BackgroundRhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides.ResultsHere we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected.ConclusionsIn this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

The textile industry has caused severe water pollution by using many toxic chemicals for producing fabric dyes. In response to this problem, indigoidine has attracted attention as an alternative natural blue dye, but it is necessary to achieve a high-level production to compete with synthetic blue dyes. Here we report a metabolically engineered Corynebacterium glutamicum capable of producing indigoidine to a high concentration with high productivity. First, the blue-pigment indigoidine synthetase (bpsA) gene from Streptomyces lavendulae was expressed in C. glutamicum, which carries strong fluxes toward L-glutamate, a precursor of indigoidine. Production performance of this base strain, already producing 7.3 ± 0.3 g/L indigoidine from the flask, was further improved by streamlining the intracellular supply of the precursors L-glutamate and L-glutamine, strengthening the phosphotransferase system-independent glucose uptake system, channeling carbon fluxes from glycolysis to the tricarboxylic acid (TCA) cycle, and minimizing byproducts formation. Fedbatch fermentation of the final strain BIRU11 produced 49.30 g/L indigoidine with a productivity of 0.96 g/L/h, the highest titer and productivity to date. Finally, indigoidine from the fed-batch fermentation of the BIRU11 strain was used to dye white cotton fabrics to examine its color and performance. This study demonstrates the potential of producing fabric dyes in a sustainable manner by using a metabolically engineered bacterium.

Synthetic sRNAs allow knockdown of target genes at translational level, but have been restricted to a limited number of bacteria. Here, we report the development of a broad-host-range synthetic sRNA (BHR-sRNA) platform employing the RoxS scaffold and the Hfq chaperone from Bacillus subtilis. BHR-sRNA is tested in 16 bacterial species including commensal, probiotic, pathogenic, and industrial bacteria, with >50% of target gene knockdown achieved in 12 bacterial species. For medical applications, virulence factors in Staphylococcus epidermidis and Klebsiella pneumoniae are knocked down to mitigate their virulence-associated phenotypes. For metabolic engineering applications, high performance Corynebacterium glutamicum strains capable of producing valerolactam (bulk chemical) and methyl anthranilate (fine chemical) are developed by combinatorial knockdown of target genes. A genome-scale sRNA library covering 2959 C. glutamicum genes is constructed for high-throughput colorimetric screening of indigoidine (natural colorant) overproducers. The BHR-sRNA platform will expedite engineering of diverse bacteria of both industrial and medical interest.

Long‐chain polyunsaturated fatty acids (LC‐PUFAs), largely obtained from fish oil, serve as valuable dietary supplements with many health benefits, especially arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. In recent years, interest in the sustainable production of LC‐PUFAs using heterologous production hosts has drastically increased because overfishing and polluted oceans have led to a gradual decline in the supply of LC‐PUFAs from fish oil in the last few decades. Some species of microalgae have been considered to be an ideal producer of LC‐PUFAs as they inherently accumulate large amounts of LC‐PUFAs and utilize CO2 using light energy. Also, a growing number of genetic toolboxes have become available, and the microalgal cultivation process is amenable to a scale‐up. In fact, tremendous progress has been achieved in the development of high‐performance strains of microalgae through metabolic engineering that has led to the efficient production of fatty acids and various derivatives in the last decade. This review discusses metabolic engineering strategies that have contributed to the enhanced production of LC‐PUFAs using microalgae, including optimizing fatty acid biosynthetic pathway and intracellular supply of precursors and cofactors, as well as engineering transcription factors.