Metabolic engineering of Streptomyces roseosporus for increased production of clinically important antibiotic daptomycin.

Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S.

Cryo-EM structure of DNA polymerase of African swine fever virus.

African swine fever virus (ASFV) is one of the most important causative agents of animal diseases and can cause highly fatal diseases in swine. ASFV DNA polymerase (DNAPol) is responsible for genome replication and highly conserved in all viral genotypes showing an ideal target for drug development. Here, we systematically determined the structures of ASFV DNAPol in apo, replicating and editing states.

A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in .

has enormous potential to produce novel natural products (NPs) as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters (BGCs). However, the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery. Here, we developed an easy-to-use, highly flexible DNA assembly toolkit for gene cluster engineering.

Building a highly efficient Streptomyces super-chassis for secondary metabolite production by reprogramming naturally-evolved multifaceted shifts.

Streptomyces has an extensive array of bioactive secondary metabolites (SMs). Nevertheless, devising a framework for the heterologous production of these SMs remains challenging. We here reprogrammed a versatile plug-and-play Streptomyces super-chassis and established a universal pipeline for production of diverse SMs via understanding of the inherent pleiotropic effects of ethanol shock on jadomycin production in Streptomyces venezuelae.

Extraction methods optimization of available heavy metals and the health risk assessment of the suburb soil in China.

Heavy metal pollution has attracted increasing concern due to its high toxicity and persistence. A suitable extraction procedure for available heavy metals in soil is necessary for assessing the ecological risk. In this work, the single extraction methods aided by shaking and microwaves were investigated and analyzed for their ability to extract available heavy metals from soil samples, and a total of 42 soil samples were collected from suburbs of Zhengzhou city in China.

Molecular mechanism of de novo replication by the Ebola virus polymerase.

Non-segmented negative-strand RNA viruses, including Ebola virus (EBOV), rabies virus, human respiratory syncytial virus and pneumoviruses, can cause respiratory infections, haemorrhagic fever and encephalitis in humans and animals, and are considered a substantial health and economic burden worldwide. Replication and transcription of the viral genome are executed by the large (L) polymerase, which is a promising target for the development of antiviral drugs. Here, using the L polymerase of EBOV as a representative, we show that de novo replication of L polymerase is controlled by the specific 3' leader sequence of the EBOV genome in an enzymatic assay, and that formation of at least three base pairs can effectively drive the elongation process of RNA synthesis independent of the specific RNA sequence.

Novel switchable ECF sigma factor transcription system for improving thaxtomin A production in .

The application of the valuable natural product thaxtomin A, a potent bioherbicide from the potato scab pathogenic strains, has been greatly hindered by the low yields from its native producers. Here, we developed an orthogonal transcription system, leveraging extra-cytoplasmic function (ECF) sigma (σ) factor 17 (ECF17) and its cognate promoter P, to express the thaxtomin gene cluster and improve the production of thaxtomin A. The minimal P promoter was determined, and a P promoter library with a wide range of strengths was constructed.

Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous Hosts.

Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination.

3-Indoleacetonitrile Is Highly Effective in Treating Influenza A Virus Infection In Vitro and In Vivo.

Influenza A viruses are serious zoonotic pathogens that continuously cause pandemics in several animal hosts, including birds, pigs, and humans. Indole derivatives containing an indole core framework have been extensively studied and developed to prevent and/or treat viral infection. This study evaluated the anti-influenza activity of several indole derivatives, including 3-indoleacetonitrile, indole-3-carboxaldehyde, 3-carboxyindole, and gramine, in A549 and MDCK cells.

AveI, an AtrA homolog of Streptomyces avermitilis, controls avermectin and oligomycin production, melanogenesis, and morphological differentiation.

Streptomyces avermitilis is well known as the producer of anthelmintic agent avermectins, which are widely used in agriculture, veterinary medicine, and human medicine. aveI encodes a TetR-family regulator, which is the homolog of AtrA. It was reported that deletion of aveI caused enhanced avermectin production.

An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters.

Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable.

d-Sedoheptulose-7-phosphate is a common precursor for the heptoses of septacidin and hygromycin B.

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics.

An external substrate-free blue/white screening system in Escherichia coli.

