Integrated Biofilm Modification and Transcriptional Analysis for Improving Fengycin Production in Bacillus amyloliquefaciens.

Although fengycin exhibits broad-spectrum antifungal properties, its application is hindered due to its low biosynthesis level and the co-existence of iturin A and surfactin in Bacillus amyloliquefaciens HM618, a probiotic strain. In this study, transcriptome analysis and gene editing were used to explore the potential mechanisms regulating fengycin production in B. amyloliquefaciens. The fengycin level of B. amyloliquefacien HM-3 (∆itu-ΔsrfAA) was 88.41 mg/L after simultaneously inhibiting the biosyntheses of iturin A and surfactin. The knockout of gene eps associated with biofilm formation significantly increased the fengycin level of the strain HM618, whereas the fengycin level decreased 32.05% after knocking out sinI, a regulator of biofilm formation. Transcriptome analysis revealed that the differentially expressed genes, involved in pathways of amino acid and fatty acid syntheses, were significantly down-regulated in the recombinant strains, which is likely associated with a decrease of fengycin production. The knockout of gene comQXPA and subsequent transcriptome analysis revealed that the ComQXPA quorum sensing system played a positive regulatory role in fengycin production. Through targeted genetic modifications and fermentation optimization, the fengycin production of the engineered strain HM-12 (∆itu-ΔsrfAA-ΔyvbJ) in a 5-L fermenter reached 1.172 g/L, a 12.26-fold increase compared to the fengycin level in the strain HM-3 (∆itu-ΔsrfAA) in the Erlenmeyer flask. Taken together, these results reveal the underlying metabolic mechanisms associated with fengycin synthesis and provide a potential strategy for improving fengycin production in B. amyloliquefaciens.

Biocatalytic Steroidal 9α-Hydroxylation and Fragmentation Enable the Concise Chemoenzymatic Synthesis of 9,10-Secosteroids.

9,10-Secosteroids are an important group of marine steroids with diverse biological activities. Herein, we report a chemoenzymatic strategy for the concise, modular, and scalable synthesis of ten naturally occurring 9,10-secosteroids from readily available steroids in three to eight steps. The key feature lies in utilizing a Rieske oxygenase-like 3-ketosteroid 9α-hydroxylase (KSH) as the biocatalyst to achieve efficient C9-C10 bond cleavage and A-ring aromatization of tetracyclic steroids through 9α-hydroxylation and fragmentation. With synthesized 9,10-secosteroides, structure-activity relationship was evaluated based on bioassays in terms of previously unexplored anti-infective activity. This study provides experimental evidence to support the hypothesis that the biosynthetic pathway through which 9,10-secosteroids are formed in nature shares a similar 9α-hydroxylation and fragmentation cascade. In addition to the development of a biomimetic approach for 9,10-secosteroid synthesis, this study highlights the great potential of chemoenzymatic strategies in chemical synthesis.

An Engineered Microbial Consortium Provides Precursors for Fengycin Production by Bacillus subtilis.

Fengycin has great potential for applications in biological control because of its biosafety and degradability. In this study, the addition of exogenous precursors increased fengycin production by Bacillus subtilis. Corynebacterium glutamicum was engineered to produce high levels of precursors (Thr, Pro, Val, and Ile) to promote the biosynthesis of fengycin. Furthermore, recombinant C. glutamicum and Yarrowia lipolytica providing amino acid and fatty acid precursors were co-cultured to improve fengycin production by B. subtilis in a three-strain artificial consortium, in which fengycin production was 2100 mg·L-1. In addition, fengycin production by the consortium in a 5 L bioreactor reached 3290 mg·L-1. Fengycin had a significant antifungal effect on Rhizoctonia solani, which illustrates its potential as a food preservative. Taken together, this work provides a new strategy for improving fengycin production by a microbial consortium and metabolic engineering.

TAT-Modified Martentoxin Displays Intravenous Antiseizure Activities.

Epilepsy is a chronic disease of brain dysfunction, which arises from imbalance between excitatory and inhibitory activities in neural circuits. Previously, we reported that peptide Martentoxin (MarTX), from scorpion Buthus martensii Karsch, displayed antiseizure activities by specifically inhibiting BK(α + ß4) channel currents. Injection of MarTX into the hippocampal region of mice significantly alleviated convulsive seizures. However, intravenous injection of MarTX had no antiepileptic efficacy due to the blood-brain barrier (BBB). To address this, here, we designed cell-penetrating peptide TAT-modified MarTX, in which the linker containing three glycines was put between TAT and the N-terminus of MarTX (forming MTX-N-TAT) or between TAT and the C-terminus of MarTX (forming MTX-C-TAT), respectively. We prepared them in a large amount through Escherichia coli overexpression system and then probed their antiseizure activities. Our results indicated that intravenous injection of MTX-C-TAT showed significant therapeutic efficacy of antiseizure. It increased seizure latency, reduced the total seizure duration and the number of seizures at stages 3, 4, and 5, inhibited hippocampal neuronal hyperexcitability, and exhibited neuroprotective effects on hippocampal neurons. These studies implied that MTX-C-TAT displayed intravenous antiseizure activities properly through crossing BBB and would be a potential antiepileptic drug in the future.

Construction and Optimization of Nonclassical Isoprenoid Biosynthetic Pathways in Yeast Peroxisomes for (+)-Valencene Production.

Isoprenoids are a kind of natural product with various activities, but their plant extraction suffers low concentration. The rapid development of synthetic biology offers a sustainable route for supply of high-value-added natural products by engineering microorganisms. However, the complexity of cellular metabolism makes engineering endogenous isoprenoid biosynthetic pathways with metabolic interaction difficult. Here, for the first time, we constructed and optimized three types of isoprenoid pathways (the Haloarchaea-type, Thermoplasma-type, and isoprenoid alcohol pathway) in yeast peroxisomes for the synthesis of sesquiterpene (+)-valencene. In yeast, the Haloarchaea-type MVA pathway is more effective than the classical MVA pathway. MVK and IPK were determined to be the rate-limiting steps of the Haloarchaea-type MVA pathway, and the production of 869 mg/L (+)-valencene under fed-batch fermentation in shake flasks was realized. This work expands isoprenoid synthesis in eukaryotes and provides a more efficient pathway for isoprenoid synthesis.

Trioxacarcin A Interactions with G-Quadruplex DNA Reveal Its Potential New Targets as an Anticancer Agent.

Trioxacarcin (TXN) A was reported to be an anticancer agent through alkylation of dsDNA. G-quadruplex DNA (G4-DNA) is frequently formed in the promoter regions of oncogenes and the ends of telomerase genes, considered as promising drug targets for anticancer therapy. There are no reports about TXN A interactions with G4-DNA. Here, we tested TXN A's interactions with several G4-DNA oligos with parallel, antiparallel, or hybrid folding, respectively. We demonstrated that TXN A preferred to alkylate one flexible guanine in the loops of parallel G4-DNA. The position of the alkylated guanine is in favor of interactions of G4-DNA with TXN A. The structure of TXN A covalently bound RET G4-DNA indicated that TXN A alkylation on RET G4-DNA stabilizes the G4-DNA conformation. These studies opened a new window of how TXN A interacted with G4-DNA, which might hint a new mode of its function as an anticancer agent.

A unique hyperdynamic dimer interface permits small molecule perturbation of the melanoma oncoprotein MITF for melanoma therapy.

Microphthalmia transcription factor (MITF) regulates melanocyte development and is the "lineage-specific survival" oncogene of melanoma. MITF is essential for melanoma initiation, progression, and relapse and has been considered an important therapeutic target; however, direct inhibition of MITF through small molecules is considered impossible, due to the absence of a ligand-binding pocket for drug design. Here, our structural analyses show that the structure of MITF is hyperdynamic because of its out-of-register leucine zipper with a 3-residue insertion. The dynamic MITF is highly vulnerable to dimer-disrupting mutations, as we observed that MITF loss-of-function mutations in human Waardenburg syndrome type 2 A are frequently located on the dimer interface and disrupt the dimer forming ability accordingly. These observations suggest a unique opportunity to inhibit MITF with small molecules capable of disrupting the MITF dimer. From a high throughput screening against 654,650 compounds, we discovered compound TT-012, which specifically binds to dynamic MITF and destroys the latter's dimer formation and DNA-binding ability. Using chromatin immunoprecipitation assay and RNA sequencing, we showed that TT-012 inhibits the transcriptional activity of MITF in B16F10 melanoma cells. In addition, TT-012 inhibits the growth of high-MITF melanoma cells, and inhibits the tumor growth and metastasis with tolerable toxicity to liver and immune cells in animal models. Together, this study demonstrates a unique hyperdynamic dimer interface in melanoma oncoprotein MITF, and reveals a novel approach to therapeutically suppress MITF activity.

