The functions of six genes in the ansamitocin biosynthetic gene cluster of Actinosynnema pretiosum have been investigated by gene inactivation and chemical analysis of the mutants. They encode a halogenase (asm12), a carbamoyltransferase (asm21), a 20-O-methyltransferase (asm7), a 3-O-acyltransferase (asm19), an epoxidase (asm11), and an N-methyltransferase (asm10), respectively, and are responsible for the six post-PKS modification steps in ansamitocin formation. Several of the enzymes have relaxed substrate specificities, resulting in multiple parallel pathways in a metabolic grid, albeit with a preferred sequence of reactions as listed above.

Pseudomonas putida KT2440 is a saprophytic, environmental microorganism that plays important roles in the biodegradation of environmental toxic compounds and production of polymers, chemicals and secondary metabolites. Gene deletion of KT2440 usually involves cloning of the flanking homologous fragments of the gene of interest into a suicide vector followed by transferring into KT2440 via triparental conjugation. Selection and counterselection steps are then employed to generate gene deletion mutant. However, these methods are tedious and are not suitable for the manipulation of multiple genes simultaneously. Herein, a two-step, markerless gene deletion method is presented. First, homologous armsflanked loxP-neo-loxP was knocked-in to replace the gene of interest, then the kanamycin resistance marker is removed by Cre recombinase catalyzed site-specific recombination. Both two-plasmid and one-plasmid gene systems were established. MekR/PmekA regulated gene expression system was found to be suitable for tight Cre expression in one-plasmid deletion system. The straightforward, time saving and highly efficient markerless gene deletion strategy has the potential to facilitate the genetics and functional genomics study of P. putida KT2440.

Molecular mechanisms and gene regulation are of interest in the area of geomicrobiology in which the interaction between microbes and minerals is studied. This paper focuses on the regulation of the expression of carbonic anhydrase (CA) genes in Bacillus mucilaginosus and the effects of the expression product of the B. mucilaginosus CA gene in Escherichia coli on calcite weathering. Real-time fluorescent quantitative PCR (RT-qPCR) was used to explore the relationship between CA gene expression in B. mucilaginosus and promotion of calcite dissolution under condition of Ca 2C deficiency. The results showed that adding calcite to the medium, which lacks Ca 2C , can up-regulate the expression of the bacterial CA genes to accelerate calcite dissolution for bacterial growth. CA genes from B. mucilaginosus were transferred into E. coli by cloning. We then employed crude enzyme extract from the resultant E. coli strain in calcite dissolution experiments. The enzyme extract promoted calcite dissolution. These findings provide direct evidence for the role of microbial CA on mineral weathering and mineral nutrition release.

Ansamitocins are potent antitumor agents produced by Actinosynnema pretiosum. As deduced from their structures, an N-methylation on the amide bond is required among the various modifications. The encoded protein by asm10 belongs to SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is 33.0 kDa in size and present as monomer as determined by gel filtration. Using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH were determined to be 32 °C and 10.0 respectively for Asm10 catalysis. Asm10 also showed broad substrate flexibility toward other N-desmethyl ansamycins and synthetic indolin-2-ones. Through site-directed mutagenesis, Asp154 and Leu155 of Asm10 were confirmed to be essential for its catalysis possibly thorough the binding of SAM. The characterization of this unique N-methyltransferase enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds, which will allow modification of known potential drugs.

A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analysis indicated the major pathway of thiacloprid (THI) metabolism by V. boronicumulans J1 involved hydrolysis of the N-cyanoimino group to form an N-carbamoylinino group containing metabolite, THI amide. Resting cells of V. boronicumulans J1 degraded 62.5% of the thiacloprid at a concentration of 200 mg/L in 60 h, and 98% of the reduced thiacloprid was converted to the final metabolite thiacloprid amide. A 2.6 kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an α-subunit of 213 amino acids and a β-subunit of 221 amino acids. The nitrile hydratase gene was recombined into plasmid pET28a and overexpressed in Escherichia coli BL21 (DE3). The resting cells of recombinant E. coli BL21 (DE3)-pET28a-NHase with overexpression of nitrile hydratase transformed thiacloprid to its amide metabolite, whereas resting cells of the control E. coli BL21 (DE3)-pET28a did not. Therefore, the major hydration pathway of thiacloprid is mediated by nitrile hydratase.

