Zengyi Shao

DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways

The assembly of large recombinant DNA encoding a whole biochemical pathway or genome represents a significant challenge. Here, we report a new method, DNA assembler, which allows the assembly of an entire biochemical pathway in a single step via in vivo homologous recombination in Saccharomyces cerevisiae. We show that DNA assembler can rapidly assemble a functional d-xylose utilization pathway (∼9 kb DNA consisting of three genes), a functional zeaxanthin biosynthesis pathway (∼11 kb DNA consisting of five genes) and a functional combined d-xylose utilization and zeaxanthin biosynthesis pathway (∼19 kb consisting of eight genes) with high efficiencies (70–100%) either on a plasmid or on a yeast chromosome. As this new method only requires simple DNA preparation and one-step yeast transformation, it represents a powerful tool in the construction of biochemical pathways for synthetic biology, metabolic engineering and functional genomics studies.

Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering

Escherichia coli only maintains a small amount of cellular malonyl-CoA, impeding its utility for overproducing natural products such as polyketides and flavonoids. Here, we report the use of various metabolic engineering strategies to redirect the carbon flux inside E. coli to pathways responsible for the generation of malonyl-CoA. Overexpression of acetyl-CoA carboxylase (Acc) resulted in 3-fold increase in cellular malonyl-CoA concentration. More importantly, overexpression of Acc showed a synergistic effect with increased acetyl-CoA availability, which was achieved by deletion of competing pathways leading to the byproducts acetate and ethanol as well as overexpression of an acetate assimilation enzyme. These engineering efforts led to the creation of an E. coli strain with 15-fold elevated cellular malonyl-CoA level. To demonstrate its utility, this engineered E. coli strain was used to produce an important polyketide, phloroglucinol, and showed near 4-fold higher titer compared with wild-type E. coli, despite the toxicity of phloroglucinol to cell growth. This engineered E. coli strain with elevated cellular malonyl-CoA level should be highly useful for improved production of important natural products where the cellular malonyl-CoA level is rate-limiting.

Engineering microbial factories for synthesis of value-added products.

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.

Activation and characterization of a cryptic polycyclic tetramate macrolactam biosynthetic gene cluster

Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of natural products with important biological activities. However, many of them have not been characterized. Here we apply a plug and play synthetic biology strategy to activate a cryptic PTM biosynthetic gene cluster SGR810-815 from Streptomyces griseus and discover three potential PTMs. This gene cluster is highly conserved in phylogenetically diverse bacterial strains and contains an unusual hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) which resembles iterative PKSs known in fungi. To further characterize this gene cluster, we use the same synthetic biology approach to create a series of gene deletion constructs and elucidate the biosynthetic steps for the formation of the polycyclic system. The strategy we employ bypasses the traditional laborious processes to elicit gene cluster expression and should be generally applicable to many other silent or cryptic gene clusters for discovery and characterization of new natural products.

Cloning and Characterization of a Panel of Constitutive Promoters for Applications in Pathway Engineering in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an important platform organism for synthesis of chemicals and fuels. However, the promoters used in most pathway engineering studies in S. cerevisiae have not been characterized and compared in parallel under multiple conditions that are routinely operated in laboratory and the number of known promoters is rather limited for the construction of large biochemical pathways. Here a total of 14 constitutive promoters from S. cerevisiae were cloned and characterized using a green fluorescent protein (GFP) as a reporter in a 2 µ vector pRS426, under varying glucose and oxygen concentrations. The strengths of these promoters varied no more than sixfold in the mean fluorescence intensity of GFP, with promoter TEF1p being the strongest and promoter PGI1p the weakest. As an example of application for these promoters in metabolic engineering, the genes involved in xylan degradation and zeaxanthin biosynthesis were subsequently cloned under the control of promoters with medium to high strength and assembled into a single pathway. The corresponding construct was transformed to a S. cerevisiae strain integrated with a D‐xylose utilizing pathway. The resulting strain produced zeaxanthin with a titer of 0.74 ± 0.02 mg/L directly from birchwood xylan. Biotechnol. Bioeng. 2012; 109:2082–2092.

Refactoring the silent spectinabilin gene cluster using a plug-and-play scaffold

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.

Rapid Characterization and Engineering of Natural Product Biosynthetic Pathways via DNA Assembler

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.

Combining Metabolic Engineering and Electrocatalysis: Application to the Production of Polyamides from Sugar.

Biorefineries aim to convert biomass into a spectrum of products ranging from biofuels to specialty chemicals. To achieve economically sustainable conversion, it is crucial to streamline the catalytic and downstream processing steps. In this work, a route that combines bio- and electrocatalysis to convert glucose into bio-based unsaturated nylon-6,6 is reported. An engineered strain of Saccharomyces cerevisiae was used as the initial biocatalyst for the conversion of glucose into muconic acid, with the highest reported muconic acid titer of 559.5 mg L(-1) in yeast. Without any separation, muconic acid was further electrocatalytically hydrogenated to 3-hexenedioic acid in 94 % yield despite the presence of biogenic impurities. Bio-based unsaturated nylon-6,6 (unsaturated polyamide-6,6) was finally obtained by polymerization of 3-hexenedioic acid with hexamethylenediamine.

Biosynthesis of 2-hydroxyethylphosphonate, an unexpected intermediate common to multiple phosphonate biosynthetic pathways.

Phosphonic acids encompass a common yet chemically diverse class of natural products that often possess potent biological activities. Here we report that, despite the significant structural differences among many of these compounds, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethyl-phosphonate, which is synthesized from phosphonoacetaldehyde by a distinct family of metal-dependent alcohol dehydrogenases (ADHs). Although the sequence identity of the ADH family members is relatively low (34–37%), in vitro biochemical characterization of the homologs involved in biosynthesis of the antibiotics fosfomycin, phosphinothricin tripeptide, and dehydrophos (formerly A53868) unequivocally confirms their enzymatic activities. These unique ADHs have exquisite substrate specificity, unusual metal requirements, and an unprecedented monomeric quaternary structure. Further, sequence analysis shows that these ADHs form a monophyletic group along with additional family members encoded by putative phosphonate biosynthetic gene clusters. Thus, the reduction of phosphonoacetaldehyde to hydroxyethyl-phosphonate may represent a common step in the biosynthesis of many phosphonate natural products, a finding that lends insight into the evolution of phosphonate biosynthetic pathways and the chemical structures of new C–P containing secondary metabolites.

DNA assembler: a synthetic biology tool for characterizing and engineering natural product gene clusters.

The majority of existing antibacterial and anticancer drugs are natural products or their derivatives. However, the characterization and engineering of these compounds are often hampered by limited ability to manipulate the corresponding biosynthetic pathways. Recently, we developed a genomics-driven, synthetic biology-based method, DNA assembler, for discovery, characterization, and engineering of natural product biosynthetic pathways (Shao, Luo, & Zhao, 2011). By taking advantage of the highly efficient yeast in vivo homologous recombination mechanism, this method synthesizes the entire expression vector containing the target biosynthetic pathway and the genetic elements needed for DNA maintenance and replication in individual hosts in a single-step manner. In this chapter, we describe the general guidelines for construct design. By using two distinct biosynthetic pathways, we demonstrate that DNA assembler can perform multiple tasks, including heterologous expression, introduction of single or multiple point mutations, scar-less gene deletion, generation of product derivatives, and creation of artificial gene clusters. As such, this method offers unprecedented flexibility and versatility in pathway manipulations.