Mutualistic microbial community of Bacillus amyloliquefaciens and recombinant Yarrowia lipolytica co-produced lipopeptides and fatty acids from food waste.

Bioconversion is an important method for transforming food waste (FW) into high value-added products, rendering it harmless, and recycling resources. An artificial microbial consortium (AMC) was constructed to produce FW-based lipopeptides in order to investigate the strategy of FW bioconversion into value-added products. Exogenous fatty acids as a precursor significantly improved the lipopeptide production of Bacillus amyloliquefaciens HM618. To enhance fatty acid synthesis and efflux in AMC, the recombinant Yarrowia lipolytica YL21 (strain YL21) was constructed by screening 12 target genes related to fatty acids to replace exogenous fatty acids in order to improve lipopeptide production. The levels of fengycin, surfactin, and iturin A in the AMC of strains HM618 and YL21 reached 76.19, 192.80, and 31.32 mg L-1, increasing 7.24-, 12.13-, and 3.23-fold compared to the results from the pure culture of strain HM618 in flask with Landy medium, respectively. Furthermore, free fatty acids were almost undetectable in the co-culture of strains HM618 and YL21, although its level was around 1.25 g L-1 in the pure culture of strain YL21 with Landy medium. Interestingly, 470.24 mg L-1 of lipopeptides and 18.11 g L-1 of fatty acids were co-produced in this AMC in a bioreactor with FW medium. To our knowledge, it is the first report of FW biotransformation into co-produce of lipopeptides and fatty acids in the AMC of B. amyloliquefaciens and Y. lipolytica. These results provide new insights into the biotransformation potential of FW for value-added co-products by AMC.

Development of charybdotoxin Q18F variant as a selective peptide blocker of neuronal BK(α + ß4) channel for the treatment of epileptic seizures.

Epilepsy is the results from the imbalance between inhibition and excitation in neural circuits, which is mainly treated by some chemical drugs with side effects. Gain-of-function of BK channels or knockout of its ß4 subunit associates with spontaneous epilepsy. Currently, few reports were published about the efficacy of BK(α + ß4) channel modulators in epilepsy prevention. Charybdotoxin is a non-specific inhibitor of BK and other K+ channels. Here, by nuclear magnetic resonance (NMR) and other biochemical techniques, we found that charybdotoxin might interact with the extracellular loop of human ß4 subunit (i.e., hß4-loop) of BK(α + ß4) channel at a molar ratio 4:1 (hß4-loop vs. charybdotoxin). Charybdotoxin enhanced its ability to prevent K+ current of BK(α + ß4 H101Y) channel. The charybdotoxin Q18F variant selectively reduced the neuronal spiking frequency and increased interspike intervals of BK(α + ß4) channel by π-π stacking interactions between its residue Phe18 and residue His101 of hß4-loop. Moreover, intrahippocampal infusion of charybdotoxin Q18F variant significantly increased latency time of seizure, reduced seizure duration and seizure numbers on pentylenetetrazole-induced pre-sensitized rats, inhibited hippocampal hyperexcitability and c-Fos expression, and displayed neuroprotective effects on hippocampal neurons. These results implied that charybdotoxin Q18F variant could be potentially used for intractable epilepsy treatment by therapeutically targeting BK(α + ß4) channel.

Aromatic disulfides as potential inhibitors against interaction between deaminase APOBEC3G and HIV infectivity factor.

APOBEC3G (A3G) is a member of cytosine deaminase family with a variety of innate immune functions. It displays activities against retrovirus and retrotransposon by inhibition of virus infectivity factor (Vif)-deficient HIV-1 replication. The interaction between A3G N-terminal domain and Vif directs the cellular Cullin 5 E3-ubiquitin ligase complex to ubiquitinate A3G, and leads to A3G proteasomal degradation, which is a potential target for anti-HIV drug. Currently, there are very few reports about stable small molecules targeting the interaction between A3G and Vif. In this study, we screened two series of small molecules containing carbamyl sulfamide bond or disulfide bond as bridges of two different aromatic rings. Five asymmetrical disulfides were successfully identified against interaction between A3G and Vif with the IC 50 values close to or smaller than 1 µM, especially, not through covalently binding with A3G or Vif. They restore the A3G expression in the presence of Vif by inhibiting Vif-induced A3G ubiquitination and degradation. This study opens a way to the discovery of new anti-HIV drugs.

