Synthetic biology: Learning the way toward high-precision biological design.

Since its inception, synthetic biology has overcome many technical barriers but is at a crossroads for high-precision biological design. Devising ways to fully utilize big biological data may be the key to achieving greater heights in synthetic biology.

Microbiome and Human Health: Current Understanding, Engineering, and Enabling Technologies.

The human microbiome is composed of a collection of dynamic microbial communities that inhabit various anatomical locations in the body. Accordingly, the coevolution of the microbiome with the host has resulted in these communities playing a profound role in promoting human health. Consequently, perturbations in the human microbiome can cause or exacerbate several diseases. In this Review, we present our current understanding of the relationship between human health and disease development, focusing on the microbiomes found across the digestive, respiratory, urinary, and reproductive systems as well as the skin. We further discuss various strategies by which the composition and function of the human microbiome can be modulated to exert a therapeutic effect on the host. Finally, we examine technologies such as multiomics approaches and cellular reprogramming of microbes that can enable significant advancements in microbiome research and engineering.

The Divergent Immunomodulatory Effects of Short Chain Fatty Acids and Medium Chain Fatty Acids.

Fatty acids are derived from diet and fermentative processes by the intestinal flora. Two to five carbon chain fatty acids, termed short chain fatty acids (SCFA) are increasingly recognized to play a role in intestinal homeostasis. However, the characteristics of slightly longer 6 to 10 carbon, medium chain fatty acids (MCFA), derived primarily from diet, are less understood. Here, we demonstrated that SCFA and MCFA have divergent immunomodulatory propensities. SCFA down-attenuated host pro-inflammatory IL-1ß, IL-6, and TNFα response predominantly through the TLR4 pathway, whereas MCFA augmented inflammation through TLR2. Butyric (C4) and decanoic (C10) acid displayed most potent modulatory effects within the SCFA and MCFA, respectively. Reduction in TRAF3, IRF3 and TRAF6 expression were observed with butyric acid. Decanoic acid induced up-regulation of GPR84 and PPARγ and altered HIF-1α/HIF-2α ratio. These variant immune characteristics of the fatty acids which differ by just several carbon atoms may be attributable to their origins, with SCFA being primarily endogenous and playing a physiological role, and MCFA exogenously from the diet.

Engineering microbes for targeted strikes against human pathogens.

Lack of pathogen specificity in antimicrobial therapy causes non-discriminant microbial cell killing that disrupts the microflora present. As a result, potentially helpful microbial cells are killed along with the pathogen, altering the biodiversity and dynamic interactions within the population. Moreover, the unwarranted exposure of antibiotics to microbes increases the likelihood of developing resistance and perpetuates the emergence of multidrug resistance. Synthetic biology offers an alternative solution where specificity can be conferred to reduce the non-specific, non-targeted activity of currently available antibiotics, and instead provides targeted therapy against specific pathogens and minimising collateral damage to the host's inherent microbiota. With a greater understanding of the microbiome and the available genetic engineering tools for microbial cells, it is possible to devise antimicrobial strategies for novel antimicrobial therapy that are able to precisely and selectively remove infectious pathogens. Herein, we review the strategies developed by unlocking some of the natural mechanisms used by the microbes and how these may be utilised in targeted antimicrobial therapy, with the promise of reducing the current global bane of multidrug antimicrobial resistance.

An oleaginous yeast platform for renewable 1-butanol synthesis based on a heterologous CoA-dependent pathway and an endogenous pathway.

