Harnessing the Endogenous 2μ Plasmid of Saccharomyces cerevisiae for Pathway Construction

pRS episomal plasmids are widely used in Saccharomyces cerevisiae, owing to their easy genetic manipulations and high plasmid copy numbers (PCNs). Nevertheless, their broader application is hampered by the instability of the pRS plasmids. In this study, we designed an episomal plasmid based on the endogenous 2μ plasmid with both improved stability and increased PCN, naming it p2μM, a 2μ-modified plasmid. In the p2μM plasmid, an insertion site between the REP1 promoter and RAF1 promoter was identified, where the replication (ori) of Escherichia coli and a selection marker gene of S. cerevisiae were inserted. As a proof of concept, the tyrosol biosynthetic pathway was constructed in the p2μM plasmid and in a pRS plasmid (pRS423). As a result, the p2μM plasmid presented lower plasmid loss rate than that of pRS423. Furthermore, higher tyrosol titers were achieved in S. cerevisiae harboring p2μM plasmid carrying the tyrosol pathway-related genes. Our study provided an improved genetic manipulation tool in S. cerevisiae for metabolic engineering applications, which may be widely applied for valuable product biosynthesis in yeast

Genomic monitoring of SARS-CoV-2 uncovers an Nsp1 deletion variant that modulates type I interferon response

The SARS-CoV-2 virus, the causative agent of COVID-19, is undergoing constant mutation. Here, we utilized an integrative approach combining epidemiology, virus genome sequencing, clinical phenotyping, and experimental validation to locate mutations of clinical importance. We identified 35 recurrent variants, some of which are associated with clinical phenotypes related to severity. One variant, containing a deletion in the Nsp1-coding region (D500-532), was found in more than 20% of our sequenced samples and associates with higher RT-PCR cycle thresholds and lower serum IFN-beta levels of infected patients. Deletion variants in this locus were found in 37 countries worldwide, and viruses isolated from clinical samples or engineered by reverse genetics with related deletions in Nsp1 also induce lower IFN-beta responses in infected Calu-3 cells. Taken together, our virologic surveillance characterizes recurrent genetic diversity and identified mutations in Nsp1 of biological and clinical importance, which collectively may aid molecular diagnostics and drug design.Peer reviewe

Discovering novel natural products from bioactive plants and bacteria

Microorganisms and plants have evolved to produce a myriad array of natural products that are of biomedical importance. One group of valuable natural products is polyketides, which are known to be antiviral, antibacterial, and anticancer. In my first project, I attempted to develop an efficient strategy for cloning and characterizing Type III PKS genes that are likely responsible for the synthesis of novel natural products from Eucalyptus species. Nested degenerate primers were designed and a total of 94 clones and 27 clones were sequenced from the cDNA libraries created from the leaves of E. camaldulensis and E. robusta, respectively. Five unique putative Type III PKSs genes were identified. Two full-length genes were cloned and the biochemical characterization of these genes indicated their functions. In my second project, I attempted to develop a synthetic biology approach for natural product discovery. Investigation into the unknown compounds synthesized by actinomycetes may lead to the discovery of novel chemotherapeutic agents. As proof of concept, one cryptic pathway from Streptomyces griseus, containing a PKS/NRPS hybrid gene, was chosen as a model system. To decipher this cluster, I have been attempting to develop a new strategy based on our recently developed “DNA assembler” method. By using this approach, the gene cluster was modified by adding different constitutive promoters in front of each gene. RT-qPCR was employed to track expression on the transcription level. HPLC and LC-MS were used to detect the products. A potential product was detected and further characterization is in progress

Natural products discovery and characterization via synthetic biology

Microorganisms and plants have evolved to produce a myriad array of natural products that are of biomedical importance. Recent advances in synthetic biology have revolutionized our ability to discover and manipulate natural products biosynthetic pathways. This thesis describes in depth our efforts to enable natural products discovery and characterization via synthetic biology approaches. Type III PKSs produce a wide array of aromatic structures in spite of their structural simplicity. An efficient strategy for cloning and characterizing Type III PKS genes that are likely responsible for the synthesis of novel natural products from Eucalyptus species were developed. Five unique putative Type III PKSs genes were identified using this approach and two full-length genes were cloned and the biochemically characterized. With the recent development in genome sequencing projects, cryptic pathways serve as a potential source for novel natural products discovery. A synthetic biology approach for cryptic pathway activation and characterization was developed. One cryptic pathway from Streptomyces griseus, containing a PKS/NRPS hybrid gene, was chosen as a model system. To decipher this cluster, we have developed a plug-and-play platform based on the “DNA assembler” method. In this platform, one constitutive promoter was inserted in front of each gene involved in the pathway. qPCR data confirmed the increased transcription levels and HPLC data showed the formation of new polycyclic tetra macrolactams (PTMs) compounds. However, for PTM biosynthesis, no clear mechanism has been reported. Therefore, Chapter 4 describes the characterization of this PTM biosynthesis pathway by applying the same synthetic biology approach. We identified the boundary of this gene cluster by assembling a seven-gene construct and proposed a biosynthesis mechanism by studying a series of single-gene deletion and multiple-gene deletion constructs. Noticing that this one single gene cluster may have the potential to produce multiple products with closely related chemical structures, we biochemically characterized the modification enzymes in vitro and studied a phylogenetically related pathway as well. Finally, after achieving the success in the applications of our newly developed natural product discovery platform, we decided to make it more generally applicable for natural product discovery in actinomycetes. Additional strong constitutive promoters were identified via RNA-seq technique. The selected strong promoters were characterized based on both qPCR data and XylE enzyme specific activity assay. In total, 10 constitutive promoters were identified to be stronger than ermE*p, a widely used strong promoter reported in literature. These promoters will be used in our genomics-driven, synthetic biology platform for high throughput discovery of novel natural products in actinomycetes

