Developing the E. coli platform for efficient production of UMP-derived chemicals.

5-Methyluridine (5-MU) is a prominent intermediate for industrial synthesis of several antiviral-drugs, however, its availability over the past decades has overwhelmingly relied on chemical and enzymatic strategies. Here, we have realized efficient production of 5-MU in E. coli, for the first time, via a designer artificial pathway consisting of a two-enzyme cascade (UMP 5-methylase and phosphatase). More importantly, we have engineered the E. coli cell factory to boost 5-MU production by systematic evaluation of multiple strategies, and as a proof of concept, we have further developed an antibiotic-free fermentation strategy to realize 5-MU production (10.71 g/L) in E. coli MB229 (a ΔthyA strain). Remarkably, we have also established a versatile and robust platform with exploitation of the engineered E. coli for efficient production of diversified UMP-derived chemicals. This study paves the way for future engineering of E. coli as a synthetic biology platform for acceleratively accessing UMP-derived chemical diversities.

A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S.

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of ß-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the ß-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron-sulfur cluster that is necessary for activity. These findings provide an example of an iron-sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)-dependent amide bond formation in a natural product biosynthetic pathway.

Unexpected Role of a Short-Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis.

We investigated the biosynthetic pathway of type II polyketide murayaquinone. The murayaquinone biosynthetic cluster contains genes for three putative short-chain dehydrogenase/reductase family enzymes including MrqF and MrqH with known functions and MrqM with unclear function. We report the functional characterization of MrqM for its role in murayaquinone biosynthesis. Our gene deletion experiment and structural elucidation of the accumulated intermediates revealed that MrqM is related with the second polyketide ring cyclization, because the inactivation of mrqM resulted in the accumulation of an off-pathway intermediate SEK43 and disrupted the second and third ring cyclization. Site-directed mutagenesis studies showed that two conserved residues in MrqM and homologous proteins Y151 and K155 are essential for its activity. The previously proposed second/third ring cyclase, MrqD, might instead play another important role in the chain releasing step of the murayaquinone biosynthesis.

Comparative Investigation into Formycin A and Pyrazofurin A Biosynthesis Reveals Branch Pathways for the Construction of C-Nucleoside Scaffolds.

Formycin A (FOR-A) and pyrazofurin A (PRF-A) are purine-related C-nucleoside antibiotics in which ribose and a pyrazole-derived base are linked by a C-glycosidic bond. However, the logic underlying the biosynthesis of these molecules has remained largely unexplored. Here, we report the discovery of the pathways for FOR-A and PRF-A biosynthesis from diverse actinobacteria and propose that their biosynthesis is likely initiated by a lysine N6-monooxygenase. Moreover, we show that forT and prfT (involved in FOR-A and PRF-A biosynthesis, respectively) mutants are correspondingly capable of accumulating the unexpected pyrazole-related intermediates 4-amino-3,5-dicarboxypyrazole and 3,5-dicarboxy-4-oxo-4,5-dihydropyrazole. We also decipher the enzymatic mechanism of ForT/PrfT for C-glycosidic bond formation in FOR-A/PRF-A biosynthesis. To our knowledge, ForT/PrfT represents an example of ß-RFA-P (ß-ribofuranosyl-aminobenzene 5'-phosphate) synthase-like enzymes governing C-nucleoside scaffold construction in natural product biosynthesis. These data establish a foundation for combinatorial biosynthesis of related purine nucleoside antibiotics and also open the way for target-directed genome mining of PRF-A/FOR-A-related antibiotics.IMPORTANCE FOR-A and PRF-A are C-nucleoside antibiotics known for their unusual chemical structures and remarkable biological activities. Deciphering the enzymatic mechanism for the construction of a C-nucleoside scaffold during FOR-A/PRF-A biosynthesis will not only expand the biochemical repertoire for novel enzymatic reactions but also permit target-oriented genome mining of FOR-A/PRF-A-related C-nucleoside antibiotics. Moreover, the availability of FOR-A/PRF-A biosynthetic gene clusters will pave the way for the rational generation of designer FOR-A/PRF-A derivatives with enhanced/selective bioactivity via synthetic biology strategies.

Coordinated Biosynthesis of the Purine Nucleoside Antibiotics Aristeromycin and Coformycin in Actinomycetes.

