strategies for combining genetic manipulation with fermentation optimization remain finite. Lincomycin A (Lin-A), a lincosamide antibiotic, is industrially fermented by Streptomyces lincolnensis. Herein, the leucine-responsive regulatory protein (Lrp)-type regulator SLCG_4846 was confirmed to directly inhibit the lincomycin biosynthesis, whereas indirectly controlled the transcription of SLCG_2919, the first reported repressor in S. lincolnensis. Inactivation of SLCG_4846 in the high-yield S. lincolnensis LA219X (LA219XΔ4846) increases the Lin-A production and deletion of SLCG_2919 in LA219XΔ4846 exhibits superimposed yield increment. Given the effect of the double deletion on cellular primary metabolism of S. lincolnensis, Plackett-Burman design, steepest ascent and response surface methodologies were utilized and employed to optimize the seed medium of this double mutant in shake flask, and Lin-A yield using optimal seed medium was significantly increased over the control. Above strategies were performed in a 15-L fermenter. The maximal yield of Lin-A in LA219XΔ4846-2919 reached 6.56 g/L at 216 h, 55.1 % higher than that in LA219X at the parental cultivation (4.23 g/L). This study not only showcases the potential of this strategy to boost lincomycin production, but also could empower the development of high-performance actinomycetes for other antibiotics.

High-efficiency genetic engineering toolkit for virus based on lambda red-mediated recombination.

PURPOSE: Viruses, such as Ebola virus (EBOV), evolve rapidly and threaten the human health. There is a great demand to exploit efficient gene-editing techniques for the identification of virus to probe virulence mechanism for drug development. METHODS: Based on lambda Red recombination in Escherichia coli (E. coli), counter-selection, and in vitro annealing, a high-efficiency genetic method was utilized here for precisely engineering viruses. EBOV trVLPs assay and dual luciferase reporter assay were used to further test the effect of mutations on virus replication. RESULTS: Considering the significance of matrix protein VP24 in EBOV replication, the types of mutations within vp24, including several single-base substitutions, one double-base substitution, two seamless deletions, and one targeted insertion, were generated on the multi-copy plasmid of E. coli. Further, the length of the homology arms for recombination and in vitro annealing, and the amount of DNA cassettes and linear plasmids were optimized to create a more elaborate and cost-efficient protocol than original approach. The effects of VP24 mutations on the expression of a reporter gene (luciferase) from the EBOV minigenome were determined, and results indicated that mutations of key sites within VP24 have significant impacts on EBOV replication. CONCLUSION: This precise mutagenesis method will facilitate effective and simple editing of viral genes in E. coli.

The leucine-responsive regulatory protein SCAB_Lrp modulates thaxtomin biosynthesis, pathogenicity, and morphological development in Streptomyces scabies.

Streptomyces scabies is the best-characterized plant-pathogenic streptomycete, which is a special species among the large genus Streptomyces. The pathogenicity of S. scabies relies on the production of the secondary metabolite thaxtomin A. Little is known about the molecular mechanisms underlying the regulation of thaxtomin biosynthesis in S. scabies beyond the pathway-specific activator TxtR and the cellulose utilization repressor CebR. The leucine-responsive regulatory protein (Lrp) family modulates secondary metabolism in nonpathogenic streptomycetes. However, the regulatory relationship between the Lrp and pathogenic streptomycetes remains unknown. In this study, we demonstrated that SCAB_Lrp (SCAB_77931) from S. scabies significantly affects thaxtomin biosynthesis, pathogenicity, and morphological development. SCAB_Lrp deletion resulted in a dramatic decline in thaxtomin A production and a low-virulence phenotype of S. scabies. An in-depth dissection of the regulatory mechanism of SCAB_Lrp revealed that it positively regulates the transcription of the thaxtomin biosynthetic gene cluster by directly binding to the promoter of the cluster-situated regulator gene txtR. SCAB_Lrp also controls the morphological development of S. scabies by directly activating the transcription of amfC, whiB, and ssgB. SCAB_Lrp directly controls the transcription of its own gene by binding a specific sequence (5'-GGACAGTCGCCGTGCTACG-3'). Moreover, phenylalanine and methionine have been characterized as SCAB_Lrp effectors by strengthening the binding affinity and complex status between SCAB_Lrp and DNA. Our findings characterize a multifunctional regulatory protein, SCAB_Lrp, that controls secondary metabolism, pathogenicity, and sporulation in S. scabies and provide new insights into the complex regulatory network that modulates thaxtomin phytotoxins in pathogenic Streptomyces.

