An unusual aromatase/cyclase programs the formation of the phenyldimethylanthrone framework in anthrabenzoxocinones and fasamycin.

Aromatic polyketides are renowned for their wide-ranging pharmaceutical activities. Their structural diversity is mainly produced via modification of limited types of basic frameworks. In this study, we characterized the biosynthesis of a unique basic aromatic framework, phenyldimethylanthrone (PDA) found in (+)/(-)-anthrabenzoxocinones (ABXs) and fasamycin (FAS). Its biosynthesis employs a methyltransferase (Abx(+)M/Abx(-)M/FasT) and an unusual TcmI-like aromatase/cyclase (ARO/CYC, Abx(+)D/Abx(-)D/FasL) as well as a nonessential helper ARO/CYC (Abx(+)C/Abx(-)C/FasD) to catalyze the aromatization/cyclization of polyketide chain, leading to the formation of all four aromatic rings of the PDA framework, including the C9 to C14 ring and a rare angular benzene ring. Biochemical and structural analysis of Abx(+)D reveals a unique loop region, giving rise to its distinct acyl carrier protein-dependent specificity compared to other conventional TcmI-type ARO/CYCs, all of which impose on free molecules. Mutagenic analysis discloses critical residues of Abx(+)D for its catalytic activity and indicates that the size and shape of its interior pocket determine the orientation of aromatization/cyclization. This study unveils the tetracyclic and non-TcmN type C9 to C14 ARO/CYC, significantly expanding our cognition of ARO/CYCs and the biosynthesis of aromatic polyketide framework.

A large conserved family of small-molecule carboxyl methyltransferases identified from microorganisms.

Small-molecule carboxyl methyltransferases (CbMTs) constitute a small proportion of the reported methyltransferases, but they have received extensive attention due to their important physiological functions. Most of the small-molecule CbMTs isolated to date originate from plants and are members of the SABATH family. In this study, we identified a type of CbMT (OPCMT) from a group of Mycobacteria, which has a distinct catalytic mechanism from the SABATH methyltransferases. The enzyme contains a large hydrophobic substrate-binding pocket (~400 Å3) and utilizes two conserved residues, Thr20 and Try194, to retain the substrate in a favorable orientation for catalytic transmethylation. The OPCMT_like MTs have a broad substrate scope and can accept diverse carboxylic acids enabling efficient production of methyl esters. They are widely (more than 10,000) distributed in microorganisms, including several well-known pathogens, whereas no related genes are found in humans. In vivo experiments implied that the OPCMT_like MTs was indispensable for M. neoaurum, suggesting that these proteins have important physiological functions.

Hepatectomy After Conversion Therapy Using Tyrosine Kinase Inhibitors Plus Anti-PD-1 Antibody Therapy for Patients with Unresectable Hepatocellular Carcinoma.

BACKGROUND: Combined treatment with tyrosine kinase inhibitors (TKI) plus anti-PD-1 antibodies showed high anti-tumor efficacy and made conversion resection possible for patients with unresectable hepatocellular carcinoma (HCC). However, long-term survival has not been reported. METHODS: A cohort of consecutive patients who received combined TKI/anti-PD-1 antibodies as first-line treatment for initially unresectable HCC at the authors' hospital between August 2018 and September 2020 was eligible for this study. Patients who were responding to systemic therapy and met the criteria for hepatectomy underwent liver resection with curative intention. The study also investigated the association of clinical factors with successful conversion resection and postoperative recurrence. RESULTS: The study enrolled 101 patients including 24 patients (23.8 %) who underwent R0 resection a median of 3.9 months (interquartile range: 2.5-5.9 months) after initiation of systemic therapy. Patients with an Eastern cooperative oncology group performance status of 0, fewer intrahepatic tumors, or a radiographic response to systemic therapy were more likely to be able to receive curative resection. After a median follow-up period of 21.5 months, hepatectomy was independently associated with a favorable overall survival (hazard ratio [HR], 0.050; 95 % confidence interval [CI], 0.007-0.365; P = 0.003). For the 24 patients who underwent surgery, the 12-month recurrence-free survival and overall survival rates were respectively 75% and 95.8%. Achieving a pathologic complete response (n = 10) to systemic therapy was associated with a favorable recurrence-free survival after resection, with a trend toward significance (HR, 0.345; 95% CI, 0.067-1.785; P = 0.187). CONCLUSIONS: Selected patients with initially unresectable HCC can undergo hepatectomy after systemic therapy with combined TKI/anti-PD-1 antibodies. In this study, conversion resection was associated with a favorable prognosis.

