High-throughput genetic engineering tools for regulating gene expression in a microbial cell factory.

With the rapid advances in biotechnological tools and strategies, microbial cell factory-constructing strategies have been established for the production of value-added compounds. However, optimizing the tradeoff between the biomass, yield, and titer remains a challenge in microbial production. Gene regulation is necessary to optimize and control metabolic fluxes in microorganisms for high-production performance. Various high-throughput genetic engineering tools have been developed for achieving rational gene regulation and genetic perturbation, diversifying the cellular phenotype and enhancing bioproduction performance. In this paper, we review the current high-throughput genetic engineering tools for gene regulation. In particular, technological approaches used in a diverse range of genetic tools for constructing microbial cell factories are introduced, and representative applications of these tools are presented. Finally, the prospects for high-throughput genetic engineering tools for gene regulation are discussed.

Identification of a gene cluster for D-tagatose utilization in Escherichia coli B2 phylogroup.

D-Tagatose is a promising low-calorie sugar-substituting sweetener in the food industry. Most ingested D-tagatose is fermented by intestinal microorganisms. Until now, Escherichia coli has been considered incapable of growing on D-tagatose. Here, we discovered a gene cluster involved in D-tagatose utilization in E. coli. The chromosome of the intestinal probiotic E. coli Nissle 1917 contains a six-gene cluster encoding the ABC transporter, D-tagatose kinase, D-tagatose-bisphosphate aldolase, and putative aldose 1-epimerase. The functionality of the gene cluster was experimentally validated. Based on single-gene deletions, D-tagatose dissimilation occurs via D-tagatose 6-phosphate to D-tagatose 1,6-bisphosphate to D-glyceraldehyde 3-phosphate plus dihydroxyacetone phosphate. Remarkably, this gene cluster was located in 93% of the completely sequenced genomes of the E. coli B2 phylogroup, which contains the majority of extraintestinal pathogenic and adherent-invasive E. coli strains prevalent in patients with inflammatory bowel disease. This highlights the importance of understanding the clinical significance of D-tagatose in microbiota alterations.

Synthetic fused sRNA for the simultaneous repression of multiple genes.

Efficient control over multiple gene expression still presents a major challenge. Synthetic sRNA enables targeted gene expression control in trans without directly modifying the chromosome, but its use to simultaneously target multiple genes can often cause cell growth defects because of the need for additional energy for transcription and lowering of their repression efficiency by limiting the amount of Hfq protein. To address these limitations, we present fusion sRNA (fsRNA) that simultaneously regulates the translation of multiple genes efficiently. It is constructed by linking the mRNA-binding modules for multiple targeted genes in one sRNA scaffold via one-pot generation using overlap extension PCR. The repression capacity of fsRNA was demonstrated by the construction of sRNAs to target four endogenous genes: caiF, hybG, ytfR and minD in Escherichia coli. Their cross-reactivity and the effect on cell growth were also investigated. As practical applications, we applied fsRNA to violacein- and protocatechuic acid-producing strains, resulting in increases of 13% violacein and 81% protocatechuic acid, respectively. The developed fsRNA-mediated multiple gene expression regulation system thus enables rapid and efficient development of optimised cell factories for valuable chemicals without cell growth defects and limiting cellular resources.Key points• Synthetic fusion sRNA (fsRNA)-based system was constructed for the repression of multiple target genes.• fsRNA repressed multiple genes by only expressing a single sRNA while minimising the cellular burden.• The application of fsRNA showed the increased production titers of violacein (13%) and protocatechuic acid (81%).

Biosensing Applications Using Nanostructure-Based Localized Surface Plasmon Resonance Sensors.

Localized surface plasmon resonance (LSPR)-based biosensors have recently garnered increasing attention due to their potential to allow label-free, portable, low-cost, and real-time monitoring of diverse analytes. Recent developments in this technology have focused on biochemical markers in clinical and environmental settings coupled with advances in nanostructure technology. Therefore, this review focuses on the recent advances in LSPR-based biosensor technology for the detection of diverse chemicals and biomolecules. Moreover, we also provide recent examples of sensing strategies based on diverse nanostructure platforms, in addition to their advantages and limitations. Finally, this review discusses potential strategies for the development of biosensors with enhanced sensing performance.

Recent advances in tuning the expression and regulation of genes for constructing microbial cell factories.

To overcome environmental problems caused by the use of fossil resources, microbial cell factories have become a promising technique for the sustainable and eco-friendly development of valuable products from renewable resources. Constructing microbial cell factories with high titers, yields, and productivity requires a balance between growth and production; to this end, tuning gene expression and regulation is necessary to optimise and precisely control complicated metabolic fluxes. In this article, we review the current trends and advances in tuning gene expression and regulation and consider their engineering at each of the three stages of gene regulation: genomic, mRNA, and protein. In particular, the technological approaches utilised in a diverse range of genetic-engineering-based tools for the construction of microbial cell factories are reviewed and representative applications of these strategies are presented. Finally, the prospects for strategies and systems for tuning gene expression and regulation are discussed.

Tunable Gene Expression System Independent of Downstream Coding Sequence.

Fine control of the expression levels of proteins constitutes a major challenge in synthetic biology and metabolic engineering. However, the dependence of translation initiation on the downstream coding sequence (CDS) obscures accurate prediction of the protein expression levels from mRNA sequences. Here, we present a tunable gene-expression system comprising 24 expression cassettes that produce predefined relative expression levels of proteins ranging from 0.001 to 1 without being influenced by the downstream CDS. To validate the practical utility of the tunable expression system, it was applied to a synthetic circuit displaying three states of fluorescence depending on the difference in protein expression levels. To demonstrate the suitability of application to metabolic engineering, this system was used to diversify the levels of key metabolic enzymes. As a result, expression-optimized strains were capable of producing 2.25 g/L of cadaverine, 2.59 g/L of L-proline, and 95.7 mg/L of 1-propanol. Collectively, the tunable expression system could be utilized to optimize genetic circuits for desired operation and to optimize metabolic fluxes through biosynthetic pathways for enhancing production yields of bioproducts. This tunable system will be useful for studying basic and applied biological sciences in addition to applications in synthetic biology and metabolic engineering.