Global Metabolic Rewiring of Yeast Enables Overproduction of Sesquiterpene (+)-Valencene.

(+)-Valencene is a bioactive sesquiterpene that can be used for flavoring and fragrances, and microbial production provides an alternative sustainable access. However, the complexity of cellular metabolism makes it challenging for its high-level production. Here, we report the global rewiring cellular metabolism for de novo production of (+)-valencene in yeast Saccharomyces cerevisiae by engineering central metabolism, mevalonate pathway, and sesquiterpenoid synthase. In particular, we show that metabolic transformation can help accelerate the strain construction process and multiple copy expression of sesquiterpenoid synthase is essential for boosting the metabolic flux for product synthesis with enhanced supply of precursors. The engineered strain produced 1.2 g/L (+)-valencene under fed-batch fermentation in shake flasks, which was increased by 549-fold and demonstrated great potential of the yeast cell factory for (+)-valencene production.

Artificial consortia of Bacillus amyloliquefaciens HM618 and Bacillus subtilis for utilizing food waste to synthetize iturin A.

Food waste is a cheap and abundant organic resource that can be used as a substrate for the production of the broad-spectrum antifungal compound iturin A. To increase the efficiency of food waste biotransformation, different artificial consortia incorporating the iturin A producer Bacillus amyloliquefaciens HM618 together with engineered Bacillus subtilis WB800N producing lipase or amylase were constructed. The results showed that recombinant B. subtilis WB-A13 had the highest amylase activity of 23406.4 U/mL, and that the lipase activity of recombinant B. subtilis WB-L01 was 57.5 U/mL. When strain HM618 was co-cultured with strain WB-A14, the higher yield of iturin A reached to 7.66 mg/L, representing a 32.9% increase compared to the pure culture of strain HM618. In the three-strain consortium comprising strains HM618, WB-L02, and WB-A14 with initial OD600 values of 0.2, 0.15, and 0.15, respectively, the yield of iturin A reached 8.12 mg/L, which was 38.6% higher than the control. Taken together, artificial consortia of B. amyloliquefaciens and recombinant B. subtilis can produce an increased yield of iturin A, which provides a new strategy for the valorization of food waste.

Two different kinds of interaction modes of deaminase APOBEC3A with single-stranded DNA in solution detected by nuclear magnetic resonance.

APOBEC3A (A3A) deaminates deoxycytidine in target motif TC in a single-stranded DNA (we termed it as TC DNA), which mortally mutates viral pathogens and immunoglobulins, and leads to the diversification and lethality of cancers. The crystal structure of A3A-DNA revealed a unique U-shaped recognition mode of target base dC0 . However, when TC DNA was titrated into 15 N-labeled A3A solution, we observed two sets of 1 H-15 N cross-peaks of A3A in HSQC spectra, and two sets of 1 H-1 H cross-peaks of DNA in two-dimensional 13 C,15 N-filtered TOCSY spectra, indicating two different kinds of conformers of either A3A or TC DNA existing in solution. Here, mainly by NMR, we demonstrated that one DNA conformer interacted with one A3A conformer, forming a specific complex A3AS -DNAS in a way almost similar to that observed in the reported crystal A3A-DNA structure, where dC0 inserted into zinc ion binding center. While the other DNA conformer bound with another A3A conformer, but dC0 did not extend into the zinc-binding pocket, forming a nonspecific A3ANS -DNANS complex. The NMR solution structure implied three sites Asn61 , His182 and Arg189 were necessary to DNA recognition. These observations indicate a distinctive way from that reported in X-ray crystal structure, suggesting an unexpected mode of deaminase APOBEC3A to identify target motif TC in DNA in solution.

Harnessing sub-organelle metabolism for biosynthesis of isoprenoids in yeast.

Current yeast metabolic engineering in isoprenoids production mainly focuses on rewiring of cytosolic metabolic pathway. However, the precursors, cofactors and the enzymes are distributed in various sub-cellular compartments, which may hamper isoprenoid biosynthesis. On the other side, pathway compartmentalization provides several advantages for improving metabolic flux toward target products. We here summarize the recent advances on harnessing sub-organelle for isoprenoids biosynthesis in yeast, and analyze the knowledge about the localization of enzymes, cofactors and metabolites for guiding the rewiring of the sub-organelle metabolism. This review may provide some insights for constructing efficient yeast cell factories for production of isoprenoids and even other natural products.