Formation and Loading of a (2S)-2-Ethylmalonamyl Starter Unit in the Assembly Line of Polyketide-Nonribosomal Peptide Hybrid Sanglifehrin A.

Sanglifehrin A (SFA) is a spirolactam-conjugated, 22-membered macrolide with remarkable immunosuppressive and antiviral activities. This macrolide is a result of a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) assembly line that utilizes (2S)-2-ethylmalonamyl as a starter unit. Here, we report that the formation and loading of this starter unit in the SFA assembly line involve two unusual enzymatic reactions that occur on a discrete acyl carrier protein (ACP), SfaO. An amide synthetase, SfaP, catalyzes the amidation of (2S)-2-ethylmalonyl in a SfaO-dependent manner. Then, a ß-ketoacyl-ACP synthase III-like protein, SfaN, transfers resultant (2S)-2-ethylmalonamyl from SfaO onto the loading ACP domain of the hybrid PKS-NRPS assembly line to prime SFA biosynthesis. Both SfaP and SfaN display promiscuous activities. This study furthers the appreciation of assembly line chemistry, as a new paradigm for unusual building block formation and incorporation is provided.

Engineering the Substrate Specificity of a P450 Dimerase Enables the Collective Biosynthesis of Heterodimeric Tryptophan-Containing Diketopiperazines.

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. Biosynthesis offers a practical opportunity to access their bioactive structural diversity, however, it is restricted by the limited substrate scopes of the HTDKPs-forming P450 dimerases. Herein, by genome mining and investigation of the sequence-product relationships, we unveiled three important residues (F387, F388 and E73) in these P450s that are pivotal for selecting different diketopiperazine (DKP) substrates in the upper binding pocket. Engineering these residues in NasF5053 significantly expanded its substrate specificity and enabled the collective biosynthesis, including 12 self-dimerized and at least 81 cross-dimerized HTDKPs. Structural and molecular dynamics analysis of F387G and E73S revealed that they control the substrate specificity via reducing steric hindrance and regulating substrate tunnels, respectively.

Biosynthesis of pyrroloindoline-containing natural products.

Covering: up to 2022Pyrroloindoline is a privileged tricyclic indoline motif widely present in many biologically active and medicinally valuable natural products. Thus, understanding the biosynthesis of this molecule is critical for developing convenient synthetic routes, which is highly challenging for its chemical synthesis due to the presence of rich chiral centers in this molecule, especially the fully substituted chiral carbon center at the C3-position of its rigid tricyclic structure. In recent years, progress has been made in elucidating the biosynthetic pathways and enzymatic mechanisms of pyrroloindoline-containing natural products (PiNPs). This article reviews the main advances in the past few decades based on the different substitutions on the C3 position of PiNPs, especially the various key enzymatic mechanisms involved in the biosynthesis of different types of PiNPs.

Expanding the Chemical Diversity of Fasamycin Via Genome Mining and Biocatalysis.

Genome mining and biocatalytic modification of chemical structures are critical methods to develop new antibiotics. In this study, eight new fasamycins (3, 4, 6, and 8-12) along with five known analogues (1, 2, 5, 7, and 13) were obtained by the overexpression of two phosphopantetheinyl transferases (PPtases) in Streptomyces kanamyceticus and biocatalytic transformation with two halogenases. These new compounds displayed significant activity against Staphylococcus aureus and Bacillus subtilis, in particular, C-29-methyl and C-2/C-22-halogen derivatives. This study increases the chemical diversity of bioactive fasamycin derivatives and provides useful halogenation tools for engineering their scaffolds.

A Pair of Atypical KAS III Homologues with Initiation and Elongation Functions Program the Polyketide Biosynthesis in Asukamycin.

ß-Ketoacyl-ACP synthase III (KAS III) is a class of important C-C bond-forming enzymes that mostly catalyze the initiation of polyketide and fatty acid biosynthesis. In this study, we elucidated an unusual polyketide synthase (PKS) system that involves two unique KAS IIIs (AsuC3 and C4) in the biosynthesis of the upper triene chain of asukamycin. Significantly, AsuC3 and C4 have both initiation and iterative elongation activity, while being functionally biased toward the elongation and initiation steps, respectively. Mutational analysis revealed that their catalytic activities rely on the catalytic triad Cys-His-Asn. Unlike other KAS IIIs, AsuC3 and C4 are very promiscuous and can accept various lengths of acyl-CoAs with either cyclic, branched or linear acyl moieties. By cooperation with the permissive ketoreductase (AsuC7) and dehydratase (AsuC8/C9), a large variety of polyenes can be efficiently synthesized. This study significantly broadens the understanding of KAS IIIs and polyketide biosynthesis.

