SynBioFromLeukipposInstitute

The foundations of synthetic biology rely on the concept that all living systems are constituted by functional and structural modules that could be rationally engineered to build organisms that are not easily generated by evolution. However, the cellular complexity and metabolic diversity found in organisms present both a significant challenge and opportunity to foster this interdisciplinary scientific area. Secondary metabolism is a group of biological processes which are dispensable for cell growth. In microorganisms such as fungi and filamentous bacteria, secondary metabolism is an important source of biologically active compounds (Leitão and Enguita, 2014). The genes encoding for the enzymes involved in secondary metabolism are frequently clustered together displaying a modular organization, constituting a perfect target for the application of synthetic biology principles with an almost unlimited potential for the construction of new cell factories, which is mainly constrained by the knowledge of the working rules of the functional blocks governing the biosynthesis of a particular metabolite.
More than 35 years after the first seminal ideas by Hopwood, who hypothesized about the possibility of a rational design of new antibiotics based on the combination of biosynthetic genes (Hopwood and Chater, 1980), secondary metabolites are gaining a new relevance. Modern genetic techniques have enabled complete genome sequences of secondary metabolite producers to be thoroughly and rapidly interrogated to uncover loci relevant for metabolite biosynthesis. The developed genome editing and engineering techniques are paving the way for rational pathway design and construction, modifying a genotype with the main objective of giving rise to a desired phenotype (Paddon et al., 2013;Paddon and Keasling, 2014;Hoynes-O'Connor and Moon, 2015). Recent applications of genome editing techniques such as TALE nucleases or CRISPR-Cas9 in the rational engineering of primary and secondary metabolites are an excellent example of the potential in this area (Huang et al., 2015;Lv et al., 2015;Wu et al., 2015).
This Frontiers research topic was proposed to compile new trends for the application of synthetic biology methods and protocols to the field of secondary metabolites. We are extremely grateful to all the authors that have contributed to enrich the contents of the research topic. The topic contributions include six articles, covering wide aspects of secondary metabolism in organisms ranging from filamentous bacteria to microalgae.
Two excellent articles by Mattern et al (Mattern et al., 2015) and Chávez et al (Chavez et al., 2015) describe applications of synthetic biology principles for the manipulation of secondary metabolite biosynthesis in filamentous fungi. Interestingly, the article by Chávez et al (Chavez et al., 2015) also explore the idea of genome mining to search and explore new metabolites in filamentous fungi isolated from extreme environments.
Several practical applications of synthetic biology principles were discussed by Giessen et al (Giessen and Marahiel, 2015). In this article, the authors present a large repository of modular catalytic enzymes able to modify cyclic dipeptides, and demonstrate how combinatorial chemistry can be applied to custom design new molecules with improved biological activities. Moreover, the work by Gressier et al (Gressler et al., 2015) also included in the research topic, describes a high-performance system for heterologous expression in Aspergillus terreus based on the tetA promoter and the DNA-binding domain of its cognate transcription factor TetR. As a proof of concept, the authors used the system to express the polyketide synthase-encoding gene osrA, and demonstrated its catalytic activity. Other interesting topic discussed by Beites and Mendes (Beites and Mendes, 2015) reflects the increasing number of genomic data generated by the bacterial genome sequencing projects which can be further exploited as an unlimited source of metabolic diversity. In fact, following the author’s ideas, several biosynthetic clusters for secondary metabolites present reduced or no expression (also known as “cryptic” clusters), constituting a pool of novel metabolites waiting to be mined and characterized. Within this context, the article described rational strategies and tools to be used in the development of chassis structures for the heterologous expression of biosynthetic gene clusters in filamentous bacteria. Finally, the topic also includes an article by Gimpel et al (Gimpel et al., 2015), which discusses the strategies for rational metabolic engineering of microalgae, a very promising tool for the production of several products including biofuels, carotenoids and bio-hydrogen.
We hope that this research topic will be interesting and useful to the general audience of Frontiers in Microbiology journal, and that the treated topics act as crystallization nuclei for new ideas and future applications of the principles of synthetic biology to the design of new secondary metabolites with improved biological activities.

The process of connecting genetic parts-DNA assembly-is a foundational technology for synthetic biology. Microfluidics present an attractive solution for minimizing use of costly reagents, enabling multiplexed reactions, and automating protocols by integrating multiple protocol steps. However, microfluidics fabrication and operation can be expensive and requires expertise, limiting access to the technology. With advances in commodity digital fabrication tools, it is now possible to directly print fluidic devices and supporting hardware. 3D printed micro- and millifluidic devices are inexpensive, easy to make and quick to produce. We demonstrate Golden Gate DNA assembly in 3D-printed fluidics with reaction volumes as small as 490 nL, channel widths as fine as 220 microns, and per unit part costs ranging from $0.61 to $5.71. A 3D-printed syringe pump with an accompanying programmable software interface was designed and fabricated to operate the devices. Quick turnaround and inexpensive materials allowed for rapid exploration of device parameters, demonstrating a manufacturing paradigm for designing and fabricating hardware for synthetic biology.

