CRISPR/Cas9 system has accelerated research across many fields since its demonstration for genome editing. CRISPR also offers vast therapeutic potential, but an important hurdle of this technology is the off-target mutations it can induce. In this viewpoint, we will discuss recent strategies for improving CRISPR specificity, emphasizing how a complete mechanistic understanding of CRISPR/Cas9 can benefit such efforts. We also propose that agreeing upon a consensus protocol with the highest specificity could benefit researchers working on CRISPR-based therapies. In addition to improving CRISPR/Cas9 specificity, accurate detection of off-target events is also crucial, and we will discuss various unbiased off-target detection methods in terms of their advantages and disadvantages. We suggest that using a combination of cell-based and cell-free methods can prove more useful. In addition, we point out that improving predictive algorithms for off-target sites would require pooling of the available off-target analysis data and standardization of the protocols used for obtaining the data. Moreover, we highlight the risk of insertional mutagenesis for gene correction applications requiring the use of donor DNA. We conclude by discussing future prospects for the field, as well as steps that can be taken to overcome the aforementioned challenges.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS) imaging has been used for rapid phenotyping of enzymatic activities, but is mainly limited to single-step conversions. Herein we report a label-free method for high-throughput engineering of multistep biochemical reactions based on optically guided MALDI-ToF MS analysis of bacterial colonies. The bacterial cells provide containment of multiple enzymes and access to substrates and cofactors via metabolism. Automated MALDI-ToF MS acquisition from randomly distributed colonies simplifies procedures to prepare strain libraries without liquid handling. MALDI-ToF MS profiling was utilized to screen both substrate and enzyme libraries for natural product biosynthesis. Computational algorithms were developed to process and visualize the resulting mass spectral data sets. For analogues of the peptidic antibiotic plantazolicin, multivariate analyses by t-distributed stochastic neighbor embedding were used to group similar spectra for rapid identification of nonisobaric variants. After MALDI-ToF MS screening, follow-up analyses using high-resolution MS and tandem MS were readily performed on the same sample target. Separately, relative ion intensities of rhamnolipid congeners with various lipid moieties were evaluated to engineer enzymatic specificity. The glycolipid profiles of each colony were overlaid with optical images to facilitate the recovery of desirable mutants. For both the antibiotic and rhamnolipid cases, large populations of colonies were rapidly surveyed at the molecular level, providing information-rich insights not easily obtained with traditional screening assays. Utilizing standard microbiological techniques with routine microscopy and MALDI-ToF MS instruments, this simple yet effective workflow is applicable for a wide range of screening campaigns targeting multistep enzymatic reactions.

Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research. However, they have limited sequence specificity and availability. Here we report a Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. Short DNA guides are used to direct PfAgo to target sites for cleavage at high temperatures (>87 °C) followed by reannealing of the cleaved single stranded DNAs. We used this platform to generate over 18 AREs for DNA fingerprinting and molecular cloning of PCR-amplified or genomic DNAs. These AREs work as efficiently as their naturally occurring counterparts, and some of them even do not have any naturally occurring counterparts, demonstrating easy programmability, generality, versatility, and high efficiency for this new technology.Keywords: DNA cloning; DNA profiling; recombinant DNA technology; restriction enzymes;

Eukaryotic transcriptional factors (TFs) typically recognize short genomic sequences alone or together with other proteins to modulate gene expression. Mapping of TF-DNA interactions in the genome is crucial for understanding the gene regulatory programs in cells. While chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is commonly used for this purpose, its application is severely limited by the availability of suitable antibodies for TFs. To overcome this limitation, we developed an efficient and scalable strategy named cmChIP-Seq that combines the clustered regularly interspaced short palindromic repeats (CRISPR) technology with microhomology mediated end joining (MMEJ) to genetically engineer a TF with an epitope tag. We demonstrated the utility of this tool by applying it to four TFs in a human colorectal cancer cell line. The highly scalable procedure makes this strategy ideal for ChIP-Seq analysis of TFs in diverse species and cell types.Keywords: ChIP-Seq; CRISPR/Cas9; FLAG tagging; genome engineering; microhomology mediated end joining;

Localization of mRNA is important in a number of cellular processes such as embryogenesis, cellular motility, polarity, and a variety of neurological processes. A synthetic device that controls cellular mRNA localization would facilitate investigations on the significance of mRNA localization in cellular function and allow an additional level of controlling gene expression. In this work, we developed the PUF (Pumilio and FBF homology domain)-assisted localization of RNA (PULR) system, which utilizes a eukaryotic cell’s cytoskeletal transport machinery to reposition mRNA within a cell. Depending on the cellular motor used, we show ligand-dependent transport of mRNA toward either pole of the microtubular network of cultured cells. In addition, implementation of the reprogrammable PUF domain allowed the transport of untagged endogenous mRNA in primary neurons.Keywords: dynein; kinesin; mRNA transport; Pumilio and fem3 mRNA-binding factor (PUF); RNA-binding proteins (RBP);

The concerted action of multiple genes in a time-dependent manner controls complex cellular phenotypes, yet the temporal regulation of gene expressions is restricted on a single-gene level, which limits our ability to control higher-order gene networks and understand the consequences of multiplex genetic perturbations. Here we developed a system for temporal regulation of multiple genes. This system combines the simplicity of CRISPR/Cas9 activators for orthogonal targeting of multiple genes and the orthogonality of chemically induced dimerizing (CID) proteins for temporal control of CRISPR/Cas9 activator function. In human cells, these transcription activators exerted simultaneous activation of multiple genes and orthogonal regulation of different genes in a ligand-dependent manner with minimal background. We envision that our system will enable the perturbation of higher-order gene networks with high temporal resolution and accelerate our understanding of gene–gene interactions in a complex biological setting.Keywords: chemically induced dimerization; CRISPR/Cas9; genetic regulation; orthogonality; synthetic transcription factor;

Genome integration is a powerful tool in both basic and applied biological research. However, traditional genome integration, which is typically mediated by homologous recombination, has been constrained by low efficiencies and limited host range. In recent years, the emergence of homing endonucleases and programmable nucleases has greatly enhanced integration efficiencies and allowed alternative integration mechanisms such as nonhomologous end joining and microhomology-mediated end joining, enabling integration in hosts deficient in homologous recombination. In this review, we will highlight recent advances and breakthroughs in genome integration methods made possible by programmable nucleases, and their new applications in synthetic biology and metabolic engineering.Keywords: genome integration; metabolic engineering; programmable nucleases; synthetic biology;