An Atypical Acyl-CoA Synthetase Enables Efficient Biosynthesis of Extender Units for Engineering a Polyketide Carbon Scaffold.

Acyl-CoAs are key precursors of primary and secondary metabolism. Their efficient biosynthesis is often impeded by the limited substrate specificity and low in vivo activity of acyl-CoA synthetases (ACSs) due to regulatory acylation of the catalytically important lysine residue in motif A10 (Lys-A10). In this study, we identified an unusual ACS (UkaQ) from the UK-2A biosynthetic pathway that naturally lacks the Lys-A10 residue and exhibits extraordinarily broad substrate specificity. Protein engineering significantly improved its stability and catalytic activity, enabling it to synthesize a large variety of acyl-CoAs with highly robust activity. By combining it with permissive carboxylases, we produced a large array of polyketide extender units and obtained six novel halobenzyl-containing antimycin analogues through an engineered biosynthetic pathway. This study significantly expands the catalytic mode of ACSs and provides a potent tool for the biosynthesis of acyl-CoA-derived natural products.

Engineering Leifsonia Alcohol Dehydrogenase for Thermostability and Catalytic Efficiency by Enhancing Subunit Interactions.

Leifsonia alcohol dehydrogenase (LnADH) is a promising biocatalyst for the synthesis of chiral alcohols. However, limitations of wild-type LnADH observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve its thermostability and catalytic efficiency by altering the subunit interfaces. Residues T100 and S148 were identified to be significant for thermostability and activity, and the melting temperature (ΔTm ) and catalytic efficiency of the mutant T100R/S148I toward ketone substrates was improved by 18.7 °C and 1.8-5.5-fold. Solving the crystal structures of the wild-type enzyme and T100R/S148L revealed beneficial effects of mutations on stability and catalytic activity. The most robust mutant T100R/S148I is promising for industrial applications and can produce 200 g liter-1 day-1 chiral alcohols at 50 °C by only a 1 : 500 ratio of enzyme to substrate.

Production of Heterodimeric Diketopiperazines Employing a Mycobacterium-Based Whole-Cell Biocatalysis System.

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. P450-mediated biocatalysis offers a practical avenue to access their structural diversity; however, many of these enzymes are insoluble in Escherichia coli and difficult to operate in Streptomyces. Through validation of the functions of two pairs Mycobacterium smegmatis sourced redox partners in vitro, and comparing the efficiency of different biocatalytic systems with tricky P450s in vivo, we herein demonstrated that M. smegmatis is much more efficient, robust, and cleaner in metabolites background than the regularly used E. coli or Streptomyces systems. The M. smegmatis-based system can completely convert 1 g L-1 of cyclodipeptide into HTDKPs within 18 h with minimal background metabolites. On the basis of this efficient system, 12 novel HTDPKs were readily obtained by using two HTDKP-forming P450s (NasbB and NASS1868). Among them, five compounds have neuroprotective properties. Our study significantly expands the bioactive chemical scope of HTDKPs and provides an excellent biocatalysis platform for dealing with problematic enzymes from Actinomycetes.

A Dual Role Reductase from Phytosterols Catabolism Enables the Efficient Production of Valuable Steroid Precursors.

4-Androstenedione (4-AD) and progesterone (PG) are two of the most important precursors for synthesis of steroid drugs, however their current manufacturing processes suffer from low efficiency and severe environmental issues. In this study, we decipher a dual-role reductase (mnOpccR) in the phytosterols catabolism, which engages in two different metabolic branches to produce the key intermediate 20-hydroxymethyl pregn-4-ene-3-one (4-HBC) through a 4-e reduction of 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA) and 2-e reduction of 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA), respectively. Inactivation or overexpression of mnOpccR in the Mycobacterium neoaurum can achieve exclusive production of either 4-AD or 4-HBC from phytosterols. By utilizing a two-step synthesis, 4-HBC can be efficiently converted into PG in a scalable manner (100 gram scale). This study deciphers a pivotal biosynthetic mechanism of phytosterol catabolism and provides very efficient production routes of 4-AD and PG.