Structural characterization and immunostimulatory activity of a glucan from Cyclina sinensis.

Cyclina sinensis is an edible clam widely distributed along the coastal waters of Asia. In the present study, a polysaccharide (CSP-1) isolated from C. sinensis was purified by a DEAE-Sepharose Fast Flow column, and it had an average molecular weight of 3.8 × 105 Da and a prevalent component monosaccharide of Glc. The results of methylation analysis and 1D/2D NMR indicated that CSP-1 was a glycogen constructed with α-1,4-Glc and branched at C-6 every 9 Glc residues. In addition, Cong red test suggests CSP-1 was not a helical conformation, and irregular and spherical lumps were observed by AFM. Moreover, CSP-1 was found to possess potent immunostimulatory activity on the basis of its significant abilities to enhance NO production and cytokines (TNF-α, IL-1ß and IL-6) secretion in RAW 264.7 macrophages.

Author Correction: Solution structure of extracellular loop of human ß4 subunit of BK channel and its biological implication on ChTX sensitivity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Colchicine selective interaction with oncogene RET G-quadruplex revealed by NMR.

G-quadruplexes (G4s) are frequently formed in the promoter regions of oncogenes, considered as promising drug targets for anticancer therapy. Due to high structure similarity of G4s, discovering ligands selectively interacting with only one G4 is extremely difficult. Here, mainly by NMR, we report that colchicine selectively binds to oncogene RET G4-DNA.

Selective Blockade of Neuronal BK (α + ß4) Channels Preventing Epileptic Seizure.

Gain-of-function of BK channels or knockout of their ß4 subunit is associated with spontaneous epilepsy. Currently, efficacy of BK (α + ß4) channel modulators in preventing epilepsy was never reported. Here, we show that martentoxin selectively inhibits BK (α + ß4) channels by interaction with the extracellular loop of the BK ß4 subunit (hß4-loop) at a molar ratio 4:1 (hß4-loop vs martentoxin). Residues Glu104, Glu122, Gln124, Lys125, and Glu128 of the hß4-loop form hydrogen bonds with residues Asp5, Glu13, Lys20, Ser24, Gln26, Lys28, and Arg35 of martentoxin, by which martentoxin reduces the neuronal spiking frequency and increases interspike intervals. Intrahippocampal infusion of martentoxin significantly increases the latency time of seizure, reduces seizure duration and seizure numbers on pentylenetetrazole-induced presensitized rats, inhibits hippocampal hyperexcitability and c-Fos expression, and displays neuroprotective effects on hippocampal neurons. These results suggest that the BK (α + ß4) channel is a novel therapeutic target of intractable epilepsy and martentoxin contributes to the rational drug design for epilepsy treatment.

A strategy to identify mixed polysaccharides through analyzing the monosaccharide composition of disaccharides released by graded acid hydrolysis.

It is still a challenge to identify mixed polysaccharides isolated from biological tissues. The present study aimed to develop a protocol that allowed for quick characterization of mixed polysaccharides by selecting Cyclina sinensis polysaccharides as test beds. Disaccharides were gradually released by graded acid hydrolysis, separated with 3 kDa molecular weight cut-off membrane, and analyzed by HPLC-ESI-MSn. Furthermore, the monosaccharide composition of the filtrate during the gradient acid hydrolysis process and the final retentate were also determined to provide composition information of these disaccharides. Thus, 6 disaccharides were detected and identified, indicating the existence of polysaccharides constructed of corresponding repeated disaccharide fragments in C. sinensis. The present study demonstrated a potential strategy to characterize mixed polysaccharides without separation and purification.

Investigating the interactions between DNA and DndE during DNA phosphorothioation.

The DNA phosphorothioate modification is a novel physiological variation in bacteria. DndE controls this modification by binding to dsDNA via a mechanism that remains unclear. Structural analysis of the wild-type DndE tetramer suggests that a positively charged region in its center is important for DNA binding. In the present study, we replaced residues G21 and G24 in this region with lysines, which increases the DNA binding affinity but does not affect the DNA degradation phenotype. Structural analysis of the mutant indicates that it forms a new tetrameric conformation and that DndE interacts with DNA as a monomer rather than as a tetramer. A structural model of the DndE-DNA complex, based on its structural homolog P22 Arc repressor, indicates that two flexible loops in DndE are determinants of DNA binding.