The activation of silent natural product gene clusters is a synthetic biology problem of great interest. As the rate at which gene clusters are identified outpaces the discovery rate of new molecules, this unknown chemical space is rapidly growing, as too are the rewards for developing technologies to exploit it. One class of natural products that has been underrepresented is phosphonic acids, which have important medical and agricultural uses. Hundreds of phosphonic acid biosynthetic gene clusters have been identified encoding for unknown molecules. Although methods exist to elicit secondary metabolite gene clusters in native hosts, they require the strain to be amenable to genetic manipulation. One method to circumvent this is pathway refactoring, which we implemented in an effort to discover new phosphonic acids from a gene cluster from Streptomyces sp. strain NRRL F-525. By reengineering this cluster for expression in the production host Streptomyces lividans, utility of refactoring is demonstrated with the isolation of a novel phosphonic acid, O-phosphonoacetic acid serine, and the characterization of its biosynthesis. In addition, a new biosynthetic branch point is identified with a phosphonoacetaldehyde dehydrogenase, which was used to identify additional phosphonic acid gene clusters that share phosphonoacetic acid as an intermediate.Keywords: natural products; pathway refactoring; phosphonic acids; phosphonoacetic acid;

•Most BGCs are silent due to their tight regulation under conventional laboratory culture conditions.•New methods have been developed to activate silent BGCs in both native hosts and heterologous hosts.•Computational tools have been developed for high-throughput identification of BGCs and natural products.Natural products have been a prolific source of antibacterial and anticancer drugs for decades. One of the major challenges in natural product discovery is that the vast majority of natural product biosynthetic gene clusters (BGCs) have not been characterized, partially due to the fact that they are either transcriptionally silent or expressed at very low levels under standard laboratory conditions. Here we describe the strategies developed in recent years (mostly between 2014–2016) for activating silent BGCs. These strategies can be broadly divided into two categories: approaches in native hosts and approaches in heterologous hosts. In addition, we briefly discuss recent advances in developing new computational tools for identification and characterization of BGCs and high-throughput methods for detection of natural products.

•Various Design tools at parts-, pathway-, and network-levels and for DNA assembly have been developed.•Both bottom-up and top-down automated strategies are used for the Build step.•Test automation should be implemented over a range of system scales including genetic constructs and genome, transcriptome, proteome, and metabolome.•Learning through DBT cycles is crucial for both fundamental science and practical applications.•As more enabling tools emerge for Design, Build, and Test, it has become important to integrate these steps to form industrialized pipelines.Engineered biological systems such as genetic circuits and microbial cell factories have promised to solve many challenges in the modern society. However, the artisanal processes of research and development are slow, expensive, and inconsistent, representing a major obstacle in biotechnology and bioengineering. In recent years, biological foundries or biofoundries have been developed to automate design-build-test engineering cycles in an effort to accelerate these processes. This review summarizes the enabling technologies for such biofoundries as well as their early successes and remaining challenges.

•A universal method was adopted to effectively identify biosensors for any metabolite.•Transcriptomic changes were analysed when S. cerevisiae was exposed to alcohols.•Promoters were identified that responded specifically to 1-butanol.•The promoter-based biosensors could distinguish varied 1-butanol production levels.The present study aimed to develop a universal methodology for the discovery of biosensors sensitive to particular stresses or metabolites by using a transcriptome analysis, in order to address the need for in vivo biosensors to drive the engineering of microbial cell factories. The method was successfully applied to the discovery of 1-butanol sensors. In particular, the genome-wide transcriptome profiling of S. cerevisiae exposed to three similar short-chain alcohols, 1-butanol, 1-propanol, and ethanol, identified genes that were differentially expressed only under the treatment of 1-butanol. From these candidates, two promoters that responded specifically to 1-butanol were characterized in a dose-dependent manner and were used to distinguish differences in production levels among different 1-butanol producer strains. This strategy opens up new opportunities for the discovery of promoter-based biosensors and can potentially be used to identify biosensors for any metabolite that causes cellular transcriptomic changes.Download high-res image (156KB)Download full-size image

Covering up to end 2015 Microbial fermentation provides an attractive alternative to chemical synthesis for the production of structurally complex natural products. In most cases, however, production titers are low and need to be improved for compound characterization and/or commercial production. Owing to advances in functional genomics and genetic engineering technologies, microbial hosts can be engineered to overproduce a desired natural product, greatly accelerating the traditionally time-consuming strain improvement process. This review covers recent developments and challenges in the engineering of native and heterologous microbial hosts for the production of bacterial natural products, focusing on the genetic tools and strategies for strain improvement. Special emphasis is placed on bioactive secondary metabolites from actinomycetes. The considerations for the choice of host systems will also be discussed in this review.

Covering: 2010 to 2015 Natural product scaffolds remain a major source and inspiration for human therapeutics. However, generation of a natural product in the post-genomic era often requires reconstruction of the corresponding biosynthetic gene cluster in a heterologous host. In the burgeoning fields of synthetic biology and metabolic engineering, a significant amount of efforts has been devoted to develop DNA assembly techniques with higher efficiency, fidelity, and modularity, and heterologous expression systems with higher productivity and yield. Here we describe recent advances in DNA assembly and host engineering and highlight their applications in natural product discovery and engineering.

Synthetic pathways and circuits have been increasingly used for microbial production of fuels and chemicals. Here, we report a flexible and versatile DNA assembly strategy that allows rapid, modular, and reliable construction of biological pathways and circuits from basic genetic parts. This strategy combines the automation-friendly ligase cycling reaction (LCR) method and the high-fidelity in vivo yeast-based DNA assembly method, DNA assembler. Briefly, LCR is used to preassemble basic genetic parts into gene expression cassettes or to preassemble small parts into larger parts to reduce the number of parts, in which many basic genetic parts can be reused. With the help of specially designed unique linkers, all preassembled parts will then be directly assembled using DNA assembler to build the target constructs. As proof of concept, three plasmids with varying sizes of 13.4, 24, and 44 kb were rapidly constructed with fidelities of 100, 88, and 71%, respectively. The yeast strain harboring the constructed 44 kb plasmid was confirmed to be capable of utilizing xylose, cellobiose, and glucose to produce zeaxanthin. This strategy should be generally applicable to any custom-designed pathways, circuits, or plasmids.Keywords: DNA assembler; ligase cycling reaction; pathway engineering; synthetic biology; yeast homologous recombination;