Crosstalk of TetR-like regulator SACE_4839 and a nitrogen regulator for erythromycin biosynthesis.

TetR family transcriptional regulators (TFRs) are widespread in actinomycetes, which exhibit diverse regulatory modes in antibiotic biosynthesis. Nitrogen regulators play vital roles in modulation of primary and secondary metabolism. However, crosstalk between TFR and nitrogen regulator has rarely been reported in actinomycetes. Herein, we demonstrated that a novel TFR, SACE_4839, was negatively correlated with erythromycin yield in Saccharopolyspora erythraea A226. SACE_4839 indirectly suppressed erythromycin synthetic gene eryAI and resistance gene ermE and directly inhibited its adjacent gene SACE_4838 encoding a homologue of nitrogen metabolite repression (NMR) regulator NmrA (herein named NmrR). The SACE_4839-binding sites within SACE_4839-nmrR intergenic region were identified. NmrR positively controlled erythromycin biosynthesis by indirectly stimulating eryAI and ermE and directly repressing SACE_4839. NmrR was found to affect growth viability under the nitrogen source supply. Furthermore, NmrR directly repressed glutamine and glutamate utilization-related genes SACE_1623, SACE_5070 and SACE_5979 but activated nitrate utilization-associated genes SACE_1163, SACE_4070 and SACE_4912 as well as nitrite utilization-associated genes SACE_1476 and SACE_4514. This is the first reported NmrA homolog for modulating antibiotic biosynthesis and nitrogen metabolism in actinomycetes. Moreover, combinatorial engineering of SACE_4839 and nmrR in the high-yield S. erythraea WB resulted in a 68.8% increase in erythromycin A production. This investigation deepens the understanding of complicated regulatory network for erythromycin biosynthesis. KEY POINTS: • SACE_4839 and NmrR had opposite contributions to erythromycin biosynthesis. • NmrR was first identified as a homolog of another nitrogen regulator NmrA. • Cross regulation between SACE_4839 and NmrR was revealed.

Sheep tail fat inhibits the proliferation of non-small-cell lung cancer cells in vitro and in vivo.

Increasing evidence suggests that numerous edible oils may function as adjuvant dietary therapies to treat cancer. We previously reported that the odd-chain saturated fatty acid (OCSFA), heptadecanoic acid (C17:0), profoundly inhibits non-small-cell lung cancer (NSCLC) cell proliferation. However, the antitumor potential of edible lipids rich in C17:0 remains unclear. Here, we determined that sheep tail fat (STF) is a dietary lipid rich in C17:0 and exhibited the greatest inhibitory effect against three NSCLC cell lines (A549, PC-9, and PC-9/GR) among common dietary lipids. Cell migration experiments demonstrated that STF could significantly inhibit the wound healing capacity of three NSCLC cell lines by promoting the generation of reactive oxygen species (ROS) and subsequent cell death. Mechanistic studies showed that STF suppressed NSCLC cell growth by downregulating the Akt/S6K signaling pathway. Furthermore, administration of STF reduced tumor growth, weight, and expression of the proliferative marker Ki-67 in nude mice bearing A549 xenografts. Collectively, our data show that STF has antitumor activity against NSCLC, implying that dietary intake of C17:0-rich STF may be a potential adjuvant therapy for NSCLC.

Arabinosyltransferase C Mediates Multiple Drugs Intrinsic Resistance by Altering Cell Envelope Permeability in Mycobacterium abscessus.

Mycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus. IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.

BRD4 Targets the KEAP1-Nrf2-G6PD Axis and Suppresses Redox Metabolism in Small Cell Lung Cancer.