Purine nucleoside antibiotic pairs, concomitantly produced by a single strain, are an important group of microbial natural products. Here, we report a target-directed genome mining approach to elucidate the biosynthesis of the purine nucleoside antibiotic pair aristeromycin (ARM) and coformycin (COF) in Micromonospora haikouensis DSM 45626 (a new producer for ARM and COF) and Streptomyces citricolor NBRC 13005 (a new COF producer). We also provide biochemical data that MacI and MacT function as unusual phosphorylases, catalyzing an irreversible reaction for the tailoring assembly of neplanocin A (NEP-A) and ARM. Moreover, we demonstrate that MacQ is shown to be an adenosine-specific deaminase, likely relieving the potential "excess adenosine" for producing cells. Finally, we report that MacR, an annotated IMP dehydrogenase, is actually an NADPH-dependent GMP reductase, which potentially plays a salvage role for the efficient supply of the precursor pool. Hence, these findings illustrate a fine-tuned pathway for the biosynthesis of ARM and also open the way for the rational search for purine antibiotic pairs.IMPORTANCE ARM and COF are well known for their prominent biological activities and unusual chemical structures; however, the logic of their biosynthesis has long been poorly understood. Actually, the new insights into the ARM and COF pathway will not only enrich the biochemical repertoire for interesting enzymatic reactions but may also lay a solid foundation for the combinatorial biosynthesis of this group of antibiotics via a target-directed genome mining strategy.

Biosynthesis of 2'-Chloropentostatin and 2'-Amino-2'-Deoxyadenosine Highlights a Single Gene Cluster Responsible for Two Independent Pathways in Actinomadura sp. Strain ATCC 39365.

2'-Chloropentostatin (2'-Cl PTN, 2'-chloro-2'-deoxycoformycin) and 2'-amino-2'-deoxyadenosine (2'-amino dA) are two adenosine-derived nucleoside antibiotics coproduced by Actinomadura sp. strain ATCC 39365. 2'-Cl PTN is a potent adenosine deaminase (ADA) inhibitor featuring an intriguing 1,3-diazepine ring, as well as a chlorination at C-2' of ribose, and 2'-amino dA is an adenosine analog showing bioactivity against RNA-type virus infection. However, the biosynthetic logic of them has remained poorly understood. Here, we report the identification of a single gene cluster (ada) essential for the biosynthesis of 2'-Cl PTN and 2'-amino dA. Further systematic genetic investigations suggest that 2'-Cl PTN and 2'-amino dA are biosynthesized by independent pathways. Moreover, we provide evidence that a predicted cation/H+ antiporter, AdaE, is involved in the chlorination step during 2'-Cl PTN biosynthesis. Notably, we demonstrate that 2'-amino dA biosynthesis is initiated by a Nudix hydrolase, AdaJ, catalyzing the hydrolysis of ATP. Finally, we reveal that the host ADA (designated ADA1), capable of converting adenosine/2'-amino dA to inosine/2'-amino dI, is not very sensitive to the powerful ADA inhibitor pentostatin. These findings provide a basis for the further rational pathway engineering of 2'-Cl PTN and 2'-amino dA production.IMPORTANCE 2'-Cl PTN/PTN and 2'-amino dA have captivated the great interests of scientists, owing to their unusual chemical structures and remarkable bioactivities. However, the precise logic for their biosynthesis has been elusive for decades. Actually, the identification and elucidation of their biosynthetic pathways not only enrich the biochemical repertoire of novel enzymatic reactions but may also lay solid foundations for the pathway engineering and combinatorial biosynthesis of this family of purine nucleoside antibiotics to generate novel hybrid analogs with improved features.

Unusual cysteine modifications in natural product biosynthesis.

l-Cysteine is a highly reactive amino acid that is modified into a variety of chemical structures, including cysteine sulfinic acid in human metabolic pathways, and sulfur-containing scaffolds of amino acids, alkaloids, and peptides in natural product biosynthesis. Among the modification enzymes responsible for these cysteine-derived compounds, metalloenzymes constitute an important family of enzymes that catalyze a wide variety of reactions. Therefore, understanding their reaction mechanisms is important for the biosynthetic production of cysteine-derived natural products. This review mainly summarizes recent mechanistic investigations of metalloenzymes, with a particular focus on recently discovered mononuclear non-heme iron (NHI) enzymes, dinuclear NHI enzymes, and radical-SAM enzymes involved in unusual cysteine modifications in natural product biosynthesis.

Data for developing computerized adaptive testing of problematic mobile phone use.

With the popularity of smart terminals and mobile Internet, mobile phone had been a necessity in everyone's daily life. Problematic mobile phone use (PMPU) also needs attention. Although there were multiple measurements to assess the PMPU, most of the tools were in traditional forms with fixed items. Therefore, the current study aimed to develop a computerized adaptive testing for problematic mobile phone use (CAT-PMPU) based on the Item Response Theory, which might help to provide an optimal solution to psychological assessments with long-scale and heterogeneous samples. The data in this article provided results of PMPU with different measurements. The dataset provided in this article includes 1619 participants, encompassing middle school students, undergraduate students, and graduate students recruited from various grades in China. The sample consists of 628 males and 991 females. Self-report measures were administrated including the Chinese version of the Nomophobia Questionnaire, Smartphone Addiction Proneness Scale, Smartphone Addiction Inventory, Mobile Phone Addiction Scale, Mobile Phone Addiction Tendency Scale, Smartphone Addiction Scale for College Students, and the Smartphone Addiction Scale for Chinese Adults. All the measurements were in the Chinese version. A .csv file consists of major variables we used was included as supplementary material on the Zenodo Repository. The discussion of the findings based on the dataset could be found in two articles: Development of a Computerized Adaptive Test for Problematic Mobile Phone Use & Generalizing computerized adaptive testing for problematic mobile phone use from Chinese adults to adolescents.