Acetyl-CoA is a key precursor for the biosynthesis of a wide range of fuels, chemicals, and value-added compounds, whose biosynthesis in Saccharomyces cerevisiae involves acetyl–CoA synthetase (ACS) and is energy intensive. Previous studies have demonstrated that functional expression of a pyruvate dehydrogenase (PDH) could fully replace the endogenous ACS-dependent pathway for cytosolic acetyl-CoA biosynthesis in an ATP-independent manner. However, the requirement for lipoic acid (LA) supplementation hinders its wide industrial applications. In the present study, we focus on the engineering of a de novo synthetic lipoylation machinery for reconstitution of a functional PDH in the cytosol of yeast. First, a LA auxotrophic yeast strain was constructed through the expression of the Escherichia coli PDH structural genes and a lipoate–protein ligase gene in an ACS deficient (acs1Δ acs2Δ) strain, based on which an in vivo acetyl-CoA reporter was developed for following studies. Then the de novo lipoylation pathway was reconstituted in the cytosol of yeast by coexpressing the yeast mitochondrial lipoylation machinery genes and the E. coli type II fatty acid synthase (FAS) genes. Alternatively, an unnatural de novo synthetic lipoylation pathway was constructed by combining the reversed β-oxidation pathway with an acyl-ACP synthetase gene. To the best of our knowledge, reconstitution of natural and unnatural de novo synthetic lipoylation pathways for functional expression of a PDH in the cytosol of yeast has never been reported. Our study has laid a solid foundation for the construction and further optimization of acetyl-CoA overproducing yeast strains.Keywords: acetyl-CoA; protein lipoylation; pyruvate dehydrogenase; synthetic biology; yeast

Colony biofilms of Bacillus subtilis are a widely used model for studying cellular differentiation. Here, we applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to examine cellular and molecular heterogeneity in B. subtilis colony biofilms. From B. subtilis cells cultivated on a biofilm-promoting medium, we detected two cannibalistic factors not found in previous MALDI MSI studies of the same strain under different culturing conditions. Given the importance of cannibalism in matrix formation of B. subtilis biofilms, we employed a transcriptional reporter to monitor matrix-producing cell subpopulations using fluorescence imaging. These two complementary imaging approaches were used to characterize three B. subtilis strains, the wild type isolate NCIB3610, and two mutants, Δspo0A and ΔabrB, with defective and enhanced biofilm phenotypes, respectively. Upon deletion of key transcriptional factors, correlated changes were observed in biofilm morphology, signaling, cannibalistic factor distribution, and matrix-related gene expression, providing new insights on cannibalism in biofilm development. This work underscores the advantages of using multimodal imaging to compare spatial patterns of selected molecules with the associated protein expression patterns, obtaining information on cellular heterogeneity and function not obtainable when using a single method to characterize biofilm formation.

We report the development of a tandem chemoenzymatic transformation that combines alkene metathesis with enzymatic epoxidation to provide aryl epoxides. The development of this one-pot reaction required substantial protein and reaction engineering to improve both selectivity and catalytic activity. Ultimately, this reaction converts a mixture of alkenes into a single epoxide product in high enantioselectivity and moderate yields and illustrates both the challenges and benefits of tandem catalysis combining organometallic and enzymatic systems.Keywords: biocatalysis; biocatalysis; chemo-enzymatic catalysis; cytochrome P450; olefin metathesis; organometallic catalysis; tandem catalysis

We report here the enzymatic biosynthesis of FR-900098 analogues and establish an in vivo platform for the biosynthesis of an N-propionyl derivative FR-900098P. FR-900098P is found to be a significantly more potent inhibitor of Plasmodium falciparum 1-deoxy-D-xylulose 5-phosphate reductoisomerase (PfDxr) than the parent compound, and thus a more promising antimalarial drug candidate.

Functionally reversing the β-oxidation cycle represents an efficient and versatile strategy for synthesis of a wide variety of fuels and chemicals. However, due to the compartmentalization of cellular metabolisms, reversing the β-oxidation cycle in eukaryotic systems remains elusive. Here, we report the first successful reversal of the β-oxidation cycle in Saccharomyces cerevisiae, an important cell factory for large-scale production of fuels and chemicals. After extensive gene cloning and enzyme activity assays, a reversed β-oxidation pathway was functionally constructed in the yeast cytosol, which led to the synthesis of n-butanol, medium-chain fatty acids (MCFAs), and medium-chain fatty acid ethyl esters (MCFAEEs). The resultant recombinant strain provides a new broadly applicable platform for synthesis of fuels and chemicals in S. cerevisiae.Keywords: advanced biofuels; fatty acid; synthetic biology; yeast; β-oxidation

A fundamental challenge in basic and applied biology is to reprogram cells with improved or novel traits on a genomic scale. However, the current ability to reprogram a cell on the genome scale is limited to bacterial cells. Here, we report RNA interference (RNAi)-assisted genome evolution (RAGE) as a generally applicable method for genome-scale engineering in the yeast Saccharomyces cerevisiae. Through iterative cycles of creating a library of RNAi induced reduction-of-function mutants coupled with high throughput screening or selection, RAGE can continuously improve target trait(s) by accumulating multiplex beneficial genetic modifications in an evolving yeast genome. To validate the RNAi library constructed with yeast genomic DNA and convergent-promoter expression cassette, we demonstrated RNAi screening in Saccharomyces cerevisiae for the first time by identifying two known and three novel suppressors of a telomerase-deficient mutation yku70Δ. We then showed the application of RAGE for improved acetic acid tolerance, a key trait for microbial production of chemicals and fuels. Three rounds of iterative RNAi screening led to the identification of three gene knockdown targets that acted synergistically to confer an engineered yeast strain with substantially improved acetic acid tolerance. RAGE should greatly accelerate the design and evolution of organisms with desired traits and provide new insights on genome structure, function, and evolution.Keywords: acetic acid tolerance; directed evolution; RNAi screening; S. cerevisiae; telomere

One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.Keywords: CRISPR−Cas; gene knockout; genome editing; multiple gene disruption; Saccharomyces cerevisiae;

