No abstract

No abstract

Methanol is a sustainable substrate for biotechnology. In addition to natural methylotrophs, metabolic engineering has gained attention for transfer of methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cut-offs, i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes.

No abstract

Members of the genus Paenibacillus are widespread facultative anaerobic, endospore-forming bacteria. Some species such as Paenibacillus riograndensis or Paenibacillus polymyxa fix nitrogen and may play an important role in agriculture to reduce mineral nitrogen fertilization in particular for non-legume plants. The genetic manipulation of Paenibacillus is an imperative for the functional characterization, e.g., of its plant growth-promoting activities and metabolism. This study showed that P. riograndensis and P. polymyxa can be readily transformed using physical permeation by magnesium aminoclays. By means of the fluorescent reporter genes gfpUV, mcherry, and crimson, a two-plasmid system consisting of a theta-replicating plasmid and a rolling circle-replicating plasmid was shown to operate in both species. Xylose-inducible and mannitol-inducible fluorescent reporter gene expression was demonstrated in the compatible two-plasmid system by fluorescence-activated cell scanning. As a metabolic engineering application, the biotin requiring P. riograndensis was converted to a biotin-prototrophic strain based on mannitol-inducible expression of the biotin biosynthesis operon bioWAFDBI from Bacillus subtilis.

Bacillus methanolicus is a Gram-positive, thermophilic, methanol-utilizing bacterium. As a facultative methylotroph, B. methanolicus is also known to utilize D -mannitol, D -glucose and, as recently discovered, sugar alcohol D -arabitol. While metabolic pathways for utilization of methanol, mannitol and glucose are known, catabolism of arabitol has not yet been characterized in B. methanolicus . In this work we present the elucidation of this hitherto uncharted pathway. In order to confirm our predictions regarding genes coding for arabitol utilization, we performed differential gene expression analysis of B. methanolicus MGA3 cells grown on arabitol as compared to mannitol via transcriptome sequencing (RNA-seq). We identified a gene cluster comprising eight genes that was up-regulated during growth with arabitol as a sole carbon source. The RNA-seq results were subsequently confirmed via qRT-PCR experiments. The transcriptional organization of the gene cluster identified via RNA-seq was analyzed and it was shown that the arabitol utilization genes are co-transcribed in an operon that spans from BMMGA3_RS07325 to BMMGA3_RS07365. Since gene deletion studies are currently not possible in B. methanolicus , two complementation experiments were performed in an arabitol negative Corynebacterium glutamicum strain using the four genes discovered via RNA-seq analysis as coding for a putative PTS for arabitol uptake (BMMGA3_RS07330, BMMGA3_RS07335, and BMMGA3_RS07340 renamed to atlABC ) and a putative arabitol phosphate dehydrogenase (BMMGA3_RS07345 renamed to atlD ). C. glutamicum is a natural D -arabitol utilizer that requires arabitol dehydrogenase MtlD for arabitol catabolism. The C. glutamicum mtlD deletion mutant was chosen for complementation experiments. Heterologous expression of atlABCD as well as the arabitol phosphate dehydrogenase gene atlD from B. methanolicus alone restored growth of the C. glutamicum Δ mtlD mutant with arabitol. Furthermore, D -arabitol phosphate dehydrogenase activities could be detected in crude extracts of B. methanolicus and these were higher in arabitol-grown cells than in methanol- or mannitol-grown cells. Thus, B. methanolicus possesses an arabitol inducible operon encoding, amongst others, a putative PTS system and an arabitol phosphate dehydrogenase for uptake and activation of arabitol as growth substrate.

Genetic perturbation systems are of great interest to redirect metabolic fluxes for value‐added production, as well as genetic screening for the development of new drugs, or to identify new targets for biotechnological applications. Here, we review CRISPR interference (CRISPRi), a method for gene expression using a catalytically inactive version of the CRISPR‐associated protein 9 (dCas9) of the widely applied CRISPR‐Cas9 genome editing system. In combination with the appropriate sgRNA, dCas9 binds to specific DNA sequences without causing double‐stranded DNA breakage but interfering with transcription initiation or elongation. Besides manifold uses to interrogate the physiology of a bacterial cell, CRISPRi is used in applications for metabolic engineering and strain development in industrial biotechnology. Albeit in its infancy, CRISPRi has already delivered the first success stories; however, we also analyze limitations of the CRISPRi system and give future perspectives.

BackgroundThe plant growth promoting rhizobacterium Paenibacillus riograndensis SBR5 is a promising candidate to serve as crop inoculant. Despite its potential in providing environmental and economic benefits, the species P. riograndensis is poorly characterized. Here, we performed for the first time a detailed transcriptome analysis of P. riograndensis SBR5 using RNA-seq technology.ResultsRNA was isolated from P. riograndensis SBR5 cultivated under 15 different growth conditions and combined together in order to analyze an RNA pool representing a large set of expressed genes. The resultant total RNA was used to generate 2 different libraries, one enriched in 5′-ends of the primary transcripts and the other representing the whole transcriptome. Both libraries were sequenced and analyzed to identify the conserved sequences of ribosome biding sites and translation start motifs, and to elucidate operon structures present in the transcriptome of P. riograndensis. Sequence analysis of the library enriched in 5′-ends of the primary transcripts was used to identify 1082 transcription start sites (TSS) belonging to novel transcripts and allowed us to determine a promoter consensus sequence and regulatory sequences in 5′ untranslated regions including riboswitches. A putative thiamine pyrophosphate dependent riboswitch upstream of the thiamine biosynthesis gene thiC was characterized by translational fusion to a fluorescent reporter gene and shown to function in P. riograndensis SBR5.ConclusionsOur RNA-seq analysis provides insight into the P. riograndensis SBR5 transcriptome at the systems level and will be a valuable basis for differential RNA-seq analysis of this bacterium.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4235-z) contains supplementary material, which is available to authorized users.