Characteristics and immune functions of the endogenous CRISPR-Cas systems in myxobacteria.

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system widely occurs in prokaryotic organisms to recognize and destruct genetic invaders. Systematic collation and characterization of endogenous CRISPR-Cas systems are conducive to our understanding and potential utilization of this natural genetic machinery. In this study, we screened 39 complete and 692 incomplete genomes of myxobacteria using a combined strategy to dispose of the abridged genome information and revealed at least 19 CRISPR-Cas subtypes, which were distributed with a taxonomic difference and often lost stochastically in intraspecies strains. The cas genes in each subtype were evolutionarily clustered but deeply separated, while most of the CRISPRs were divided into four types based on the motif characteristics of repeat sequences. The spacers recorded in myxobacterial CRISPRs were in high G+C content, matching lots of phages, tiny amounts of plasmids, and, surprisingly, massive organismic genomes. We experimentally demonstrated the immune and self-target immune activities of three endogenous systems in Myxococcus xanthus DK1622 against artificial genetic invaders and revealed the microhomology-mediated end-joining mechanism for the immunity-induced DNA repair but not homology-directed repair. The panoramic view and immune activities imply potential omnipotent immune functions and applications of the endogenous CRISPR-Cas machinery. IMPORTANCE: Serving as an adaptive immune system, clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) empower prokaryotes to fend off the intrusion of external genetic materials. Myxobacteria are a collective of swarming Gram-stain-negative predatory bacteria distinguished by intricate multicellular social behavior. An in-depth analysis of their intrinsic CRISPR-Cas systems is beneficial for our understanding of the survival strategies employed by host cells within their environmental niches. Moreover, the experimental findings presented in this study not only suggest the robust immune functions of CRISPR-Cas in myxobacteria but also their potential applications.

Determination of the chromosomal position effects for plug-and-play application in the Myxococcus xanthus chassis cells.

The chromosomal position effect can significantly affect the transgene expression, which may provide an efficient strategy for the inauguration of alien genes in new hosts, but has been less explored rationally. The bacterium Myxococcus xanthus harbors a large circular high-GC genome, and the position effect in this chassis may result in a thousand-fold expression variation of alien natural products. In this study, we conducted transposon insertion at TA sites on the M. xanthus genome, and used enrichment and dilution indexes to respectively appraise high and low expression potentials of alien genes at insertion sites. The enrichment sites are characteristically distributed along the genome, and the dilution sites are overlapped well with the horizontal transfer genes. We experimentally demonstrated the enrichment sites as high expression integration sites (HEISs), and the dilution sites unsuitable for gene integration expression. This work highlights that HEISs are the plug-and-play sites for efficient expression of integrated genes.

Genome-wide analysis of lipolytic enzymes and characterization of a high-tolerant carboxylesterase from Sorangium cellulosum.

Microorganisms are important sources of lipolytic enzymes with characteristics for wide promising usages in the specific industrial biotechnology. The cellulolytic myxobacterium Sorangium cellulosum is rich of lipolytic enzymes in the genome, but little has been investigated. Here, we discerned 406 potential lipolytic enzymes in 13 sequenced S. cellulosum genomes. These lipolytic enzymes belonged to 12 families, and most are novel with low identities (14-37%) to those reported. We characterized a new carboxylesterase, LipB, from the alkaline-adaptive So0157-2. This enzyme, belonging to family VIII, hydrolyzed glyceryl tributyrate and p-nitrophenyl esters with short chain fatty acids (≤C12), and exhibited the highest activity against p-nitrophenyl butyrate. It retained over 50% of the activities in a broad temperature range (from 20°C to 60°C), alkaline conditions (pH 8.0-9.5), and the enzymatic activity was stable with methanol, ethanol and isopropanol, and stimulated significantly in the presence of 5 mM Ni2+. LipB also exhibited ß-lactamase activity on nitrocefin, but not ampicillin, cefotaxime and imipenem. The bioinformatic analysis and specific enzymatic characteristics indicate that S. cellulosum is a promising resource to explore lipolytic enzymes for industrial adaptations.

Internal Promoters and Their Effects on the Transcription of Operon Genes for Epothilone Production in Myxococcus xanthus.