Accumulating evidence has witnessed the Kelch-like ECH-associated protein 1(KEAP1)- nuclear factor (erythroid-derived 2)-like 2 (Nrf2) axis is the main regulatory factor of cell resistance to endogenous and exogenous oxidative assaults. However, there are few studies addressing the upstream regulatory factors of KEAP1. Herein, bioinformatic analysis suggests bromodomain-containing protein 4 (BRD4) as a potential top transcriptional regulator of KEAP1 in lung cancer. Using molecular and pharmacological approaches, we then discovered that BRD4 can directly bind to the promoter of KEAP1 to activate its transcription and down-regulate the stability of Nrf2 which in turn transcriptionally suppresses glucose-6-phosphate dehydrogenase (G6PD) in small cell lung cancer (SCLC), a highly proliferative and aggressive disease with limited treatment options. In addition, BRD4 could associate with the Nrf2 protein in a non-KEAP1-dependent manner to inhibit Nrf2 activity. Furthermore, simultaneous application of JQ1 and ATRA or RRx-001 yielded synergistic inhibition both in vitro and in vivo. These data suggest metabolic reprogramming by JQ1 treatment improves cell resistance to oxidative stress and might be a resistance mechanism to bromodomain and extra-terminal domain (BET) inhibition therapy. Altogether, our findings provide novel insight into the transcriptional regulatory network of BRD4 and KEAP1 and transcriptional regulation of the pentose phosphate pathway in SCLC.

Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea.

Erythromycins produced by Saccharopolyspora erythraea have broad-spectrum antibacterial activities. Recently, several TetR-family transcriptional regulators (TFRs) were identified to control erythromycin production by multiplex control modes; however, their regulatory network remains poorly understood. In this study, we report a novel TFR, SACE_0303, positively correlated with erythromycin production in Sac. erythraea. It directly represses its adjacent gene SACE_0304 encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene eryAI and resistance gene ermE. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting SACE_0303 as well as eryAI and indirectly repressing ermE. Then, the SACE_0303 binding site within the SACE_0303-SACE_0304 intergenic region was defined. Through genome scanning combined with in vivo and in vitro experiments, three additional SACE_0303 target genes (SACE_2467 encoding cation-transporting ATPase, SACE_3156 encoding a large transcriptional regulator, SACE_5222 encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with SACE_0304 deletion and SACE_0303 overexpression, we performed stepwise engineering of the SACE_0303-mediated mini-regulatory network in a high-yield strain, resulting in enhanced erythromycin production by 67%. In conclusion, the present study uncovered the regulatory network of a novel TFR for control of erythromycin production and provides a multiplex tactic to facilitate the engineering of industrial actinomycetes for yield improvement of antibiotics.

An Anti-EGFR/anti- HER2 Bispecific Antibody with Enhanced Antitumor Activity Against Acquired Gefitinib-Resistant NSCLC Cells.

BACKGROUND: Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is a recurrent phenomenon during clinical therapy of non-small-cell lung cancer (NSCLC). Studies have shown that HER2 is a key factor contributing to drug resistance in a variety of cancers. Furthermore, we have observed that HER2 is overexpressed in PC-9 NSCLC cells with acquired gefitinib-resistance (PC-9/GR) as compared to that in PC-9 cells. OBJECTIVE: We hypothesized that blocking both EGFR and HER2 may serve as a potential strategy for the treatment of NSCLC with acquired gefitinib-resistance. METHODS: To target both EGFR and HER2 simultaneously, we developed a bispecific antibody HECrossMAb, which was derived from a humanized Cetuximab and Trastuzumab. The binding affinity of HECrossMAb for EGFR and HER2 was measured using an enzyme-linked immunosorbent assay. The MTT assay was used to determine the effect of HECrossMAb on the proliferation of PC-9 and PC-9/GR cells in vitro. Finally, the effect of HECrossMAb on PI3K/AKT signaling and associated transcription factors was measured using western blot analysis. RESULTS: Our results showed that HECrossMAb exerts enhanced cytotoxicity in both PC-9 and PC-9/GR cells by inhibiting the activation of PI3K/AKT signaling and expression of relevant transcription factors such as AEG-1, c-Myc, and c-Fos. CONCLUSION: Our results suggest that HECrossMAb may function as a potential therapeutic agent for treating NSCLC overexpressing EGFR and HER2.

Polyketide Starter and Extender Units Serve as Regulatory Ligands to Coordinate the Biosynthesis of Antibiotics in Actinomycetes.

