CRISPR/Cas9 advances engineering of microbial cell factories

One of the key drivers for successful metabolic engineering in microbes is the efficacy by which genomes can be edited. As such there are many methods to choose from when aiming to modify genomes, especially those of model organisms like yeast and bacteria. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing with special emphasis on their application for metabolic engineering of yeast and bacteria. Also, examples of how nuclease-deficient Cas9 has been applied for RNA-guided transcriptional regulation of target genes will be reviewed, as well as tools available for computer-aided design of guide-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering

Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity.

The ability to generate RNA aptamers for synthetic biology using in vitro selection depends on the informational complexity (IC) needed to specify functional structures that bind target ligands with desired affinities in physiological concentrations of magnesium. We investigate how selection for high-affinity aptamers is constrained by chemical properties of the ligand and the need to bind in low magnesium. We select two sets of RNA aptamers that bind planar ligands with dissociation constants (K(d)s) ranging from 65 nM to 100 microM in physiological buffer conditions. Aptamers selected to bind the non-proteinogenic amino acid, p-amino phenylalanine (pAF), are larger and more informationally complex (i.e., rarer in a pool of random sequences) than aptamers selected to bind a larger fluorescent dye, tetramethylrhodamine (TMR). Interestingly, tighter binding aptamers show less dependence on magnesium than weaker-binding aptamers. Thus, selection for high-affinity binding may automatically lead to structures that are functional in physiological conditions (1-2.5 mM Mg(2+)). We hypothesize that selection for high-affinity binding in physiological conditions is primarily constrained by ligand characteristics such as molecular weight (MW) and the number of rotatable bonds. We suggest that it may be possible to estimate aptamer-ligand affinities and predict whether a particular aptamer-based design goal is achievable before performing the selection

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin

Utilizing a highly responsive gene, yhjX, in E. coli based production of 1,4-butanediol

AbstractThe role of yhjX, a predicted major facilitator superfamily protein, was examined in context of E. coli response to 1,4-butanediol (1,4-BDO). E. coli DH1 and MG1655, two commonly used metabolic engineering hosts, were both sensitive to the presence of 1,4-BDO in the growth medium, but to different extents. The strains also showed differences in the transcriptional response of the yhjX gene that was highly induced in response to 1,4-BDO. yhjX deletion improved growth of the E. coli strains in the control defined medium but did not significantly impact 1,4-BDO sensitivity. Overexpression of yhjX using a plasmid-borne copy and lactose-inducible promoter also did not result in an improvement in 1,4-BDO tolerance. However, the large differential expression of yhjX in response to this diol provided the foundation to develop a biosensor for the detection of 1,4-BDO using a fluorescent gene under the control of the yhjX promoter. A basic PyhjX:GFP biosensor in E. coli DH1 allows the detection of 4–7% 1,4-BDO in the extracellular medium and provides a tool for high throughput engineering for improving 1,4-BDO production strains