The biosynthetic genes for secondary metabolites are often clustered into giant operons with no transcription terminator before the end. The long transcripts are frangible and the transcription efficiency declines along with the process. Internal promoters might occur in operons to coordinate the transcription of individual genes, but their effects on the transcription of operon genes and the yield of metabolites have been less investigated. Epothilones are a kind of antitumor polyketides synthesized by seven multifunctional enzymes encoded by a 56-kb operon. In this study, we identified multiple internal promoters in the epothilone operon. We performed CRISPR-dCas9-mediated transcription activation of internal promoters, combined activation of different promoters, and activation in different epothilone-producing M. xanthus strains. We found that activation of internal promoters in the operon was able to promote the gene transcription, but the activation efficiency was distinct from the activation of separate promoters. The transcription of genes in the operon was influenced by not only the starting promoter but also internal promoters of the operon; internal promoters affected the transcription of the following and neighboring upstream/downstream genes. Multiple interferences between internal promoters thus changed the transcriptional profile of operon genes and the production of epothilones. Better activation efficiency for the gene transcription and the epothilone production was obtained in the low epothilone-producing strains. Our results highlight that interactions between promoters in the operon are critical for the gene transcription and the metabolite production efficiency.

Characterization of Constitutive Promoters for the Elicitation of Secondary Metabolites in Myxobacteria.

Genome mining has revealed that myxobacteria contain a myriad of cryptic biosynthetic gene clusters (BGCs). Here, we report the characterization of a panel of myxobacterial promoters with variable strength that are applicable in the engineering of BGCs in myxobacteria. The screened strongest constitutive promoter was used to efficiently enhance the expression of two complex BGCs governing the biosynthesis of myxochromide and DKxanthene in the model myxobacterium Myxococcus xanthus DK1622. We also showcased the combination of promoter engineering and MS2-based spectral networking as an effective strategy to shed light on the previously overlooked chemistry in the family of myxochromide-type lipopeptides. The enriched promoter library substantially expanded the synthetic biology toolkit available for myxobacteria.

Effects of glycosylation on the bioactivity of rapamycin.

The macrolactone rapamycin (RAP) presents a broad range of bioactivities, but its clinical applications are compromised due to the poor water solubility and low bioavailability, which could probably be overcome by glycosylation. In this study, we tested a set of promiscuous glycosyltransferases (GTs) to modify rapamycin with four different sugar donors. BsGT-1 displayed the best glycosylation activity with a preference for UDP-glucose, and the glycosylation happened at C-28 or C-40 of rapamycin, producing rapamycin-40-O-ß-D-glucoside (RG1), and two new compounds rapamycin-28-O-ß-D-glucoside (RG2) and rapamycin-28,40-O-ß-D-diglucoside (RG3). The glycosylation remarkably improved water solubility and almost completely abolished cytotoxicity but simultaneously attenuated the antifungal, antitumor, and immunosuppression bioactivities of rapamycin. We found the glycosylation at C-40 had less effect on the bioactivities than that at C-28. The molecular docking analysis revealed that the glycosylation, especially the glycosylation at C-28, weakened the hydrophobic and hydrogen bonding contacts between the rapamycin glucosides and the binding proteins: the FK506-binding protein (FKBP12) and the FKBP12-rapamycin binding (FRB) domain. This study highlights a succinct approach to expand the chemical diversity of the therapeutically important molecule rapamycin by using promiscuous glycosyltransferases. Moreover, the fact that glycosyl moieties at different positions of rapamycin affect bioactivity to different extents inspires further glycosylation engineering to improve properties of rapamycin. KEY POINTS: • Rapamycin was glycosylated efficiently by some promiscuous GTs. • Glycosylation improved water solubility, attenuated cytotoxicity, and bioactivities. • Glycosylation affected the interactions between ligand and binding proteins.

Functional Division Between the RecA1 and RecA2 Proteins in Myxococcus xanthus.

Myxococcus xanthus DK1622 has two RecA genes, recA1 (MXAN_1441) and recA2 (MXAN_1388), with unknown functional differentiation. Herein, we showed that both recA genes were induced by ultraviolet (UV) irradiation but that the induction of recA1 was more delayed than that of recA2. Deletion of recA1 did not affect the growth but significantly decreased the UV-radiation survival, homologous recombination (HR) ability, and induction of LexA-dependent SOS genes. In contrast, the deletion of recA2 markedly prolonged the lag phase of bacterial growth and increased the sensitivity to DNA damage caused by hydrogen peroxide but did not change the UV-radiation resistance or SOS gene inducibility. Protein activity analysis demonstrated that RecA1, but not RecA2, catalyzed DNA strand exchange (DSE) and LexA autocleavage in vitro. Transcriptomic analysis indicated that RecA2 has evolved mainly to regulate gene expression for cellular transportation and antioxidation. This is the first report of functional divergence of duplicated bacterial recA genes. The results highlight the evolutionary strategy of M. xanthus cells for DNA HR and genome sophistication.

