The Genomic-Driven Discovery of Glutarimide-Containing Derivatives from Burkholderia gladioli.

Glutarimide-containing polyketides exhibiting potent antitumor and antimicrobial activities were encoded via conserved module blocks in various strains that favor the genomic mining of these family compounds. The bioinformatic analysis of the genome of Burkholderia gladioli ATCC 10248 showed a silent trans-AT PKS biosynthetic gene cluster (BGC) on chromosome 2 (Chr2C8), which was predicted to produce new glutarimide-containing derivatives. Then, the silent polyketide synthase gene cluster was successfully activated via in situ promoter insertion and heterologous expression. As a result, seven glutarimide-containing analogs, including five new ones, gladiofungins D-H (3-7), and two known gladiofungin A/gladiostatin (1) and 2 (named gladiofungin C), were isolated from the fermentation of the activated mutant. Their structures were elucidated through the analysis of HR-ESI-MS and NMR spectroscopy. The structural diversities of gladiofungins may be due to the degradation of the butenolide group in gladiofungin A (1) during the fermentation and extraction process. Bioactivity screening showed that 2 and 4 had moderate anti-inflammatory activities. Thus, genome mining combined with promoter engineering and heterologous expression were proved to be effective strategies for the pathway-specific activation of the silent BGCs for the directional discovery of new natural products.

Biosynthesis and engineering of the nonribosomal peptides with a C-terminal putrescine.

The broad bioactivities of nonribosomal peptides rely on increasing structural diversity. Genome mining of the Burkholderiales strain Schlegelella brevitalea DSM 7029 leads to the identification of a class of dodecapeptides, glidonins, that feature diverse N-terminal modifications and a uniform putrescine moiety at the C-terminus. The N-terminal diversity originates from the wide substrate selectivity of the initiation module. The C-terminal putrescine moiety is introduced by the unusual termination module 13, the condensation domain directly catalyzes the assembly of putrescine into the peptidyl backbone, and other domains are essential for stabilizing the protein structure. Swapping of this module to another two nonribosomal peptide synthetases leads to the addition of a putrescine to the C-terminus of related nonribosomal peptides, improving their hydrophilicity and bioactivity. This study elucidates the mechanism for putrescine addition and provides further insights to generate diverse and improved nonribosomal peptides by introducing a C-terminal putrescine.

Heterologous Biosynthesis of Complex Bacterial Natural Products in Burkholderia gladioli.

Bacterial natural products (NPs) are an indispensable source of drugs and biopesticides. Heterologous expression is an essential method for discovering bacterial NPs and the efficient biosynthesis of valuable NPs, but the chassis for Gram-negative bacterial NPs remains inadequate. In this study, we built a Burkholderiales mutant Burkholderia gladioli Δgbn::attB by introducing an integrated site (attB) to inactivate the native gladiolin (gbn) biosynthetic gene cluster, which stabilizes large foreign gene clusters and reduces the native metabolite profile. The growth and successful heterologous production of high-value NPs such as phylogenetically close Burkholderiales-derived antitumor polyketides (PKs) rhizoxins, phylogenetically distant Gammaproteobacteria-derived anti-MRSA (methicillin-resistant Staphylococcus aureus) antibiotics WAP-8294As, and Deltaproteobacteria-derived antitumor PKs disorazols demonstrate that this strain is a potential chassis for Gram-negative bacterial NPs. We further improved the yields of WAP-8294As through promoter insertions and precursor pathway overexpression based on heterologous expression in this strain. This study provides a robust bacterial chassis for genome mining, efficient production, and molecular engineering of bacterial NPs.

Biosynthesis of Glidomides and Elucidation of Different Mechanisms for Formation of ß-OH Amino Acid Building Blocks.

Nonribosomal peptide synthetases (NRPSs) can incorporate nonproteinogenic amino acids into peptidyl backbones to increase structural diversity. Genome mining of Schlegelella brevitalea led to the identification of a class of linear lipoheptapeptides, glidomides, featuring two unusual residues: threo-ß-OH-L-His and threo-ß-OH-D-Asp. The ß-hydroxylation of Asp and His is catalyzed by the nonheme FeII /α-ketoglutarate-dependent ß-hydroxylases GlmD and GlmF, respectively. GlmD independently catalyzes the hydroxylation of L-Asp to primarily produce threo-ß-OH-L-Asp on the thiolation domain, and then undergoes epimerization to form threo-ß-OH-D-Asp in the final products. However, ß-hydroxylation of His requires the concerted action of GlmF and the interface (I) domain, a novel condensation domain family clade. The key sites of I domain for interaction with GlmF were identified, suggesting that the mechanism for hydroxylation of His depends on the collaboration between hydroxylase and NRPS.