BACKGROUND: Microbial biofuel production provides a promising sustainable alternative to fossil fuels. 1-Butanol is recognized as an advanced biofuel and is gaining attention as an ideal green replacement for gasoline. In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was first engineered with a heterologous CoA-dependent pathway and an endogenous pathway, respectively. RESULTS: The co-overexpression of two heterologous genes ETR1 and EutE resulted in the production of 1-butanol at a concentration of 65 µg/L. Through the overexpression of multiple 1-butanol pathway genes, the titer was increased to 92 µg/L. Cofactor engineering through endogenous overexpression of a glyceraldehyde-3-phosphate dehydrogenase and a malate dehydrogenase further led to titer improvements to 121 µg/L and 110 µg/L, respectively. In addition, the presence of an endogenous 1-butanol production pathway and a gene involved in the regulation of 1-butanol production was successfully identified in Y. lipolytica. The highest titer of 123.0 mg/L was obtained through this endogenous route by combining a pathway gene overexpression strategy. CONCLUSIONS: This study represents the first report on 1-butanol biosynthesis in Y. lipolytica. The results obtained in this work lay the foundation for future engineering of the pathways to optimize 1-butanol production in Y. lipolytica.

Whole-cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae.

Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio-alkane has gained attention as an ideal drop-in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof-of-principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α-dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C12-18 free fatty acids to C11-17 aldehydes. Co-expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole-cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232-237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Isolated Reporter Bacteria in Supramolecular Hydrogel Microwell Arrays.

The combination of supramolecular hydrogels formed by low molecular weight gelator self-assembly via noncovalent interactions within a scaffold derived from polyethylene glycol (PEG) affords an interesting approach to immobilize fully functional, isolated reporter bacteria in novel microwell arrays. The PEG-based scaffold serves as a stabilizing element and provides physical support for the self-assembly of the C2-phenyl-derived gelator on the micrometer scale. Supramolecular hydrogel microwell arrays with various shapes and sizes were used to isolate single or small numbers of Escherichia coli TOP10 pTetR-LasR-pLuxR-GFP. In the presence of the autoinducer N-(3-oxododecanoyl) homoserine lactone, the entrapped E. coli in the hydrogel microwell arrays showed an increased GFP expression. The shape and size of microwell arrays did not influence the fluorescence intensity and the projected size of the bacteria markedly, while the population density of seeded bacteria affected the number of bacteria expressing GFP per well. The hydrogel microwell arrays can be further used to investigate quorum sensing, reflecting communication in inter- and intraspecies bacterial communities for biology applications in the field of biosensors. In the future, these self-assembled hydrogel microwell arrays can also be used as a substrate to detect bacteria via secreted autoinducers.

Reprogrammable microbial cell-based therapeutics against antibiotic-resistant bacteria.

The discovery of antimicrobial drugs and their subsequent use has offered an effective treatment option for bacterial infections, reducing morbidity and mortality over the past 60 years. However, the indiscriminate use of antimicrobials in the clinical, community and agricultural settings has resulted in selection for multidrug-resistant bacteria, which has led to the prediction of possible re-entrance to the pre-antibiotic era. The situation is further exacerbated by significantly reduced antimicrobial drug discovery efforts by large pharmaceutical companies, resulting in a steady decline in the number of new antimicrobial agents brought to the market in the past several decades. Consequently, there is a pressing need for new antimicrobial therapies that can be readily designed and implemented. Recently, it has become clear that the administration of broad-spectrum antibiotics can lead to collateral damage to the human commensal microbiota, which plays several key roles in host health. Advances in genetic engineering have opened the possibility of reprogramming commensal bacteria that are in symbiotic existence throughout the human body to implement antimicrobial drugs with high versatility and efficacy against pathogenic bacteria. In this review, we discuss recent advances and potentialities of engineered bacteria in providing a novel antimicrobial strategy against antibiotic resistance.

The Fungal Mycobiome and Its Interaction with Gut Bacteria in the Host.

The advent of sequencing technology has endowed us with the capacity to study microbes constituting the human commensal community that were previously non-culturable. Much of the initial works have concentrated on the bacterial flora constituting the gut microbiome, since specimens are readily accessible in health and disease. Less, however, is understood of the "silent population"-the fungal species, also known as the mycobiome. Living in symbiosis with bacteria as commensals in our body, it is perceivable that the mycobiome exerts an inadvertent influence on the microbiome. We review here the recent knowledge gained from study of the interaction between the mycobiome and microbiome in health and disease susceptibility, immunity, and consequences from antimicrobial treatment.

Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids.

Short branched-chain fatty acids (SBCFAs, C4-6) are versatile platform intermediates for the production of value-added products in the chemical industry. Currently, SBCFAs are mainly synthesized chemically, which can be costly and may cause environmental pollution. In order to develop an economical and environmentally friendly route for SBCFA production, we engineered Saccharomyces cerevisiae, a model eukaryotic microorganism of industrial significance, for the overproduction of SBCFAs. In particular, we employed a combinatorial metabolic engineering approach to optimize the native Ehrlich pathway in S. cerevisiae. First, chromosome-based combinatorial gene overexpression led to a 28.7-fold increase in the titer of SBCFAs. Second, deletion of key genes in competing pathways improved the production of SBCFAs to 387.4 mg/L, a 31.2-fold increase compared to the wild-type. Third, overexpression of the ATP-binding cassette (ABC) transporter PDR12 increased the secretion of SBCFAs. Taken together, we demonstrated that the combinatorial metabolic engineering approach used in this study effectively improved SBCFA biosynthesis in S. cerevisiae through the incorporation of a chromosome-based combinatorial gene overexpression strategy, elimination of genes in competitive pathways and overexpression of a native transporter. We envision that this strategy could also be applied to the production of other chemicals in S. cerevisiae and may be extended to other microbes for strain improvement.

Engineering Saccharomyces cerevisiae to produce odd chain-length fatty alcohols.

Fatty aldehydes and alcohols are valuable precursors used in the industrial manufacturing of a myriad of specialty products. Herein, we demonstrate the de novo production of odd chain-length fatty aldehydes and fatty alcohols in Saccharomyces cerevisiae by expressing a novel biosynthetic pathway involving cytosolic thioesterase, rice α-dioxygenase and endogenous aldehyde reductases. We attained production titers of ∼20 mg/l fatty aldehydes and ∼20 mg/l fatty alcohols in shake flask cultures after 48 and 60 h respectively without extensive fine-tuning of metabolic fluxes. In contrast to prior studies which relied on bi-functional fatty acyl-CoA reductase to produce even chain-length fatty alcohols, our biosynthetic route exploits α-oxidation reaction to produce odd chain-length fatty aldehyde intermediates without using NAD(P)H cofactor, thereby conserving cellular resource during the overall synthesis of odd chain-length fatty alcohols. The biosynthetic pathway presented in this study has the potential to enable sustainable and efficient synthesis of fatty acid-derived chemicals from processed biomass.

Genome-scale metabolic modeling and in silico analysis of lipid accumulating yeast Candida tropicalis for dicarboxylic acid production.

Recently, the bio-production of α,ω-dicarboxylic acids (DCAs) has gained significant attention, which potentially leads to the replacement of the conventional petroleum-based products. In this regard, the lipid accumulating yeast Candida tropicalis, has been recognized as a promising microbial host for DCA biosynthesis: it possess the unique ω-oxidation pathway where the terminal carbon of α-fatty acids is oxidized to form DCAs with varying chain lengths. However, despite such industrial importance, its cellular physiology and lipid accumulation capability remain largely uncharacterized. Thus, it is imperative to better understand the metabolic behavior of this lipogenic yeast, which could be achieved by a systems biological approach. To this end, herein, we reconstructed the genome-scale metabolic model of C. tropicalis, iCT646, accounting for 646 unique genes, 945 metabolic reactions, and 712 metabolites. Initially, the comparative network analysis of iCT646 with other yeasts revealed several distinctive metabolic reactions, mainly within the amino acid and lipid metabolism including the ω-oxidation pathway. Constraints-based flux analysis was, then, employed to predict the in silico growth rates of C. tropicalis which are highly consistent with the cellular phenotype observed in glucose and xylose minimal media chemostat cultures. Subsequently, the lipid accumulation capability of C. tropicalis was explored in comparison with Saccharomyces cerevisiae, indicating that the formation of "citrate pyruvate cycle" is essential to the lipid accumulation in oleaginous yeasts. The in silico flux analysis also highlighted the enhanced ability of pentose phosphate pathway as NADPH source rather than malic enzyme during lipogenesis. Finally, iCT646 was successfully utilized to highlight the key directions of C. tropicalis strain design for the whole cell biotransformation application to produce long-chain DCAs from alkanes. Biotechnol. Bioeng. 2016;113: 1993-2004. © 2016 Wiley Periodicals, Inc.

Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production.

Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae.

Development and characterization of AND-gate dynamic controllers with a modular synthetic GAL1 core promoter in Saccharomyces cerevisiae.

Expression of heterologous proteins in metabolic engineering endeavors can be detrimental to host cells due to increased usage of cellular resources. Dynamic controls, where protein expression can be triggered on-demand, are effective for the engineering and optimization of bio-catalysts towards robust cell growth and enhanced biochemical productivity. Here, we describe the development and characterization of AND-gate dynamic controllers in Saccharomyces cerevisiae which combine two dynamic control strategies, inducible promoters and sensing-regulation. These dynamic controllers were constructed based on synthetic hybrid promoters. Promoter enhancer sequences were fused to a synthetic GAL1 core promoter containing DNA binding sites for the binding of a repressor that reduced DNA affinity upon interaction with key intermediates in a biochemical pathway. As fatty acids are key intermediates for production of fatty alcohols, fatty acid esters, alkenes, and alkanes, which are advanced biofuels, we used the fatty acid responsive FadR repressor and its operator sequence to demonstrate the functionality of the dynamic controllers. We established that the synthetic GAL1 core promoter can be used as a modular promoter part for constructing synthetic hybrid promoters and conferring fatty acid inducibility. We further showed the performance of the AND-gate dynamic controllers, where two inputs (fatty acid and copper presence/phosphate starvation) were required to switch the AND-gate ON. This work provides a convenient platform for constructing AND-gate dynamic controllers, that is, promoters that combine inducible functionality with regulation of protein expression levels upon detection of key intermediates towards the engineering and optimization of bio-catalytic yeast cells.

Prodrug-conjugated tumor-seeking commensals for targeted cancer therapy.

Prodrugs have been explored as an alternative to conventional chemotherapy; however, their target specificity remains limited. The tumor microenvironment harbors a range of microorganisms that potentially serve as tumor-targeting vectors for delivering prodrugs. In this study, we harness bacteria-cancer interactions native to the tumor microbiome to achieve high target specificity for prodrug delivery. We identify an oral commensal strain of Lactobacillus plantarum with an intrinsic cancer-binding mechanism and engineer the strain to enable the surface loading of anticancer prodrugs, with nasopharyngeal carcinoma (NPC) as a model cancer. The engineered commensals show specific binding to NPC via OppA-mediated recognition of surface heparan sulfate, and the loaded prodrugs are activated by tumor-associated biosignals to release SN-38, a chemotherapy compound, near NPC. In vitro experiments demonstrate that the prodrug-loaded microbes significantly increase the potency of SN-38 against NPC cell lines, up to 10-fold. In a mouse xenograft model, intravenous injection of the engineered L. plantarum leads to bacterial colonization in NPC tumors and a 67% inhibition in tumor growth, enhancing the efficacy of SN-38 by 54%.

MFSD7c functions as a transporter of choline at the blood-brain barrier.

