Anaerobic production of medium-chain fatty alcohols via a ß-reduction pathway.

In this report, we identify the relevant factors to increase production of medium chain n-alcohols through an expanded view of the reverse ß-oxidation pathway. We began by creating a base strain capable of producing medium chain n-alcohols from glucose using a redox-balanced and growth-coupled metabolic engineering strategy. By dividing the heterologous enzymes in the pathway into different modules, we were able to identify and evaluate homologs of each enzyme within the pathway and identify several capable of enhancing medium chain alcohol titers and/or selectivity. In general, the identity of the trans-2-enoyl-CoA reductase (TER) and the direct overexpression of the thiolase (FadA) and ß-hydroxy-acyl-CoA reductase (FadB) improved alcohol titer and the identity of the FadBA complex influenced the dominant chain length. Next, we linked the anaerobically induced VHb promoter from Vitreoscilla hemoglobin to each gene to remove the need for chemical inducers and ensure robust expression. The highest performing strain with the autoinduced reverse ß-oxidation pathway produced n-alcohols at titers of 1.8 g/L with an apparent molar yield of 0.2 on glucose consumed in rich medium (52% of theoretical yield).

A transcription activator-like effector (TALE) induction system mediated by proteolysis.

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes.

The last several years have witnessed an explosion in the understanding and use of novel, versatile trans-acting elements. TALEs, CRISPR/Cas, and sRNAs can be easily fashioned to bind any specific sequence of DNA (TALEs, CRISPR/Cas) or RNA (sRNAs) because of the simple rules governing their interactions with nucleic acids. This unique property enables these tools to repress the expression of genes at the transcriptional or post-transcriptional levels, respectively, without prior manipulation of cis-acting and/or chromosomal target DNA sequences. These tools are now being harnessed by synthetic biologists, particularly those in the eukaryotic community, for genome-wide regulation, editing, or epigenetic studies. Here we discuss the exciting opportunities for using TALEs, CRISPR/Cas, and sRNAs as synthetic trans-acting regulators in prokaryotes.

Production of medium chain length fatty alcohols from glucose in Escherichia coli.

Metabolic engineering offers the opportunity to produce a wide range of commodity chemicals that are currently derived from petroleum or other non-renewable resources. Microbial synthesis of fatty alcohols is an attractive process because it can control the distribution of chain lengths and utilize low cost fermentation substrates. Specifically, primary alcohols with chain lengths of 12 to 14 carbons have many uses in the production of detergents, surfactants, and personal care products. The current challenge is to produce these compounds at titers and yields that would make them economically competitive. Here, we demonstrate a metabolic engineering strategy for producing fatty alcohols from glucose. To produce a high level of 1-dodecanol and 1-tetradecanol, an acyl-ACP thioesterase (BTE), an acyl-CoA ligase (FadD), and an acyl-CoA/aldehyde reductase (MAACR) were overexpressed in an engineered strain of Escherichia coli. Yields were improved by balancing expression levels of each gene, using a fed-batch cultivation strategy, and adding a solvent to the culture for extracting the product from cells. Using these strategies, a titer of over 1.6 g/L fatty alcohol with a yield of over 0.13 g fatty alcohol/g carbon source was achieved. These are the highest reported yield of fatty alcohols produced from glucose in E. coli.

Artificial repressors for controlling gene expression in bacteria.

Transcriptional repression is a common approach to control gene expression in synthetic biology applications. Here, an engineered DNA binding protein based upon a transcription activator-like effector (TALE) scaffold was shown to outperform LacI in blocking transcription from a promoter and to repress expression of a downstream gene in an operon.