Knockout of pde gene in Arthrobacter sp. CGMCC 3584 and transcriptomic analysis of its effects on cAMP production.

Arthrobacter sp. CGMCC 3584 is used for the industrial production of cyclic adenosine monophosphate (cAMP). However, because of the paucity of genetic engineering tools for genetic manipulation on Arthrobacter species, only a few metabolically engineered Arthrobacter have been constructed and investigated. In this study, for the first time, we constructed an arpde knockout mutant of Arthrobacter without any antibiotic resistance marker by a PCR-targeting-based homologous recombination method. Our results revealed that the deletion of arpde had little effect on biomass production and improved cAMP production by 31.1%. Furthermore, we compared the transcriptomes of the arpde knockout strain and the wild strain, aiming to understand the capacities of cAMP production due to arpde inactivation at the molecular level. Comparative transcriptomic analysis revealed that arpde inactivation had two major effects on metabolism: inhibition of glycolysis, PP pathway, and amino acid metabolism (phenylalanine, tryptophan, branched-chain amino acids, and glutamate metabolism); promotion of the purine metabolism and carbon flux from the precursor 5'-phosphoribosyl 1-pyrophosphate, which benefited cAMP production.

Arsenic trioxide inhibits EMT in hepatocellular carcinoma by promoting lncRNA MEG3 via PKM2.

Hepatocellular carcinoma (HCC) presents a great burden for patients worldwide, and metastasis of HCC remains problematic. Arsenic trioxide is a traditional drug that has shown excellent efficacy when applied as cancer therapy. Our study explored the antimetastatic mechanism of arsenic trioxide in HCC. We investigated changes in pyruvate kinase muscle isoform 2 (PKM2) and maternal expression gene 3 (MEG3) following treatment with arsenic trioxide in HCC cells. Consequently, arsenic trioxide negatively regulated PKM2 and positively regulated MEG3. We explored migration ability and the expression of the epithelial to mesenchymal transition (EMT)-related biomarkers E-cadherin, N-cadherin and Vimentin by silencing MEG3 under arsenic trioxide treatment. The wound healing assay showed that arsenic trioxide inhibited the migration of HCC, but silencing MEG3 partially reversed this effect. On the other hand, the EMT-related biomarkers are alleviated under the treatment of arsenic trioxide, but this effect deteriorated when MEG3 is silenced. In conclusion, our study demonstrates a novel mechanism by which arsenic trioxide inhibits EMT in hepatocellular carcinoma by promoting lncRNA MEG3 and PKM2 negatively regulating MEG3.

Characterization of the Biosynthetic Gene Cluster for the Antibiotic Armeniaspirols in Streptomyces armeniacus.

Armeniaspirols (1-3) are potent antibiotics against Gram-positive pathogens. Through a biosynthetic investigation, we identified four enzymes involved in the structural modification of 1-3. Manipulation of their activity led to the generation of 4-6 and nine novel analogues, 7-15. Bioactivity assessments revealed that the pyrrole chloro group and the methyl group are important for the antimicrobial activities of armeniaspirols, which lays the foundation for future structure optimization and mechanism of action studies of armeniaspirols.

Hypoxia-induced HMGB1 expression of HCC promotes tumor invasiveness and metastasis via regulating macrophage-derived IL-6.

Hypoxia is associated with the progression of hepatocellular carcinoma through promotion of spontaneous metastasis but the mechanism remains unclear. Here, we hypothesis that tumor cell-derived HMGB1 orchestrates macrophages infiltration and promotes metastasis of HCC via enhancing macrophage-secreted IL-6 under hypoxia. HMGB1 expression was robustly exacerbated in tumors of HCC patients with PVTT. Meanwhile, hypoxia exposure gave rise to HMGB1 expression in hepatoma cells of human and mouse in a HIF-1α-dependent manner and subsequently induced the infiltration and reprogramming of macrophages to augment the expression of Il-6. Further study demonstrated macrophage-derived IL-6 enhanced the invasiveness and metastasis of murine HCC cells. Therefore, our study provides a novel understanding of the relationship between tumor cells and tumor associated macrophages (TAMs) in the context of hypoxia.

Structural Basis of a Broadly Selective Acyltransferase from the Polyketide Synthase of Splenocin.