The concept of "Morphology Engineering" is proposed here. There are many genes involved in maintaining the bacterial shapes. The manipulations of these genes allow us to change the bacterial shapes from rod to fibers or to small spheres or large spheres. The advantages of morphology engineered bacteria for bio-production including accelerated growth, high cell density, simplification of downstream separation, enlarged space for more inclusion body accumulation and reduction on the cost of bio-production, has recently started to be exploited. So far only a few shape related genes have been manipulated for bioprocess benefits, much more genes are to be exploited for various cell morphology. The limits of bacterial lengths and diameters may depend on how we manipulate relevant genes. Over time, these limits can be broken to enhance the bioprocess competitiveness including improvements on the effectiveness of up- and downstream. Morphology engineering is just starting to show its promises.

The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology.

Early this year, synthetic biologist Christina Smolke was in a dead-heat race with a handful of other labs to engineer a yeast strain capable of making opioids. These powerful pain-killing drugs are crucial in medicine, but they come solely from opium poppy crops that can have unpredictable yields. Scientists were seeking a more stable production method but faced a daunting hurdle: no one had been able to identify an enzyme that converts reticuline — a chemical building block of morphine and other narcotics — from one form to another.

Most other labs hunting for the enzyme were working to isolate it from poppies directly. But Smolke and her team at Stanford University, California, took a different approach: they combed through genetic databases, looking for snippets of sequence that looked as if they might be involved in reticuline metabolism. When they found a hit from several different poppy species, they ordered a synthetic version of the gene that had been built letter-by-letter by a machine. They plugged it into yeast and it worked. “I was super excited, really proud and also relieved,” Smolke says. “It was a bit of a Hail Mary.”

The discovery enabled Smolke’s lab to stitch together a pathway of 23 different genes from plants, mammals, bacteria and yeast to produce the world’s first narcotic through synthetic biology (S. Galanie et al. Science 349, 1095–1100; 2015). It was a crowning achievement for a biological wunderkind who started her own lab at the California Institute of Technology in Pasadena at the age of just 28. The opioid-producing yeast cells contain the most complex synthetic-biology pathway developed so far, and mark a turning point for the field by showing how step-by-step engineering can turn microbes into drug factories. “This will significantly impact our future ability to produce many more chemicals through biotech­nology,” says Jens Nielsen, a synthetic biologist at the Chalmers University of Technology in Gothenburg, Sweden.


Drugs: Regulate 'home-brew' opiates
Much of the news coverage of the work, however, stirred fears about how it could foster new ways to easily manufacture illegal drugs — and some scientists have argued for tighter regulation of the growing field. Smolke counters that existing regulations already restrict the production and distribution of narcotics; any lab that wishes to work with the yeast strain reported in her paper, for instance, must be licensed by the US Drug Enforcement Administration. So far, no one has requested the strain.

In a bid to ground the debate in reality, Smolke, her husband — fellow Stanford synthetic biologist Drew Endy — and another colleague this year attempted to brew opioids using her lab’s strain and standard beer-making equipment (D. Endy et al. Preprint at bioRxiv http://doi.org/9t2; 2015). The set-up produced only a trace amount of reticuline and none of the downstream chemical, thebaine, that is used to synthesize commercial drugs such as oxycodone and oxymorphone — suggesting that it would be difficult for the average home-brewer to start making these pharmaceuticals. (The scientists’ positive fermentation control, an English ale, was “palatable”, the manuscript notes.)

Smolke co-founded a company, Antheia, based in Palo Alto, to produce opiate drugs in yeast commercially, and specialists in the field suspect that more will follow. But some onlookers are circumspect. Plant biologist Ian Graham at the University of York, UK, says that it will be hard to beat poppies. “Where plants already do it very well, the arguments for taking a synthetic-biology route are much less convincing,” he says.

For Smolke, the goal is not merely to copy plants, but to engineer opioids that are free of side effects such as dependency and addiction. Sitting in the office of a Palo Alto incubator space, wearing jeans and grey Converse sneakers to a meeting with the co-founders of Antheia, Smolke can appear casual — but the intensity that has propelled her to the pinnacle of her field is tangible. For her, the year’s accomplishments are just part of a quest to understand and improve on opioids, which are among the most complex natural chemicals . “It’s a very powerful approach to take inspiration from nature and go beyond it,” she says.

The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology.