Since the lacZα-based blue/white screening system was introduced to molecular biology, several different visual reporter systems were developed and used for various purposes in Escherichia coli. A common limit to the existent visual reporter systems is that an extracellular chromogenic substrate has to be added for the visible pigment production. In this study, we developed a new blue/white screening system based on a non-ribosomal peptide synthetase encoded by idgS from Streptomyces and a phosphopantetheinyl transferase encoded by sfp from Bacillus.

Paired Design of dCas9 as a Systematic Platform for the Detection of Featured Nucleic Acid Sequences in Pathogenic Strains.

We developed an in vitro DNA detection system using a pair of dCas9 proteins linked to split halves of luciferase. Luminescence was induced upon colocalization of the reporter pair to a ∼44 bp target sequence defined by sgRNAs. We used the system to detect Mycobacterium tuberculosis DNA with high specificity and sensitivity.

The NS1 gene from bat-derived influenza-like virus H17N10 can be rescued in influenza A PR8 backbone.

Influenza A viruses have the potential to cause pandemics due to the introduction of novel subtypes against which human hosts have little or no preexisting immunity. Such viruses may result from reassortment between human and animal influenza viruses. Recently, new influenza-like viruses were identified in bats, raising the concern for a new reservoir of potentially harmful influenza viruses that could form reassortants with categorized human influenza A viruses.

Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate) Accumulation in Escherichia coli by Combining One-Step Library Construction and High-Throughput Screening.

As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product.

A novel approach for metabolic pathway optimization: Oligo-linker mediated assembly (OLMA) method.

Background: Imbalances in gene expression of a metabolic pathway can result in less-yield of the desired products. Several targets were intensively investigated to balance the gene expression, such as promoter, ribosome binding site (RBS), the order of genes, as well as the species of the enzymes. However, the capability of simultaneous manipulation of multiple targets still needs to be explored.

Intermediate-sensor assisted push-pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli.

Because high-throughput screening tools are typically unavailable when using the pathway-engineering approach, we developed a new strategy, named intermediate sensor-assisted push-pull strategy, which enables sequential pathway optimization by incorporating a biosensor targeting a key pathway intermediate. As proof of concept, we constructed an L-Trp biosensor and used it to optimize the deoxyviolacein biosynthetic pathway, which we divided into two modules with L-Trp being the product of the upstream and the substrate of the downstream module for deoxyviolacein synthesis. Using the biosensor and fluorescence-activated cell sorting, the activities of the two modules were sequentially and independently optimized in Escherichia coli to achieve the desired phenotypes.

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces.

There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining.

Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters.

The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step.

Overexpression of metK shows different effects on avermectin production in various Streptomyces avermitilis strains.

The S-adenosylmethionine synthetase gene (metK) from Streptomyces avermitilis was cloned into multi-copy vector pIJ653 and integrative vector pSET152 yielding two metK expression plasmids pYJ02 and pYJ03, respectively. When wild-type strain ATCC31267 was transformed with these two plasmids, avermectin production was increased about 2.0-fold and 5.

Complete genomic sequence analysis of the temperate bacteriophage phiSASD1 of Streptomyces avermitilis.

The bacteriophage phiSASD1, isolated from a failed industrial avermectin fermentation, belongs to the Siphoviridae family. Its four predominant structural proteins, which include the major capsid, portal and two tail-related proteins, were separated and identified by SDS-PAGE and N-terminal sequence analysis. The entire double-stranded DNA genome of phiSASD1 consists of 37,068 bp, with 3'-protruding cohesive ends of nine nucleotides.

Construction of a doramectin producer mutant from an avermectin-overproducing industrial strain of Streptomyces avermitilis.

The avermectin analogue doramectin (CHC-B1), which is produced in mutants that have an altered biosynthesis pathway of avermectin, is one of the most effective agricultural pesticides and antiparasitics. We report here the construction of a bkdF olmA double-deletion mutant lacking one of the branched-chain alpha-keto acid dehydrogenase encoding genes (bkdF) and the oligomycin PKS encoding gene cluster (olmA) in Streptomyces avermitilis 76-05. We then characterized the production of various antibiotics in cultures of the deletion mutant.