Blossom of CRISPR technologies and applications in disease treatment

Since 2013, the CRISPR-based bacterial antiviral defense systems have revolutionized the genome editing field. In addition to genome editing, CRISPR has been developed as a variety of tools for gene expression regulations, live cell chromatin imaging, base editing, epigenome editing, and nucleic acid detection. Moreover, in the context of further boosting the usability and feasibility of CRISPR systems, novel CRISPR systems and engineered CRISPR protein mutants have been explored and studied actively. With the flourish of CRISPR technologies, they have been applied in disease treatment recently, as in gene therapy, cell therapy, immunotherapy, and antimicrobial therapy. Here we present the developments of CRISPR technologies and describe the applications of these CRISPR-based technologies in disease treatment. Keywords: CRISPR technologies, CRISPR-based tools, CRISPR-based therap

Coordinated regulation for nature products discovery and overproduction in Streptomyces

Streptomyces is an important treasure trove for natural products discovery. In recent years, many scientists focused on the genetic modification and metabolic regulation of Streptomyces to obtain diverse bioactive compounds with high yields. This review summarized the commonly used regulatory strategies for natural products discovery and overproduction in Streptomyces from three main aspects, including regulator-related strategies, promoter engineering, as well as other strategies employing transposons, signal factors, or feedback regulations. It is expected that the metabolic regulation network of Streptomyces will be elucidated more comprehensively to shed light on natural products research in the future

Recent Advances in the Biosynthesis of Natural Sugar Substitutes in Yeast

Natural sugar substitutes are safe, stable, and nearly calorie-free. Thus, they are gradually replacing the traditional high-calorie and artificial sweeteners in the food industry. Currently, the majority of natural sugar substitutes are extracted from plants, which often requires high levels of energy and causes environmental pollution. Recently, biosynthesis via engineered microbial cell factories has emerged as a green alternative for producing natural sugar substitutes. In this review, recent advances in the biosynthesis of natural sugar substitutes in yeasts are summarized. The metabolic engineering approaches reported for the biosynthesis of oligosaccharides, sugar alcohols, glycosides, and rare monosaccharides in various yeast strains are described. Meanwhile, some unresolved challenges in the bioproduction of natural sugar substitutes in yeast are discussed to offer guidance for future engineering

Rational construction of genome-minimized Streptomyces host for the expression of secondary metabolite gene clusters

Streptomyces offer a wealth of naturally occurring compounds with diverse structures, many of which possess significant pharmaceutical values. However, new product exploration and increased yield of specific compounds in Streptomyces have been technically challenging due to their slow growth rate, complex culture conditions and intricate genetic backgrounds. In this study, we screened dozens of Streptomyces strains inhabiting in a plant rhizosphere for fast-growing candidates, and further employed CRISPR/Cas-based engineering techniques for stepwise refinement of a particular strain, Streptomyces sp. A-14 that harbors a 7.47 Mb genome. After strategic removal of nonessential genomic regions and most gene clusters, we reduced its genome size to 6.13 Mb, while preserving its growth rate to the greatest extent. We further demonstrated that cleaner metabolic background of this engineered strain was well suited for the expression and characterization of heterologous gene clusters, including the biosynthetic pathways of actinorhodin and polycyclic tetramate macrolactams. Moreover, this streamlined genome is anticipated to facilitate directing the metabolic flux towards the production of desired compounds and increasing their yields

A method for Absolute Protein Expression Quantity Measurement Employing Insulator RiboJ

Measuring the absolute protein expression quantity for a specific promoter is necessary in the fields of both molecular biology and synthetic biology. The strength of a promoter is traditionally characterized by measuring the fluorescent intensity of the fluorescent protein downstream of the promoter. Until now, measurement of the absolute protein expression quantity for a promoter, however, has been unsuccessful in synthetic biology. The fact that the protein coding sequence influences the expression level for different proteins, and the inconvenience of measuring the absolute protein expression level, present a challenge to absolute quantitative measurement. Here, we introduce a new method that combines the insulator RiboJ with the standard fluorescence curve in order to measure the absolute protein expression quantity quickly; this method has been validated by modeling verification. Using this method, we successfully measured nine constitutive promoters in the Anderson promoter family. Our method provides data with higher accuracy for pathway design and is a straightforward way to standardize the strength of different promoters. Keywords: RiboJ, Promoter measurement, Synthetic biolog

RNAi-Assisted Genome Evolution in <i>Saccharomyces cerevisiae</i> for Complex Phenotype Engineering

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 <i>Saccharomyces cerevisiae</i>. 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 <i>Saccharomyces cerevisiae</i> for the first time by identifying two known and three novel suppressors of a telomerase-deficient mutation <i>yku70</i>Δ. 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