Multicellular organisms depend on the exchange of information between specialized cells. This communication is often difficult to decipher in its native context, but synthetic biology provides tools to engineer well-defined systems that allow the convenient study and manipulation of intercellular communication networks.
RESULTS:
Here, we present the first mammalian synthetic network for reciprocal cell-cell communication to compute the border between a sender/receiver and a processing cell population. The two populations communicate via L-tryptophan and interleukin-4 to highlight the population border by the production of a fluorescent protein. The sharpness of that visualized edge can be adjusted by modulating key parameters of the network.
CONCLUSIONS:
We anticipate that this network will on the one hand be a useful tool to gain deeper insights into the mechanisms of tissue formation in nature and will on the other hand contribute to our ability to engineer artificial tissues.

The Synthetic Yeast Genome Project (Sc2.0), an ongoing effort to synthesize the genome of Saccharomyces cerevisiae, addresses fundamental questions about genome function. To generate phenotypic diversity, Jeff Boeke and his collaborators at Sc2.0 developed SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) by systematically introducing loxP sites—which undergo recombination in the presence of Cre—in certain chromosomal regions. Although the method was promising, concerns about specificity and decreased diversity remained

Designing and producing novel proteins that fold into stable structures and provide essential biological functions are key goals in synthetic biology. In initial steps toward achieving these goals, we constructed a combinatorial library of de novo proteins designed to fold into 4-helix bundles. As described previously, screening this library for sequences that function in vivo to rescue conditionally lethal mutants of E. coli (auxotrophs) yielded several de novo sequences, termed SynRescue proteins, which rescued four different E. coli auxotrophs. In an effort to understand the structural requirements necessary for auxotroph rescue, we investigated the biophysical properties of the SynRescue proteins, using both computational and experimental approaches. Results from circular dichroism, size exclusion chromatography, and NMR demonstrate that the SynRescue proteins are α-helical and relatively stable. Surprisingly, however, they do not form well-ordered structures. Instead, they form dynamic structures that fluctuate between monomeric and dimeric states. These findings show that a well-ordered structure is not a prerequisite for life-sustaining functions, and suggest that dynamic structures may have been important in the early evolution of protein function.

Nutraceuticals are food substances with medical and health benefits for humans. Limited by complicated procedures, high cost, low yield, insufficient raw materials, resource waste, and environment pollution, chemical synthesis and extraction are being replaced by microbial synthesis of nutraceuticals. Many microbial strains that are generally regarded as safe (GRAS) have been identified and developed for the synthesis of nutraceuticals, and significant nutraceutical production by these strains has been achieved. In this review, we systematically summarize recent advances in nutraceutical research in terms of physiological effects on health, potential applications, drawbacks of traditional production processes, characteristics of production strains, and progress in microbial fermentation. Recent advances in systems and synthetic biology techniques have enabled comprehensive understanding of GRAS strains and its wider applications. Thus, these microbial strains are promising cell factories for the commercial production of nutraceuticals.

Standards are important to synthetic biology because they enable exchange and reproducibility of genetic designs. This paper describes a procedure for converting between two standards: the Systems Biology Markup Language (SBML) and the Synthetic Biology Open Language (SBOL). SBML is a standard for behavioral models of biological systems at the molecular level. SBOL describes structural and basic qualitative behavioral aspects of a biological design. Converting SBML to SBOL enables a consistent connection between behavioral and structural information for a biological design. The conversion process described in this paper leverages Systems Biology Ontology (SBO) annotations to enable inference of a designs qualitative function.

Nano-bioelectronics represents a rapidly expanding interdisciplinary field that combines nanomaterials with biology and electronics and, in so doing, offers the potential to overcome existing challenges in bioelectronics. In particular, shrinking electronic transducer dimensions to the nanoscale and making their properties appear more biological can yield significant improvements in the sensitivity and biocompatibility and thereby open up opportunities in fundamental biology and healthcare. This review emphasizes recent advances in nano-bioelectronics enabled with semiconductor nanostructures, including silicon nanowires, carbon nanotubes, and graphene. First, the synthesis and electrical properties of these nanomaterials are discussed in the context of bioelectronics. Second, affinity-based nano-bioelectronic sensors for highly sensitive analysis of biomolecules are reviewed. In these studies, semiconductor nanostructures as transistor-based biosensors are discussed from fundamental device behavior through sensing applications and future challenges. Third, the complex interface between nanoelectronics and living biological systems, from single cells to live animals, is reviewed. This discussion focuses on representative advances in electrophysiology enabled using semiconductor nanostructures and their nanoelectronic devices for cellular measurements through emerging work where arrays of nanoelectronic devices are incorporated within three-dimensional cell networks that define synthetic and natural tissues. Last, some challenges and exciting future opportunities are discussed.