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.

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.

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.

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.

Establishing chromosomal design-build-test-learn through a synthetic chromosome and its combinatorial reconfiguration.

Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic Saccharomyces cerevisiae chromosome XV, synXV. We designed and built synXV to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged synXV as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between synXV and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics in vivo. Overall, we established synXV as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains.

Transporter-Driven Engineering of a Genetic Biosensor for the Detection and Production of Short-Branched Chain Fatty Acids in Saccharomyces cerevisiae.

Biosensors can be used for real-time monitoring of metabolites and high-throughput screening of producer strains. Use of biosensors has facilitated strain engineering to efficiently produce value-added compounds. Following our recent work on the production of short branched-chain fatty acids (SBCFAs) in engineered Saccharomyces cerevisiae, here we harnessed a weak organic acid transporter Pdr12p, engineered a whole-cell biosensor to detect exogenous and intracellular SBCFAs and optimized the biosensor's performance by varying PDR12 expression. We firstly constructed the biosensor and evaluated its response to a range of short-chain carboxylic acids. Next, we optimized its sensitivity and operational range by deletion and overexpression of PDR12. We found that the biosensor responded to exogenous SBCFAs including isovaleric acid, isobutyric acid and 2-methylbutanoic acid. PDR12 deletion enhanced the biosensor's sensitivity to isovaleric acid at a low concentration and PDR12 overexpression shifted the operational range towards a higher concentration. Lastly, the deletion of PDR12 improved the biosensor's sensitivity to the SBCFAs produced in our previously engineered SBCFA-overproducing strain. To our knowledge, our work represents the first study on employing an ATP-binding-cassette transporter to engineer a transcription-factor-based genetic biosensor for sensing SBCFAs in S. cerevisiae. Our findings provide useful insights into SBCFA detection by a genetic biosensor that will facilitate the screening of SBCFA-overproducing strains.

Improving a natural enzyme activity through incorporation of unnatural amino acids.

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

Mutation of outer-shell residues modulates metal ion co-ordination strength in a metalloenzyme.

The metal ion co-ordination sites of many metalloproteins have been characterized by a variety of spectroscopic techniques and small-molecule model systems, revealing many important insights into the structural determinants of metal ion co-ordination. However, our understanding of this fundamentally and practically important phenomenon remains frustratingly simplistic; in many proteins it is essentially impossible to predict metal ion specificity and the effects of remote 'outer-shell' residues on metal ion co-ordination strength are also poorly defined. This is exemplified by our inability to explain why metalloenzymes with identical metal ion co-ordination spheres, such as the closely related orthologues of bacterial PTE (phosphotriesterase) from Agrobacterium radiobacter and Pseudomonas diminuta, display different metal ion specificity and co-ordination strength. In the present study, we present a series of PTE variants that all possess identical metal ion co-ordination spheres, yet display large differences in their metal ion co-ordination strength. Using measurement of the rates of metal ion dissociation from the active site alongside analysis of structural data obtained through X-ray crystallography, we show that 'outer-shell' residues provide essential support for the metal ion ligands, in effect buttressing them in their optimal orientation. Remote mutations appear to modulate metal ion interactions by increasing or decreasing the stabilizing effects of these networks. The present study therefore provides a description of how the greater protein fold can be modified to 'tune' the strength of metal ion co-ordination and metal ion specificity, as well as reinforcing the concept of proteins as ensembles of conformational states with unique structures and biochemical properties.

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study.

BACKGROUND: In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. RESULTS: In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1-30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica. CONCLUSION: We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

Simultaneous Improvement of Limonene Production and Tolerance in Yarrowia lipolytica through Tolerance Engineering and Evolutionary Engineering.

Limonene is an important plant natural product widely used in food and cosmetics production as well as in the pharmaceutical and chemical industries. However, low efficiency of plant extraction and high energy consumption in chemical synthesis limit the sustainability of industrial limonene production. Recently, the advancement of metabolic engineering and synthetic biology has facilitated the engineering of microbes into microbial cell factories for producing limonene. However, the deleterious effects on cellular activity by the toxicity of limonene is the major obstacle in achieving high-titer production of limonene in engineered microbes. In this study, by using transcriptomics, we identified 82 genes from the nonconventional yeast Yarrowia lipolytica that were up-regulated when exposed to limonene. When overexpressed, 8 of the gene candidates improved tolerance of this yeast to exogenously added limonene. To determine whether overexpression of these genes could also improve limonene production, we individually coexpressed the tolerance-enhancing genes with a limonene synthase gene. Indeed, expression of 5 of the 8 candidate genes enhanced limonene production in Y. lipolytica. Particularly, overexpressing YALI0F19492p led to an 8-fold improvement in product titer. Furthermore, through short-term adaptive laboratory evolution strategy, in combination with morphological and cytoplasmic membrane integrity analysis, we shed light on the underlying mechanism of limonene cytotoxicity to Y. lipolytica. This study demonstrated an effective strategy for improving limonene tolerance of Y. lipolytica and limonene titer in the host strain through the combinatorial use of tolerance engineering and evolutionary engineering.