Polyketides are a large family of pharmaceutically important natural products, and the structural modification of their scaffolds is significant for drug development. Herein, we report high-resolution X-ray crystal structures of the broadly selective acyltransferase (AT) from the splenocin polyketide synthase (SpnD-AT) in the apo form and in complex with benzylmalonyl and pentynylmalonyl extender unit mimics. These structures revealed the molecular basis for the stereoselectivity and substrate specificity of SpnD-AT, and enabled the engineering of the industrially important Ery-AT6 to broaden its substrate scope to include three new types of extender units.

Stabilization of Multimeric Proteins via Intersubunit Cyclization.

Proteins with high catalytic efficiency and selectivity under mild conditions have long been appreciated by industrial and medicinal fields. These proteins, which are commonly multimeric, often possess low stability, impeding wider application. Currently, strategies to improve the stability of multimeric proteins concentrate on enhancing the interaction at internal interface of the subunits. In this report, we confirmed that the largely underestimated subunit terminal ends are as significant as the internal interface for protein stability. By connecting both the terminal ends and internal interface of subunits, the tetrameric Leifsonia alcohol dehydrogenase (LnADH) protein can been cyclized into a rigid form with significantly improved thermostability and resilience. The improvement in the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment (T5015) and melting temperature (Tm ) of the modified protein was 18°C and 23.3°C, respectively, which is superior to the results achieved by normal protein engineering. Our study provided a novel strategy to effectively improve the stability of multimeric proteins, which is suitable not only for the short-chain dehydrogenase/reductase (SDR) family but also other classes of proteins with close terminal ends.IMPORTANCE Industrially interesting proteins are generally multimeric proteins; however, their applications are often restricted due to low stability caused by the natural tendency of subunit disassociation. Current approaches targeting this problem mainly focus on enhancing the internal interfaces of the subunits to avoid their disassociation. In this study, we identified and confirmed the external interface to be significant for improving the stability of multimeric proteins. By connecting the terminal ends and internal interface with disulfide bonds, we found that the multimeric protein LnADH cyclized into a robust monomeric-like form, resulting in superior thermostability compared to traditional protein engineering. This intersubunit cyclization approach is efficient and easy to perform, providing a novel method for engineering many important classes of multimeric proteins.

Prospective Study of Transcatheter Arterial Chemoembolization (TACE) with Ginsenoside Rg3 versus TACE Alone for the Treatment of Patients with Advanced Hepatocellular Carcinoma.

Purpose To conduct a single-center, open-label, randomized, controlled trial to compare the effectiveness and safety of (a) ginsenoside Rg3 combined with transcatheter arterial chemoembolization (TACE) and (b) TACE alone in patients with advanced hepatocellular carcinoma (HCC). Materials and Methods This trial was approved by the Fudan University Zhongshan Hospital ethics committee and was registered with the Chinese Clinical Trial Registry (ChiCTR-TRC-11001643). After informed consent was obtained, 228 patients with advanced HCC (Barcelona Clinic Liver Cancer stage C) were randomly assigned to receive an Rg3 capsule and undergo TACE (n = 152; mean age ± standard deviation, 52.4 years ± 11.8; 84.2% men) or undergo TACE alone (n = 76; mean age, 52.4 years ± 10.4; 82.9% men). TACE was performed by using iodized oil with epirubicin and gelatin sponge after oxaliplatin and 5-fluorouracil were infused. The primary end point was overall survival. Secondary end points included time to progression, time to untreatable progression, disease control rate, and safety. Data were compared with the log-rank test, and survival curves were generated with the Kaplan-Meier method. Results Median overall survival was 13.2 months (95% confidence interval [CI]: 11.15, 15.26) in the TACE with Rg3 group and 10.1 months (95% CI: 9.14, 11.06) in the control group (hazard ratio, 0.63 [95% CI: 0.46, 0.85]; P = .002). Median time to progression (4.3 vs 3.2 months, respectively; P = .151) and median time to untreatable progression (8.3 vs 7.3 months, respectively; P = .063) were similar in the two groups. Disease control rate was 69.7% in the TACE with Rg3 group versus 51.3% in the control group (P = .012). Constipation and epistaxis were more frequent in the Rg3 with TACE group (P < .05). Importantly, Rg3 alleviated some TACE-related adverse syndromes and blood anomalies. Conclusion In patients with advanced HCC and adequate liver function, the combination of TACE and ginsenoside Rg3 may prolong overall survival when compared with TACE alone. (©) RSNA, 2016.