Mutations in the orphan transporter MFSD7c (also known as Flvcr2), are linked to Fowler syndrome. Here, we used Mfsd7c knockout (Mfsd7c-/-) mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood-brain barrier (BBB). We performed comprehensive metabolomics analysis and detected differential changes of metabolites in the brains and livers of Mfsd7c-/-embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c-/- embryos. Thus, we hypothesized that MFSD7c regulates the level of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch clamp analysis showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c was shown to be conserved in vertebrates, but not in yeasts. We demonstrated that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. We also showed that several missense mutations identified in patients exhibiting Fowler syndrome had abolished or reduced choline transport activity. Mice lacking Mfsd7c in endothelial cells of the central nervous system suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c was required for exporting choline derived from lysophosphatidylcholine in the brain. Collectively, our work identifies MFSD7c as a choline exporter at the BBB and provides a foundation for future work to reveal the disease mechanisms of Fowler syndrome.

FELICX: A robust nucleic acid detection method using flap endonuclease and CRISPR-Cas12.

Nucleic acid detection is crucial for monitoring diseases for which rapid, sensitive, and easy-to-deploy diagnostic tools are needed. CRISPR-based technologies can potentially fulfill this need for nucleic acid detection. However, their widespread use has been restricted by the requirement of a protospacer adjacent motif in the target and extensive guide RNA optimization. In this study, we developed FELICX, a technique that can overcome these limitations and provide a useful alternative to existing technologies. FELICX comprises flap endonuclease, Taq ligase and CRISPR-Cas for diagnostics (X) and can be used for detecting nucleic acids and single-nucleotide polymorphisms. This method can be deployed as a point-of-care test, as only two temperatures are needed without thermocycling for its functionality, with the result generated on lateral flow strips. As a proof-of-concept, we showed that up to 0.6 copies/µL of DNA and RNA could be detected by FELICX in 60 min and 90 min, respectively, using simulated samples. Additionally, FELICX could be used to probe any base pair, unlike other CRISPR-based technologies. Finally, we demonstrated the versatility of FELICX by employing it for virus detection in infected human cells, the identification of antibiotic-resistant bacteria, and cancer diagnostics using simulated samples. Based on its unique advantages, we envision the use of FELICX as a next-generation CRISPR-based technology in nucleic acid diagnostics.

Engineering of a probiotic yeast for the production and secretion of medium-chain fatty acids antagonistic to an opportunistic pathogen Candida albicans.

Candida albicans is an opportunistic pathogen, with its infection as one of the causes of morbidity or mortality. Notably, the probiotic yeast Saccharomyces cerevisiae var. boulardii has shown the potential to fight against Candida infections. In this study, we aimed to engineer a commercial boulardii strain to produce medium-chain fatty acids (MCFAs) with antagonistic effects against C. albicans. First, we identified and characterized a boulardii strain and created its auxotrophic strain Δura3. Next, we constructed and expressed a heterologous MCFA biosynthetic pathway under the control of inducible and constitutive promoters. Aside from examining MCFA production and secretion, we confirmed MCFAs' effects on C. albicans' anti-biofilm and anti-hyphal formations and the immunomodulatory effect of MCFA-containing supernatants on Caco-2 cells. We found that under constitutive promoters, the engineered boulardii strain constitutively produced and secreted a mixture of C6:0, C8:0, and C10:0. The secreted MCFAs then reduced biofilm and hyphal formations in C. albicans SC5314. We also confirmed that MCFAs upregulated the expression of virulence-related genes in SC5314. Furthermore, we found that the constitutively produced MCFAs in the supernatant induced the upregulation of immune response genes in Caco-2 cells co-cultured with SC5314, indicating MCFAs' roles in immunomodulation. Overall, the engineered boulardii strain produced and secreted MCFAs, as well as demonstrated antagonistic effects against C. albicans SC5314 and immune-modulatory effects in Caco-2. To our knowledge, this represents the first study tackling the metabolic engineering of a commercial probiotic yeast strain to constitutively produce and secrete MCFAs showing anti-Candida effects. Our study forms the basis of the potential development of a live biotherapeutics probiotic yeast against Candida infections through metabolic engineering strategies.