Metabolic engineering of microbes for monoterpenoid production.

Monoterpenoids are an important class of natural products that are derived from the condensation of two five­carbon isoprene subunits. They are widely used for flavouring, fragrances, colourants, cosmetics, fuels, chemicals, and pharmaceuticals in various industries. They can also serve as precursors for the production of many industrially important products. Currently, monoterpenoids are produced predominantly through extraction from plant sources. However, the small quantity of monoterpenoids in nature renders this method of isolation non-economically viable. Similarly impractical is the chemical synthesis of these compounds as they suffer from high energy consumption and pollutant discharge. Microbial biosynthesis, however, exists as a potential solution to these hindrances, but the transformation of cells into efficient factories remains a major impediment. Here, we critically review the recent advances in engineering microbes for monoterpenoid production, with an emphasis on categorized strategies, and discuss the challenges and perspectives to offer guidance for future engineering.

Mechanism-Driven Metabolic Engineering for Bio-Based Production of Free R-Lipoic Acid in Saccharomyces cerevisiae Mitochondria.

Lipoic acid is a valuable organosulfur compound used as an antioxidant for dietary supplementation, and potentially anti-diabetic and anti-cancer. Currently, lipoic acid is obtained mainly through chemical synthesis, which requires toxic reagents and organic solvents, thus causing environmental issues. Moreover, chemically synthesized lipoic acid is conventionally a racemic mixture. To obtain enantiomerically pure R-lipoic acid, which has superior bioactivity than the S form, chiral resolution and asymmetric synthesis methods require additional reagents and solvents, and often lead to wastage of S-lipoic acid or precursors with undesired chirality. Toward sustainable production of R-lipoic acid, we aim to develop a synthetic biology-based method using engineered yeast. Here, we deepened mechanistic understanding of lipoic acid biosynthesis and protein lipoylation in the model yeast Saccharomyces cerevisiae to facilitate metabolic engineering of the microbe for producing free R-lipoic acid. In brief, we studied the biosynthesis and confirmed the availability of protein-bound lipoate in yeast cells through LC-MS/MS. We then characterized in vitro the activity of a lipoamidase from Enterococcus faecalis for releasing free R-lipoic acid from lipoate-modified yeast proteins. Overexpression of the lipoamidase in yeast mitochondria enabled de novo free R-lipoic acid production in vivo. By overexpressing pathway enzymes and regenerating the cofactor, the production titer was increased ∼2.9-fold. This study represents the first report of free R-lipoic acid biosynthesis in S. cerevisiae. We envision that these results could provide insights into lipoic acid biosynthesis in eukaryotic cells and drive development of sustainable R-lipoic acid production.

Next-generation metabolic engineering of non-conventional microbial cell factories for carboxylic acid platform chemicals.

Carboxylic acids contain carboxyl groups that can undergo a wide range of chemical transformation. Therefore, they serve as key platform chemicals for the production of high value-added industrial products. Currently, the majority of carboxylic acid platform chemicals is produced predominantly through traditional chemical synthesis. However, these chemical conversion processes are heavily dependent on fossil fuels and often lead to serious environmental pollution. Recently, the rapid development in metabolic engineering of microbes provide a new and promising alternative route for producing carboxylic acids as platform chemicals. We envision that these bio-based manufacturing processes using microbial cell factories will help move the industrial production of carboxylic acid platform chemicals towards a more sustainable, environmentally friendly and economically competitive direction. While Escherichia coli and Saccharomyces cerevisiae have been the workhorses for biochemical production through metabolic engineering, non-conventional microbes are emerging as suitable hosts for producing carboxylic acids to meet the needs of the industries. Here, we review the employment of metabolic engineering strategies on non-conventional microbes to serve as microbial cell factories for the production of industrially important carboxylic acid platform chemicals.

Engineering an Alcohol-Forming Fatty Acyl-CoA Reductase for Aldehyde and Hydrocarbon Biosynthesis in Saccharomyces cerevisiae.