Polyketides are one of the largest categories of secondary metabolites, and their biosynthesis is initiated by polyketide synthases (PKSs) using coenzyme A esters of short fatty acids (acyl-CoAs) as starter and extender units. In this study, we discover a universal regulatory mechanism in which the starter and extender units, beyond direct precursors of polyketides, function as ligands to coordinate the biosynthesis of antibiotics in actinomycetes. A novel acyl-CoA responsive TetR-like regulator (AcrT) is identified in an erythromycin-producing strain of Saccharopolyspora erythraea. AcrT shows the highest binding affinity to the promoter of the PKS-encoding gene eryAI in the DNA affinity capture assay (DACA) and directly represses the biosynthesis of erythromycin. Propionyl-CoA (P-CoA) and methylmalonyl-CoA (MM-CoA) as the starter and extender units for erythromycin biosynthesis can serve as the ligands to release AcrT from PeryAI, resulting in an improved erythromycin yield. Intriguingly, anabolic pathways of the two acyl-CoAs are also suppressed by AcrT through inhibition of the transcription of acetyl-CoA (A-CoA) and P-CoA carboxylase genes and stimulation of the transcription of citrate synthase genes, which is beneficial to bacterial growth. As P-CoA and MM-CoA accumulate, they act as ligands in turn to release AcrT from those targets, resulting in a redistribution of more A-CoA to P-CoA and MM-CoA against citrate. Furthermore, based on analyses of AcrT homologs in Streptomyces avermitilis and Streptomyces coelicolor, it is believed that polyketide starter and extender units have a prevalent, crucial role as ligands in modulating antibiotic biosynthesis in actinomycetes. IMPORTANCE Numerous antibiotics are derived from polyketides, whose biosynthesis is accurately controlled by transcriptional regulators that respond to diverse physiological or environmental signals. It is generally accepted that antibiotics or biosynthetic intermediates serve as effectors to modulate their production in actinomycetes. Our study unprecedentedly demonstrates that the direct precursors of polyketide, propionyl-CoA and methylmalonyl-CoA, play a role as ligands to modulate erythromycin biosynthesis in Saccharopolyspora erythraea. More importantly, the two acyl-CoAs as ligands could adjust their own supplies by regulating the acetyl-CoA metabolic pathway so as to well settle the relationship between cellular growth and secondary metabolism. Significantly, polyketide starter and extender units have a universal role as ligands to coordinate antibiotic biosynthesis in actinomycetes. These findings not only expand the understanding of ligand-mediated regulation for antibiotic biosynthesis but also provide new insights into the physiological functions of polyketide starter and extender units in actinomycetes.

Hyper-Synergistic Antifungal Activity of Rapamycin and Peptide-Like Compounds against Candida albicans Orthogonally via Tor1 Kinase.

Candida albicans is a life-threatening, opportunistic fungal pathogen with a high mortality rate, especially within the immunocompromised populations. Multidrug resistance combined with limited antifungal drugs even worsens the situation. Given the facts that the current drug discovery strategies fail to deliver sufficient antifungals for the emerging multidrug resistance, we urgently need to develop novel approaches. By systematically investigating what caused the different antifungal activity of rapamycin in RPMI 1640 and YPD, we discovered that peptide-like compounds can generate a hyper-synergistic antifungal effect with rapamycin on both azole-resistant and sensitive clinical C. albicans isolates. The minimum inhibitory concentration (MIC) of rapamycin reaches as low as 2.14 nM (2-9 µg/mL), distinguishing this drug combination as a hyper-synergism by having a fractional inhibitory concentration (FIC) index ≤ 0.05 from the traditional defined synergism with an FIC index < 0.5. Further studies reveal that this hyper-synergism orthogonally targets the protein Tor1 and affects the TOR signaling pathway in C. albicans, very likely without crosstalk to the stress response, Ras/cAMP/PKA, or calcineurin signaling pathways. These results lead to a novel strategy of controlling drug resistant C. albicans infection in the immunocompromised populations. Instead of prophylactically administering other antifungals with undesirable side-effects for extended durations, we now only need to coadminister some nontoxic peptide additives. The novel antifungal strategy approached in this study not only provides a new therapeutic method to control fungal infections in rapamycin-taking immunocompromised patients but also mitigates the immunosuppressive side-effects of rapamycin, repurposing rapamycin as an antifungal agent with wide applications.

