In This Issue pubs.acs.org/synthbio
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THE IMPACT OF DNA TOPOLOGY AND GUIDE LENGTH ON TARGET SELECTION BY A CYTOSINE-SPECIFIC CAS9
The engineering of microbes to act as cell factories has enabled the production of fuels, pharmaceuticals and fine chemicals in a sustainable and clean manner. To date, large libraries of genetic parts have been created to allow for fine control of gene expression, including regulatory elements such as promoters, terminators, and ribosome binding sites. The methylotrophic yeast Pichia pastoris is a well-established expression host that has the ability to perform post-translational modifications and is generally regarded as safe (GRAS). Nevertheless, optimization of protein secretion in this host remains a challenge due to the multiple steps involved during secretion and a lack of genetic tools to tune this process. In this issue, Obst et al. (DOI: 10.1021/acssynbio.6b00337) develop a toolkit of standardized regulatory elements specific for Pichia pastoris allowing the tuning of gene expression and choice of protein secretion tag. To assess the performance of these parts, the authors built and characterized the expression and secretion efficiency of 124 constructs that combined different regulatory elements with two fluorescent reporter proteins (RFP, yEGFP). Intracellular expression from our promoters was comparatively independent of whether RFP or yEGFP, and whether plasmidbased expression or genomically integrated expression, was used. In contrast, secretion efficiency significantly varied for different genes expressed using identical regulatory elements, with differences in secretion efficiency of >10-fold observed. The results from this study highlight the importance of generating diverse secretion libraries when searching for optimal expression conditions, and demonstrate that the newly developed toolkit is a valuable asset for the creation of efficient microbial cell factories.
Cas9 is an RNA-guided DNA cleavage enzyme utilized by some bacteria to defend against invading species, such as bacteriophages, through the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-mediated pathway. A functional Cas9 is comprised of a polypeptide and two short RNAs, a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). In this issue, Tsui et al. (DOI: 10.1021/acssynbio.7b00050) report the identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable the development of versatile and safe Cas9-based technologies. In this study, the authors found that AceCas9 depends on a 5′-NNNCC-3′ PAM and is more efficient in cleaving negative supercoils than relaxed DNA. It was shown that kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.
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DEVELOPMENT OF A BIOSENSOR CONCEPT TO DETECT THE PRODUCTION OF CLUSTER-SPECIFIC SECONDARY METABOLITES
A MODULAR TOOLKIT FOR GENERATING PICHIA PASTORIS SECRETION LIBRARIES
Natural products from Streptomyces and other actinomycete bacteria remain a rich source for the discovery of new bioactive compounds that may have a potential to be developed into human medicines. Recent advances in genomics of these important bacteria revealed their unprecedented capacity to Received: June 5, 2017 Published: June 16, 2017 © 2017 American Chemical Society
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DOI: 10.1021/acssynbio.7b00187 ACS Synth. Biol. 2017, 6, 913−914
ACS Synthetic Biology
In This Issue
control gene activation, signal transduction, and cytoskeletal remodeling in response to Fe2+. This technology was utilized to design signal circuitry incorporating “AND” and “OR” biologic gates that enables mammalian cells to translate different combinations of Fe2+ and H2O2 signals into predefined biological outputs. The authors believe that these observations can provide useful information for further optimization and future design of similar molecules. This study presents a unique strategy that specific sensor units can be integrated into orthogonal CIP inducers to build custom signaling pathways and logic gates responding to physiologically relevant signal cues. This new strategy should provide alternative approaches to engineer cellular signaling and responses in mammalian cells.
synthesize structurally diverse secondary metabolites, while only a few of those are actually produced in standard laboratory conditions. Currently, genome mining still appears to be unpredictable in terms of outcomes and may yield unexpected results. The most laborious part of genome mining remains analytics aimed at detection and identification of a compound expected to be produced as a result of activation or heterologous expression of a targeted gene cluster. Hence, the development of a robust biosensor that could be used to directly assess actual production of a cluster-specific compound would be of great advantage, also with respect to high-throughput screening after random mutagenesis. In this issue, Sun et al. (DOI: 10.1021/ acssynbio.6b00353) develop a new repressor-based biosensor to detect activated secondary metabolite biosynthesis gene clusters in Streptomyces. Biosynthetic gene clusters for undecylprodigiosin and coelimycin in the genome of Streptomyces lividans TK24, which encoded TetR-like repressors and appeared to be almost “silent” based on the RNA-seq data, were chosen for the proof-ofprinciple studies. The bpsA reporter gene for indigoidine synthetase was placed under control of the promotor/operator regions presumed to be controlled by the cluster-associated TetR-like repressors. While the biosensor for undecylprodigiosin turned out to be nonfunctional, the coelimycin biosensor was shown to perform as expected, turning on biosynthesis of indigoidine in response to the concomitant production of coelimycin. The developed reporter system concept can be applied to those cryptic gene clusters that encode metabolitesensing repressors to speed up discovery of novel bioactive compounds in Streptomyces using synthetic biology techniques.
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ENGINEERING IRON RESPONSES IN MAMMALIAN CELLS BY SIGNAL-INDUCED PROTEIN PROXIMITY
The introduction of artificial signaling networks using synthetic biology methods has facilitated the investigation of signaling mechanisms and the identification of essential components in signaling pathways. It is proposed that the combination of elevated levels of Fe2+ and H2O2 (under oxidative stress) leads to the production of free radicals which promote neuron death in Alzheimer’s disease (AD) and Parkinson’s disease (PD). The availability of an engineered system that enables cells to detect elevated levels of H2O2 and Fe2+ in local environments and respond with specific therapeutic outputs would contribute to the development of novel therapies for AD and PD. In this issue, Zeng et al. (DOI: 10.1021/acssynbio.6b00255) describe a method to engineer artificial Fe2+ signaling that responds to elevated Fe2+ levels and to build “AND” and “OR” Boolean logic-based signaling circuitries that respond to a combination of H2O2 and Fe2+ in a predefined manner. The dual function probe ABA-FE18 (Fe2+-sensing and protein dimerization) derived from ABA was developed and used to 914
DOI: 10.1021/acssynbio.7b00187 ACS Synth. Biol. 2017, 6, 913−914