Actinobacteria, particularly those of genus Streptomyces, remain invaluable hosts for the discovery and engineering of natural products and their cognate biosynthetic pathways. However, genetic manipulation of these bacteria is often labor and time intensive. Here, we present an engineered CRISPR/Cas system for rapid multiplex genome editing of Streptomyces strains, demonstrating targeted chromosomal deletions in three different Streptomyces species and of various sizes (ranging from 20 bp to 30 kb) with efficiency ranging from 70 to 100%. The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly (or traditional digestion/ligation), respectively, allowing rapid plasmid construction to target any genomic locus of interest. As such, the pCRISPomyces system represents a powerful new tool for genome editing in Streptomyces.Keywords: Cas9; CRISPR; genome engineering; Streptomyces; synthetic guide RNA;

Fatty acid ethyl esters (FAEEs) are a form of biodiesel that can be microbially produced via a transesterification reaction of fatty acids with ethanol. The titer of microbially produced FAEEs can be greatly reduced by unbalanced metabolism and an insufficient supply of fatty acids, resulting in a commercially inviable process. Here, we report on a pathway engineering strategy in Saccharomyces cerevisiae for enhancing the titer of microbially produced FAEEs by providing the cells with an orthogonal route for fatty acid synthesis. The fatty acids generated from this heterologous pathway would supply the FAEE production, safeguarding endogenous fatty acids for cellular metabolism and growth. We investigated the heterologous expression of a Type-I fatty acid synthase (FAS) from Brevibacterium ammoniagenes coupled with WS/DGAT, the wax ester synthase/acyl-coenzyme that catalyzes the transesterification reaction with ethanol. Strains harboring the orthologous fatty acid synthesis yielded a 6.3-fold increase in FAEE titer compared to strains without the heterologous FAS. Variations in fatty acid chain length and degree of saturation can affect the quality of the biodiesel; therefore, we also investigated the diversity of the fatty acid production profile of FAS enzymes from other Actinomyces organisms.Keywords: advanced biofuels; biodiesel; fatty acid ethyl esters (FAEEs); fatty acid synthase; orthogonal fatty acid synthesis; pathway and metabolic engineering;

Actinomycetes are important organisms for the biosynthesis of valuable natural products. However, only a limited number of well-characterized native constitutive promoters from actinomycetes are available for the construction and engineering of large biochemical pathways. Here, we report the discovery and characterization of 32 candidate promoters identified from Streptomyces albus J1074 by RNA-seq analysis. These 32 promoters were cloned and characterized using a streptomycete reporter gene, xylE, encoding catechol 2,3-dioxygenase. The strengths of the identified strong promoters varied from 200 to 1300% of the strength of the well-known ermE*p in MYG medium, and the strongest of these promoters was by far the strongest actinomycete promoter ever reported in the literature. To further confirm the strengths of these promoters, qPCR was employed to determine the transcriptional levels of the xylE reporter. In total, 10 strong promoters were identified and four constitutive promoters were characterized via a time-course study. These promoters were used in a plug-and-play platform to activate a cryptic gene cluster from Streptomyces griseus, and successful activation of the target pathway was observed in three widely used Streptomyces strains. Therefore, these promoters should be highly useful in current synthetic biology platforms for activation and characterization of silent natural product biosynthetic pathways as well as the optimization of pathways for the synthesis of important natural products in actinomycetes.Keywords: actinomycetes; qPCR; RNA-seq; Streptomyces promoters; synthetic biology; XylE assay;

Genetic sensors capable of converting key metabolite levels to fluorescence signals enable the monitoring of intracellular compound concentrations in living cells, and emerge as an efficient tool in high-throughput genetic screening. However, the development of genetic sensors in yeasts lags far behind their development in bacteria. Here we report the design of a malonyl-CoA sensor in Saccharomyces cerevisiae using an adapted bacterial transcription factor FapR and its corresponding operator fapO to gauge intracellular malonyl-CoA levels. By combining this sensor with a genome-wide overexpression library, we identified two novel gene targets that improved intracellular malonyl-CoA concentration. We further utilized the resulting recombinant yeast strain to produce a valuable compound, 3-hydroxypropionic acid, from malonyl-CoA and enhanced its titer by 120%. Such a genetic sensor provides a powerful approach for genome-wide screening and could further improve the synthesis of a large range of chemicals derived from malonyl-CoA in yeast.Keywords: 3-hydroxypropionic acid; genetic sensor; genome-wide library; high-throughput screening; malonyl-CoA;

Synthetic biology is a relatively new field with the key aim of designing and constructing biological systems with novel functionalities. Today, synthetic biology devices are making their first steps in contributing new solutions to a number of biomedical challenges, such as emerging bacterial antibiotic resistance and cancer therapy. This review discusses some synthetic biology approaches and applications that were recently used in disease mechanism investigation and disease modeling, drug discovery and production, as well as vaccine development and treatment of infectious diseases, cancer, and metabolic disorders.

Successful evolutionary enzyme engineering requires a high throughput screening or selection method, which considerably increases the chance of obtaining desired properties and reduces the time and cost. In this review, a series of high throughput screening and selection methods are illustrated with significant and recent examples. These high throughput strategies are also discussed with an emphasis on compatibility with phenotypic analysis during directed enzyme evolution. Lastly, certain limitations of current methods, as well as future developments, are briefly summarized.

With the expanding interest in RNA biology, interest in artificial RNA-binding proteins (RBPs) is likewise increasing. RBPs can be designed in a modular fashion, whereby effector and RNA-binding domains are combined in chimeric proteins that exhibit both functions and can be applied for regulation of a broad range of biological processes. The elucidation of the RNA recognition code for Pumilio and fem-3 mRNA-binding factor (PUF) homology proteins allowed engineering of artificial RBPs for targeting endogenous mRNAs. In this review, we will focus on the recent advances in elucidating and reprogramming PUF domain specificity, update on several promising applications of PUF-based designer RBPs, and discuss some other domains that hold the potential to be used as the RNA-binding scaffolds for designer RBP engineering.

Microbial long chain alcohols and alkanes are renewable biofuels that could one day replace petroleum-derived fuels. Here we report a novel pathway for high efficiency production of these products in Escherichia coli strain BL21(DE3). We first identified the acyl-ACP reductase/aldehyde deformylase combinations with the highest activity in this strain. Next, we used catalase coexpression to remove toxic byproducts and increase the overall titer. Finally, by introducing the type-I fatty acid synthase from Corynebacterium ammoniagenes, we were able to bypass host regulatory mechanisms of fatty acid synthesis that have thus far hampered efforts to optimize the yield of acyl-ACP-derived products in BL21(DE3). When all these engineering strategies were combined with subsequent optimization of fermentation conditions, we were able to achieve a final titer around 100 mg L−1 long chain alcohol/alkane products including a 57 mg L−1 titer of pentadecane, the highest titer reported in E. coli BL21(DE3) to date. The expression of prokaryotic type-I fatty acid synthases offer a unique strategy to produce fatty acid-derived products in E. coli that does not rely exclusively on the endogenous type-II fatty acid synthase system.