Challenges of functional expression of complex polyketide biosynthetic gene clusters.

Polyketide natural products are valuable sources of bioactive molecules such as nutraceuticals and pharmaceuticals. The tremendous development of the genome sequence database revealed that the majority of the biosynthetic gene clusters (BGCs) are cryptic. Activation of these cryptic BGCs and identification of the related products is essential for finding more lead compounds for pharmaceuticals. On the other hand, 99% of microbes in nature cannot be cultured in regular conditions, which greatly hinders the efforts to explore their biosynthetic potentials. Expression of polyketide BGCs in heterologous hosts with better growth, good genetic characteristics, and amenable molecular tools is a robust approach to identify new polyketides and characterize their biosynthesis. This review outlines the challenges in the heterologous production of polyketide BGCs of bacterial origins.

Offloading Role of a Discrete Thioesterase in Type II Polyketide Biosynthesis.

Type II polyketides are a group of secondary metabolites with various biological activities. In nature, biosynthesis of type II polyketides involves multiple enzymatic steps whereby key enzymes, including ketoacyl-synthase (KSα), chain length factor (KSß), and acyl carrier protein (ACP), are utilized to elongate the polyketide chain through a repetitive condensation reaction. During each condensation, the biosynthesis intermediates are covalently attached to KSα or ACP via a thioester bond and are then cleaved to release an elongated polyketide chain for successive postmodification. Despite its critical role in type II polyketide biosynthesis, the enzyme and its corresponding mechanism for type II polyketide chain release through thioester bond breakage have yet to be determined. Here, kinamycin was used as a model compound to investigate the chain release step of type II polyketide biosynthesis. Using a genetic knockout strategy, we confirmed that AlpS is required for the complete biosynthesis of kinamycins. Further in vitro biochemical assays revealed high hydrolytic activity of AlpS toward a thioester bond in an aromatic polyketide-ACP analog, suggesting its distinct role in offloading the polyketide chain from ACP during the kinamycin biosynthesis. Finally, we successfully utilized AlpS to enhance the heterologous production of dehydrorabelomycin in Escherichia coli by nearly 25-fold, which resulted in 0.50 g/liter dehydrorabelomycin in a simple batch-mode shake flask culture. Taken together, our results provide critical knowledge to gain an insightful understanding of the chain-releasing process during type II polyketide synthesis, which, in turn, lays a solid foundation for future new applications in type II polyketide bioproduction.

An Unusual Protector-Protégé Strategy for the Biosynthesis of Purine Nucleoside Antibiotics.

Pentostatin (PTN, deoxycoformycin) and arabinofuranosyladenine (Ara-A, vidarabine) are purine nucleoside antibiotics used clinically to treat hematological cancers and human DNA virus infections, respectively. PTN has a 1,3-diazepine ring, and Ara-A is an adenosine analog with an intriguing epimerization at the C-2' hydroxyl group. However, the logic underlying the biosynthesis of these interesting molecules has long remained elusive. Here, we report that the biosynthesis of PTN and Ara-A employs an unusual protector-protégé strategy. To our surprise, we determined that a single gene cluster governs PTN and Ara-A biosynthesis via two independent pathways. Moreover, we verified that PenB functions as a reversible oxidoreductase for the final step of PTN. Remarkably, we provided the first direct biochemical evidence that PTN can protect Ara-A from deamination by selective inhibition of the host adenosine deaminase. These findings expand our knowledge of natural product biosynthesis and open the way for target-directed genome mining of Ara-A/PTN-related antibiotics.

Construction of an octosyl acid backbone catalyzed by a radical S-adenosylmethionine enzyme and a phosphatase in the biosynthesis of high-carbon sugar nucleoside antibiotics.

Unique bicyclic octosyl uronic acid nucleosides include ezomycin, malayamycin, and octosyl acid (OA). They are structurally characterized by OA, an unusual 8-carbon furanosyl nucleoside core proposed to be the precursor to polyoxin and nikkomycin. Despite the well-known bioactivity of these nucleoside antibiotics, the biosynthesis of OA has not been elucidated yet. Here we report the two pivotal enzymatic steps in the polyoxin biosynthetic pathway leading to the identification of OA as a key intermediate. Our data suggest that this intermediate is formed via a free radical reaction catalyzed by the radical S-adenosylmethionine (SAM) enzyme, PolH, and using 3'-enolpyruvyl uridine 5'-monophosphate (3'-EUMP) as a substrate. Subsequent dephosphorylation catalyzed by phosphatase PolJ converts the resulting octosyl acid 5'-phosphate (OAP) to OA. These results provide, for the first time, significant in vitro evidence for the biosynthetic origins of the C8 backbone of OA.