Phylogeny-guided characterization of glycosyltransferases for epothilone glycosylation.

Glycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti-tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC-subbranch with 160 GT sequences. We demonstrated that the YjiC-subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC-subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high-active GTs (Q66 and P77) but changed in two low-efficient GTs. Site-directed residues swapping at the two positions in the two low-active GTs (BssGT and BamGT) and the high-active GT BsGT-1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.

Effects of transcriptional mode on promoter substitution and tandem engineering for the production of epothilones in Myxococcus xanthus.

Promoter optimization is an economical and effective approach to overexpress heterologous genes and improve the biosynthesis of valuable products. In this study, we swapped the original promoter of the epothilone biosynthetic gene cluster in Myxococcus xanthus with two endogenous strong promoters P pilA and P groEL1 , respectively, which, however, decreased the epothilone production ability. The transcriptional abilities by the two promoters were found to be bloomed in the growth stage but markedly decreased after the growth, whereas the original promoter P epo functioned majorly after the exponential growth stage. Tandem repeat engineering on the original promoter P epo remarkably increased epothilone production. The tandem promoter exerted similar expressional pattern as P epo did in M. xanthus. We demonstrated that differential transcriptional modes markedly affected the efficiency of promoters in controlling the gene expressions for the production of the secondary metabolite epothilones. Our study provides an insight into exploiting powerful promoters to produce valuable secondary metabolites, especially in host with limited known promoters.

CRISPR/dCas9-mediated transcriptional improvement of the biosynthetic gene cluster for the epothilone production in Myxococcus xanthus.

BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.

A bacterial negative transcription regulator binding on an inverted repeat in the promoter for epothilone biosynthesis.

BACKGROUND: Microbial secondary metabolism is regulated by a complex and mostly-unknown network of global and pathway-specific regulators. A dozen biosynthetic gene clusters for secondary metabolites have been reported in myxobacteria, but a few regulation factors have been identified. RESULTS: We identified a transcription regulator Esi for the biosynthesis of epothilones. Inactivation of esi promoted the epothilone production, while overexpression of the gene suppressed the production. The regulation was determined to be resulted from the transcriptional changes of epothilone genes. Esi was able to bind, probably via the N-terminus of the protein, to an inverted repeat sequence in the promoter of the epothilone biosynthetic gene cluster. The Esi-homologous sequences retrieved from the RefSeq database are all of the Proteobacteria. However, the Esi regulation is not universal in myxobacteria, because the esi gene exists only in a few myxobacterial genomes. CONCLUSIONS: Esi binds to the epothilone promoter and down-regulates the transcriptional level of the whole gene cluster to affect the biosynthesis of epothilone. This is the first transcription regulator identified for epothilone biosynthesis.

Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus.

BACKGROUND: Exotic genes, especially clustered multiple-genes for a complex pathway, are normally integrated into chromosome for heterologous expression. The influences of insertion sites on heterologous expression and allotropic expressions of exotic genes on host remain mostly unclear. RESULTS: We compared the integration and expression efficiencies of single and multiple exotic genes that were inserted into Myxococcus xanthus genome by transposition and attB-site-directed recombination. While the site-directed integration had a rather stable chloramphenicol acetyl transferase (CAT) activity, the transposition produced varied CAT enzyme activities. We attempted to integrate the 56-kb gene cluster for the biosynthesis of antitumor polyketides epothilones into M. xanthus genome by site-direction but failed, which was determined to be due to the insertion size limitation at the attB site. The transposition technique produced many recombinants with varied production capabilities of epothilones, which, however, were not paralleled to the transcriptional characteristics of the local sites where the genes were integrated. Comparative transcriptomics analysis demonstrated that the allopatric integrations caused selective changes of host transcriptomes, leading to varied expressions of epothilone genes in different mutants. CONCLUSIONS: With the increase of insertion fragment size, transposition is a more practicable integration method for the expression of exotic genes. Allopatric integrations selectively change host transcriptomes, which lead to varied expression efficiencies of exotic genes.