Fatty acids or their activated forms, fatty acyl-CoAs and fatty acyl-ACPs, are important precursors to synthesize a wide variety of fuels and chemicals, including but not limited to free fatty acids (FFAs), fatty alcohols (FALs), fatty acid ethyl esters (FAEEs), and alkanes. However, Saccharomyces cerevisiae, an important cell factory, does not naturally accumulate fatty acids in large quantities. Therefore, metabolic engineering strategies were carried out to increase the glycolytic fluxes to fatty acid biosynthesis in yeast, specifically to enhance the supply of precursors, eliminate competing pathways, and bypass the host regulatory network. This review will focus on the genetic manipulation of both structural and regulatory genes in each step for fatty acids overproduction in S. cerevisiae, including from sugar to acetyl-CoA, from acetyl-CoA to malonyl-CoA, and from malonyl-CoA to fatty acyl-CoAs. The downstream pathways for the conversion of fatty acyl-CoAs to the desired products will also be discussed.

Synthetic biology is an interdisciplinary field that takes top-down approaches to understand and engineer biological systems through design-build-test cycles. A number of advances in this relatively young field have greatly accelerated such engineering cycles. Specifically, various innovative tools were developed for in silico biosystems design, DNA de novo synthesis and assembly, construct verification, as well as metabolite analysis, which have laid a solid foundation for building biological foundries for rapid prototyping of improved or novel biosystems. This review summarizes the state-of-the-art technologies for synthetic biology and discusses the challenges to establish such biological foundries.

The adenylation (A) domain acts as the first “gate-keeper” to ensure the activation and thioesterification of the correct monomer to nonribosomal peptide synthetases (NRPSs). Our understanding of the specificity-conferring code and our ability to engineer A domains are critical for increasing the chemical diversity of nonribosomal peptides (NRPs). We recently discovered a novel NRPS-like protein (ATEG_03630) that can activate 5-methyl orsellinic acid (5-MOA) and reduce it to 2,4-dihydroxy-5,6-dimethyl benzaldehyde. A NRPS-like protein is much smaller than multidomain NRPSs, but it still represents the thioesterification half-reaction, which is otherwise missed from a stand-alone A domain. Therefore, a NRPS-like protein may serve as a better model system for A domain engineering. Here, we characterize the substrate specificity of ATEG_03630 and conclude that the hydrogen-bond donor at the 4-position is crucial for substrate recognition. Next, we show that the substrate specificity of ATEG_03630 can be engineered toward our target substrate anthranilate via bioinformatics analysis and mutagenesis. The resultant mutant H358A increased its activity toward anthranilate by 10.9-fold, which led to a 26-fold improvement in specificity. Finally, we demonstrate one-pot chemoenzymatic synthesis of 4-hydroxybenzaldoxime from 4-hydroxybenzoic acid with high yield.Keywords: adenylation domain; aldehyde; NRPS-like protein; one-pot synthesis; substrate specificity engineering

Conversion of cellulosic biomass to renewable chemicals such as 5-hydroxymethylfurfural (HMF) is of high current interest. Herein, we report a rare example of one-pot synthesis of HMF from glucose by tandem catalysis. The system is composed of a thermophilic glucose isomerase enzyme for glucose isomerization to fructose and a solid acid catalyst for fructose dehydration to HMF. A base (−NH2) functionalized mesoporous silica (aminopropyl-FMS) with large pore size was deployed successfully to immobilize and protect the thermophilic glucose isomerase in organic solvents at high temperature. The combination of this catalyst with a Brønsted acid (−SO3H) functionalized mesoporous silica (propylsulfonic acid-FMS) allowed us to conduct a one-pot transformation of glucose to HMF directly in a monophasic solvent system composed of tetrahydrofuran (THF) and H2O (4:1 v/v) with 61% yield of fructose and 30% yield of HMF at temperatures >363 K in 24 h.Keywords: biocatalysis; biomass conversion; functionalized ordered mesoporous silica; one-pot reaction; tandem catalysis

Recombinant transcription activator-like effectors (TALEs) have been effectively used for genome editing and gene regulation applications. Due to their remarkable modularity, TALEs can be tailored to specifically target almost any user-defined DNA sequences. Here, we introduce fairyTALE, a liquid phase high-throughput TALE synthesis platform capable of producing TALE-nucleases, activators, and repressors that recognize DNA sequences between 14 and 31 bp. It features a highly efficient reaction scheme, a flexible functionalization platform, and fully automated robotic liquid handling that enable the production of hundreds of expression-ready TALEs within a single day with over 98% assembly efficiency at a material cost of just $5 per TALE. As proof of concept, we synthesized and tested 90 TALEs, each recognizing 27 bp, without restrictions on their sequence composition. 96% of these TALEs were found to be functional, while sequencing confirmation revealed that the nonfunctional constructs were all correctly assembled.Keywords: genome editing; genome engineering; synthetic biology; TAL effector; TALEN

•We identify a distinct mechanism for aryl-aldehyde generation in polyketides•An NRPS-like protein can activate and reduce an aryl-acid to an aryl-aldehyde•Such a distinct mechanism may be widely used throughout the fungi kingdomAryl-aldehydes are a common feature in fungal polyketides, which are considered to be exclusively generated by the R domain of nonreducing polyketide synthases (NR-PKSs). However, by cloning and heterologous expression of both cryptic NR-PKS and nonribosomal peptide synthase (NRPS)-like genes from Aspergillus terreus in Saccharomyces cerevisiae, we identified a distinct mechanism for aryl-aldehyde formation in which a NRPS-like protein activates and reduces an aryl-acid produced by the accompanying NR-PKS to an aryl-aldehyde. Bioinformatics study indicates that such a mechanism may be widely used throughout the fungi kingdom.Figure optionsDownload full-size imageDownload high-quality image (177 K)Download as PowerPoint slide

Transcription-activator like effector nucleases (TALENs) are tailor-made DNA endonucleases and serve as a powerful tool for genome engineering. Site-specific DNA cleavage can be made by the dimerization of FokI nuclease domains at custom-targeted genomic loci, where a pair of TALENs must be positioned in close proximity with an appropriate orientation. However, the simultaneous delivery and coordinated expression of two bulky TALEN monomers (>100 kDa) in cells may be problematic to implement for certain applications. Here, we report the development of a single-chain TALEN (scTALEN) architecture, in which two FokI nuclease domains are fused on a single polypeptide. The scTALEN was created by connecting two FokI nuclease domains with a 95 amino acid polypeptide linker, which was isolated from a linker library by high-throughput screening. We demonstrated that scTALENs were catalytically active as monomers in yeast and human cells. The use of this novel scTALEN architecture should reduce protein payload, simplify design and decrease production cost.

AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing ∼10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a Km of 14.7 ± 1.1 μM, a kcat of 47.5 ± 5.4 min−1 and a kcat/Km of 3.2 ± 0.4 μM−1 min−1. PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H2O2 produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the Km is the main determinant of AurF activity toward alternative substrates.

In a continuous effort to emulate the efficiency of biosynthetic pathways, considerable progress has been made in developing one-pot chemoenzymatic processes that take full advantage of the chemo-, regio-, and stereoselectivity of biocatalysts and the productivity of chemical catalysts. Over the last 20 years, research in this area has provided us with proof of concept examples in which chemical and biological transformations occur in one vessel, sequentially or concurrently. These transformations typically access products with high enantiopurity and chemical diversity. In this perspective, we present some of the most successful reports in this field.Keywords: artificial metalloenzyme; biocatalysis; chemoenzymatic; dynamic kinetic resolution; supramolecular assembly; tandem catalysis

Natural products (secondary metabolites) are a rich source of compounds with important biological activities. Eliciting pathway expression is always challenging but extremely important in natural product discovery because an individual pathway is tightly controlled through a unique regulation mechanism and hence often remains silent under the routine culturing conditions. To overcome the drawbacks of the traditional approaches that lack general applicability, we developed a simple synthetic biology approach that decouples pathway expression from complex native regulations. Briefly, the entire silent biosynthetic pathway is refactored using a plug-and-play scaffold and a set of heterologous promoters that are functional in a heterologous host under the target culturing condition. Using this strategy, we successfully awakened the silent spectinabilin pathway from Streptomyces orinoci. This strategy bypasses the traditional laborious processes to elicit pathway expression and represents a new platform for discovering novel natural products.Keywords: genome mining; natural products; pathway assembly; plug-and-play scaffold; silent pathways; synthetic biology;

Phloroglucinol synthase PhlD is a type III polyketide synthase capable of directly converting three molecules of malonyl-CoA to an industrially important chemical—phloroglucinol (1, 3, 5-trihydroxylbenzene). Although this enzymatic process provides an attractive biosynthetic route to phloroglucinol, the low productivity of PhlD limits its further practical application. Here we used protein engineering coupled with in situ product removal to improve the productivity of phoroglucinol biosynthesis in recombinant Escherichia coli. Specifically, directed evolution was used to obtain a series of thermostable PhlD mutants with the best one showing over 24-fold longer half-life of thermal inactivation than the wild-type enzyme at 37 °C. When introduced into a malonyl-CoA overproducing E. coli strain, one of the mutants showed 30 % improvement in phloroglucinol productivity compared to the wild-type enzyme in a shake-flask study and the final phloroglucinol concentration reached 2.35 g/L with 25 % of theoretical yield. A continuous product extraction strategy was designed to remove the toxic phloroglucinol product from the cell media, which further increased the titer of phloroglucinol to 3.65 g/L, which is the highest phloroglucinol titer ever reported to date.

Synthetic biology, with its goal of designing biological entities for wide-ranging purposes, remains a field of intensive research interest. However, the vast complexity of biological systems has heretofore rendered rational design prohibitively difficult. As a result, directed evolution remains a valuable tool for synthetic biology, enabling the identification of desired functionalities from large libraries of variants. This review highlights the most recent advances in the use of directed evolution in synthetic biology, focusing on new techniques and applications at the pathway and genome scale.Highlights► Recent advances in directed evolution applied to synthetic biology are reviewed. ► New strategies facilitate library creation and in vivo evolution. ► Recent applications target biosynthetic and signal transduction pathways. ► New tools enable multiplex genome evolution.

We report the mechanistic studies of a FAD:NADH reductase (PrnF) involved in arylamine oxygenation. PrnF catalyzes the reduction of FAD via a sequential ordered bi-bi mechanism with NADH as the first substrate to bind and FADH2 as the first product to be released. The residues Asp145 and His146 are proposed as catalytic acid/base residues for PrnF based on pH profile and molecular dynamics simulation studies. These studies provide the first detailed account of the mechanism of the flavin reductase involved in arylamine oxygenation.

The Fe(II) and α-ketoglutarate-dependent hydroxylase FrbJ was previously demonstrated to utilize FR-900098 synthesizing a second phosphonate FR-33289. Here we assessed its ability to hydroxylate other possible substrates, generating a library of potential antimalarial compounds. Through a series of bioassays and in vitro experiments, we identified two new antimalarials.

We report a synthetic biology strategy for rapid genetic manipulation of natural product biosynthetic pathways. Based on DNA assembler, this method synthesizes the entire expression vector containing the target biosynthetic pathway and the genetic elements required for DNA maintenance and replication in various hosts in a single-step manner through yeast homologous recombination, offering unprecedented flexibility and versatility in pathway manipulations.

Type III polyketide synthases (PKSs) are the condensing enzymes that catalyze the formation of a myriad of aromatic polyketides in plant, bacteria, and fungi. Here we report the cloning and characterization of a putative type III PKS from Aspergillusniger, AnPKS. This enzyme catalyzes the synthesis of alkyl pyrones from C2 to C18 starter CoA thioesters with malonyl-CoA as an extender CoA through decaboxylative condensation and cyclization. It displays broad substrate specificity toward fatty acyl-CoA starters to yield triketide and tetraketide pyrones, with benzoyl-CoA as the most preferred starter. The optimal temperature and pH of AnPKS are 50 °C and 8, respectively. Under optimal conditions, the enzyme shows the highest catalytic efficiency (kcat/Km) of 7.4 × 105 s−1 M−1 toward benzoyl-CoA. Homology modeling and site-directed mutagenesis were used to probe the molecular basis of its substrate specificity. This study should open doors for further engineering of AnPKS as a biocatalyst for synthesis of value-added polyketides.

Active site modeling of dimerization interface in combination with site-directed mutagenesis indicates that the electron in the PrnD Rieske oxygenase can be transferred by either of two pathways, one involving Asp183′ and the other involving Asn180′. In addition, the overexpression of the isc operon involved in the assembly of iron–sulfur clusters increased the catalytic activity of PrnD in Escherichia coli by a factor of at least 4.The electron in the PrnD Rieske oxygenase can be transferred by either of two pathways, one involving Asp183′ and the other involving Asn180′.

Microorganisms have become an increasingly important platform for the production of drugs, chemicals, and biofuels from renewable resources. Advances in protein engineering, metabolic engineering, and synthetic biology enable redesigning microbial cellular networks and fine-tuning physiological capabilities, thus generating industrially viable strains for the production of natural and unnatural value-added compounds. In this review, we describe the recent progress on engineering microbial factories for synthesis of valued-added products including alkaloids, terpenoids, flavonoids, polyketides, non-ribosomal peptides, biofuels, and chemicals. Related topics on lignocellulose degradation, sugar utilization, and microbial tolerance improvement will also be discussed.

We report the first example of directed evolution of a P450 monooxygenase with inverted enantioselectivity for asymmetric biohydroxylation. The biohydroxylation product of the best mutant 1AF4A has an ee of 83% (R) compared to the wild type's ee of 43% (S).

FR-900098 is a potent chemotherapeutic agent for the treatment of malaria. Here we report the heterologous production of this compound in Escherichia coli by reconstructing the entire biosynthetic pathway using a three-plasmid system. Based on this system, whole-cell feeding assays in combination with in vitro enzymatic activity assays reveal an unusual functional role of nucleotide conjugation and lead to the complete elucidation of the previously unassigned late biosynthetic steps. These studies also suggest a biosynthetic route to a second phosphonate antibiotic, FR-33289. A thorough understanding of the FR-900098 biosynthetic pathway now opens possibilities for metabolic engineering in E. coli to increase production of the antimalarial antibiotic and combinatorial biosynthesis to generate novel derivatives of FR-900098.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (162 K)Download as PowerPoint slide

Saccharomyces cerevisiae is considered one of the most promising organisms for ethanol production from lignocellulosic feedstock. Unfortunately, pentose sugars, which comprise up to 30% of lignocellulose, cannot be utilized by wild type S. cerevisiae. Heterologous pathways were introduced into S. cerevisiae to enable utilization of D-xylose, the most abundant pentose sugar. However, the resulting recombinant S. cerevisiae strains exhibited a slow growth rate and poor sugar utilization efficiency when grown on D-xylose as the sole carbon source. D-xylose uptake is the first step of D-xylose utilization. D-xylose can only enter yeast cells through hexose transporters, which have two orders of magnitude lower affinity towards D-xylose compared to hexoses. It was also shown that inefficient pentose uptake is the limiting step in some D-xylose metabolizing yeast strains. Here we report the cloning and characterization of two novel D-xylose-specific transporters from Neurospora crassa and Pichia stipitis. These two transporters were identified from a total of 18 putative pentose transporters. They were functionally expressed and properly localized in S. cerevisiae as indicated by HPLC analysis and fluorescence confocal microscopy, respectively. Kinetic parameters of the D-xylose-specific transporters were determined using a 14C-labeled sugar uptake assay. Use of pentose-specific transporters should improve D-xylose consumption and ethanol production in fast D-xylose assimilating strains, thereby lowering the cost of lignocellulosic ethanol production.

Glucose repression is one of the main limitations in mixed lignocellulosic sugar fermentation for cost-effective production of fuels and chemicals. Here we report a novel strategy to overcome glucose repression by co-expressing a cellobiose transporter and a β-glucosidase in an engineered D-xylose-utilizing Saccharomyces cerevisiae strain. The resulting strain can simultaneously utilize cellobiose and D-xylose for ethanol production.

Eucalyptus species synthesize a wealth of polyketide natural products, but no relevant biosynthetic enzyme has been identified. Degenerate primers designed from conserved regions of fourteen chalcone synthase superfamily enzymes were used to isolate gene fragments from at least five different Type III polyketide synthases (PKSs) in E. camaldulensis and E. robusta.

Spectinabilin is a rare nitrophenyl-substituted polyketidemetabolite. Here we report the cloning and heterologous expression of the spectinabilin gene cluster from Streptomyces spectabilis. Unexpectedly, this gene cluster is evolutionarily closer to the aureothin gene cluster than to the spectinabilin gene cluster from Streptomyces orinoci. Moreover, the two nearly identical spectinabilin gene clusters use a distinctly different regulation mechanism.

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD+ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD+-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP+ was increased by 2.5 × 104-fold, whereas the cofactor preference toward NADP+ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds.

The United States is a leading nation in the development of synthetic biology, an emerging engineering discipline to create, control and reprogram biological systems. With strategic investment from its government agencies, the U.S. has established numerous research centers and programs in synthetic biology, enabling significant advances in foundational tool development and practical applications ranging from bioenergy, biomanufacturing, to biomedicine. To maintain its leadership in synthetic biology, U.S, has conducted several roadmap studies to provide strategic visions and action recommendations. Here we will provide a brief overview of the major research programs and roadmap studies of synthetic biology in the U.S.

•Natural products are discovered through top-down and bottom-up approaches.•Diverse sampling provides diverse natural products.•Powerful analytical techniques enable robust product detection.•Sophisticated genetic manipulation unlocks silent gene clusters.•Heterologous expression bypasses native host regulation of silent genes.Natural products have been and continue to be the source and inspiration for a substantial fraction of human therapeutics. Although the pharmaceutical industry has largely turned its back on natural product discovery efforts, such efforts continue to flourish in academia with promising results. Natural products have traditionally been identified from a top-down perspective, but more recently genomics- and bioinformatics-guided bottom-up approaches have provided powerful alternative strategies. Here we review recent advances in natural product discovery from both angles, including diverse sampling and innovative culturing and screening approaches, as well as genomics-driven discovery and genetic manipulation techniques for both native and heterologous expression.Download high-res image (193KB)Download full-size image

Lignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. However, the recalcitrant nature of lignocellulose poses technical hurdles to an economically viable biorefinery. Low enzymatic hydrolysis efficiency and low productivity, yield, and titer of biofuels are among the top cost contributors. Protein engineering has been used to improve the performance of lignocellulose-degrading enzymes, as well as proteins involved in biofuel synthesis pathways. Unlike its great success seen in other industrial applications, protein engineering has achieved only modest results in improving the lignocellulose-to-biofuels efficiency. This review will discuss the unique challenges that protein engineering faces in the process of converting lignocellulose to biofuels and how they are addressed by recent advances in this field.

Synthetic biology provides numerous great opportunities for chemical engineers in the development of new processes for large-scale production of biofuels, value-added chemicals, and protein therapeutics. However, challenges across all scales abound. In particular, the modularization and standardization of the components in a biological system, so-called biological parts, remain the biggest obstacle in synthetic biology. In this perspective, we will discuss the main challenges and opportunities in the rapidly growing synthetic biology field and the important roles that chemical engineers can play in its advancement.Highlights► Main challenges and opportunities for chemical engineers in synthetic biology are discussed. ► Standardization of biological parts represents a key challenge in synthetic biology. ► Chemical engineers play a leading role in engineering cellular factories.

At the heart of synthetic biology lies the goal of rationally engineering a complete biological system to achieve a specific objective, such as bioremediation and synthesis of a valuable drug, chemical, or biofuel molecule. However, the inherent complexity of natural biological systems has heretofore precluded generalized application of this approach. Directed evolution, a process which mimics Darwinian selection on a laboratory scale, has allowed significant strides to be made in the field of synthetic biology by allowing rapid identification of desired properties from large libraries of variants. Improvement in biocatalyst activity and stability, engineering of biosynthetic pathways, tuning of functional regulatory systems and logic circuits, and development of desired complex phenotypes in industrial host organisms have all been achieved by way of directed evolution. Here, we review recent contributions of directed evolution to synthetic biology at the protein, pathway, network, and whole cell levels.

l-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of l-arabinitol into l-xylulose with concomitant NAD+ reduction. It is an essential enzyme in the development of recombinant organisms that convert l-arabinose into fuels and chemicals using the fungal l-arabinose catabolic pathway. Here we report the crystal structure of LAD from the filamentous fungus Neurospora crassa at 2.6 Å resolution. In addition, we created a number of site-directed variants of N. crassa LAD that are capable of utilizing NADP+ as cofactor, yielding the first example of LAD with an almost completely switched cofactor specificity. This work represents the first structural data on any LAD and provides a molecular basis for understanding the existing literature on the substrate specificity and cofactor specificity of this enzyme. The engineered LAD mutants with altered cofactor specificity should be useful for applications in industrial biotechnology.

•It reports characterization of a galactitol dehydrogenase (RlGDH) from Rhizobium leguminosarum.•RlGDH catalyzes the oxidation of polyvalent alcohols and polyols.•RlGDH is distinguished from other GDHs by its higher specific activity for galactitol.•RlGDH can be a good option for d-tagatose production from galactitol.Galactitol 2-dehydrogenase (GDH) belongs to the protein subfamily of short-chain dehydrogenases/reductases and can be used to produce optically pure building blocks and for the bioconversion of bioactive compounds. An NAD+-dependent GDH from Rhizobium leguminosarum bv. viciae 3841 (RlGDH) was cloned and overexpressed in Escherichia coli. The RlGDH protein was purified as an active soluble form using His-tag affinity chromatography. The molecular mass of the purified enzyme was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 114 kDa by gel filtration chromatography, suggesting that the enzyme is a homotetramer. The enzyme has an optimal pH and temperature of 9.5 and 35 °C, respectively. The purified recombinant RlGDH catalyzed the oxidation of a wide range of substrates, including polyvalent aliphatic alcohols and polyols, to the corresponding ketones and ketoses. Among various polyols, galactitol was the preferred substrate of RlGDH with a Km of 8.8 mM, kcat of 835 min−1 and a kcat/Km of 94.9 min−1 mM−1. Although GDHs have been characterized from a few other sources, RlGDH is distinguished from other GDHs by its higher specific activity for galactitol and broad substrate spectrum, making RlGDH a good choice for practical applications.

•It reports characterization of a galactitol dehydrogenase (RlGDH) from Rhizobium leguminosarum.•RlGDH catalyzes the oxidation of polyvalent alcohols and polyols.•RlGDH is distinguished from other GDHs by its higher specific activity for galactitol.•RlGDH can be a good option for d-tagatose production from galactitol.Galactitol 2-dehydrogenase (GDH) belongs to the protein subfamily of short-chain dehydrogenases/reductases and can be used to produce optically pure building blocks and for the bioconversion of bioactive compounds. An NAD+-dependent GDH from Rhizobium leguminosarum bv. viciae 3841 (RlGDH) was cloned and overexpressed in Escherichia coli. The RlGDH protein was purified as an active soluble form using His-tag affinity chromatography. The molecular mass of the purified enzyme was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 114 kDa by gel filtration chromatography, suggesting that the enzyme is a homotetramer. The enzyme has an optimal pH and temperature of 9.5 and 35 °C, respectively. The purified recombinant RlGDH catalyzed the oxidation of a wide range of substrates, including polyvalent aliphatic alcohols and polyols, to the corresponding ketones and ketoses. Among various polyols, galactitol was the preferred substrate of RlGDH with a Km of 8.8 mM, kcat of 835 min−1 and a kcat/Km of 94.9 min−1 mM−1. Although GDHs have been characterized from a few other sources, RlGDH is distinguished from other GDHs by its higher specific activity for galactitol and broad substrate spectrum, making RlGDH a good choice for practical applications.

We report here the enzymatic biosynthesis of FR-900098 analogues and establish an in vivo platform for the biosynthesis of an N-propionyl derivative FR-900098P. FR-900098P is found to be a significantly more potent inhibitor of Plasmodium falciparum 1-deoxy-D-xylulose 5-phosphate reductoisomerase (PfDxr) than the parent compound, and thus a more promising antimalarial drug candidate.

We report the first example of directed evolution of a P450 monooxygenase with inverted enantioselectivity for asymmetric biohydroxylation. The biohydroxylation product of the best mutant 1AF4A has an ee of 83% (R) compared to the wild type's ee of 43% (S).

The Fe(II) and α-ketoglutarate-dependent hydroxylase FrbJ was previously demonstrated to utilize FR-900098 synthesizing a second phosphonate FR-33289. Here we assessed its ability to hydroxylate other possible substrates, generating a library of potential antimalarial compounds. Through a series of bioassays and in vitro experiments, we identified two new antimalarials.