[Ligands of TetR family transcriptional regulators: a review].

TetR family transcriptional regulators (TFRs) are widely distributed in bacteria and archaea, and the first discovered TFR was confirmed to control the expression of tetracycline efflux pump in Escherichia coli. TFRs can bind DNAs and ligands. Small molecule ligands can induce conformational changes of TFRs, inhibiting or promoting TFRs to control target gene expression. Currently, TFRs have a wide variety of ligands, including carbohydrates, proteins, fatty acids and their derivatives, metal ions, and so on. Due to the diversity of ligands, TFRs regulate a wide range of physiological processes, from basic carbon metabolism and nitrogen metabolism to quorum sensing and antibiotic biosynthesis. On the basis of the recent studies in our laboratory and the literature, we review here the regulatory mechanism mediated by ligands of TFRs in primary and secondary metabolism, as well as the application of ligands for TFRs in the development of gene route and the activation of antibiotic biosynthesis.

Stable overexpression of mutated PTEN in Chinese hamster ovary cells enhances their performance and therapeutic antibody production.

Chinese hamster ovary (CHO) cells with a high viable cell density (VCD), resilience to culture stress, and the capacity to continuously express recombinant proteins are highly desirable. Phosphatase and tension homology deleted on chromosome ten (PTEN) functions as a key negative regulator of the PI3K/Akt signaling pathway, mediating cell growth and survival. Its oncogenic mutant endows cells with an enhanced proliferation rate and resistance to death. In this study, the role of oncogenic PTEN C124S or G129E on the performance of CHO-K1 and CHO-IgG cells was investigated. Our results showed that CHO-K1 cells stably expressing PTEN C124S or G129E exhibited enhanced proliferation, reduced apoptosis rate, and increased transient expression of therapeutic antibodies compared to the control cells. Moreover, the stable overexpression of PTEN C124S or G129E endowed CHO-IgG cells with higher cell viability, VCD, and antibody titers (yield increased by approximately 0.77-fold) in the fed-batch culture process and enhanced their performance in response to the addition of sodium lactate. Moreover, the engineering of mutated PTEN in CHO-IgG cells did not alter antibody quality. Collectively, our data suggest that mutated PTEN is a potential target for improving the manufacture of therapeutic antibodies.

Joint engineering of SACE_Lrp and its target MarR enhances the biosynthesis and export of erythromycin in Saccharopolyspora erythraea.

The Lrp and MarR families are two groups of transcriptional regulators widely distributed among prokaryotes. However, the hierarchical-regulatory relationship between the Lrp family and the MarR family remains unknown. Our previous study found that an Lrp (SACE_Lrp) from Saccharopolyspora erythraea indirectly repressed the biosynthesis of erythromycin. In this study, we characterized a novel MarR family protein (SACE_6745) from S. erythraea, which is controlled by SACE_Lrp and plays a direct regulatory role in erythromycin biosynthesis and export. SACE_Lrp directly regulated the expression of marR by specifically binding a precise site OM (5'-CTCCGGGAACCATT-3'). Gene disruption of marR increased the production of erythromycin by 45% in S. erythraea A226. We found that MarR has direct DNA-binding activity for the promoter regions of the erythromycin biosynthetic genes, as well as an ABC exporter SACE_2701-2702 which was genetically proved to be responsible for erythromycin efflux. Disruption of SACE_Lrp in industrial S. erythraea WB was an efficient strategy to enhance erythromycin production. Herein, we jointly engineered SACE_Lrp and its target MarR by deleting marR in WBΔSACE_Lrp, resulting in 20% increase in erythromycin yield in mutant WBΔLrpΔmarR compared to WBΔSACE_Lrp, and 39% to WB. Overall, our findings provide new insights into the hierarchical-regulatory relationship of Lrp and MarR proteins and new avenues for coordinating antibiotic biosynthesis and export by joint engineering regulators in actinomycetes. KEY POINTS: • The hierarchical-regulatory relationship between SACE_Lrp and MarR was identified. • MarR directly controlled the expression of erythromycin biosynthesis and export genes. • Joint engineering of SACE_Lrp-MarR regulatory element enhanced erythromycin production.

Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p.

Clinical use of antimicrobials faces great challenges from the emergence of multidrug-resistant pathogens. The overexpression of drug efflux pumps is one of the major contributors to multidrug resistance (MDR). Reversing the function of drug efflux pumps is a promising approach to overcome MDR. In the life-threatening fungal pathogen Candida albicans, the major facilitator superfamily (MFS) transporter Mdr1p can excrete many structurally unrelated antifungals, leading to MDR. Here we report a counterintuitive case of reversing MDR in C. albicans by using a natural product berberine to hijack the overexpressed Mdr1p for its own importation. Moreover, we illustrate that the imported berberine accumulates in mitochondria and compromises the mitochondrial function by impairing mitochondrial membrane potential and mitochondrial Complex I. This results in the selective elimination of Mdr1p overexpressed C. albicans cells. Furthermore, we show that berberine treatment can prolong the mean survival time of mice with blood-borne dissemination of Mdr1p overexpressed multidrug-resistant candidiasis. This study provides a potential direction of novel anti-MDR drug discovery by screening for multidrug efflux pump converters.

The variable oligomeric state of Amuc_1100 from Akkermansia muciniphila.

Akkermansia muciniphila is a beneficial microorganism colonized in the human gut that can reverse many intestinal metabolic-related diseases. Amuc_1100 is an outer-membrane protein of A. muciniphila. Oral administration of Amuc_1100 can reduce fat mass development, insulin resistance, and dyslipidemia in mice and activated the toll-like receptor 2 (TLR2) to regulate the immune response of the host, but the molecular mechanism remains unclear. Here we report the crystal structure of the extramembranous domain of Amuc_1100, which consists of a four-stranded antiparallel ß-sheet and four α-helices. Two C-terminal helices and the four-stranded antiparallel ß-sheet formed two "αßß" motifs and constituted the core domain, which shared a similar fold with type IV pili and type II Secretion system protein. Although the full-length of the extramembranous domain of Amuc_1100 existed as a monomer in solution, they formed trimer in the crystal. Elimination of the N-terminal coiled-coil helix α1 led to dimerization of Amuc_1100 both in solution and in crystal, indicating that the oligomeric state of Amuc_1100 was variable and could be influenced by α1. In addition, we identified that Amuc_1100 could directly bind human TLR2 (hTRL2) in vitro, suggesting that Amuc_1100 may serve as a new ligand for hTLR2. Dimerization of Amuc_1100 improved its hTLR2-binding affinity, suggesting that the α1-truncated Amuc_1100 could be a beneficial candidate for the development of A. muciniphila related drugs.

Transcriptional regulation of a leucine-responsive regulatory protein for directly controlling lincomycin biosynthesis in Streptomyces lincolnensis.

Leucine-responsive regulatory proteins (Lrps) are a family of transcription factors involved in diverse biological processes in bacteria. So far, molecular mechanism of Lrps for regulating antibiotics biosynthesis in actinomycetes remains largely unexplored. This study, for the first time in Streptomyces lincolnensis, identified an Lrp (named as SLCG_Lrp) associated with lincomycin production. SLCG_Lrp was validated to be a positive regulator for lincomycin biosynthesis by directly stimulating transcription of two structural genes (lmbA and lmbV), three resistance genes (lmrA, lmrB and lmrC), and a regulatory gene (lmbU) within the lincomycin biosynthetic gene (lin) cluster. SLCG_Lrp was transcriptionally self-inhibited and triggered the expression of its adjacent gene SLCG_3127 encoding a LysE superfamily protein. Further, the binding site of SLCG_Lrp in the intergenic region of SLCG_3127 and SLCG_Lrp was precisely identified. Inactivation of SLCG_3127 in S. lincolnensis resulted in yield improvement of lincomycin, which was caused by intracellular accumulation of proline and cysteine. Arginine and phenylalanine were identified as specific regulatory ligands, respectively, to reduce and promote DNA-binding affinity of SLCG_Lrp. We further found that SLCG_Lrp was directly repressed by SLCG_2919, the first identified transcription factor outside lin cluster for lincomycin production. Therefore, our findings revealed SLCG_Lrp-mediated transcriptional regulation of lincomycin biosynthesis. This study extends the understanding of molecular mechanisms underlying lincomycin biosynthetic regulation.