Genetic defects in bone morphogenetic protein type II receptor (BMPRII) signalling and inflammation contribute to the pathogenesis of pulmonary arterial hypertension (PAH). The receptor is activated by bone morphogenetic protein (BMP) ligands, which also enhance transcription. A small-molecule BMP upregulator with selectivity on vascular endothelium would be a desirable therapeutic intervention for PAH.We assayed compounds identified in the screening of BMP2 upregulators for their ability to increase the expression of inhibitor of DNA binding 1 (Id1), using a dual reporter driven specifically in human embryonic stem cell-derived endothelial cells. These assays identified a novel piperidine, BMP upregulator 1 (BUR1), that increased endothelial Id1 expression with a half-maximal effective concentration of 0.098 μmol·L Microarray analyses and immunoblotting showed that BUR1 induced BMP2 and prostaglandin-endoperoxide synthase 2 (PTGS2) expression. BUR1 effectively rescued deficient angiogenesis in autologous endothelial cells generated by CRISPR/Cas9 and patient cells.BUR1 prevented and reversed PAH in monocrotaline rats, and restored BMPRII downstream signalling and modulated the arachidonic acid pathway in the pulmonary arterial endothelium in the Sugen 5416/hypoxia PAH mouse model.In conclusion, using stem cell technology we have provided a novel small-molecule compound which regulates BMP2 and PTGS2 levels that might be useful for the treatment of PAH.

N-Acetyl-d-neuraminic acid (Neu5Ac) is a potential baby nutrient and the key precursor of antiflu medicine Zanamivir. The Neu5Ac chemoenzymatic synthesis consists of N-acetyl-d-glucosamine epimerase (AGE)-catalyzed epimerization of N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc) and aldolase-catalyzed condensation between ManNAc and pyruvate. Herein, we cloned and characterized BT0453, a novel AGE, from a human gut symbiont Bacteroides thetaiotaomicron. BT0453 shows the highest soluble fraction among the AGEs tested. With GlcNAc and sodium pyruvate as substrates, Neu5Ac production by coupling whole cells expressing BT0453 and Escherichia coli N-acetyl-d-neuraminic acid aldolase was explored. After 36 h, a 53.6% molar yield, 3.6 g L–1 h–1 productivity and 42.9 mM titer of Neu5Ac were obtained. Furthermore, for the first time, the T7-BT0453-T7-nanA polycistronic unit was integrated into the E. coli genome, generating a chromosome-based biotransformation system. BT0453 protein engineering and metabolic engineering studies hold potential for the industrial production of Neu5Ac.

Pseudomonas putida KT2440 is a saprophytic, generally recognized as safe microorganism that plays important roles in the biodegradation and production of value-added chemicals. Chromosomal gene deletion of P. putida KT2440 usually involves time-consuming gene coning, conjugal transfer and counterselection. Recently, we developed a P. putida KT2440 markerless gene deletion method based on recombineering and Cre/loxP site-specific recombination. PCR-based λ Red recombineering circumvents the tedious cloning steps and is more amenable to high-throughput manipulation. Herein we report an improved scarless gene deletion strategy based on recombineering and I-SceI-mediated double-strand break repair. Sixteen drug exporter gene(s) were deleted and the minimal inhibition concentrations of the mutants to a variety of antibiotics were determined. The robustness of the procedure was also demonstrated by sequential deletion of five large genomic regions. Up to 59% recombination efficiency was achieved for 54.8 kb deletion, and the efficiency of RecA mediated double-strand break repair, which was boosted by λ Red recombinase, was nearly 100%. The strain with a 3.76% genome reduction showed an improved growth rate and transformation efficiency. The straightforward, time-saving and highly efficient scarless deletion approach has the potential to facilitate the genetic study and biotechnological and environmental applications of P. putida KT2440.