Recent advances of metabolic engineering strategies in natural isoprenoid production using cell factories.

Covering: up to 2019As abundant natural products, isoprenoids have many useful industrial applications in the manufacturing of drugs, fragrances, food additives, colorants, rubber and advanced biofuels. The microbial production of isoprenoids has received much attention in recent years. Metabolic engineering approaches and synthetic biology have been utilized to reconstruct and optimize the metabolic pathways for isoprenoid production in cell factories. In this review, the recent advances in isoprenoid production using microbes are summarized, with a focus on MEP and MVA pathway engineering, downstream isoprenoid pathway engineering and microbial host engineering, which mainly includes central carbon pathway engineering. Finally, future perspectives for the improvement of isoprenoid production are discussed.

A flow cytometric approach to engineering Escherichia coli for improved eukaryotic protein glycosylation.

A synthetic pathway for production of the eukaryotic trimannosyl chitobiose glycan (mannose3-N-acetylglucosamine2, Man3GlcNAc2) and its transfer to specific asparagine residues in target proteins was previously engineered in Escherichia coli, providing this simple microbe with the ability to perform a complex post-translational protein modification. Here, we leveraged a flow cytometric fluorescence-based assay to improve Man3GlcNAc2 glycan biosynthesis in E. coli cells. Specifically, pathway improvements were identified, including reducing pathway enzyme expression levels and overexpressing nucleotide sugar biosynthesis genes, which enhanced production of lipid-linked Man3GlcNAc2 by nearly 50-fold to 13.9 µg/L. In turn, cells producing higher levels of the Man3GlcNAc2 substrate yielded up to 10 times more glycosylated acceptor protein (to ~ 14 mg/L) than their non-optimized counterparts. These results demonstrate the use of flow cytometry screening as a powerful tool for interrogating the surfaces of glyco-engineered bacteria and identifying meaningful improvements in glycan biosynthesis. We anticipate this approach will enable further optimization of bacterial glycan biosynthesis pathways using new strain engineering tools from metabolic engineering and synthetic biology.

High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli.

Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in Escherichia coli mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). Growth rates were severely decreased, to approximately 50% of those of wild type cells, depending on the expression levels of dCas9. Cell shape was changed to abnormal filamentous morphology, indicating that dCas9 affects bacterial cell division. RNA-Seq revealed that 574 genes were differentially transcribed in the presence of high expression levels of dCas9. Genes associated with cell division were upregulated, which was consistent with the observed atypical morphologies. In contrast, 221 genes were downregulated, and these mostly encoded proteins located in the cell membrane. Further, ChIP-Seq results showed that dCas9 directly binds upstream of 37 genes without single-guide RNA, including fimA, which encodes bacterial fimbriae. These results support the fact that dCas9 has critical effects on cell division as well as inner and outer membrane structure. Thus, to precisely understand gene functions using dCas9-driven transcriptional modulation, the regulation of intracellular levels of dCas9 is pivotal to avoid unexpected morphological changes in E. coli.

Peyer's Patches as a Portal for DNA Delivery by Lactococcus lactis in Vivo.

Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner.