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Alternative nucleotides, gene editing technology and humanized model organisms are advancing our ability to manipulate the building blocks of life.

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Advancing synthetic biology across all fronts

16 Jul 2021

From expanding the genetic alphabet to uncovering antibiotics hidden in silent genes, A*STAR’s researchers are wielding the tools of synthetic biology to improve our lives in surprising ways.

In the early 20th century, pioneering molecular biologists sought to understand the intricacies of living systems by breaking them down into their most basic units—the building blocks of life that we now know as nucleic acids, proteins, lipids and carbohydrates.

Today, not only do scientists have a deep understanding of these building blocks, but with molecular biology tools like recombinant DNA, polymerase chain reaction (PCR), DNA synthesis and genome editing, they can redesign living systems to gain new or improved functions and even construct novel biological entities from scratch. Accordingly, applying engineering principles to biological systems has quickly become a full-fledged field of its own: synthetic biology.

As Nobel Prize-winning technologies like directed evolution and CRISPR/Cas9 make molecular biology techniques more powerful than ever, the synthetic biology approach and its applications are slowly permeating different aspects of society.

From using microbes to sustainably produce high-value molecules to cells engineered to fight their diseased counterparts, it’s only a matter of time before such products become widespread in our daily lives. With the growing spotlight on synthetic biology, here’s an overview of A*STAR’s ongoing efforts in this developing field.


From A to Z

One emerging tool in synthetic biology comes from a millennia-old war waged between bacteria and the viruses that infect them, known as bacteriophages or phages. To prevent bacterial enzymes from chopping up their DNA, phages have evolved to write their genomes using a chemical base called 2-aminoadenine or Z, instead of adenosine (A).

This unique version of genetic material, known as Z-DNA, was first discovered by Soviet scientists in 1977 in a phage called S-2L, which infects photosynthetic bacteria. Their experiments revealed that the phage replaced A with Z, which formed three hydrogen bonds with thymine (T) instead of the usual two.

“Because there is an extra hydrogen bond between the Z-T base pair than the canonical A-T base pair, Z-DNA is more stable at higher temperatures and more specific for sequence recognition,” shared Huimin Zhao from the Singapore Institute of Food and Biotechnology Innovation (SIFBI). “Also, because A is replaced by Z, Z-DNA is resistant to degradation by nucleases that recognize and cut specific DNA sequences containing A.”

However, four decades since this discovery, scientists still couldn’t explain how the S-2L phage synthesized its Z-containing genome. But in a recent Science publication, an international team of researchers including Zhao and his SIFBI colleagues Ee Lui Ang and Yifeng Wei reported a breakthrough: the discovery of a multi-enzyme system responsible for the synthesis of the Z-containing phage genome1.

As it turns out, these enzymes are widespread in nature, with dozens of other phages found to harbor them. In a phage known as Acinetobacter phage SH-Ab 15497, the team even verified the presence of Z-DNA.

Given its hardiness compared to conventional genetic material, Z-DNA is more than just a biological oddity. It could be the key to overcoming current limitations in emerging techniques that harness DNA’s distinctive properties. Consider DNA data storage, which offers a density of up to 1,018 bytes per mm3—far surpassing the capacity of today’s hard drives and magnetic or optical data storage systems.

Thanks to the advent of sequencing, storing data as DNA is remarkably straightforward: digital information is first encoded into DNA sequences, which are synthesized into actual DNA molecules that are stored. To read the information, the DNA molecules are sequenced and converted back into digital data. Numerous copies can be simultaneously made from a simple PCR run.

When kept away from light, humidity and extreme temperatures, DNA can last up to millennia compared to the typical decades-long lifetime of archival storage media like tape and optical disks. By using Z-DNA, DNA data storage can be made stable and long-lasting even when exposed to higher temperatures.

Another technique that could benefit from Z-DNA is DNA origami, where long, single-stranded DNA molecules fold into two- or three-dimensional shapes through crossover base pairing. Therapeutic molecules can then be readily loaded into the cavities and docking sites of DNA origami, turning the tiny nanomachines into formidable drug delivery systems.

Given Z-DNA’s stability and binding strength, applying it to DNA origami could result in better-folded nanostructures that are more resistant to enzymatic degradation. Sure enough, certain DNA origami structures have been shown to successfully cross biological barriers like mature plant cell walls.


New characters in the genetic cast

Aside from the naturally-occurring Z base, synthetic biologists are also expanding the ‘vocabulary’ of the genetic alphabet by creating ‘unnatural’ base pairs. Since Watson and Crick first delineated the structure of DNA almost 70 years ago, conventional wisdom held that the complementary binding of bases A and T, as well as cytosine (C) and guanine (G) is fundamental to the flow of genetic information.

“However, the limited letters of the genetic alphabet restrict the further improvement of biomaterials and biosystems with increased functionalities,” explained Ichiro Hirao, a Senior Group Leader at A*STAR's Institute of Bioengineering and Bioimaging (IBB). Institute of Bioengineering and Bioimaging (IBB) For instance, only 20 amino acids are possible with the current four-letter alphabet. By adding just two more letters, up to 216 different amino acids can be formed.

In 2009, Hirao and IBB Senior Research Scientist Michiko Kimoto did exactly that: creating two new genetic letters—Ds and Px—which combine to form a third, artificial base pair2. Along with collaborator Andreas Marx from the University of Konstanz in Germany, the A*STAR team determined that the tertiary structure of Ds-Px when complexed with DNA polymerase was strikingly similar to the A-T and G-C pairs3—allowing for high accuracy in DNA replication.

Beyond new amino acids, unnatural base pairs like Ds and Px can be used in myriad ways. For instance, functional groups of interest can be attached to unnatural bases, endowing nucleic acids with desired new functionalities.

The unnatural base pairs can be used as well to generate DNA aptamers—short, single - stranded nucleic acid molecules that selectively bind to targets ranging from simple inorganic molecules to large protein complexes. Introducing Ds bases to DNA aptamers also dramatically enhances their affinities4.

While they operate much like antibodies, generating aptamers is significantly easier and cheaper. Moreover, aptamers are neither immunogenic nor toxic, making them ideal candidates for diagnostic and therapeutic applications, like purifying target molecules from complex mixtures or designing biosensors, among others.

However, generating DNA aptamers with unnatural bases requires a method that can reliably determine their sequence. Accordingly, the A*STAR team has been developing a modified Sanger sequencing method for DNA containing unnatural bases5—further simplifying this in 2019 by refining conventional deep sequencing methods6.

In their 2019 update, the team used replacement PCR to replace unnatural bases with their natural counterparts, creating an expansive encyclopedia detailing natural base replacement patterns. By comparing the natural base composition in the actual and encyclopedic data, scientists could easily pinpoint the original positions of the unnatural bases.

Ultimately, their refined sequencing method paves the way for quicker and more efficient methods of generating aptamers. Since then, Hirao and his team have already used the improved method to create novel diagnostics for dengue infection, with more use cases sure to arise over the years.


Synthetic biology techniques for natural products

Despite its name, there’s more to synthetic biology than exploring unnatural bases and products. At its core, the discipline seeks to take existing systems to the next level through frontier tools like the CRISPR/Cas9 genetic scissors, which won its inventors the Nobel Prize in Chemistry last year.

At A*STAR, Yee Hwee Lim at the Institute of Chemical and Engineering Sciences (ICES), Fong Tian Wong at the Molecular Engineering Lab (MEL), Institute of Cell and Molecular Biology (IMCB), and their colleagues harness CRISPR/Cas9 to better predict, design and build pathways for synthesizing beneficial natural products.

In 2017, for instance, their team—along with SIFBI’s Zhao—unearthed a new antibiotic from a silent biosynthetic gene cluster found in Streptomyces roseosporus7. “50 percent of current commercial drugs are derivations or mimics of natural products,” said Lim. “For a long time, the discovery of new natural products has been hindered by our inability to activate silent gene clusters and produce the encoded molecules under lab conditions.”

To work around this setback, the team leveraged CRISPR/Cas9 to replace native repressed promoters with strong constitutive ones in the bacterial genome—activating the expression of the whole biosynthetic gene cluster. Notably, the gene cluster was found to encode auroramycin, which has potent activity against Gram-positive bacteria, including the notorious superbug methicillin-resistant Staphylococcus aureus, as well as antifungal properties.

More recently, in 2020, Lim and Wong’s team used CRISPR/Cas9 to accelerate the combinatorial engineering of auroramycin’s native biosynthetic pathways8. In their study, they employed different engineering strategies to target different pathways, producing 12 strains of auroromycin with variations in methylation, hydroxylation, among others. By comparing the bioactivity profiles of various analogs, the team managed to pinpoint the key to auroramycin’s antifungal activity: an additional methyl group in its 3,5-epi-lemonose outer sugar unit.

In the same vein, Fan Hao, a Senior Principal Investigator of the Bioinformatics Institute (BII) is applying his experience in computational modeling and docking to accelerate the synthesis of natural products through enzyme engineering. In collaboration with the National University of Singapore, BII is developing enhanced enzymes that catalyze the synthesis of therapeutic agents like cannabinoids, which have potential antibacterial, anti-epileptic and anti-tumor effects9.

The team does this by suggesting mutations in the enzyme active site through computational approaches. Through modeling and docking, they can then effectively screen in silico the binding of a series of chemically related substrates onto a large collection of mutated enzymes10. They are also improving their methods with the aid of machine learning models. All in all, with the help of synthetic biology techniques, researchers across A*STAR are unlocking a treasure trove of new, enhanced natural products.


Tiny factors with a big impact

Even as scientists find novel antibiotics hidden in unassuming bacteria, other microbes like Escherichia coli and the baker’s yeast Saccharomyces cerevisiae have emerged as two prominent workhorses in the field. By modifying specific synthesis pathways, researchers can transform the two microorganisms into cellular factories that generate useful products far more efficiently compared to traditional techniques.

Consider SiNOPSEE Therapeutics, an A*STAR spinoff company led by IMCB Research Director Uttam Surana that tackles unwanted blood vessel growth—a characteristic symptom associated with diseases like cancer and blindness—using small molecule drugs. Such drugs were produced based on the principles of synthetic biology, according to Surana.

First, the team developed ‘humanized’ S. cerevisiae strains by integrating human proteins and pathways into the yeast’s genome through genetic engineering. This allowed them to use the yeast as a platform to design drug screening strategies that could rapidly identify inhibitors against disease-causing proteins or pathways—in other words, potential drug candidates. This platform is now being used to develop a pipeline of novel anti-cancer compounds.

But S. cerevisiae isn’t the only yeast that’s making a mark in synthetic biology. Also at the IMCB, Research Director, Yue Wang is studying Candida albicans—a common fungal pathogen in humans that causes life-threatening infections. As multi-drug resistant Candida strains emerge worldwide—threatening to render existing antifungals obsolete—researchers like Wang are looking for novel ways to study these fungal foes.

Until recently, C. albicans was thought to be diploid, meaning that it had two sets of genes. However, its diploid nature made the fungus tricky to study within the laboratory. Producing C. albicans mutants resistant to antifungals like echinocandin, for instance, would take about 100 days on average as both copies of the target gene had to be inactivated.

To accelerate the process, Wang and his team created the first haploid strains of this pathogen—designing a one-step protocol to delete genes in haploid C. albicans strains that takes only 11 days, as well as another method to quickly construct mutant libraries of the haploid strains in a week11,12.

The latter relies on introducing genetic elements called transposons, which are capable of “jumping” around the genome to cause mutations. Since then, the team has applied the new technologies to two other Candida species, including the superbug C. auris, discovering new drug resistance and antifungal mechanisms along the way13,14.


Synthetic biology dawns at A*STAR

Given the field’s vast scope, A*STAR is now pioneering a synthetic biology program to successfully bring the various themes discussed together. Set to helm the program is none other than Shawn Hoon, Director of IMCB’s MEL, who is known for his work on biomimetic materials inspired by nature.

According to Hoon, the program aims to develop end-to-end capabilities for biomanufacturing—integrating A*STAR’s existing capabilities in synthetic biology, bioinformatics, artificial intelligence and automation for biosystems design across multiple levels. “The resulting platform will be based on the design-build-test-learn cycle, and aspires to be a fully automated, scalable and high-throughput platform, that goes from discovery to engineering to production,” he explained.

Armed with this platform, the synthetic biology program is set to explore the possibilities offered in areas like the biomanufacturing of bioactive molecules, specialty chemicals, biologics and materials as well as food. Together, these areas are predicted to make up around 80 percent of synthetic biology’s potential impact on society.

As they continue to tinker with these natural systems, A*STAR scientists are standing at the brink of the next biomedical revolution—pushing the boundaries of life as we know it to great effect.

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References

[1] Zhou, Y., Xu, X., Wei, Y., Cheng, Y., Guo, Y., et al. A widespread pathway for substitution of adenine by diaminopurine in phage genomes. Science 372, 6541 (2021) | article

[2] Kimoto, M., Kawai, R., Mitsui, T., Yokoyama, S., Hirao, I. An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules. Nucleic Acids Research 37, 2 (2009) | article

[3] Betz, K., Kimoto, M., Diederichs, K., Hirao, I. Marx, A. Structural basis for expansion of the genetic alphabet with an artificial nucleobase pair. Angewandte Chemie 56, 39 (2017) | article

[4] Kimoto, M., Yamashige, R., Matsunaga, K., Yokoyama, S., Hirao, I. Generation of high-affinity DNA aptamers using an expanded genetic alphabet. Nature Biotechnology 31, 453-457 (2013) | article

[5] M. Kimoto, I. Hirao, Genetic alphabet expansion technology by creating unnatural base pairs. Chemical Society Reviews 49, 7602-7626 (2020) | article

[6] Hamashima, K., Soong, Y. Y., Matsunaga, K., Kimoto, M., Hirao, I. DNA sequencing method including unnatural bases for DNA aptamer generation by genetic alphabet expansion. ACS Synthetic Biology 8 (2019) | article

[7] Zhang, M.M., Wong, F., Wang, Y., Luo, S., Lim, Y.H., et al. CRISPR-Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters. Nature Chemical Biology 13, 607-609 (2017) | article

[8] Yeo, W.L., Heng, E., Tan, L.L., Lim, Y.W., Ching, K.C., et al. Biosynthetic engineering of the antifungal, anti-MRSA auroramycin. Microbial Cell Factories 19, 3 (2020) | article

[9] Lim. K.J.H., Lim, Y.P., Hartono, Y.D., Go, M.K., Fan, H., et al.. Biosynthesis of nature-inspired unnatural cannabinoids. Molecules 26, 10 (2021) | article

[10] Go, M.K., Zhao, L.N., Xue, B., Supekar, S., Robinson, R., et al. Directed computational evolution of quorum-quenching lactonases from the amidohydrolase superfamily. Structure 28 (6), 635-642 (2020) | article

[11] Zeng, G., Wang, Y., Chan, F.Y.,Wang, Y. One-step targeted gene deletion in Candida albicans haploids. Nature Protocols 9, 464-473 (2014) | article

[12] Li, Z., Wang, H., Cai, C., Wong, A.H., Wang, J., et al. Genome-wide piggyBac transposon-based mutagenesis and quantitative insertion-site analysis in haploid Candida species. Nature Protocols 15, 2705-2727 (2020) | article

[13] Gao, J., Chow, E.W.L., Wang, H., Xu, X., Cai, C., et al. LncRNA DINOR is a virulence factor and global regulator of stress responses in Candida auris. Nature Microbiology 6, 842–851 (2021) | article

[14] Truong, T., Suriyanarayanan, T., Zeng, G., Le, T.D., Liu, L., et al. Use of haploid model of Candida albicans to uncover mechanism of action of a novel antifungal agent. Frontiers in Cellular and Infection Microbiology 8, 164 (2018). | article

This article was made for A*STAR Research by Wildtype Media Group