Developmental regulator BldD directly regulates lincomycin biosynthesis in Streptomyces lincolnensis.

The regulatory mechanism of lincomycin biosynthesis remains largely unknown, although lincomycin and its derivatives have been of great application in pharmaceutical industry. As a global regulator, BldD is widespread in Streptomyces, and functions as an on-off switch to regulate the transition from morphological differentiation to secondary metabolism, inspiring us to explore scarcely regulatory realm of lincomycin biosynthesis. In this work, deletion of bldD gene (SLCG_1664) in Streptomyces lincolnensis blocked the sporulation and nearly abolished lincomycin production, while the morphological phenotype and lincomycin production were restored when introducing a functional bldD gene into the ΔbldD mutant. S. lincolnensis BldD (BldDSL) was validated to bind to upstream regions of lincomycin biosynthetic structural genes lmbA, lmbC-lmbD, lmbE, lmbV-lmbW, resistant genes lmrA, lmrB, lmrC, and regulatory gene lmbU. Disruption of bldD significantly decreased the transcription of genes in lincomycin biosynthetic cluster, thus resulting in the sharply loss of lincomycin production. These findings indicate that BldDSL, similar to Saccharopolyspora erythraea BldD (BldDSE), directly regulates the biosynthesis of lincomycin. What's more, we discovered that BldDSE could bind to upstream regions of lmbA, lmbV-lmbW, lmrA and lmrC. Corresponding to this, S. lincolnensis BldD can bind to upstream region of eryAI-eryBIV, revealing an interactional regulation of the two BldDs. In summary, our data indicated that the developmental regulator BldD played a vital role in directly regulating the biosynthesis of lincomycin, and expanded the knowledge on lincomycin biosynthetic regulation in S. lincolnensis.

A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules.

Besides genome editing, CRISPR-Cas12a has recently been used for DNA detection applications with attomolar sensitivity but, to our knowledge, it has not been used for the detection of small molecules. Bacterial allosteric transcription factors (aTFs) have evolved to sense and respond sensitively to a variety of small molecules to benefit bacterial survival. By combining the single-stranded DNA cleavage ability of CRISPR-Cas12a and the competitive binding activities of aTFs for small molecules and double-stranded DNA, here we develop a simple, supersensitive, fast and high-throughput platform for the detection of small molecules, designated CaT-SMelor (CRISPR-Cas12a- and aTF-mediated small molecule detector). CaT-SMelor is successfully evaluated by detecting nanomolar levels of various small molecules, including uric acid and p-hydroxybenzoic acid among their structurally similar analogues. We also demonstrate that our CaT-SMelor directly measured the uric acid concentration in clinical human blood samples, indicating a great potential of CaT-SMelor in the detection of small molecules.

MiR-106b promotes therapeutic antibody expression in CHO cells by targeting deubiquitinase CYLD.

MicroRNAs (miRNAs) function as important regulators of major cellular processes, such as cell cycle, proliferation, development, and apoptosis. Recently, miRNA engineering of Chinese hamster ovary (CHO) cells has emerged as a promising strategy for enhancing therapeutic antibody production. Previously, we have reported that inhibition of deubiquitinase cylindromatosis (CYLD) remarkably enhanced the therapeutic antibody production in CHO cells. However, the mechanisms regulating CYLD in CHO cells remain elusive. Herein, we demonstrated that miR-106b targets CYLD directly, as shown by a series of bioinformatics analyses and experimental assays. Stable overexpression of miR-106b in CHO cells promoted CHO cell viability and subsequent antibody expression in transient transfection assay. Furthermore, the results in fed-batch culture showed that stable overexpression of miR-106b in a CHO-IgG cell line achieved about 0.66-fold promotion in product titer compared to the parental cells. Meanwhile, overexpression of miR-106b did not affect the quality of antibody. Taken together, our findings highlight the effect of miR-106b inhibition in CYLD synthesis and its function in antibody expression as a new target for improving CHO manufacturing cells.