Genomics-Driven Activation of Silent Biosynthetic Gene Clusters in Burkholderia gladioli by Screening Recombineering System.

The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product discovery. However, exploitation of the metabolomic potential of Burkholderia is limited by the absence of efficient genetic manipulation tools. Here, we screened a bacteriophage recombinase system Redγ-BAS, which was functional for genome modification in the plant pathogen Burkholderia gladioli ATCC 10248. By using this recombineering tool, the constitutive promoters were precisely inserted in the genome, leading to activation of two silent nonribosomal peptide synthetase gene clusters (bgdd and hgdd) and production of corresponding new classes of lipopeptides, burriogladiodins A-H (1-8) and haereogladiodins A-B (9-10). Structure elucidation revealed an unnatural amino acid Z- dehydrobutyrine (Dhb) in 1-8 and an E-Dhb in 9-10. Notably, compounds 2-4 and 9 feature an unusual threonine tag that is longer than the predicted collinearity assembly lines. The structural diversity of burriogladiodins was derived from the relaxed substrate specificity of the fifth adenylation domain as well as chain termination conducted by water or threonine. The recombinase-mediating genome editing system is not only applicable in B. gladioli, but also possesses great potential for mining meaningful silent gene clusters from other Burkholderia species.

Engineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis.

Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.

Impact of Ultraviolet Radiation on the Aging Properties of SBS-Modified Asphalt Binders.

Styrene Butadiene Styrene (SBS) polymer-modified asphalt binders have become widely used in asphalt pavement because of their advantages in high- and low-temperature performance and fatigue resistance. Asphalt pavement is inevitably exposed to sunlight and ultraviolet (UV) radiation during its construction and service life. However, consideration of the aging effect of UV radiation is still limited in current pavement design and evaluation systems. In order to evaluate the impact of UV radiation on the aging properties of SBS-modified asphalt binders, UV aging tests were performed on Rolling Thin Film Oven Test (RTFOT)-aged samples with different UV radiation intensities and aging times. Sixteen different groups of tests were conducted to compare the rheological properties and functional group characteristics of SBS-modified asphalt binders. Dynamic Shear Rheometer (DSR), Bending Beam Rheometer (BBR), FTIR, and SEM tests were conducted to evaluate the aging mechanisms in various UV aging conditions. The results found that UV radiation seriously destroys the network structure formed by the cross-linking effect in SBS-modified asphalt binders, which aggravates the degradation of SBS and results in a great change of rheological properties after UV aging. The nature of SBS-modified asphalt binder aging resulted from the degradation of SBS and the changes of asphalt binder base composition, which lead to the transformation of colloidal structure and the deterioration of asphalt binder performance. The tests also found that continuous UV radiation can increase shrinkage stress in the asphalt binder surface and leads to surface cracking of the asphalt binder.

Aging Mechanism and Properties of SBS Modified Bitumen under Complex Environmental Conditions.

Bitumen aging can lead to the deterioration of asphalt pavement performance, shortening the service life of road. In order to solve the problem that current studies on the ultraviolet (UV) aging of bitumen either ignore the effects of natural environmental conditions or only consider the effects of water. In this study, different aqueous media and UV coupled simulated aging tests were carried out on virgin bitumen and styrene butadiene styrene (SBS) modified bitumen in a UV environment chamber. The combination of macroscopic performance tests and microstructure tests was used to analyze the physical, rheological, and microstructure changes of virgin bitumen and SBS modified bitumen after The film oven test (TFOT) aging and UV aging in different environments (UV, UV + Water, UV + Acid, UV + Salt). Dynamic shear rheometer (DSR) results indicated that UV aging results in the increase of rutting factor and the improvement of rutting resistance at high temperature. The Fourier transform infrared spectrum (FTIR) results illustrated that the bitumen would be oxidized and SBS would be degraded under ultraviolet radiation. The four-component analysis test results showed that light component migrated to the heavy component during the aging process. Moreover, water will aggravate the UV aging of bitumen, and the presence of acid or salt worsens ultraviolet aging.