Aldehydes are a class of highly versatile chemicals that can undergo a wide range of chemical reactions and are in high demand as starting materials for chemical manufacturing. Biologically, fatty aldehydes can be produced from fatty acyl-CoA by the action of fatty acyl-CoA reductases. The aldehydes produced can be further converted enzymatically to other valuable derivatives. Thus, metabolic engineering of microorganisms for biosynthesizing aldehydes and their derivatives could provide an economical and sustainable platform for key aldehyde precursor production and subsequent conversion to various value-added chemicals. Saccharomyces cerevisiae is an excellent host for this purpose because it is a robust organism that has been used extensively for industrial biochemical production. However, fatty acyl-CoA-dependent aldehyde-forming enzymes expressed in S. cerevisiae thus far have extremely low activities, hence limiting direct utilization of fatty acyl-CoA as substrate for aldehyde biosynthesis. Toward overcoming this challenge, we successfully engineered an alcohol-forming fatty acyl-CoA reductase for aldehyde production through rational design. We further improved aldehyde production through strain engineering by deleting competing pathways and increasing substrate availability. Subsequently, we demonstrated alkane and alkene production as one of the many possible applications of the aldehyde-producing strain. Overall, by protein engineering of a fatty acyl-CoA reductase to alter its activity and metabolic engineering of S. cerevisiae, we generated strains with the highest reported cytosolic aliphatic aldehyde and alkane/alkene production to date in S. cerevisiae from fatty acyl-CoA.

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria.

Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.

Synthetic genetic circuits for programmable biological functionalities.

Living organisms evolve complex genetic networks to interact with the environment. Due to the rapid development of synthetic biology, various modularized genetic parts and units have been identified from these networks. They have been employed to construct synthetic genetic circuits, including toggle switches, oscillators, feedback loops and Boolean logic gates. Building on these circuits, complex genetic machines with capabilities in programmable decision-making could be created. Consequently, these accomplishments have led to novel applications, such as dynamic and autonomous modulation of metabolic networks, directed evolution of biological units, remote and targeted diagnostics and therapies, as well as biological containment methods to prevent release of engineered microorganisms and genetic materials. Herein, we outline the principles in genetic circuit design that have initiated a new chapter in transforming concepts to realistic applications. The features of modularized building blocks and circuit architecture that facilitate realization of circuits for a variety of novel applications are discussed. Furthermore, recent advances and challenges in employing genetic circuits to impart microorganisms with distinct and programmable functionalities are highlighted. We envision that this review gives new insights into the design of synthetic genetic circuits and offers a guideline for the implementation of different circuits in various aspects of biotechnology and bioengineering.

Rewriting the Metabolic Blueprint: Advances in Pathway Diversification in Microorganisms.

Living organisms have evolved over millions of years to fine tune their metabolism to create efficient pathways for producing metabolites necessary for their survival. Advancement in the field of synthetic biology has enabled the exploitation of these metabolic pathways for the production of desired compounds by creating microbial cell factories through metabolic engineering, thus providing sustainable routes to obtain value-added chemicals. Following the past success in metabolic engineering, there is increasing interest in diversifying natural metabolic pathways to construct non-natural biosynthesis routes, thereby creating possibilities for producing novel valuable compounds that are non-natural or without elucidated biosynthesis pathways. Thus, the range of chemicals that can be produced by biological systems can be expanded to meet the demands of industries for compounds such as plastic precursors and new antibiotics, most of which can only be obtained through chemical synthesis currently. Herein, we review and discuss novel strategies that have been developed to rewrite natural metabolic blueprints in a bid to broaden the chemical repertoire achievable in microorganisms. This review aims to provide insights on recent approaches taken to open new avenues for achieving biochemical production that are beyond currently available inventions.

Synthetic biology toolkits and applications in Saccharomyces cerevisiae.

Synthetic biologists construct biological components and systems to look into biological phenomena and drive a myriad of practical applications that aim to tackle current global challenges in energy, healthcare and the environment. While most tools have been established in bacteria, particularly Escherichia coli, recent years have seen parallel developments in the model yeast strain Saccharomyces cerevisiae, one of the most well-understood eukaryotic biological system. Here, we outline the latest advances in yeast synthetic biology tools based on a framework of abstraction hierarchies of parts, circuits and genomes. In brief, the creation and characterization of biological parts are explored at the transcriptional, translational and post-translational levels. Using characterized parts as building block units, the designing of functional circuits is elaborated with examples. In addition, the status and potential applications of synthetic genomes as a genome level platform for biological system construction are also discussed. In addition to the development of a toolkit, we describe how those tools have been applied in the areas of drug production and screening, study of disease mechanisms, pollutant sensing and bioremediation. Finally, we provide a future outlook of yeast as a workhorse of eukaryotic genetics and a chosen chassis in this field.